The complex is responsible for the degradation of UNC-45, a co-chaperone of the protein myosin B, required for proper assembly of myosin filaments in the ...
University of Freiburg Laboratory for Bioinformatics and Molecular Genetics
Suppressors of developmental myogenesis defects resulting in Duchenne muscular dystrophy, genes and mechanisms
Inaugural-Dissertation zur Erlangung der Doktorw¨ urde der Fakult¨at f¨ ur Biologie der Albert-Ludwigs-Universit¨at Freiburg im Breisgau
vorgelegt von Davide Faggionato aus Padua Freiburg im Breisgau 2010
Dekan der Fakult¨ at f¨ ur Biologie: Prof. Dr. Gunther Neuhaus Promotionsausschussvorsitzender: Prof. Dr. Karl-Friedrich Fischbach 1◦ Pr¨ ufer - Betreuer der Arbeit: Prof. Dr. Ralf Baumeister 2◦ Pr¨ ufer - Koreferent: P.D. Dr. Marcus Frank 3◦ Pr¨ ufer: Prof. Dr. Leena Bruckner-Tuderman Tag der m¨ undlichen Pr¨ ufung: 4 M¨arz 2011
c Copyright - 2010 Davide Faggionato
Ai miei genitori
In memoria di Giuseppe, Francesca e Terenzio
e believe we have now shown that there is a tendency in nature to the continued progression of certain classes of varieties further and further from the original type – a progression to which there appears no reason to assign any definite limits – and that the same principle which produces this result in a state of nature will also explain why domestic varieties have a tendency to revert to the original type. This progression, by minute steps, in various directions, but always checked and balanced by the necessary conditions, subject to which alone existence can be preserved, may, it is believed, be followed out so as to agree with all the phenomena presented by organized beings, their extinction and succession in past ages, and all the extraordinary modifications of form, instinct, and habits which they exhibit. Alfred Russel Wallace - On the Tendency of Varieties to Depart Indefinitely from the Original Type (1858) Journal of the Proceedings of the Linnean Society (Zoology), Vol.3, pp.53-62
W
se nel fossile di un cranio atavico riscopro forme che a me somigliano allora Adamo no non pu` o pi` u esistere e sette giorni soli son pochi per creare e ora ditemi se la mia genesi fu d’altri uomini o di quadrumani. Banco del Mutuo Soccorso – Darwin! (1972), L’evoluzione
E
ife has emerged in the universe without requiring special intervention from a Creator God. Should that fact lessen our wonder at the emergence and evolution of life and the evolution of the biosphere? No. Since we hold life to be sacred, we are stepping towards the reinvention of the sacred as the creativity in nature. Stuart Alan Kauffman - Reinventing the sacred (2008), Chap. The Origin of Life
L
Die folgenden praktischen Teile dieser Arbeit wurden von mir eigenst¨andig ausgef¨ uhrt: Chapter 3: – Section 3.1: ? ? ? ?
Subsection Subsection Subsection Subsection
3.1.1 3.1.2 3.1.4 3.1.5
– Section 3.2: ? Subsection 3.2.5 ? Subsection 3.2.6 ? Subsection 3.2.7 – Section 3.3 – Section 3.4: ? Subsection 3.4.1 (cloning of bait plasmids) ? Subsection 3.4.2 – Section 3.5 – Section 3.6 – Section 3.7 – Section 3.8
Table of Contents List of Figures
VI
List of Tables
IX
List of Videos
XI
List of Sequences
XII
Acknowledgements
XIII
Summary
XIV
1 Introduction 1.1
1.2
1
Caenorhabditis elegans as model organism . . . . . . . . . . . . . . . . . . .
1
1.1.1
C. elegans life cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2
1.1.2
Evolution of C. elegans and its significance in biomedical studies . .
3
Protein homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
1.2.1
Protein folding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
1.2.2
Protein degradation and the ubiquitin-proteasome degradation pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7
U-box E4 ligases connecting protein folding and protein degradation
11
Muscles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15
1.4
C. elegans musculature and myogenesis . . . . . . . . . . . . . . . . . . . . .
16
1.5
Duchenne Muscular Dystrophy – a historical perspective . . . . . . . . . . .
17
1.6
Dystrophin and the Dystrophin Glycoprotein Complex . . . . . . . . . . . .
18
1.7
Pathophysiology of Duchenne Muscular Dystrophy . . . . . . . . . . . . . .
21
1.8
C. elegans model of Duchenne Muscular Dystrophy . . . . . . . . . . . . . .
23
1.9
Thesis question . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
26
1.2.3 1.3
2 Methods 2.1
28
Molecular biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
28
2.1.1
28
Determination of Nucleic acid concentration . . . . . . . . . . . . . . I
II
TABLE OF CONTENTS
2.2
2.3
2.4
2.1.2
Polymerase chain reaction (PCR) . . . . . . . . . . . . . . . . . . . .
28
2.1.3
Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
29
2.1.4
Gateway
cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . .
30
2.1.5
DNA preparation and purification . . . . . . . . . . . . . . . . . . .
32
2.1.6
Total RNA preparation from C. elegans . . . . . . . . . . . . . . . .
32
2.1.7
Reverse Transcription and RT-PCR . . . . . . . . . . . . . . . . . .
33
Protein techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
34
2.2.1
34
Protein separation and visualisation . . . . . . . . . . . . . . . . . .
2.2.2
Expression and purification of recombinant proteins from E. coli . .
35
2.2.3
GST pull-down assay . . . . . . . . . . . . . . . . . . . . . . . . . . .
36
Microbiological techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . .
36
2.3.1
Preparation of chemically competent bacteria . . . . . . . . . . . . .
36
2.3.2
Transformation of E. coli
. . . . . . . . . . . . . . . . . . . . . . . .
37
2.3.3
Transformation of S. cerevisiae . . . . . . . . . . . . . . . . . . . . .
37
2.3.4
Split-ubiquitin system . . . . . . . . . . . . . . . . . . . . . . . . . .
38
Cell culture techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
40
2.4.1
Maintenance of HEK293 cell line . . . . . . . . . . . . . . . . . . . .
40
2.4.2
Cryopreservation and storage of HEK293 cells . . . . . . . . . . . . .
40
2.4.3
Overexpression of target protein in HEK293 cells upon transient transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
41
Harvesting of overexpressed target proteins from HEK293 cells . . .
41
C. elegans methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
41
2.5.1
Maintenance of C. elegans strains . . . . . . . . . . . . . . . . . . . .
41
2.5.2
Freezing of C. elegans strains . . . . . . . . . . . . . . . . . . . . . .
42
2.5.3
Crossing of C. elegans strains . . . . . . . . . . . . . . . . . . . . . .
42
2.4.4 2.5
TM
2.5.4
Outcrossing of C. elegans strains . . . . . . . . . . . . . . . . . . . .
42
2.5.5
Worm lysis for single worm PCR (SW-PCR) . . . . . . . . . . . . .
43
2.5.6
Decontamination and synchronization of C. elegans strains . . . . . .
43
2.5.7
Generation of transgenic C. elegans strains
43
2.5.7.1
. . . . . . . . . . . . . .
Neuronal chn-1 rescue strains of chn-1(by155) dys-1(cx18); hlh-1(cc561) . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5.7.2
2.5.8 2.5.9
44
Muscular chn-1 rescue strains of chn-1(by155) dys-1(cx18); hlh-1(cc561) . . . . . . . . . . . . . . . . . . . . . . . . . .
44
2.5.7.3
Reporter strains of genomic chn-1 under aex-3 promoter .
44
2.5.7.4
Reporter strains of genomic chn-1 under unc-54 promoter
45
RNA interference (RNAi) feeding experiments . . . . . . . . . . . . 0
45
2 -deoxy-5-fluorouridine (FUdR) treatment . . . . . . . . . . . . . .
46
2.5.10 Counts of eggs in uterus . . . . . . . . . . . . . . . . . . . . . . . . .
46
2.5.11 Food race assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
46
2.5.12 Motility assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
47
TABLE OF CONTENTS
III
2.5.13 Treatment with proteasome inhibitor MG132 . . . . . . . . . . . . .
48
2.5.14 Phalloidin staining . . . . . . . . . . . . . . . . . . . . . . . . . . . .
48
2.5.15 Lifespan analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
48
2.6
Statistical analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
48
2.7
Microscopy and imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
49
2.8
Bioinformatics tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
49
3 Results 3.1
50
Characterisation of Pdys-1 ::gfp, dys-1(cx18), and dys-1(cx18); hlh-1(cc561) 50 3.1.1
Expression pattern of Pdys-1 ::gfp . . . . . . . . . . . . . . . . . . . .
50
3.1.2
Aging experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . .
51
3.1.3
dys-1(cx18); hlh-1(cc561) body wall muscle cells show necrotic anomalies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
51
3.1.4
RT-PCR expression analysis of caveolins in dys-1(cx18); hlh-1(cc561) worms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.5
RT-PCR
expression
analysis
of
three
E3
ligases
in
dys-
1(cx18); hlh-1(cc561) worms . . . . . . . . . . . . . . . . . . . . . . . 3.2
53
Analysis of CHN-1/CHIP and UPS roles in the manifestation of muscle wasting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1 3.2.2
56
chn-1(by155) strongly ameliorates motility in the C. elegans DMD model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
56
chn-1(by155) strongly improves body wall muscle actin distribution in the C. elegans DMD model . . . . . . . . . . . . . . . . . . . . . .
57
3.2.3
chn-1(by155) strongly improves muscular myosin distribution in the
3.2.4
Loss of nuclear integrity in dys-1(cx18); hlh-1(cc561) body wall muscles 61
3.2.5
Expression of chn-1 in muscles but not in neurons is sufficient to
C. elegans DMD model . . . . . . . . . . . . . . . . . . . . . . . . . .
trigger muscle degeneration in the DMD model of C. elegans
60
. . . .
62
3.2.6
Contribution to muscle wasting of CHN-1 known E3 partners . . . .
65
3.2.7
Proteasome inhibition phenocopies motility amelioration of chn-1(by155) dys-1(cx18); hlh-1(cc561)
3.3
52
. . . . . . . . . . . . . . . . . . . . . . . .
66
RNAi suppression screening of motility defects of dys-1(cx18); hlh-1(cc561) worms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
67
3.3.1
67
RNAi feeding of 76 E3 ligases from the Ahringer RNAi library . . . 3.3.1.1
Speed measurements of dys-1(cx18); hlh-1(cc561) fed with nhl-3, sli-1, or F33H1.4 RNAi . . . . . . . . . . . . . . . .
67
3.3.1.2
Bioinformatics analysis of F33H1.4
68
3.3.1.3
dys-1(cx18); hlh-1(cc561); F33H1.4(RNAi) animals show an
. . . . . . . . . . . . .
ameliorate muscular actin distribution in the dorsal mid body region . . . . . . . . . . . . . . . . . . . . . . . . . . .
70
IV
TABLE OF CONTENTS 3.3.1.4
dys-1(cx18); hlh-1(cc561); F33H1.4(RNAi) worms have fewer eggs in utero than dys-1(cx18); hlh-1(cc561) worms
3.3.1.5
. . . .
Contribution of FUdR treatment and F33H1.4 RNAi to fertility and worm motility . . . . . . . . . . . . . . . . . .
3.3.2
71 72
Selection of 108 E2 and E3 ubiquitin ligase clones from the ORFeome RNAi library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2.1
Speed measurements of F08G12.5, C32D5.10, vps-11, and lin-41 RNAi . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.2.2
74 75
Sequencing of the positive clones and confirmation of motility amelioration of dys-1(cx18); hlh-1(cc561) fed with lin-41
3.4
RNAi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
76
Biochemical analysis of HsCHIP and CHN-1 protein . . . . . . . . . . . . .
77
3.4.1
Split-ubiquitin yeast-two-hybrid protein interaction studies of HsCHIP 77
3.4.2
Bioinformatics comparison of Rpn13 and C56G2.7 – GST-Pull down experiment of CHN-1 and C56G2.7 . . . . . . . . . . . . . . . . . . .
81
3.5
Expression pattern of C56G2.7 . . . . . . . . . . . . . . . . . . . . . . . . .
83
3.6
Genomic characterisation of the C56G2.7(tm3442) deletion mutant . . . . .
84
3.7
C56G2.7(tm3442) does not suppress motility loss of dystrophic animals . .
85
3.8
Synthetic phenotypes of C56G2.7(tm3442) and rpn-10(RNAi) . . . . . . .
86
4 Discussion 4.1
88
Characterisation of the C. elegans DMD model . . . . . . . . . . . . . . . .
88
4.1.1
Dystrophin is broadly expressed in C. elegans . . . . . . . . . . . . .
88
4.1.2
Dystrophin deficiency shortens lifespan in C. elegans . . . . . . . . .
88
4.1.3
Muscle degeneration in the C. elegans DMD model is a progressive phenomenon showing necrotic hallmarks . . . . . . . . . . . . . . . .
4.1.4
Caveolin expression in the C. elegans DMD model is comparable to wild type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.5
90
The RING gene trim-9 is down regulated in the C. elegans DMD model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
91
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
92
A mutation in CHN-1/CHIP suppresses muscle degeneration in C. elegans .
93
4.1.6 4.2
89
4.2.1
Loss of chn-1 activity ameliorates motility in the C. elegans DMD
4.2.2
Loss of chn-1 activity strongly improves general muscular histology
model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . in the C. elegans DMD model . . . . . . . . . . . . . . . . . . . . . . 4.2.3
93 94
chn-1 activity in muscles is necessary to trigger muscle degeneration in the C. elegans DMD model . . . . . . . . . . . . . . . . . . . . . .
95
4.2.4
chn-1 plays a major role in muscle wasting . . . . . . . . . . . . . .
96
4.2.5
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
97
TABLE OF CONTENTS 4.3
RNAi suppressor screenings . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1
98
F33H1.4 RNAi ameliorates worm motility and muscular histology of the C. elegans DMD model . . . . . . . . . . . . . . . . . . . . . .
4.4
V
99
4.3.2
vps-11 is a putative suppressor of locomotory defects of dys-1(cx18);
4.3.3
hlh-1(cc561) worms . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Protein interactions of CHN-1/CHIP . . . . . . . . . . . . . . . . . . . . . . 103 4.4.1
C56G2.7, the C. elegans homologue of Rpn13, interacts with CHN-1 104
4.4.2
Loss of C56G2.7 cannot suppress motility defects of the C. elegans DMD model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
4.4.3
Loss of C56G2.7 and rpn-10 triggers a severe pleiotropic effect in-
4.4.4
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
fluencing development and egg laying . . . . . . . . . . . . . . . . . 106
5 Materials 5.1
110
Chemicals and consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 5.1.1
Enzymes and chemicals used for molecular and biochemical analysis 111
5.1.2
Kits, resins, and chemicals used for protein analysis . . . . . . . . . 112
5.1.3
Kits and components used for molecular analysis . . . . . . . . . . . 113
5.1.4
Chemicals and solutions used for cell culture and transient transfection113
5.1.5
Chemicals used for yeast culture . . . . . . . . . . . . . . . . . . . . 114
5.2
Devices and equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
5.3
Buffers, media and solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
5.4 5.5
Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
5.6
Plasmids and vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
5.7
Strains and cell lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
5.8
Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
A Appendix
134
A.1 Vector maps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 A.2 Videos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 A.3 Nucleotide and amino acid sequences . . . . . . . . . . . . . . . . . . . . . . 142 A.4 Ahringer RNAi clones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 A.5 ORFeome RNAi clones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 A.6 Split-ubiquitin hits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 B Abbreviations
160
References
165
List of Figures 1.1
Life cycle of hermaphrodite C. elegans at 22◦ C . . . . . . . . . . . . . . . .
3
1.2
The two cladistics trees suggested by recent evolutionary analyses . . . . . .
4
1.3
Schematic view of the ubiquitin-proteasome system (UPS) . . . . . . . . . .
11
1.4
Dot plot matrixes of C. elegans CHN-1 versus Homo sapiens (H. sapiens) CHIP and C. elegans UFD-2 versus H. sapiens UFD2 . . . . . . . . . . . . .
14
1.5
Dot plot matrix of C. elegans HLH-1 versus H. sapiens MyoD1 . . . . . . .
17
1.6
Schematic view of the DGC in vertebrates . . . . . . . . . . . . . . . . . . .
20
1.7
Dot plot matrix of C. elegans DYS-1 (DYS-1) versus H. sapiens dystrophin (HsDYS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24
1.8
Schematic view of the DGC in C. elegans
25
2.1
Schematic view of the BR and LR reaction occurring during the Gateway
cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
31
2.2
Schematic view of the cloning site for the bait fragment in the pPCup1 ubc9 -CRU plasmid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
39
2.3
. . . . . . . . . . . . . . . . . . . TM
Example of motility analysis of two wild type animals with the DIAS 3.4.2 software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
47
3.1
Expression analysis of Pdys-1 ::gfp . . . . . . . . . . . . . . . . . . . . . . . .
50
3.2
Aging graph of dys-1(cx18) . . . . . . . . . . . . . . . . . . . . . . . . . . .
51
3.3
Representative DIC picture of mid-body region of dys-1(cx18); hlh-1(cc561) mutant worm two days after the adult moult . . . . . . . . . . . . . . . . .
52
3.4
RT-PCR expression analysis of cav-1 and cav-2 . . . . . . . . . . . . . . . .
52
3.5
Dot plot matrix of TRIM-9a versus MuRF1 . . . . . . . . . . . . . . . . . .
54
3.6
Dot plot matrix of C32E8.11 versus E3αII . . . . . . . . . . . . . . . . . . .
55
3.7
RT-PCR expression analysis of C32E8.11, mfb-1, and trim-9a/b . . . . . . .
55
3.8
Suppression of the motility defects of dystrophic worms by chn-1(by155) mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.9
57
Suppression of muscle degeneration defects of dystrophic worms by chn1(by155) mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI
59
LIST OF FIGURES
VII
3.10 Improvement of myosin patterns in chn-1(by155) dys-1(cx18); hlh-1(cc561) mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
60
3.11 Degradation of muscle cell nuclei in dystrophin-deficient worms . . . . . . .
61
3.12 Gchn-1::gfp is properly expressed in neurons and body wall muscles . . . .
63
3.13 Expression of chn-1 in the musculature is sufficient to rescue the chn1(by155) suppression of dystrophic animals . . . . . . . . . . . . . . . . . .
64
3.14 Suppression of motility defects of dystrophic worms by mutants of chn-1 interactor genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
65
3.15 MG132 treatment improves motility of dys-1(cx18); hlh-1(cc561) dystrophic animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
66
3.16 Speed measurement of RNAi fed worms . . . . . . . . . . . . . . . . . . . .
68
3.17 Bioinformatics analysis of F33H1.4 . . . . . . . . . . . . . . . . . . . . . . .
69
3.18 Actin patterns of dorsal body wall muscle cells after F33H1.4 RNAi feeding
70
3.19 Eggs in uterus of dys-1(cx18); hlh-1(cc561) and dys-1(cx18); hlh-1(cc561); F33H1.4(RNAi) worms . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
71
3.20 Effect of 100 µM FUdR treatment and F33H1.4 RNAi on the amount of eggs in uterus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
72
3.21 Speed measurement of worms treated with FUdR and fed with F33H1.4 RNAi vector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
73
3.22 Speed measurement of dys-1(cx18); hlh-1(cc561) worms fed with different ORFeome RNAi clones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
75
3.23 Length of second day adult dys-1(cx18); hlh-1(cc561) worms fed with lin-41 RNAi from the Ahringer library . . . . . . . . . . . . . . . . . . . . . . . . .
76
3.24 Schematic view of HsCHIP and the two truncated variants HsCHIP(∆Ubox) and HsCHIP(∆TPR) . . . . . . . . . . . . . . . . . . . . . . . . . . . .
77
3.25 Expression test of HsCHIP(∆TPR) construct . . . . . . . . . . . . . . . . .
78
3.26 Biological functions of candidate HsCHIP(∆TPR) interactors . . . . . . . .
78
3.27 Schematic representation of the subcellular localisation of candidate HsCHIP (∆TPR) interactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
79
3.28 Pairwise transformation of HsCHIP(∆TPR) with Rpn13 and CFL1 . . . .
80
3.29 Dot plot matrix of C56G2.7 versus HsRpn13
. . . . . . . . . . . . . . . . .
81
3.30 Confirmation of the interaction between CHN-1 and C56G2.7 . . . . . . . .
82
3.31 Expression analysis of C56G2.7 . . . . . . . . . . . . . . . . . . . . . . . . .
83
3.32 Schematic view of wild type C56G2.7 and the C56G2.7(tm3442) allele . . .
84
3.33 Influence of C56G2.7(tm3442) and rpn-10(RNAi) on motility of dys-1(cx18); hlh1(cc561) worms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
85
3.34 The proteasomal genes C56G2.7 and rpn-10 contribute to normal worms development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
86
3.35 The proteasomal components C56G2.7 and rpn-10 influence egg laying . .
87
VIII
LIST OF FIGURES
A.1 Plasmid-map of RNAi feeding vector L4440 . . . . . . . . . . . . . . . . . . 134 A.2 Plasmid-map of split-ubiquitin bait vector pCub . . . . . . . . . . . . . . . 135 A.3 Plasmid-map of split-ubiquitin bait vector pNub . . . . . . . . . . . . . . . 135 A.4 Plasmid-map of pDONR221 . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 A.5 Plasmid-map of pPD30.38 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 A.6 Plasmid-map of pPD117.01 . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 A.7 Plasmid-map of pBY2085 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 A.8 Plasmid-map of pcDNA3.1/nV5-DEST . . . . . . . . . . . . . . . . . . . . . 138 A.9 Plasmid-map of pBY2912 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 A.10 Plasmid-map of pPD114.95 . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
List of Tables 2.1
Exemplary PCR conditions . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2
PCR settings for expression analysis of cav-1, cav-2, and housekeeping gene eft-3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3
29 33
PCR settings for expression analysis of C32E8.11, trim-9a/b, mfb-1, and housekeeping gene eft-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
33
2.4
Composition of 12% separating gel . . . . . . . . . . . . . . . . . . . . . . .
34
2.5
Composition of 4% stacking gels . . . . . . . . . . . . . . . . . . . . . . . .
34
3.1
Food race assay of second-day adult animals . . . . . . . . . . . . . . . . . .
56
3.2
Body wall muscle cell count of second-day adult animals . . . . . . . . . . .
58
5.1
List of enzymes and chemicals used for molecular and biochemical analysis . 111
5.2
List of kits, resins, and chemicals used for protein analysis . . . . . . . . . . 112
5.3
List of kits and components used for molecular analysis . . . . . . . . . . . 113
5.4
List of chemicals and solutions used for cell culture and transient transfection113
5.5
List of chemicals used for yeast culture . . . . . . . . . . . . . . . . . . . . . 114
5.6
List of C. elegans buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
5.7
List of buffers for competent cells preparation and transformation . . . . . . 116
5.8
List of buffers for protein extraction and precipitation . . . . . . . . . . . . 117
5.9
List of buffers for protein analysis . . . . . . . . . . . . . . . . . . . . . . . . 117
5.10 List of buffers for DNA analysis . . . . . . . . . . . . . . . . . . . . . . . . . 118 5.11 List of media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 5.12 List of primary antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 5.13 List of secondary antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 5.14 List of oligonucleotides used in this study . . . . . . . . . . . . . . . . . . . 120 5.15 List of plasmids and vectors used in this study . . . . . . . . . . . . . . . . 124 5.16 List of E. coli strains used in this study . . . . . . . . . . . . . . . . . . . . 126 5.17 List of S. cerevisiae strains used in this study . . . . . . . . . . . . . . . . . 126 5.18 List of H. sapiens cell lines used in this study . . . . . . . . . . . . . . . . . 126 5.19 List of C. elegans strains used in this study . . . . . . . . . . . . . . . . . . 127 IX
X
LIST OF TABLES 5.20 List of software used in this study . . . . . . . . . . . . . . . . . . . . . . . 132 A.1 List of Ahringer RNAi clones used in this study . . . . . . . . . . . . . . . . 149 A.2 List of ORFeome RNAi clones used in this study . . . . . . . . . . . . . . . 153 A.3 Split ubiquitin hits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
List of Videos A.1 2-day-old adult dys-1(cx18); hlh-1(cc561) hermaphrodite on an NGM plate without OP50 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 A.2 2-day-old adult chn-1(by155) dys-1(cx18); hlh-1(cc561) hermaphrodite on an NGM plate without OP50 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
XI
List of Sequences A.1 Genomic sequence of chn-1 used as template for rescue experiments of chn-1(by155) dys-1(cx18); hlh-1(cc561) . . . . . . . . . . . . . . . . . . . . . 142 A.2 Coding sequence of chn-1 used as template for the generation of the pulldown bait vector pBY1875 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 A.3 Coding sequence of HsCHIP used as template for the generation of the split-ubiquitin bait vector pBY2555 . . . . . . . . . . . . . . . . . . . . . . . 143 A.4 Amino acid sequence of HsCHIP coded by the split-ubiquitin bait vector pBY2555 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 A.5 Coding sequence of HsCHIP(∆U-box) used as template for the generation of the split-ubiquitin bait vector pBY2577 . . . . . . . . . . . . . . . . . . . 144 A.6 Amino acid sequence of HsCHIP(∆U-box) coded by the split-ubiquitin bait vector pBY2577 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 A.7 Coding sequence of HsCHIP(∆TPR) used as template for the generation of the split-ubiquitin bait vector pBY2578 . . . . . . . . . . . . . . . . . . . . 144 A.8 Amino acid sequence of HsCHIP(∆TPR) coded by the split-ubiquitin bait vector pBY2578 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 A.9 Coding sequence of C56G2.7 used as template for the generation of the donor vector pBY2666 and the pulldown prey vector pBY2912 . . . . . . . 145 A.10 Amino acid sequence of C56G2.7 coded by the pulldown prey vector pBY2912145 A.11 Partial sequence of the F33H1.4 Ahringer RNAi vector . . . . . . . . . . . 145 A.12 Partial sequence of the vps-11 ORFeome RNAi vector . . . . . . . . . . . . 146 A.13 Partial sequence of the lin-41 ORFeome RNAi vector . . . . . . . . . . . . 146 A.14 Partial sequence of the lin-41 Ahringer RNAi vector . . . . . . . . . . . . . 147 A.15 Partial sequence of the rpn-10 Ahringer RNAi vector . . . . . . . . . . . . . 147 A.16 Genomic sequence of wild type C56G2.7 and mutant allele C56G2.7(tm3442)148
XII
Acknowledgements
F
irst of all, I would like to thank Prof. Dr. Ralf Baumeister for giving me the opportunity to conduct my PhD thesis in his lab.
I would like to thank my supervising committee Prof. Dr. Ralf Baumeister, Prof. Dr. Heimut Omran, and Dr. Verdon Taylor for their guidance and useful advices they provided. I thank GRK1104 for funding and all the members of the Graduiertenkolleg for support and scientific discussions. In particular I express my gratitude to Prof. Dr. Annette Neub¨ user for excellent coordination of the Research Training Program and to Cornelia Wagner for her administrative management of the GRK1104. Thanks to all the members of Baumeister lab, former and current, especially to P.D. Dr. Ekkehard Schulze to whom I owe a debt of gratitude for the high valuable scientific advices it gave me during my graduation and writing of this thesis. Thanks to Monika Messerschmidt for performing the split-ubiquitin screening and for her exceptional technical assistance and philosophical chatting as bench mate in the last period of my graduation. A big thanks to Dr. Oyunbileg Nyamsuren-von Stackelberg, Dr. Wiebke Loch, and Angelika Sch¨ afer for their precious collaboration on the experiments concerning CHN-1/CHIP. I would like to thank Katrin Pansa for her excellent job in the secretary of the Baumeister lab. To Malikmohamed Yousuf and Polychronis Fatouros goes my gratitude for the intense hours spent in an eternal l∞p inside and outside the lab. Thanks to Luca Rizzini for the constant provision of caffeine (or was it theine?), scientific discussions, and for giving me the opportunity to uncover the practical meaning of “serendipity”.
Eine liebliche kostbare Einzigkeit
XIII
Summary
P
rotein quality control exerts an essential role in the development and maintenance of living cells. An alteration in the balance of protein homeostasis can lead to
the manifestation of different pathologies. Components of a certain class of protein ubiquitin ligases (E3/E4) have been recently identified as pivotal proteins linking the proteasome degradation pathway to protein quality control. In particular, the highly conserved E3/E4 ligases CHN-1/CHIP was shown to be part of a multiprotein complex in muscles, interacting with molecular chaperons and other E3/E4 ubiquitin ligases. This protein complex is required for proper myosin assembly and is therefore crucial for myogenesis. In this study, the Duchenne Muscular Dystrophy (DMD) model of C. elegans was further characterised and a relevant contribution of CHN-1/CHIP in the manifestation of DMD was revealed. Detailed analysis of the C. elegans DMD model evidenced necrotic degradation of muscle cells and a shorter lifespan compared to wild type. These features recapitulate the mammalian DMD model and the human muscular pathology. The data presented in this study show that chn-1 mutation specifically suppresses the age related muscle degeneration of DMD worms. Blockage of the proteasome with the chemical compound MG132 phenocopied the motility amelioration triggered by the chn-1 mutation. This proves that the proteasome exerts a major role in the progression of muscle loss. Analysis of transgenic lines overexpressing chn-1 exclusively in muscles or in neurons provided a direct link between CHN-1 muscular activity and muscle wasting. Moreover, the results show that a role in muscle degeneration of the CHN-1/CHIP interacting partner PDR-1/Parkin cannot be excluded. To identify new genetic factors involved in muscle wasting, two independent RNAi screenings with selected candidate genes were performed. The RNAi experiments identified the two genes F33H1.4 and vps-11 as possible suppressors of muscle degeneration. Detailed RNAi feeding experiments with F33H1.4 confirmed its suppression activity and identified this gene as a bona fide suppressor of muscle wasting in the C. elegans DMD model. Additionally, an interaction screening based on the split ubiquitin technology – a modified method of the yeast two hybrid system – was performed to find new partners and substrates of CHN-1/CHIP. Further biochemical characterisation of promising candidates led to the identification of the proteasomal component C56G2.7/Rpn13 as interaction XIV
XV partner of CHN-1/CHIP. Altogether, these data disclose a critical role for CHN-1/CHIP in the control of muscle wasting and degeneration, designating CHN-1/CHIP as a potential drug target for the treatment of DMD. In addition, the study provides new candidate genes possibly involved in the process of muscle wasting and expands the CHN-1/CHIP interactome.
1. Introduction
D
uchenne muscular dystrophy (DMD) affects one in 3,300 newborn males and occupies a prominent position among monogenic human disorders. In Duchenne
muscular dystrophy, due to catastrophic muscular decay, muscle integrity is being lost successively from early adulthood onwards eventually leading to premature death of affected patients. Although the last decades have seen significant progresses in the characterisation of this muscular dystrophy, the molecular factors involved had been poorly understood. In particular, possible implications in the pathology of factors that actively control and promote muscle maintenance through the quality control of muscular proteins had not been intensely investigated. Recently, such factors have been shown to actively participate in proper muscular development and integrity. Moreover, the perturbation of targets of this molecular machinery appears to be related to the development and to the manifestation of some muscular pathogenic processes. Taking advantage of the simplicity of the nematode C. elegans and its prominence as biomedical model organism, this work further characterizes the C. elegans DMD model. Moreover, this study explores and elucidates the contribution to muscle degeneration of factors responsible for protein quality control and involved in muscle development and maintenance.
1.1
Caenorhabditis elegans as model organism
Caenorhabditis elegans (C. elegans) is a free living soil nematode that feeds on bacteria. Under natural conditions C. elegans occurs in two sexes: hermaphrodite and male. Hermaphrodites are self fertilising organisms with 5 autosomal chromosomes and 2 sexual chromosomes (XX). Males have 5 autosomal chromosomes and are hemizygous for the sexual chromosome (XO). Males show a low spontaneous occurrence (0,1%) in a population at laboratory conditions. However, the abundance can approach 50% once male/hermaphrodite mating occurs (Altun & Hall, 2008a). In the laboratory C. elegans is typically maintained in Petri dishes on the surface of an agar medium seeded with Escherichia coli (E. coli ). Stocks of C. elegans strains can be 1
2
1. INTRODUCTION
preserved for long periods of time, typically many years, by cryoconservation in ultra low freezers at -80◦ C. It has been predicted that C. elegans genome codes for about 20,000 genes (Claverie, 2001). Among these genes, 83% are clear homologues of human genes as inferred by a comparative analysis of expressed sequence tag (EST) (Lai et al., 2000; Rubin et al., 2000). This high degree of conservation places C. elegans at a privileged position to study genetics of human hereditary diseases. Its relatively small genome of roughly 97 million base pairs was sequenced in 1998 as the first genome of a multicellular organism (The C. elegans Sequencing Consortium, 1998). All informations about C. elegans genome are available online from the website http://wormbase.org/ (Chen et al., 2005). The life-long maintained body transparency of C. elegans allows microscopic observation of its body anatomy both in vivo and after fixation. One peculiarity of C. elegans is the predetermination of the fate of its cells. The hermaphrodite body bears exactly 959 somatic cells whereas the male consist of 1031 somatic cells. Since the lineage is largely invariant, the fate of each single cell can be followed through the whole animal life (Sulston & Horvitz, 1977; Sulston et al., 1980). The small size of about 1 mm in length of adult individuals, its short life cycle of 3 days, its short lifespan of 14 days under laboratory conditions, and its straightforward manipulation and inexpensive handling makes it one of the most prominent model organism in genetics and molecular biology. In order to study genetics of muscle degeneration in detail, advantage has been taken in this work from the straightforward way to manipulate the organism C. elegans.
1.1.1
C. elegans life cycle
C. elegans life cycle can be divided into three major steps, i.e. embryogenesis, larval development, and adulthood. Embryogenesis takes place after egg fertilisation in the hermaphrodite uterus. After the egg has been laid the embryo undergoes organogenesis and the young larva (stage L1) finally hatches from the egg. The hatched nematode passes four larval stages (L1, L2, L3, and L4) until it reaches adulthood. Shifts between the different stages are marked by moulting processes during which the new cuticle surrounding the animal replaces the old one. A particular larval stage, called the dauer larva follows the L2 stage when the environmental conditions are not favourable to a proper development. Unfavourable conditions may be determined by scarcity of food resources, overcrowded population or an adverse environment in general. In this dauer larval stage, the animal stops feeding and reduces movements compared to a non stressed animal. Apart from a change in morphology this particular larval stage has a lifespan that is overwhelmingly longer than the adult worm (Cassada & Russell, 1975). In case environmental conditions will eventually change, the dauer larvae can re-enter the development program and reach the L3/L4 moult.
1.1. Caenorhabditis elegans as model organism
3
The last moult after the L4 stage leads to an adult animal. Adult animals reach sexual maturity and start laying eggs or search for mature hermaphrodites in the case of hermaphrodite or male, respectively. Under standard laboratory conditions, C. elegans reproductive life cycle from egg to fertile animal takes about three days. Once having reached fertility, the adult hermaphrodite animal is fertile for 3-4 days, has a lifespan of 10-15 days and generates about 300 progenies (Fig. 1.1) (Altun & Hall, 2008a).
Figure 1.1 – Life cycle of hermaphrodite C. elegans at 22◦ C. The egg is laid at approximately the 30 cells stage. Dramatic changes in the embryo occur during extra utero development until the egg hatch releases an L1 larva. Post hatching development passes through four larval stages (L1, L2, L3, and L4) before the worm reaches adulthood. A conditional developmental choice before the L1/L2 moult is offered by the dauer larva stage. The names of the stages and the dimension in µm of the animal are indicated in black. The time required for the transition from one stage to the next is expressed in hours (hr) and specified in light blue. Adapted from Altun & Hall (2008a).
1.1.2
Evolution of C. elegans and its significance in biomedical studies
Since the introduction of C. elegans as model organism by Sydney Brenner (Brenner, 1974), the small nematode has been broadly analysed.
4
1. INTRODUCTION From a phylogenetic point of view, there are two relevant cladistics trees that can be
produced by an evolutional comparison of the following three clades: vertebrates, nematodes, and arthropods like the fruit fly Drosophila melanogaster (D. melanogaster ). The first scenario called “Coelomata hypothesis”, focuses on the timing of appearance of a body cavity (coelom) in metazoans during development. Following this working hypothesis, arthropods and vertebrates cluster together on one branch of the phylogenetic tree and the nematodes on the other branch. The second scenario, namely “Ecdysozoa hypothesis” stresses on the similarity between animals undergoing moult. According to this hypothesis, nematodes and arthropods cluster on one branch whereas vertebrates diverge to another branch. Analysis of the 18S rRNA supported this idea. However, the Coelomata hypothesis has recently gained relevance once comparative genomics have reinforced the underlying assumptions. Although more phylogenetic data are required to reinforce this hypothesis, it is currently assumed that insects (arthropods) are evolutionarily and genetically closer to vertebrates than to nematodes (Fig. 1.2) (Blair et al., 2002; Fitch, 2005; Telford, 2004).
Figure 1.2 – The two cladistics trees suggested by recent evolutionary analyses. On the left side, the ecdysozoa hypothesis clusters arthropods and nematodes together based on the moulting process common to both groups. On the right side, the coelomata hypothesis highlights the early formation of a hollow tube during embryogenesis. The latter hypothesis is regarded as the most correct and was reinforced by comparative genomics data. Adapted from Blair et al. (2002).
As suggested by the Coelomata hypothesis C. elegans diverged utterly in the past from the phylogenetic branches that later developed to the arthropods and the vertebrates clades. In the perspective of this hypothesis C. elegans gains a privileged place among model organisms relevant for biomedical studies. Shared molecular or physiological features between vertebrates and nematodes could be derived either by a common ancestor or by convergent evolution. In case that conserved characteristics originated before the separation of the nematodes from the vertebrate and arthropod clades, their conservation reflects the key role that they have for the processes they control. This suggests that conserved features could underlie basic biological mechanisms developed early in the evolutionary history. From this perspective, C. elegans’s place in the evolutionary tree offers a privileged insight into crucial mechanisms that build metazoans bodies, associate to diseases, and
1.2. Protein homeostasis
5
underlie aging processes.
1.2
Protein homeostasis
The tight regulation of catabolic and anabolic proteolytic processes is a key requirement for most cells of living organisms. The two contenders in protein homeostasis are the protein folding and protein maturation processes on one side and the anabolic pathways for the degradation of cellular constituents on the other side. The balance between anabolic and catabolic processes is a general hallmark of cellular functions and it is fine tuned to respond to the physiological necessities. Conversely many pathological distresses are characterized by an imbalance between the two forces (Malgaroli et al., 2006).
1.2.1
Protein folding
Protein folding is a continuous process in most living cells. It allows the right packaging of nascent polypeptide chains and avoids misfolding under physiological and denaturing conditions. Many human pathologies are connected to protein misfolding and to the so called Unfolded Protein Response (UPR), such as phenylketonuria, desmin-related myopathy, Parkinson’s disease, amyotrophic lateral sclerosis, and Huntington disease (Bauer & Nukina, 2009; Bruijn et al., 2004; Gamez et al., 2000; Moore et al., 2005; Vicart et al., 1998). This critical duty is accomplished by chaperone proteins, shortly chaperons. Chaperon proteins are divided into two classes, i.e. monomeric chaperons and chaperonins. The main difference between the two groups is that the monomeric chaperons usually work alone in the first steps of protein folding (secondary structures) and have low target specificity, whereas the chaperonins form oligomeric barrel structures, have higher affinity for specific target proteins, and intervene later in the folding process (tertiary structures). The high conservation of core steps in the protein folding system among prokaryotes and eukaryotes points out its evolutionary relevance and conserved significance for all organisms (Archibald et al., 2000). The different affinity for unfolded proteins and the different time of action divide the folding process into two crucial steps (Young et al., 2004). The first step begins directly after the newly formed polypeptide emerges from the ribosome. These first moments are crucial as hydrophobic stretches could interact with each other leading to protein aggregates that could have a cytotoxic effect. In the eukaryotic cytosol the chaperone protein related to the 70-kDa heat-shock protein (Hsp70) known as 70-kDa heat-shock cognate protein (Hsc70) is responsible for the first steps of protein folding. Hsc70 is ubiquitously expressed and the most abundant chaperon in the cytosol (Kawagoe et al., 1992). It recognises short stretches of hydrophobic amino acids of nascent polypeptides and binds to them preventing unspecific interactions (Eggers et al., 1997). Hsc70 interacts with
6
1. INTRODUCTION
several proteins and cofactors like the 40-kDa heat-shock protein (Hsp40) and HDJ2, that in turn influences its activity (Eggers et al., 1997; Terada et al., 1997). The interaction of Hsc70 with the target peptide is regulated by the cofactor ATP. In the ATP bound state Hsc70 releases the peptide, whereas the incorporation of ADP tightens the affinity to the nascent protein. Nucleotide exchange is facilitated by accessory proteins like Hsp70interacting protein (HIP), which favours the ATP bound state facilitating the polypeptide release, and BCL2-associated athanogene-1 (BAG1) which favours the interaction with ADP, and therefore, the polypeptide bound state (Nollen et al., 2001). The second step of protein folding begins immediately after the polypeptide binds to Hsc70 and its cofactor Hsp40. At this time point the stabilized nascent polypeptide has to be properly processed in the right secondary and tertiary structure in order to reach its native conformation. At this step the cyotosolic polypeptide can be usually processed by either the chaperonins TCP1 ring complex (TRiC) or the 90-kDa heat-shock protein (Hsp90) folding pathway. TCP1 belongs to the 60-kDa Heat-Shock Protein (Hsp60) class of chaperonins and is formed by two rings juxtaposed to each other. Each ring is composed of eight different subunits. Typically, Hsc70 interacts with TCP1 by presenting the stretched polypeptide to be folded (Cuellar et al., 2008; Frydman et al., 1994). Through cycles of ATP hydrolysis TCP1 first incorporates the polypeptide in the central cavity of one of its rings and finally the internal structure of the ring helps the target polypeptide to be folded correctly (Valpuesta et al., 2002). In the cytosol, Hsp90 is present as a homodimer. Like the TCP1 complex it mediates an ATP dependent protein folding process. The single Hsp90 monomer has an ATP binding N-terminus, a central region and a C-terminus responsible for protein dimerisation. Once Hsp90 binds ATP to its N-terminus, a transient dimerisation of the N-terminus takes place. This transient dimerisation event seems to trigger the proper folding of the target protein. After ATP is hydrolyzed, the whole structure opens and releases the folded target protein (Grenert et al., 1999; Panaretou et al., 1998; Young et al., 2001). Although less studied than the TCP1 complex, Hsp90 has recently gained importance as many studies addressed its relevance in medical context for its role in cancer development and viral capsid protein maturation. Moreover, it was shown that Hsp90 and Hsp90related proteins contribute to a buffering system responsible to suppress the influence on the phenotype of some genetic mutations. Guiding the folding of some aberrant form of proteins to the correct conformation, Hsp90 could have an important role in evolutionary processes (Mayer et al., 2009; Sangster et al., 2004). One of the most relevant characteristics of Hsp90 is that it vastly increases its arrays of targets and specificity by differential cooperation with diverse cofactors. One of the advantages for the cell is that through the combination of a relatively low number of starting components it can gain extremely vast chaperon specificity for virtually each of its constituent proteins. A specific class of interacting motif, the tetratricopeptide repeat (TPR) is present
1.2. Protein homeostasis
7
in Hsp90 and Hsc70. It was shown that the TPR motif allows interaction of different cochaperons with the Hsp90 and the Hsc70 chaperons (Scheufler et al., 2000). The first strong evidence of the crucial role of TPR domains in Hsp90 and Hsc70 chaperons functions was provided by the mammalian co-chaperon Hsp-organizing protein (HOP). HOP interacts with Hsp90 through its TPR domains and has proven to influence Hsp90 substrates release by affecting ATP hydrolysis and to mediate substrate presenting from Hsc70 to Hsp90 (Prodromou et al., 1999).
1.2.2
Protein degradation and the ubiquitin-proteasome degradation pathway
Three main routes contribute to protein clearance in mammalian cells: the lysosom, the protein degradation exerted by the mitochondrial proteases, and the cyotosolic proteases including the ubiquitin-proteasome pathway (Ding & Yin, 2008; Rubinsztein, 2006). Lysosoms are intracellular acidic organelles that enclose special acid-optimized proteases and hydrolases. Lysosomal activity is of particular importance during the immunological responses. The immunogenic polypeptide is degraded in the lysosom into little antigenic fragments which are then presented by the major histocompatibility complex II (Colbert et al., 2009). Protein degradation in the organelles occurs via ATP mediated proteolysis exerted by high molecular weight proteases without the involvement of ubiquitin. These proteases are equivalent to those found in bacteria and can degrade short polypeptides as well as whole proteins to single amino acids (Koppen & Langer, 2007). In the crowded cyotosolic environment a vast array of proteins is available for the continuous recycling of protein constituents. The gross part of protein degradation in the cytosol is conducted by the fine tuned ubiquitin-proteasome pathway, whereas the calpains and caspases, two major groups of cytosolic proteases, have predominant roles in particular cellular states (Jung et al., 2009). Calpains are Calcium (Ca2+ ) activated cysteine proteases that increase their activity when the calcium content increases as in the case of pathological states like tissue injury, necrosis or autolysis (Goll et al., 1992). Caspases are responsible for the activation of the apoptotic pathway in eukaryotes (Salvesen & Dixit, 1997). The ubiquitin-proteasome pathway (UPS) is the degradative pathway that mostly affects cytosolic and nuclear protein lifetime. It requires an array of proteins that organise the concerted degradation of target proteins through ATP consuming catalytic activity. Most of the proteins in eukaryotic cells are degraded through this pathway (Jung et al., 2009). The UPS can be divided into two distinct steps: the labelling of the target protein with a polyubiquitin chain, and the degradation by the proteasome. The UPS pathway as a whole has a high energetic demand. It requires energy a) for the recruitment and the tagging and b) for the degradation of the target proteins. The high energy costs are
8
1. INTRODUCTION
required to maintain the high sensitivity and specificity of the UPS that converges to one of the biggest and most refined protein machinery of the cells: the proteasome (Tanaka, 2009). The proteasome is composed of a catalytic core particle (CP) and one or two regulatory particle(s) (RP). The CP, also known as 20S proteasome, consists of two outer α rings and two inner β rings (Dahlmann et al., 1989). Both rings are formed by seven structurally related proteins. The superimposition of the four rings confers a barrel shape to the CP with a hollow space traversing the whole structure (Tanaka, 2009). Protein degradation takes place inside the hollow channel. The two inner rings are the catalytic core of the CP that mediates the unspecific degradation of the target polypeptides through Caspase-like, Trypsin-like, and Chymotrypsin-like hydrolysis (Tanaka, 2009). This barrel shape of the CP confines the unspecific hydrolysis activity to a controlled environment and delegates most of the specificity of the whole UPS system to the recruitment and the recognition of target proteins. The RP, also known as 19S proteasome, is responsible for the recognition of target proteins, their deubiquitylation and presentation to the CP for destruction. The RP, formed by 20 components divided in catalytic and non catalytic, can be present at one or both openings of the CP. The catalytic components have ATPase properties and use ATP hydrolysis as energy source for the deubiquitylation and the unfolding of the target proteins. The non catalytic components are assigned prevalently to structural roles and act as “receptors” of the target proteins (Glickman et al., 1998). The RP itself can be divided into two subunits, the lid and the base subcomplex. The main function of the lid is to receive the target protein and deubiquitylate it. The base helps in the deubiquitylation process and is responsible for the unfolding and the presentation of the target proteins to the α ring of the CP (Smith et al., 2006; Rosenzweig et al., 2008; Verma et al., 2002). The targeting of candidate proteins to the proteasome requires a complex cascade of specific events that culminate in the conjugation of the target protein with several moieties of ubiquitin. Ubiquitin is a 76 amino acids long protein highly conserved among all eukaryotic organisms. It is expressed as a polyprotein that, after cleavage, matures into single ubiquitin monomers (Ozkaynak et al., 1984). Conjugation of ubiquitin to the target protein occurs by a peptide bound between the -amino group of a lysine residue of the target protein and the terminal glycine of ubiquitin. The addition of subsequent ubiquitin moieties, called polyubiquitination, occurs through the conjugation of other ubiquitin moieties to the first ubiquitin. These ubiquitin moieties are bound through the terminal glycine to one of the five lysine residues of the ubiquitin moiety. Depending on how many ubiquitin moieties are covalently tagged to the protein and at which position they are linked to each other, they can induce protein degradation or regulate the protein’s functions. Moreover, the protein’s destiny is determined by the lysine of the target protein initiating the ubiquitination
1.2. Protein homeostasis
9
process (Spence et al., 1995). The priming of target proteins with ubiquitin moieties and the polyubiquitination of primed proteins requires the involvement of three different enzymes in a strictly hierarchical order. These enzymes are responsible for the three steps culminating in ubiquitin conjugation: ubiquitin activation mediated by E1 enzymes, ubiquitin transportation mediated by E2 enzymes and, finally, ubiquitin ligation to the target protein mediated by E3 enzymes. Ubiquitin ligation to the target protein can include a transient binding of the ubiquitin molecule to the E3 or alternatively the direct ligation by the E2 enzyme. Typically the E1 enzymes class is not numerous. The E2s are characterized by many different proteins, whereas the E3 family is represented by many proteins able to form vast variety of multi protein complexes. This increase in effectors involved in ubiquitination towards the final steps of the pathway enables the UPS to gain target specificity and sensitivity in the last step of the pathway, where it is most required (Hershko & Ciechanover, 1998) (Fig. 1.3). Generally the E1 enzyme has a molecular mass of about 115-130 kDa and is responsible for ubiquitin activation. E1 enzymes covalently bind a single ubiquitin moiety via a thioester bond that involves the C-terminal glycine of ubiquitin and a cysteine on the C-terminus of the E1 enzyme (Hershko et al., 1983; Lee & Schindelin, 2008). The E2 enzyme interacts with the E1 enzyme and accepts the activated ubiquitin moiety. The ubiquitin moiety is transferred to a specialized domain of the E2 enzyme, the ubiquitin-conjugating (Ubc) domain. This particular domain of 150 amino acids has a catalytic cysteine that binds ubiquitin by a thioester bond (Hurley et al., 2006). Once ubiquitin is bound to the E2 it is carried into proximity of the target protein in order to be transferred. The transfer of ubiquitin to the target protein is mediated by the E3 ligase. The E3 enzyme has to be in close proximity to the target protein and to ubiquitin. Therefore a close interaction of the E2 conjugated to ubiquitin, the E3, and the target protein is required. For most proteins the signal that triggers ubiquitin priming of the target protein and protein polyubiquitination is not well known and thought to require interaction of E enzymes with other cofactors. One of the best characterised degradation pathway is represented by the N-end rule pathway that implies proteasomemediated degradation of short living proteins through the reception by dedicated E3 ligases that recognise specific features on the N-terminus of the target protein (Tasaki et al., 2009). Usually protein polyubiquitination proceeds with polymerisation of ubiquitin moieties conjugated through a covalent bound between lysine 48 and glycine 76, and although ubiquitin polymerisation can proceed with other residues, the lysine 48 to glycine 76 bound seems the most common for targeting to proteasome degradation (Chau et al., 1989; Mastrandrea et al., 1999) (Fig. 1.3). So far E3 ligases have been divided into three major classes on the basis of the catalytic domain: the Homologous to E6-AP Carboxyl Terminus (HECT) domain, the Really Interesting New Gene (RING) domain, which are the most abundant E3 ligases in eukaryotes,
10
1. INTRODUCTION
and the U-box domain. In the HECT domain E3 ligases, the HECT domain actively mediates the enzymatic reaction required for the conjugation of ubiquitin to its target. The HECT domain is a region of approximately 350 residues with a core cysteine circa 35 amino acids upstream of the C-terminus (Huibregtse et al., 1995). This cysteine is required for the formation of a thioester link with ubiquitin essential for the ubiquitination of the substrate protein. Although the HECT class of E3 ligases is represented by 28 genes in human (Li et al., 2008), its members are involved in many different physiological roles spanning from cell survival over hormonal response to Human papillomavirus (HPV)-induced cervical cancers, amongst others (Rotin & Kumar, 2009). Typically, protein-specific domains outside the HECT region allow its binding to the substrate, suggesting that other domains than the HECT itself determine the target specificity (Schwarz et al., 1998). The RING domain is characterised by a cluster of histidines and cysteines that allows the coordination of zinc ions in a typical cross-brace structure. Different lines of evidence have shown that the RING domain has a role as organizer of the E2 enzyme whereas other domains of the RING protein are responsible for the recognition of the ubiquitination signal (Haupt et al., 1997; Joazeiro et al., 1999; Kubbutat et al., 1997). The zinc ions and the ligand that interact with the RING domain are chemically inert. This feature suggests that the RING E3 ligases have a structural rather than chemical role during protein ubiquitination. The RING domain could therefore have much broader functions beyond the involvement as protein interacting motif in the ubiquitination pathway (Borden, 2000). Supporting this hypothesis, the RING domain does not necessarily participate in the formation of the thiol link with ubiquitin for the conjugation of ubiquitin with the target protein (Seol et al., 1999). The RING class of ubiquitin ligases is represented by 300 predicted proteins in human and is therefore the most numerous family of E3 ligases (Li et al., 2008). The first discoveries of a U-box protein and its connection with the UPS arose from studies in Saccharomyces cerevisiae, where the genetic dissection of the UPS led to the identification of the U-box E3 ligase UFD2 (Ub fusion degradation protein 2) as ubiquitinating protein (Johnson et al., 1995). The 75 amino acids long U-box domain is extremely similar to the RING domain. The main difference lies in its lack of cysteines necessary in the RING domain for the coordination with the zinc ion. The high degree of similarity suggests that the U-box domain could have originated early in the evolutionary history from the RING domain. From bioinformatics data it seems plausible that the U-box domain folds similarly to the RING domain. However, instead of the coordination with zinc it is thought to form salt bonds that keep the structure in proper shape (Aravind & Koonin, 2000). It was indeed shown that U-box domain proteins in combination with an E2 enzyme can trigger ubiquitination of substrate proteins (Hatakeyama et al., 2001). Moreover U-box proteins have the capability to drive the polymerisation of polyubiquitin chains in lysine other than lysine 48. This feature differentiates them from RING and HECT E3
1.2. Protein homeostasis
11
ligases (Hatakeyama et al., 2001; Patterson, 2002). This work will focus on the role of the ubiquitin-proteasome pathway in muscle maintenance and degeneration.
1.2.3
U-box E4 ligases connecting protein folding and protein degradation
Recently it was shown that the U-box protein UFD2 has the unprecedented capability of specifically elongate ubiquitin chains of target proteins primed with ubiquitin by an E3 ligase. This novel kind of enzymatic protein was named E4 ligase (Fig. 1.3) (Koegl et al., 1999).
Figure 1.3 – Schematic view of the ubiquitin-proteasome system (UPS). The first step for protein degradation is the activation of a moiety of ubiquitin (grey dot) from the free ubiquitin pool by an E1 enzyme (light yellow) through a covalent thioester bound. The E1 enzyme transfers the ubiquitin moiety to an E2 enzyme (light green) that forms a direct thioester bond with ubiquitin and releases a free E1 enzyme. An E3 enzyme (red) finally catalyses the formation of a peptide bond between the -amino group of a lysine in the target protein (light blue) and the terminal glycine of ubiquitin releasing uncoupled E2 and E3 enzymes. Polyubiquitination occurs through repeated cycles of interactions between the ubiquitin bound E2 ligase and the target specific E3 ligase. Recently a new class of ubiquitin ligase enzymes (E4) (blue) has been discovered. E4 ligases are a specialized class of enzymes that efficiently polyubiquitinate target proteins. As in the case of the UFD-2-CHN-1 E3/E4 complex, the two enzymes alone have E3 activity for their target protein UNC-45 leading to UNC-45 monoubiquitination. Once the two enzymes are simultaneously present they steadily polyubiquitinate UNC-45 (see text for details). Once the target protein has been polyubiquitinated, it can be degraded by the proteasome multiprotein complex (yellow, regulatory part of the proteasome; brown, α rings; orange, β rings; the α and β rings together form the proteasome core particle). After degradation the proteasome releases free ubiquitin moieties that join the ubiquitin pool, and short polypeptides (small light blue squares) that are further degraded by cyotosolic or nuclear proteases.
UFD2 recognises protein primed with short polyubiquitin chains, interacts with the
12
1. INTRODUCTION
ubiquitin moieties and elongates the ubiquitin chain through a mechanism that does not seem to require a stable interaction with E2 enzymes. Although E4 polyubiquitination normally targets candidate proteins to the proteasome, E4 polyubiquitination can proceed with other lysine than the canonical lysine 48. This suggests an involvement of E4 ligases in other biological function not related to the UPS (Hatakeyama et al., 2001). In the last years new proteins with E4 enzymatic activity that do not bear the U-box domain were discovered, like p300, and the yeast RSP5-BUL1-BUL2 complex. Both proteins do not share obvious domain similarity with RING, HECT, or U-box. Hence, E4 ligases activity might not be restricted to U-box proteins (Grossman et al., 2003; Helliwell et al., 2001). Another significant U-box E3 ligase with E4 activity is the Carboxy terminus of Hsc70 Interacting Protein (CHIP). A search for TPR domain bearing proteins resulted in the discovery of CHIP in humans (Ballinger et al., 1999). It has been subsequently clarified that CHIP presents two clear domains: three tandem TPR motifs at its N-terminus and a U-box domain at its C-terminus. The TPR domains allow CHIP to interact with mammalian Hsc70. It was shown that this interaction negatively regulates Hsc70 substrate binding (Ballinger et al., 1999). Moreover, the U-box domain at the C-terminus is responsible for its E3 ligase activity on unfolded proteins bound to Hsc70 (Jiang et al., 2001). Subsequent discoveries confirmed the involvement of CHIP in the fate of unfolded or unstable proteins (Murata et al., 2001). Other work revealed a significant role for CHIP in the progression of pathologies triggered by aberrant protein folding like Huntington’s disease (HD) and other polyglutamine (polyQ) neurodegenerative diseases (Miller et al., 2005). The involvement of CHIP was found to be relevant in the oncogenic pathway, where it is involved in the degradation of phosphorylated Tau, a protein relevant in the pathology of Alzheimer disease, and to interact with Parkin, an E3 ligase involved in the progression of juvenile Parkinson disease (Hatakeyama et al., 2004; Imai et al., 2002; Kajiro et al., 2009; Shimura et al., 2004). These new discoveries lead to a profound increase in knowledge of the connections between protein degradation and protein folding revealing that CHIP functions are relevant for a plethora of different cellular functions. A convincing proof of the newly discovered role of the U-box protein CHIP in the fate of newly synthesized proteins comes along with its interaction with Hsp90. It was shown that CHIP is involved in the steady state of the glucocorticoid receptor, a target of Hsp90, by inducing its ubiquitylation and degradation through the proteasome. These results suggest that CHIP is part of a protein complex that plays an important role in protein folding and protein degradation (Connell et al., 2001; Rosser et al., 2007). Clear evidences of the importance of E3/E4 ligase complexes in the fine tuning of UPS and folding pathway in protein homeostasis were provided by two works on C. elegans body wall muscles (Hoppe et al., 2004; Janiesch et al., 2007). The results of these studies clearly showed that CHN-1 and UFD-2, the worm homologues of CHIP and UFD2 (Hoppe et al., 2004; Khan & Nukina, 2004) (Fig. 1.4), interact and form a new functional E3/E4 ligases
1.2. Protein homeostasis
13
complex. The complex is responsible for the degradation of UNC-45, a co-chaperone of the protein myosin B, required for proper assembly of myosin filaments in the worm (Barral et al., 2002). Hoppe et al. (2004) showed that the two proteins alone trigger the addition of one to three ubiquitin moieties to UNC-45. Once the two proteins have been added simultaneously in the presence of an E1 enzyme, an E2 enzyme, ubiquitin, ATP, and their target UNC-45, the amount of polyubiquitinated UNC-45 with more than three ubiquitin increases dramatically. The polyubiquitinated UNC-45 is thought to be consequentially digested by the proteasome in vivo. The fact that CHN-1 expression increases in muscles at the end of the L4 stage when the musculature of the worm has acquired its final shape and size, suggests that the mechanism could indeed regulate UNC-45 homeostasis in vivo (Hoppe et al., 2004). Moreover, in the case of the uncoordinated movement phenotype triggered by the temperature sensitive (ts) unc-45 point mutants like unc-45(m94) and unc-45(e286), a combination with the loss of function mutant chn-1(by155) restores the motility of the worms at the non permissive temperature (Hoppe et al., 2004). The genetic suppression exerted by chn-1(by155) can be interpreted in terms of suppression of a UPR response triggered by the mutated form of unc-45. This model suggests that once unc-45(ts) worms are kept at the non permissive temperature, the sensitive allele, although molecularly functional, is recognised as aberrant by components of the protein quality control and thus degraded. In turn, loss of degrading component as chn-1 specifically blocks the proteasomal degradation of the aberrant UNC-45 protein restoring a muscular development close to normality (Hoppe, 2008; Kim et al., 2008). Further experiments have shown that the CHN-1−UFD-2 complex could indeed interact with other cofactors like the ubiquitin-selective co-chaperon CDC-48, the C. elegans homologue of human p97. It was shown that the p97-CHIP-UFD2 complex is relevant for the modulation of myosin assembly and myofibril organisation in human and C. elegans, suggesting a high degree of evolutionary conservation in the mechanisms involved in muscle development and maintenance (Janiesch et al., 2007). Furthermore, CHN-1 was shown to associate with HSP-1, the C. elegans homologue of Hsp70 and to interact and co-localize in muscles with the E2 ligases UBC-25, supporting the view that CHN-1/CHIP molecular interactions and possibly its functions are conserved among metazoans (Sch¨ affer et al., 2010). These results uncover a new kind of cellular apparatus called the triage system. In this system, decision of refolding and degradation of proteins are made by a concerted interplay between chaperons, chaperonins and the degradation machinery. Recently, a triage system formed by CHIP−Hsp70/Hsp90−UBC4/UBC5 was shown to be responsible for the quality control of transgenic mutated luciferase under denaturing conditions. These in vitro experiments showed that CHIP functions could be relevant for the quality control of a vast array of unrelated proteins (Marques et al., 2006).
14
1. INTRODUCTION
Figure 1.4 – Dot plot matrixes of C. elegans CHN-1 versus Homo sapiens (H. sapiens) CHIP and C. elegans UFD-2 versus H. sapiens UFD2. a – The three diagonal lines highlight the regions of similarity between CHN-1 (horizontal) and CHIP (vertical). The line close to the black square reflects the similarity of three TPR domains conserved among the two proteins. The line close to the red square represents a central region of similarity between the two proteins with no homology with annotated protein domains. The line close to the gray square reflects the similarity among the C-terminus of the two proteins where the U-box domain is located. Alignment between the two proteins showed that 40% of the amino acids are identical and 75% are similar (Khan & Nukina, 2004). b – The diagonal lines highlight the region of similarity between UFD-2 (horizontal) and UFD2 (vertical). The line close to the black square emphasises the high degree of similarity among the C-terminus of the two proteins where the U-box domain is located. The two proteins share 26% of identity at the amino acidic level (Hoppe et al., 2004). a – CHN-1, C. elegans CHN-1 protein (horizontal); CHIP, H. sapiens CHIP protein (vertical). Sliding window length: 23; Greyramp tool min cutoff score: 55; Greyramp tool max cutoff score: 57. b – UFD-2, C. elegans UFD-2 protein (horizontal); UFD2, H. sapiens UFD2 protein (vertical). Sliding window length: 20; Greyramp tool min cutoff score: 35; Greyramp tool max cutoff score: 73. Numbers on axis of abscissae and ordinates indicate single amino acid positions. These dot plot matrixes are based on the BLOSUM62 alignment score matrix (Eddy, 2004).
It is now clear that the selectivity of protein ubiquitination grants its target specificity in the final steps of protein ubiquitination, where E3 and E3/E4 proteins collaborate together. Thanks to the vast amount of possible protein-protein interactions of its components, the degradation machinery increases its range of target specificity. Summarising these new findings, the Hsc70−Hsp90 machinery and its satellite co-chaperons do not only participate in the protein folding processes but, in connection with specific ubiquitin ligases, they connect the folding processes with the degradation machinery. In this way the cell can tightly control the balance between protein degradation and protein folding. These protein folding/degradation complexes offer an interesting array of putative drug
1.3. Muscles
15
target proteins especially in the context of pathologies characterised by the specific impairment of components of the triage systems or pathologies triggered by cellular events controlled by the triage system.
1.3
Muscles
Muscles can be defined as molecular machines that convert biochemical energy into labour. They are present in most metazoans. Through contraction these organs absolve different functions in the organism. Muscles permit movements, facilitate work of internal organs and allow body fluid movement. In vertebrates, muscles can be divided into three categories: cardiac, smooth and striated skeletal muscles. Cardiac muscles are found only in the heart and are involuntary; smooth muscles are involuntary like cardiac muscles and responsible for movements of internal organs and contraction of blood vessels. Skeletal muscles are the only voluntary muscles in vertebrates and are formed - in contrast to cardiac and smooth muscles - by multinucleated cells. The functional contractile unit of muscles, the sarcomere, is formed by the two proteins myosin and actin. Monomers of the two proteins are organized in long filaments, thin filaments in the case of actin and thick filaments in the case of myosin, that interdigitate with each other. During hydrolysis of ATP, chemical energy is converted into sliding movements of the two filaments along each other. The synchronised sliding of numerous sarcomere units is transmitted to the muscular membrane (sarcolemma) through anchoring structures (the Z line for actin and the M line for myosin) leading to muscle contraction. Despite dramatic morphological differences in size between the metazoan clade, muscles display an astonishing similarity at the molecular level among animals. Seen from an evolutionary perspective the high degree of similarity amongst metazoan striated muscles can be explained by the presence of highly specialized protein complexes (OOta & Saitou, 1999). The functionality of each protein subunit depends on its interactions with other proteins of the complex. Therefore a mutation in one of these proteins can be detrimental for the functionality of the whole complex. Accordingly, maintenance of muscles has been under strong evolutive pressure from the paradigm “no muscle no movements”, leading to a strongly conserved homology in muscle functions and molecular composition (OOta & Saitou, 1999). The two most common invertebrate models for genetic studies in muscles are the fruit fly D. melanogaster and the soil nematode C. elegans. D. melanogaster adults have muscles of all three kinds that are divided into somatic gonad muscles, visceral muscles, cardiac muscles and somatic muscles. In contrast, C. elegans hermaphrodites’ muscles belong to two categories: striated somatic and nonstriated muscles (Altun & Hall, 2008b; Hartenstein, 2006; Taylor, 1995). D. melanogaster and C. elegans are proven to be reliable model organisms for studies
16
1. INTRODUCTION
on muscle development (Baylies & Michelson, 2001), muscle pathologies (Baumeister & Ge, 2002; Sparrow et al., 2008), and genetic diseases (Rubin et al., 2000). In C. elegans, in contrast to D. melanogaster, muscles cells do not undergo dramatic changes during the transition from the larval to the adult stage. Hence, it is possible to follow the fate of each single muscle cell throughout development in a deterministic manner.
1.4
C. elegans musculature and myogenesis
Thanks to the astonishing conservation of the basic muscular functions throughout the evolution of metazoans, many major contributions in the understanding of fundamental issues in muscles raise from genetics studies of muscle development and muscle maintenance in invertebrates (Altun & Hall, 2008b; Epstein & Bernstein, 1992). Being the most abundant muscle type in C. elegans, striated muscles are represented by 95 body wall muscles cells organized in two arrays (dorsal and ventral), each array arranged in two adjacent muscle rows (left and right). The body wall muscles are multisarcomere mononucleated cells devoted to locomotion. Non striated muscles in the hermaphrodite comprise the contractile gonadal sheath, 1 anal sphincter muscle and 1 anal depressor muscle, 2 stomato-intestinal muscles, 8 vulva muscles, 8 uterine muscles, and 20 pharyngeal muscles responsible for feeding. In the male the vulva, the uterine muscles, and the gonadal sheath are replaced by 41 specialized mating muscles (Altun & Hall, 2008b). Striated and non striated muscles in C. elegans differ mainly in their sarcomere orientation and in the attachments to the cuticle. Striated muscles show attachment points evenly distributed on their surface, whereas nonstriated muscles attach to the cuticle mainly by focal adhesions (Hooper & Thuma, 2005). Most of these muscles develop in the embryo, except 14 body wall muscles, 8 vulva muscles, and 8 uterine muscles that develop post embryonically (Altun & Hall, 2008b). Compared to mammals the process of muscle development in C. elegans is dramatically different. Skeletal muscle myogenesis in mouse is a developmental event affecting some particular stem cells, the myoblast. In the myoblast the whole network of factors required for myogenesis converge to a few transcription factors called the myogenic regulatory factors (MRFs), all belonging to the basic helix-loop-helix domain-containing family. These factors are: myogenic factor 5 (Myf5 ), myogenic differentiation 1 (MyoD1, also known as MyoD), myogenic factor 6 (Myf6 also known as Mrf4 ) and myogenin (Myog). These transcription factors, although being the ultimate effectors for the developmental program of muscle formation, interact with other cofactors and with each other. Myf5 and Myf6 effects on myogenesis were found to be both dependent and independent of MyoD in triggering the early steps of myogenesis. On the other side the myoblasts terminal differentiation was shown to be controlled by Myf6 and Myog, suggesting that a complex cross regulating network is required in mammals for proper skeletal muscle development
1.5. Duchenne Muscular Dystrophy – a historical perspective
17
(Bryson-Richardson & Currie, 2008). In C. elegans the complexity of the helix-loop-helix domain-containing transcription factors family responsible for skeletal muscle myogenesis is represented by a single gene, hlh-1. HLH-1 is 79% identical to mouse MyoD within the functional domain required for myogenic activation, the helix-loop-helix motif (Krause et al., 1990) (Fig. 1.5).
Figure 1.5 – Dot plot matrix of C. elegans HLH-1 versus H. sapiens MyoD1. The diagonal line close to the black square highlights the region of similarity between HLH-1 (horizontal) and MyoD1 (vertical). This region corresponds to the central helix-loop-helix motif, hallmark of this family of myogenic transcription factors. HLH-1, C. elegans HLH-1 protein (horizontal); MyoD1, H. sapiens MyoD1 protein (vertical). Numbers on axis of abscissae and ordinates indicate single amino acid positions. This dot plot matrix is based on the BLOSUM62 alignment score matrix (Eddy, 2004). Sliding window length: 22 – Greyramp tool min cutoff score: 60 – Greyramp tool max cutoff score: 61.
Similarly to vertebrate MyoD, hlh-1 shows a specific pattern of expression characterised by activation in body wall muscle progenitors and in adult body wall muscles. It was shown that hlh-1 is required for proper morphogenesis and proper functioning of body wall muscles (Chen et al., 1994). Although myogenesis in C. elegans is an interplay of two other myogenic factors: unc-120/SRF and hnd-1/HAND, overexpression of hlh-1 alone during early embryogenesis generates muscle-like fate of almost all cells, highlighting hlh-1 major role in myogenesis (Fukushige & Krause, 2005).
1.5
Duchenne Muscular Dystrophy – a historical perspective
Duchenne Muscular Dystrophy (DMD, OMIM#310200) is a congenital neuromuscular disorder leading to a progressive and irreversible loss of motility and muscle strength that eventually ends with the patient’s death. One of the first descriptions of this myopathy
18
1. INTRODUCTION
originates from Duchenne de Boulogne. Duchenne for the first time described the male predominance, the time dependent loss of muscular strength, the pseudo-hypertrophy and the early death associated with the pathology in 1858 (Pearce, 2005). Currently DMD is the most severe and most common type among the muscular dystrophies. It affects one in 3,300 males’ births. In the typical patient affected by this disorder, the first symptoms manifest at 3-5 years. Characteristic symptoms are an increase of creatine kinase activity above physiological level, a pseudo hypertrophy of calf muscles, a progressive loss of physical ability due to muscular fibre necrosis, and finally patients display a mild non-progressive cognitive impairment. Altogether, these symptoms highlight DMD as a neuromuscular human disorder (O’Brien & Kunkel, 2001). Most of the patients are dependent on a wheelchair at the age of 12 and many of them die of respiratory failure in their twenties (O’Brien & Kunkel, 2001). In 1886 W. Gowers described the correlation between the inheritance of the pathology and a common maternal lineage especially in the case of children with the same mother but with different paternity. Although not clearly recognised at that time this pattern of hereditability is a hallmark of X chromosome linked recessive diseases (Gowers, 1886). In 1953 P. E. Becker proposed a form of familial X-linked muscular dystrophy with a similar hereditary pattern, but with a much milder aetiology, a later age of onset and slower progression (Becker & Kiener, 1955). This kind of muscular dystrophy is now known as Becker Muscular Dystrophy (BMD), that was found to be an allelic variant of DMD (O’Brien & Kunkel, 2001).
1.6
Dystrophin and the Dystrophin Glycoprotein Complex
For over more than a century, the aetiological genetic agent responsible for the pathology of DMD had been elusive. In 1988, when the entire 14-kb DNA sequence of the affected gene was finally identified, the protein product of 3685 amino acids was named dystrophin (Hoffman et al., 1987; K¨ onig et al., 1988). Since these pioneering molecular works, a detailed analysis of the gene and its function begun and a considerable amount of data has been generated on DMD and its aetiolant agent: dystrophin. The dystrophin gene is now known to be among the largest and most complex genes in human genome, spanning over more than 2.5 million bp corresponding to 0.1% of the total human genome and 1.5% of the X chromosome (Kunkel & Hoffman, 1989; Mandel, 1989; Manole, 1995). The effort to study the dystrophin gene in detail has revealed its complexity. It is well known that the human dystrophin gene is made up of 99% introns and the coding sequences spans over 86 exons. So far seven promoters linked to seven unique first exons have been identified. The best characterised messenger RNA (mRNA) is the 14 kb long
1.6. Dystrophin and the Dystrophin Glycoprotein Complex
19
messenger that was found to be the most common splicing variant, detected predominantly in skeletal and cardiac muscles and less in the brain (Lambert et al., 1993; Sironi et al., 2001; Torelli et al., 1999). The full-length dystrophin gene product is a large protein bearing four different domains with specific functions, with a total molecular weight of 427 kDa. The amino terminal domain is structurally homologous to α-actinin and shows similarities with the members of the actin cross linking family of proteins. This domain is assumed to interact with the inner cytoskeleton and sarcomere components. It spans the first 232-240 amino acids, depending on the isoform. The following central rod domain with a length of about 3000 amino acids is a succession of 25 triple-helical repeats similar to spectrin. After the spectrin-like domain lays a cysteine rich domain that was shown essential for the association of dystrophin with the transmembrane complex called dystroglycan complex. Finally, the last 420 residues are unique to dystrophin and necessary for the interaction with several intracellular proteins. This domain is known as the C-terminus domain (Muntoni et al., 2003; O’Brien & Kunkel, 2001). Along with the first data available on dystrophin, this protein was suggested to have a primary role as mechanical bridge between the inner cytoplasm cytoskeleton and the outer membrane (Ibraghimov-Beskrovnaya et al., 1992). Consequently, dystrophin was assumed to exert predominantly structural roles in muscles (Ibraghimov-Beskrovnaya et al., 1992). The first biochemical isolation of dystrophin from muscles provided evidence for the role of dystrophin as stabilisation and organisation of a particular protein complex on the membrane. In these studies, dystrophin was found tightly associated with a particular set of proteins named the Dystrophin Glycoprotein Complex (DGC). This first evidence suggested that the cysteine rich domain and the first part of the C-terminal domain are responsible for this interaction (Campbell & Kahl, 1989; Ervasti et al., 1990; Suzuki et al., 1992). In the mid nineties this complex was found to be formed by three sub complexes: a first group composed by α- and β-dystroglycan (dystroglycan complex), a second group of dystrophin-associated glycoproteins (sarcoglycan complex), and a third complex of dystrophin-associated proteins (dystrophin/dystrobrevin/syntrophin complex) (Yoshida et al., 1994) (Fig. 1.6). Results from these studies suggested that the whole glycoprotein complex could be responsible for the correct attachment of the sarcolemma to the extracellular matrix. This theory would reinforce the role of dystrophin as stabiliser of the DGC and in turn of the muscular cell membrane. Consistently with the structural role of the DGC, it was shown that α-dystroglycan directly interacts with laminin, perlecan, agrin, and neurexins. Moreover, it was shown that this binding requires the glycosilation of α-dystroglycan (Ruegg, 2006). Another indication for the protein interactions involving dystrophin is given by the homology with spectrin in the central rod domain. New insights demonstrated an important role of spectrin in protein accumulation at the membrane level (Beck & Nelson,
20
1. INTRODUCTION
Figure 1.6 – Schematic view of the DGC in vertebrates. In vertebrate muscles, the DGC is typically coordinated by dystrophin that interacts at the N-terminus (N) with structural proteins of the cytoskeleton and associates at the C-terminus (C) with specific plasmalemma-associated proteins. The C-terminus on one side interacts with cytosolic proteins like syntrophin (SYN) and dystrobrevin (DYB). In turn, syntrophin interacts with the enzymatic protein nNOS (NOS). The C-terminus of dystrophin further interacts with the transmembrane glycoprotein β-dystroglycan (βDG). β-dystroglycan interacts with the glycoprotein α-dystroglycan (αDG) and with a group of structurally related single-pass transmembrane glycoproteins, the sarcoglycans (Betto et al., 1999; Henry & Campbell, 1996). α-sarcoglycan (αSG), β-sarcoglycan (βSG), γ-sarcoglycan (γSG), and δ-sarcoglycan (δSG) are tightly associated with each other and predominantly expressed in muscles. Finally, the sarcoglycans target and stabilise the sarcospan (SP) transmembrane protein to the plasmalemma (Crosbie et al., 1999).
1996; Dubreuil et al., 2000; Hammarlund et al., 2000; Moorthy et al., 2000). Therefore, a role for dystrophin as important protein stabiliser/accumulator to the sarcolemma cannot be excluded. New evidences on the neurobiology of vertebrate DMD models highlight other structural roles of dystrophin in the brain. In these studies it was shown that dystrophin loss and DGC instability affect presynaptic components and in turn extracellular communication and synaptic transmission (Blake & Kroger, 2000; Waite et al., 2009). Finally, the involvement in intracellular signalling was postulated for dystrophin and the DGC. It was found that dystrophin facilitates the localisation of signalling molecules to the membrane (Rando, 2001). DGC members like β-dystroglycan and syntrophin are involved in signalling cascades and can be modified by signalling proteins. It was shown that tyrosine phosphorilation of the C-terminal tail of β-dystroglycan enables its interaction with caveolin-3 and several Src homology 2 (SH2) domain containing proteins. On the other hand, syntrophin interacts directly with neuronal Nitric Oxide Synthase (nNOS)
1.7. Pathophysiology of Duchenne Muscular Dystrophy
21
(Fig. 1.6), some serine/threonine kinases, calmodulin, acquaporin-4, and a voltage-gated sodium channel (Abmayr & Chamberlain, 2006). One of the most evident proofs that DGC related proteins are involved in signalling and promote physiological changes is clarified by the role of nNOS. It was found that nNOS at the sarcolemma is required for skeletal muscular function since it modulates α-adrenergic receptor-mediated vasoconstriction that in turn regulates blood flow in exercised muscles. As expected in skeletal muscles of DMD patients and mdx mice, i.e. the mouse model of DMD, the modulation of α-adrenergic mediated blood vessel constriction is disrupted (Thomas et al., 1998; Sander et al., 2000). An intriguing role postulated for dystrophin in striated muscles is the transduction of mechanical stimuli that leads to a change in gene expression (Goldspink, 1999). This hypothesis is currently at the beginning of investigation, and so far important evidence for a primary direct role of dystrophin in the regulation of gene expression is lacking. However, a clear link between misregulation of some genes and the muscular pathology was proven in utrophin-dystrophin mutant mice (Nakamura et al., 2001). In turn, the sarcoglycans are necessary for targeting and stabilizing the sarcospan (SP) transmembrane protein to the plasmalemma (Crosbie et al., 1999).
1.7
Pathophysiology of Duchenne Muscular Dystrophy
Two recent review articles offer a wide insight into molecular and physiological mechanisms that trigger muscle degeneration in DMD patients (Batchelor & Winder, 2006; Deconinck & Dan, 2007). DMD is characterised by a progressive decay and loss of muscle fibres. This loss of muscular integrity occurs typically via necrotic processes affecting clusters of fibres. Biopsies showed that the formation of small muscular fibres accompany the degenerating muscles proving that a regenerative response from the myoblast is activated (Schmalbruch, 1984). The effective replacement of necrotic muscle fibres with newly formed myocytes from the satellite cells occurs in the first phases of muscle wasting. Once the pathology advances, the replacement of dying fibres does not occur efficiently and hence causes a net accumulation of adipose and connective tissue (Laguens, 1963; Schmalbruch, 1984). Lack of mechanical connection between muscular fibres, and muscular membrane instability provoked by dystrophin loss seem to be two of the major mechanical damages triggering muscular necrosis. In vertebrate striated muscles dystrophin might have an important mechanical function in proximity of costamers. Costamers are transverse muscular structures responsible for accurate force transmission to the neighbouring tissues and muscle fibres. Loss of dystrophin impairs the proper distribution of muscle force along the sarcolemma. This uncoupling of the sarcomere with sarcolemma ultimately could lead to severe muscle fibre dissociation and cellular membrane laceration (Rybakova et al., 2000). It was indeed shown that CD4+ lymphocytes and macrophages generally infiltrate and
22
1. INTRODUCTION
surround necrotic fibres. It is not yet well understood though if this infiltration is part of the pathology or solely a secondary effect (McDouall et al., 1990). Membrane leakage was experimentally proven in both DMD patients and mouse model by the use of the specific dye orange Procion yellow (Bradley & Fulthorpe, 1978). A hallmark of damaged membranes of muscles cells is albumin infiltration that was observed in affected muscle cells. Since albumin is normally not present in muscle fibres, this observation provides a proof for severe impairment of membrane integrity in DMD patients (Clarke et al., 1993). This loss of membrane integrity was shown to be greatly enhanced by motility and muscle usage in mice model (Mizuno, 1992; Mokhtarian et al., 1999) suggesting that intense exercise could worsen instead of ameliorate muscular integrity. Other lines of evidence point to a strong contribution of calcium imbalance in the aetiology of DMD pathology. Regulation of calcium homeostasis is crucial in muscular functions. There is clear evidence for abnormal intracellular calcium accumulation in striated muscles of DMD patients (Bodensteiner & Engel, 1978). Impairment of calcium homeostasis can be linked to the detrimental effect of dystrophin loss on the correct clustering of acetylcholine receptors (Kong & Anderson, 1999). Moreover, evidence for calcium impairment reinforces the role of calcium activated protease like calpains in muscle necrosis, a contribution that was corroborated by the amelioration of muscle wasting in mouse model after treatment with calpastatin, an inhibitor of calpains (Spencer & Mellgren, 2002). Another player in the imbalance of muscle physiology is possibly represented by nNOS, the neuronal isoform of NOS that is also expressed in muscles. NO is known to be a vasodilatation agent that, once generated by nNOS enzymes, helps the blood irroration of muscles. nNOS interacts with components of the DGC complex like dystrobrevin and syntrophin (Sec. 1.6). In the case of DMD pathology it was found to be reduced in amount and to be mislocalised in the sarcoplasmatic space (Brenman et al., 1995). Although nNOS does not seem to play a major role in DMD pathology, it could have a function in the extension and the severity of muscle wasting. Recent findings suggest that dystrophin might have remarkable roles in intracellular signalling (Sec. 1.6) and that dystrophin loss determines an imbalance in these signalling cascades that eventually contribute to the pathology (Evans et al., 2009). Evidence from mouse models emphasises on the possible connection of the DGC with the MAPK signalling pathway, suggesting that imbalances in DGC stability trigger a pathological response that converges to the MAPK and AKT pathway (Langenbach & Rando, 2002; Nakamura et al., 2002). Furthermore, the stability of the DGC could play a pivotal role in muscle maintenance and its instability could lead to the activation of two muscle specific E3 ligases: MuRF1 and atrogin-1/MAFbx (Glass, 2005). These E3 ligases are known to be activated under atrophic conditions (Bodine et al., 2001; Gomes et al., 2001). Finally, experiments in the mouse model have confirmed that the DGC plays an important role during cachexia onset (Acharyya et al., 2005). Cachexia is a pathological loss of muscular integrity occurring during cancer or high debilitating infectious diseases such
1.8. C. elegans model of Duchenne Muscular Dystrophy
23
as AIDS. These results provide hints for a role of dystrophin in the AKT pathway in the control of muscle maintenance. In this perspective impairment of the DGC integrity could lead to an imbalance of the equilibrium in favour of irreversible muscle degeneration (Glass, 2005). An interaction of the DGC complex with the GTPase Rac1 further promotes an involvement of dystrophin function and JNK signalling pathway (Oak et al., 2003).
1.8
C. elegans model of Duchenne Muscular Dystrophy
Dystrophin and dystrophin-related proteins are widely represented in metazoans. So far, three dystrophin-like proteins have been described: dystrophin, utrophin, dystrophin related protein 2 (DRP2), and the more distant related dystrobrevin. Among the first three proteins, utrophin is mainly expressed in neurons and dystrophin is the most conserved between the different phyla, suggesting an important conserved role leading to more stringent constrains to variation (Roberts & Bobrow, 1998). In C. elegans, different mutants of the dystrophin gene (dys-1 ) have been isolated and characterised at the molecular level. All mutants show a characteristic hyperactive locomotion, over bending of the anterior part of the body when moving forwards and a peculiar sensitivity to aldicarb, an inhibitor of acetylcholine esterase. These phenotypes were correlated to an impairment of cholinergic neurotransmission (Bessou et al., 1998). The dys-1 gene is 31 kb long and has 46 exons coding for a 3,674 amino acid long protein. The protein shares significant similarities with its human counterpart in its sequence presenting an actinin-type actin-binding domain at the N-terminus, a cysteine rich C-terminus domain, and a long rod domain connecting N- and C-terminus (Bessou et al., 1998) (Fig. 1.7). Genetic analyses in the worm pointed out that C. elegans has one dystrophin gene (dys-1 ), 3 sarcoglycan genes (sgca-1, sgca-2 and sgn-1 ), 2 syntrophin genes (stn-1 and stn-2 ), one dystrobrevin gene (dyb-1 ), three dystroglycan gene (dgn-1, dgn-2, and dgn-3 ), no sarcospan gene, and no nNOS. These findings suggest that, although more simple than the vertebrate counterpart, the core components of the DGC are conserved in C. elegans (Fox et al., 2007; Grisoni et al., 2002; Johnson et al., 2006). Molecular interactions between DYS-1, DYB-1, and SNT-1 were confirmed experimentally (Gieseler et al., 1999a). They reinforce the idea that C. elegans DGC does not diverge dramatically from the vertebrate counterpart (Fig. 1.8 and 1.6). Worms defective for the dys-1 gene alone do not show any severe phenotype assimilable to the dramatic human DMD pathology (Bessou et al., 1998). Massive muscle degeneration occurs only once the missense allele dys-1(cx18), a G to A point mutation truncating DYS-1 at the 2,721 amino acid is coupled to the MyoD temperature sensitive mutant hlh-1(cc561) (Gieseler et al., 2000).
24
1. INTRODUCTION
Figure 1.7 – Dot plot matrix of C. elegans DYS-1 (DYS-1) versus H. sapiens dystrophin (HsDYS). The black lines next to the black square highlight the similarity between the conserved N-terminal region of the two dystrophin proteins. The black lines close to the red square emphasise the 37% similarity among the C-terminal region (Bessou et al., 1998). The central region of the two proteins is less conserved. DYS-1, C. elegans DYS-1 protein (horizontal); HsDYS, H. sapiens dystrophin protein (vertical). Numbers on axis of abscissae and ordinate indicate single amino acid positions. This dot plot matrix is based on BLOSUM62 alignment score matrix (Eddy, 2004). Sliding window length: 28 – Greyramp tool min cutoff score: 40 – Greyramp tool max cutoff score: 100.
dys-1(cx18); hlh-1(cc561) double mutants develop like wild type worms until the L4 stage. However, once the worms reach adulthood, a dramatic loss of muscle integrity occurs, leading to paralysis on the second day of adulthood and eventually the worms die prematurely. In parallel to the progression observed in the human pathology, the synthetic myopathy of dys-1(cx18); hlh-1(cc561) worms affects only striated muscles and among them mainly body wall muscles. Currently dys-1(cx18); hlh-1(cc561) worms are considered as C. elegans DMD model (Gieseler et al., 2000). Not only mutations in dys-1 lead to muscle degeneration and defects in movements coordination. Other mutations of genes belonging to the DGC, like dyb-1 and stn-1, show
1.8. C. elegans model of Duchenne Muscular Dystrophy
25
Figure 1.8 – Schematic view of the DGC in C. elegans. The DGC complex in C. elegans muscles is thought to be similar to the one present in vertebrate muscles. Dystrophin links the internal components of the cytoskeleton with its N-terminus (N). The C-terminus (C) interacts with syntrophin (STN-1) and dystrobrevin (DYB-1). Moreover, it is assumed to interact with one of the three dystroglycan genes present in C. elegans (DGN-X). The interaction with STN-1 and DYB-1 has been confirmed (Grisoni et al., 2003), whereas it is not clear which one of the dystroglycan isoforms (DGN-1, DGN-2, or DGN-3) are expressed in muscles. A recent study proposed that DGN-1 might not to be expressed in muscles (Johnson et al., 2006). According to genetic studies, the sarcoglycan family of proteins is represented by three proteins (SGCA-1, SGCB-1, and SGN-1) that could interact with the dystroglycans (Fox et al., 2007; Grisoni et al., 2002). Sarcospan and nNOS have not been found in C. elegans (Grisoni et al., 2002). Intriguingly, STN-1 was found to interact with SNF-6, a muscular acetylcholine/choline transporter located at the neuromuscular junction (Kim et al., 2004).
similar primary phenotypes such as overcontraction, overbending, as well as synthetic phenotype when combined with hlh-1(cc561), like time dependent muscle degeneration, although less severe. These similarities suggest a functional relationship between these genes (Gieseler et al., 1999b; Grisoni et al., 2003). The molecular mechanisms underlying the time dependent muscle degeneration of C. elegans dys-1(cx18); hlh-1(cc561) double mutants are still enigmatic. However, it was shown that physiological calcium imbalance of body wall muscles could play a significant role (Carre-Pierrat et al., 2006a; Mariol & S´egalat, 2001). Intriguingly, a loss of function of snf-6 (allele eg28 ) shows an identical hyper contraction phenotype as dys-1(cx18). Furthermore, hlh-1(cc561); snf-6(eg28) mutant worms undergo a muscle degeneration similar to the muscle degeneration observed in dys-1(cx18); hlh-1(cc561) mutant (Kim et al., 2004). SNF-6 is a newly identified muscular acetyl-
26
1. INTRODUCTION
choline/choline transporter located at the neuromuscular junctions that interacts with the DGC through a physical interaction with STN-1 (Kim et al., 2004). These results additionally support the idea that imbalance in muscular physiology due by an instable DGC or its interacting partners contribute to muscle degeneration in the C. elegans DMD model. From the mdx mouse model a similar picture emerges. In mdx the two phenomena of membrane fragility and activity-induced damage lead to an increase in calcium level. Increased calcium in turn could disturb the balance of some proteases such as calpains, triggering the necrosis responsible for muscle loss (Gillis, 1999; Tidball & Spencer, 2000).
1.9
Thesis question
Two decades of intense molecular research on DMD have passed, but the details of the molecular events leading to muscle wasting are still elusive. Conversely, pharmaceutical research on new potential treatments has been one of the main fields of investigation. Indeed progresses were made on palliative treatments effectively boosting life quality and prolonging life expectancy of affected patients (Ciafaloni & Moxley, 2008). Even though in C. elegans some lines of evidence suggest that the treatment of C. elegans DMD model with the neurotransmitter serotonin ameliorates muscular condition (Carre-Pierrat et al., 2006b), so far a clear-cut test in vertebrates based on these results has not be performed. Medical-oriented research on vertebrate and invertebrate models helped to elucidate the role of physical constrains in the course of the pathology. These works proved that denervation and physical blockage of muscular activity slow down the progression of the pathology and exert a palliative effect (Kimura et al., 2006; Mokhtarian et al., 1999; Moschella & Ontell, 1987). In parallel, it was found that muscle degeneration of dys-1(cx18); hlh-1(cc561) worms occurs only if the muscle is provided with a fully functional wild type contraction machinery (Mariol et al., 2007). These studies lead the research of a cure to a fundamental question: is possible to radically delay degeneration in dystrophic muscles and at the same time prolong muscular activity to near-normal levels? To clarify this question a more satisfactory model of DMD pathology at the molecular level is needed. In particular, the connections between the processes of protein homeostasis and muscle degeneration have been poorly investigated and deserve more attention. Encouraging recent discoveries showed that molecular processes occurring in myogenesis link protein quality control mechanisms to myopathy (Hoppe et al., 2004; Janiesch et al., 2007). These studies provided new insights into the importance of a tight regulation of muscular protein homeostasis under healthy and pathological conditions. Moreover, these findings helped to highlight in the components of the protein quality control a new class of potential pharmaceutical targets. In this perspective, this study focuses on the contribution of the UPS in muscle degeneration and particularly on components of the triage system: CHN-1/CHIP and its partners UFD-2/Ufd2 and PDR-1/Parkin. Through genetic manipulation of the C. elegans DMD
1.9. Thesis question
27
model and biochemical analyses in heterologous systems this work tried to identify new possible factors involved in muscle wasting. The results provided in this study could eventually help to expand the knowledge of the basic molecular mechanisms underlying muscle degeneration and possibly provide hints for new targets suitable for the development of convenient pharmaceutical treatments of muscle degeneration.
2. Methods 2.1
D
Molecular biology na and rna handling, including cloning, agarose gel runs, restriction, digestion, dephosphorylation, and ligation were performed as previously described (Sambrook
et al., 2001). Standard polymerase chain reaction (PCR) was performed using the commercially available Taq or Pfu polymerases or a mixture of both according to manufacturers’ instructions. Purified RNA was stored at -80◦ C in RNase-free ddH2 O whereas purified DNA was stored at -20◦ C until the day of use in Tris•Cl, pH 8.0 buffer. DNA markers “GeneRuler 1 kb Plus DNA Ladder” and “GeneRuler 100 bp Plus DNA Ladder” (Fermentas, St. Leon-Rot, Germany) were used as marker for the DNA electrophoresis gels in conformity with the expected fragment size.
2.1.1
Determination of Nucleic acid concentration
The concentration of nucleic acid was calculated by measuring the absorbance at 260 nm and 320 nm (A260 and A320 ) following Lambert and Beer law. This measurement was conducted using BioRad and Amersham spectrophotometers. The following equation was used to calculate final concentrations of nucleic acid: nucleic acid concentration [µg/ml]
=
(A260 − A320 ) × absorbance factor × dilution factor
Absorbance factors are approximations derived from the molecular extinction coefficient and have the following values: 50 µg/ml for double-stranded DNA, 40 µg/ml for singlestranded DNA and RNA, and 20 µg/ml for oligonucleotides.
2.1.2
Polymerase chain reaction (PCR)
Amplification of target DNA was performed by PCR (Tab. 2.1). This procedure was used for the genotypization of single C. elegans mutants during worm crosses (SW-PCR) and for the specific amplification of DNA fragments from H. sapiens, C. elegans, or bacteria. 28
2.1. Molecular biology
29
For genotypic analysis the standard Taq polymerase was used (Genaxxon, Biberach, Germany and ABgene, Hamburg, Germany). For cloning, where a higher fidelity was required; the KOD DNA Polymerase (Novagen, Darmstadt, Germany) with its 30 to 50 exonucleotidic activity that enhances the fidelity of DNA synthesis or the Expand Long Template PCR System (Roche Diagnostics, Mannheim, Germany), a mix of Taq and Tgo DNA polymerases, were the enzyme mixtures of choice. The melting temperature (Tm) of the oligonuclotide primers (Tab. 5.14) was calculated according to the following empirical equation (Newton & Graham, 1997): Tm
=
[(number of A + T) × 2◦ C] + [(number of G + C) × 4◦ C]
For genotypic analysis of the single nucleotide polymorphism of the dys-1(cx18) allele the procedure indicated in Ye et al. (2001) was followed. Table 2.1 – Exemplary PCR conditions. § Melting temperature was calculated for each oligonuclotide primers pair. ∗ Elongation temperature depends on the DNA polymerase of choice. ‡ Elongation time depends on the expected size of the fragment.
Reaction mix
Volume (µl)
PCR settings
Forward primer (10 pmol/µl)
2.0
1. 95◦ C 5 min
Reverse primer (10 pmol/µl)
2.0
2. 95◦ C 30 sec
dNTPs (2mM each)
2.0
3. Tm§ 30 sec
10× PCR buffer with MgCl2
2.0
4. 68◦ C/72◦ C∗ X‡ min
Taq polymerase
0.2
5. Repeat steps 2 to 4 30 times
Template
2.0
6. 72◦ C 5 min
ddH2 O
9.8
7. 10◦ C
Total volume
20.0
2.1.3
∞
Cloning
Molecular cloning of specific fragments in suitable vectors was used for sequencing of nucleotide sequences or expression of protein of interest in yeast, bacteria, HEK cells and C. elegans. The procedure was done following the standardised molecular methods (Sambrook et al., 2001). After PCR amplification of the fragment of interest with high fidelity DNA polymerases “Expand Long Template PCR System” (Roche Diagnostics, Mannheim, Germany) in a total volume of 50 µl, one fifth of the PCR product was run on a 0.8% agarose gel. In case the analysis of the PCR on the gel resulted in an unambiguous fragment of the expected length the remaining PCR reaction was purified with the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) following manufacturer’s instructions. In case of some
30
2. METHODS
unspecific bands, the remaining PCR product was run on a 0.8% gel until a clear definition of each single band was reached. The band of interest was then excised from the gel with a blade and the DNA was extracted from the agarose using DNA Extraction Kit (Fermentas, St. Leon-Rot, Germany) following manufacturer’s instructions. The amplified fragment and the destination vector were cut with the appropriate endonuclease enzyme(s) (Fermentas, St. Leon-Rot, Germany) using the temperature and buffer as recommended on http://www.fermentas.com/en/tools/doubledigest. After restriction the vector DNA was subsequently dephosphorylated with alkaline phosphatase (Roche Diagnostics, Mannheim, Germany) according to manufacturer’s instructions in order to avoid religation. Ligation was the last step leading to the integration of the fragment of interest into the vector of choice. Ligation was performed using T4 ligase (MBI Fermentas, Vilnius, Lithuania) or the Rapid Ligation Kit (Roche, Mannheim, Germany) according to manufacturer’s instructions. For an optimal ligation the w:w ratio between vector and insert ranged from 1:1 to 1:5 depending on insert and vector size. Typically 10 to 100 ng of vector DNA were incubated in the reaction mixture with an excess of insert DNA. The reaction mixture was prepared in advance mixing 1 µl (5 U/µl) T4 ligase, the appropriate volume of ligation buffer, and ddH2 O up to 19 µl. Finally the DNA was added to the reaction mixture up to 20 µl. The whole volume was brewed following an optimized incubation protocol from our laboratory: 30 minutes at 37◦ C followed by 2 hours at 25◦ C, at last the ligase enzyme was inactivated by exposition to 68◦ C for 20 minutes. An aliquot of the ligation reaction was directly used for bacterial transformation. Aliquots of chemically competent cells TOP10 or DH5α (Invitrogen, Karlsruhe, Germany) (Tab. 5.16) were used for transformation as described in section 2.3.2.
2.1.4
Gateway
The Gateway
TM
TM
cloning
technology (Invitrogen, Karlsruhe, Germany) allows an easy cloning of
a desired insert into different vector backbones. The system is based on the site-specific recombination properties of the recombinase from bacteriophage lambda (Landy, 1989). Out of a single cloning event, it is possible to generate a construct (entry clone) from which the fragment of interest can be subcloned to a vast array of vectors. Thanks to the lambda recombination system a whole array of vectors is available for the functional and biochemical analysis of specific DNA sequences and their products. Once the fragment of interest is properly cloned using the Gateway
TM
technology, the insert will always keep the
same frame allowing directional cloning and easy tagging with the tag of interest. TM
Gateway
cloning is based on the specificity of the recombination sites (att sites)
integrated in the vector. This recombination does not lead to loss of genetic material and sharply combine the replacement of a given sequence in the vector of interest with the inserted sequence without the need for canonical ligation or restriction steps (Fig. 2.1).
2.1. Molecular biology
31
In case of an insert generated from PCR amplification, it was necessary that the specific primers contained a 50 adaptor region called attB site (Fig. 2.1a). Once the fragment was amplified, a first BP reaction could be performed. The reaction occurred in a total volume of 20 µl: 1 to 10 µl of the linear PCR product were incubated with 2 µl of the donor vector with the attP sites (e.g. Fig. A.4), 4 µl of BP Clonase Reaction Buffer, 4 µL of BP clonase, and TE buffer pH 8 up to a total volume of 20 µl. The mixture was then incubated at 25◦ C for 1 hour. To stop the reaction, 2 µl Proteinase K were added followed by incubation for 10 minutes at 37◦ C. The BP reaction led to the integration of the fragment into the donor vector by specific recombination of the attB cassette of the insert with the attP cassette of the donor vector. For easy selection of true recombinants the donor vector contained the toxic ccd B cassette that allowed a negative selection of the bacteria transformed with donor vectors that do not come from a recombination event. Once the insert was integrated into the donor vector, the new clone was referred to as “entry clone”. In the whole process of recombination a by-product containing the ccd B cassette flanked by two attR sequences was generated (Fig. 2.1a). This newly generated entry clone was used for bacterial transformation and transformed cells were selected for the antibiotic resistance carried by the donor vector. The entry clone was transformed for amplification in bacterial cells as described in section 2.3.2 and purified as described in section 2.1.5. After purification the new construct was stored in TE buffer at -20◦ C or -80◦ C.
TM
Figure 2.1 – Schematic view of the BR and LR reaction occurring during the Gateway cloning. a – Schematic view of the generation of an entry vector. A PCR fragment flanked by the two attB sites is combined with the donor vector. A recombination event takes place in presence of BP clonase generating an entry clone with the fragment of interest and a by-product composed of the ccd B cassette flanked by two attR sequences. b – Schematic view of the generation of an expression clone. An entry clone flanked by the two attL sites recombine with the attR sites of the destination vector in presence of LR clonase forming an expression clone and a by-product. Note that the expression clone insert is flanked by two attB sites allowing a recloning in a donor vector. TM Adapted from Gateway technology manual.
32
2. METHODS Once obtained the entry clone, the Gateway
TM
technology allows the insertion of the
fragment of interest into the functional vector of choice (e.g. Fig. A.8) through an LR reaction. In this work the LR reaction was performed as follows. In a total volume of 20 µl, 100 to 300 ng of the entry clone with the fragment of interest were incubated at 25◦ C for one hour with 300 ng of the destination vector, 4 µl of LR Clonase Reaction Buffer, 4 µl of LR enzyme, and TE buffer up to a total volume of 20 µl. To stop the reaction the mixture was shifted for 10 minutes to 37◦ C and 2 µl of Proteinase K were added. The reaction generated the proper expression vector with the parallel production of a by-product containing the backbone of the entry clone and the ccd B cassette from the destination vector (Fig. 2.1b). Once the LR reaction was completed, the reaction was transformed in DH5α or TOP10 cells (Invitrogen, Karlsruhe, Germany) as described in section 2.3.2 to increase the amount of plasmid. Newly generated plasmids were sent for sequencing to GATC biotech, Konstanz, Germany. Once the sequence of the insert had been confirmed the expression clone of interest was transfected, transformed, or used for the required molecular technology.
2.1.5
DNA preparation and purification
DNA purification from 0.8% agarose gel (w/v) was performed following a method based on the solubilisation of the agarose gel followed by ethanol precipitation of DNA using the DNA Extraction Kit (Fermentas, St. Leon-Rot, Germany) and following manufacturer’s instructions. DNA fragments from PCR reactions were purified following a method based on silica-membrane: QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) following manufacturer’s instructions. Plasmid purification from E. coli was performed according to a protocol based on alkali lysis of bacterial cells (Birnboim & Doly, 1979) using QIAprep Spin Miniprep Kit (QIAGEN, Hilden, Germany).
2.1.6
Total RNA preparation from C. elegans
For RNA extraction from C. elegans, mixed populations of worms were grown on 4 Nematode Growth Medium (NGM) (Tab. 5.11) plates (∅ 9 cm) and harvested before the worms ran out of food. Worms were washed off the plate with prechilled M9 buffer (Tab. 5.6) (15◦ C) and collected in 15 ml falcon tubes. After 2 washes with the prechilled M9 buffer spaced out by centrifugation at 1000 g for 2 minutes at 4◦ C, worms where placed in a new falcon tube. After the second wash the worms were shock frozen in liquid nitrogen and conserved at -80◦ C until RNA extraction. Frozen worm pellet was homogenised in a mortar with a sterile pestle both chilled with liquid nitrogen and processed with RNeasy Kit (QIAGEN, Hilden, Germany) following manufacturer’s instructions. The method consist of resuspension of the homogenate in 450 µl RLT buffer containing β-mercaptoethanol to allow cell disruption, followed by centrifugation for 10 minutes at 16,000 g in a QIAGEN shredder column that allow the separation of the cells debris
2.1. Molecular biology
33
from the cellular contents. Subsequent washing steps allowed the purification of RNA by binding to another column and finally the elution of the total RNA in two aliquots (50 µl and 30 µl) of RNAse-free ddH2 O in a 1.5 ml tube. All buffers and columns were provided with the RNeasy Kit (QIAGEN, Hilden, Germany). To avoid DNA contamination, the total extracted RNA was treated with 1 U of DNase (Roche, Mannheim, Germany) for 20 minutes at 37◦ C. Since the experiment was performed using N2 and dys-1(cx18); hlh-1(cc561) strains, the temperature the worms were exposed to did not exceed 15◦ C during all preparation steps.
2.1.7
Reverse Transcription and RT-PCR
For generation of cDNA, 1 µg of total RNA for each worm strain was used. According to SuperScript III First-Strand Synthesis SuperMix (Invitrogen, Karlsruhe, Germany), 1 µg of total RNA was mixed with 1 µl 10 mM dNTP mix, 1 µl of 10 pmol/µl oligo (dT)18 (Fermentas, St. Leon-Rot, Germany), and RNase/DNase-free ddH2 O to 10 µl. The whole mixture was incubated in a thermal cycler at 65◦ C for 5 minutes to allow disruption of secondary RNA structures and then immediately placed on ice for at least 1 minute. After incubation in ice, 2 µl 10× RT buffer, 4 µl 25 mM MgCl2 , 2 µl 0.1 M DTT, and 1 µl RNaseOUT Recombinant RNase Inhibitor (Invitrogen, Karlsruhe, Germany) were added to the mixture, mixed gently and incubated at 42◦ C for 2 minutes. Finally 1 µl (50 U) of SuperScript III RT was added and the whole mixture was incubated at 42◦ C for 50 minutes. To terminate the reaction an incubation of 15 minutes at 70◦ C followed. The cDNA mixtures were kept at -20◦ C until used.
Table 2.2 – PCR settings for expression analysis of cav-1, cav-2, and housekeeping gene eft-3. Elongation steps (X) were repeated 26 times for eft-3, 28 times for cav-1, and 30 times for cav-2.
Table 2.3 – PCR settings for expression analysis of C32E8.11, trim-9a/b, mfb-1, and housekeeping gene eft-1. Elongation steps (X) were repeated 26 times for eft-3, 28 times for C32E8.11, 30 times for trim-9a/b, and 32 times for mfb-1.
PCR settings
PCR settings
1. 95◦ C 2 min
1. 95◦ C 5 min
2. 95◦ C 45 sec
2. 95◦ C 30 sec
3. 58◦ C 30 sec
3. 58◦ C 30 sec
4. 72◦ C 45 sec
4. 72◦ C 45 sec
5. Repeat steps 2 to 4 X times
5. Repeat steps 2 to 4 X times
◦
6. 72 C 2 min 7. 10◦ C
∞
6. 72◦ C 5 min 7. 10◦ C
∞
34
2. METHODS For Reverse-Transcriptase PCR (RT-PCR) analysis the PCR conditions (Tm, elonga-
tion time) of the individual transcripts of interest were first defined using 150 ng of total cDNA as template (Tab. 2.2 and 2.3). RT-PCR analysis of differential expressed transcripts was performed in triplicate using 50 ng, 150 ng, and 300 ng of total cDNA as template. Specific primers for the house keeping gene translation elongation factor eft-3 were used as internal control to verify homogeneous loading and cDNA integrity (Tab. 5.14).
2.2 2.2.1
Protein techniques Protein separation and visualisation
Proteins were analysed by the standard SDS-PAGE method that allows a visualisation of denatured proteins separated according to their molecular weight (Laemmli, 1970). Gel casting, protein visualisation from gels with the standard coomassie staining, and immune blotting was performed according to standard methods (Sambrook et al., 2001). All buffers are reported in tables 5.8 and 5.9. In this study 12% separating gels were used and the specific composition of the separating and stacking gels are listed in table 2.4 and 2.5 respectively. Table 2.4 – Composition of 12% separating gel.
Table 2.5 – Composition of 4% stacking gels.
Components
Volumes
Components
Volumes
ddH2 0
5.25 ml
ddH2 0
3.05 ml
6.50 ml
30% acrylamide/ bisacrylamide
Tris-HCl/SDS, pH 8.8 (4×)
3.75 ml
Tris-HCl/SDS, pH 8.8 (4×)
1.25 ml
10% APS
50 µl
10% APS
25 µl
TEMED
10 µl
TEMED
5 µl
30% acrylamide/ bisacrylamide
0.8%
0.8%
650 µl
After extraction, proteins were denatured at 94◦ C in Laemmli buffer solution (Tab. 5.9) containing the denaturing agents β-mercaptoethanol, Dithiothreitol (DTT), and Sodium Dodecyl Sulfate (SDS). Both β-mercaptoethanol and SDS cause protein denaturation. β-mercaptoethanol reduces the intra- and intermolecular bounds allowing a linearization of the protein whereas SDS-anions bind to the positively charged amino acids. Hence the negatively charged polypeptides were separated electrophoretically according to their mass. Protein samples were then loaded onto the gel and a current of 20 mA was applied constantly during separation, using Biorad Mini Protean II system (Biorad, M¨ unchen, Germany). PageRuler Prestained Protein Ladder marker (Fermentas, St. Leon-Rot, Germany) ran in parallel to determine protein masses. Once the electrophoresis was completed
2.2. Protein techniques
35
the chamber was disassembled and the gel was either stained with Coomassie to visualize the proteins or used for blotting. For Coomassie staining the gel was incubated in Coomassie G250 Staining solution (Biorad, M¨ unchen, Germany) for 3 to 4 hours, destained with Coomassie destaining solution (Biorad, M¨ unchen, Germany) and then incubated overnight in ddH2 O. For western blotting the gel was mounted in a Trans-Blot semidry transfer cell (Biorad, M¨ unchen, Germany). The device consists of a base covered by a lid. 5 Whatman blotting papers soaked in anode buffer (Carl Roth, Karlsruhe, Germany) were placed on top of the base, followed by a Polyvinyldifluoride (PVDF) membrane (Millipore, Schwalbach, Germany) soaked in anode buffer, and the gel of interest. On top of the gel 5 Whatman blotting papers soaked in cathode buffer (Carl Roth, Karlsruhe, Germany) were placed and covered by the lid. An electric current of 1 mA/cm2 was applied for 2-3 hours according to the size of the biggest expected protein to blot. Once the blotting was completed the device was dismantled. The PVDF membrane was incubated with Ponceau S staining solution (Sigma-Aldrich, Hamburg, Germany) to verify proper protein blotting. After rinsing with 1× Phosphate Buffered Saline (PBS) (Tab. 5.9) the membrane was incubated in Odyssey blocking solution for 30 minutes at 4◦ C (LI-COR, Bad Homburg, Germany). After blocking, the appropriate amount of primary antibody was applied to the blocking solution and incubated overnight at 4◦ C. Thereafter the membrane was washed 3 times for 10 minutes with 1× PBS and incubated with Odyssey blocking solution and the appropriate amount of secondary antibody for 1 hour. After the incubation with the secondary antibody the membrane was washed 3 times for 10 minutes with 1× PBS buffer and scanned using the Odyssey LI-COR system (LI-COR, Bad Homburg, Germany). All antibodies used in this study are listed in section 5.4.
2.2.2
Expression and purification of recombinant proteins from E. coli
For overexpression of protein in bacteria the chemically competent strains Rosetta and BL21DE3 were used (Tab. 5.16). These two strains offer the possibility to gain a great amount of recombinant protein. Bacterial cells were transformed as described in section 2.3.2. Transformed cells were grown overnight at 37◦ C in 4 ml aliquot of Luria-Bertani Broth (LB) medium (Tab. 5.11) with the specific antibiotic. An aliquot of this starting culture was diluted 1:1,000 in 250 ml of fresh LB medium with the specific antibiotic. The whole culture was grown at 37◦ C and shaked at 250 rpm until it reached an OD600 value of 0.6-0.7. Protein expression was induced with Isopropyl β-D-1-thiogalactopyranoside (IPTG) in a final concentration of 0.1-0.25 mM. The whole culture was incubated for 4 hours at 30◦ C and then centrifuged at 6,000 g in order to precipitate the bacterial cells in suspension. After centrifugation the bacterial pellet was carefully resuspended in 5 ml GST lysis buffer (Tab. 5.8). Cells
36
2. METHODS
were disrupted by three subsequent ultrasonic pulses of 20 seconds with a power of 50% amplitude. After sonication the lysate was centrifuged at 10,000 g for 15 minutes in order to sediment the debris. At this step the aliquoted supernatant lysate was stored at -80◦ C for several weeks.
2.2.3
GST pull-down assay
Lysate aliquots of bacteria overexpressing GST tagged protein or GST alone were incubated overnight at 4◦ C with 50 µl GSH-Sepharose beads (Amersham Biosciences Europe, Freiburg, Germany). Before incubation the GSH-Sepharose beads were first washed 10 minutes with 1× PBS buffer (Tab. 5.9) and then equilibrated by washing three times for 10 minutes with buffer A (Tab. 5.8). Beads were sedimented by centrifugation for 2 minutes at 0.1 g at 4◦ C. After overnight incubation with the lysate of interest the beads were washed three times for 10 minutes with buffer A to remove unbound GST-tagged proteins and excess GST protein. The Sepharose beads with immobilized “bait” protein were then incubated overnight with a defined amount of HEK293 cells lysate containing the “prey” protein tagged with V5 tag. After overnight incubation an aliquot of the supernatant was stored as control for the “unbound” fraction. The beads were subsequently washed four times for 5 minutes with buffer A. After the final washing step the beads were resuspended in 1× SDS Laemmli solution (Tab. 5.9) and boiled at 94◦ C for 5 minutes. The proteins in the supernatant were then separated by SDS-PAGE electrophoresis. Finally the gel was blotted following the western blot procedure (Sec. 2.2.1).
2.3
Microbiological techniques
All E. coli and S. cerevisiae strains used in this study are listed in tables 5.16 and 5.17, respectively.
General methods for handling, culture, and conservation of E. coli and
S. cerevisiae were performed as described in Sambrook et al. (2001) and according to manufacturer’s instructions.
2.3.1
Preparation of chemically competent bacteria
For the preparation of chemically competent bacterial cells (Hanahan, 1983) a 4 ml culture of E. coli strains DH5α or TOP10 (Invitrogen, Karlsruhe, Germany) was grown in LB medium at 37◦ C overnight. The following day 1 ml of this culture was used to inoculate 500 ml of LB medium and grown at 37◦ C until the culture had reached an OD600 value of 0.6-0.7. Once the correct optical density was reached, cells were incubated on ice for 15 minutes and then centrifuged at 4,000 g for 10 minutes at 4◦ C to precipitate the cells. After washing twice with ddH2 O, the pellet was resuspended in 80 ml ice-cold TrafoI buffer (Tab. 5.7) and the suspension was kept on ice for 25 minutes. Subsequently the
2.3. Microbiological techniques
37
suspension was centrifuged at 4,000 g and the pellet resuspended in 16 ml TrafoII buffer (Tab. 5.7). After resuspension the whole volume was incubated on ice for 15 minutes and 520 µl Dimethyl Sulfoxide (DMSO) were added. Finally the suspension was frozen in aliquots of 100 µl and kept at -80◦ C until use.
2.3.2
Transformation of E. coli
Typically 50 µl aliquots of the chemical competent DH5α or TOP10 cells (Invitrogen, Karlsruhe, Germany) were melted on ice in a 1.5 ml sterile reaction tube and mixed 1:1 with the transformation buffer (Tab. 5.7). 20 µl of the ligation reaction were added to the mixture and the reaction tube was gently tapped and kept for 5 minutes on ice. After 60 seconds of heat shock at 42◦ C the mixture was incubated on ice. After 5 minutes incubation 800 µl of LB medium without antibiotics were added to the cells and gently shaken at 800 rpm at 37◦ C for 1 hour. After incubation the cells were sedimented by centrifugation at 800 rpm for 2 minutes in a table top centrifuge and resuspended in 200 µl LB medium. The cells were finally spread on plates containing the required antibiotic according to the resistance gene in the plasmid and incubate overnight at 37◦ C for positive selection of the right transformants (Sambrook et al., 2001). Final confirmation of the right insert was performed by “colony PCR” using TaqS DNA polymerase (Genaxxon, Biberach, Germany). Once the right colony was identified, a culture was gown overnight using the same antibiotic. On the next day, the plasmid was extracted with QIAprep Spin Miniprep Kit (QIAGEN, Hilden, Germany) and sequenced at GATC biotech (Konstanz, Germany). All vectors and plasmids used and generated in this study are listed in Materials section 5.6.
2.3.3
Transformation of S. cerevisiae
For the screening of a fetal brain human cDNA library in S. cerevisiae the LiAc method was used according to the protocol described by Gietz & Schiestl (2007). An aliquot of yeast cells from a single yeast colony was incubated overnight at 30◦ C and 200 rpm in 5 ml aliquot of fresh Yeast extract Peptone Dextrose (YPD) medium (Tab. 5.11). After overnight incubation 2.5 × 108 yeast cells were inoculated in 50 ml of pre warmed 2× YPD medium and incubated at 30◦ C and 200 rpm until after about 4 hours the cell titer reached 2 × 107 cells ml-1 (an OD600 of 0.1 corresponds to ≈ 1 × 106 cells ml-1 ). Cells were precipitated by centrifugation at 3,000 g for 5 minutes and directly resuspended in 25 ml of sterile ddH2 O. After a second centrifugation and a second wash in 25 ml ddH2 O a final centrifugation at 3,000 g for 5 minutes at 20◦ C followed. The harvested cells were resuspended in 1 ml of ddH2 O. The cell suspension was transferred to a 1.5 ml microcentrifuge tube and centrifuged for 30 sec at 13,000 g. The pellet was resuspended in 1 ml of ddH2 O and the suspension divided into 100 µl aliquots, centrifuged at 13,000 g for 30 seconds and
38
2. METHODS
the supernatant removed. 50 µl of 2 mg/ml denaturated carrier DNA (herring sperm, Invitrogen, Karlsruhe, Germany) were added to 240 µl of PEG (50% w/v), 36 µl LiAc 1.0 M, and 34 µl of plasmid of interest (e.g. pNubI-LEU, Fig. A.3). The whole transformation mix was added to the cell pellet and resuspended by vigorous mixing. The tubes were incubated at 42◦ C for 40 minutes. After a brief centrifugation at 13,000 g for 30 seconds the transformed pellet was resuspended in 1 ml of YPD, incubated 2-3 hours at 30◦ C and plated on the appropriate Synthetic Complete (SC) medium followed by an incubation for 2-3 days at 30◦ C.
2.3.4
Split-ubiquitin system
The protein interaction screening was performed using the split-ubiquitin method; a modified version of the yeast two hybrid system that allows testing of interactions occurring in the cytosol or at the inner face of the cytoplasmic membrane. The procedure was performed according to the methods described elsewhere (Dirnberger et al., 2006, 2008; Johnsson & Varshavsky, 1994). Three different constructs were generated from human E3/E4 ligase CHIP (HsCHIP): a full length construct (plasmid pBY2555, seq. A.3 and A.4), a construct coding for the first 150 amino acids bearing the three TPR domains (plasmid pBY2577, seq. A.5 and A.6), and a third construct coding for amino acids 150 to 303 and bearing the U-box domain (plasmid pBY2578, seq. A.7 and A.8) (Tab 5.15). Only plasmid pBY2578 bearing the U-box domain was properly expressed and therefore used as bait. All sequences were cloned introducing unique Sal I and NotI restriction sites at their extremities. Digestion with the two restriction sites excised gene ubc9 from the vector allowing directional cloning of the fragment of interest in plasmid pPCup1 -ubc9 -CRU (Fig. A.2) (Dirnberger et al., 2006). This led to an in-frame C-terminal fusion of the insert with the C-terminus of Ubiquitin (Cub) followed by the complementation gene URA3 (Fig. 2.2). In order to facilitate the expression of the bait protein, some nucleotides around the starting ATG were substituted according to the Kozak consensus sequence (Kozak, 1987). These modifications were the addition of the ACC sequence 50 to the starting codon and the substitution of the first A 30 to the ATG with a Guanidine. The yeast strain used, JD53, is a strain mutant for URA3 (orotidine 5-phosphate decarboxylase) and HIS3 (midazoleglycerol-phosphate dehydratase) resulting in heterotrophy for Uracil and Histidine (Tab 5.17). The vector in which the bait fragment was inserted (pPCup1 -ubc9 -CRU) carried a gene for Histidine (HIS3 ) and Uracil (URA3 ) complementation. HIS3 was lying in the vector’s backbone and did not need induction for expression, whereas URA3 was lying under the PCup1 promoter. The insert was cloned between Sal I (50 ) and NotI (30 ) in frame with Cub (Ubiquitin C-terminus) and R-ura (URA3 ) under the PCup1 promoter whose activation was dependent on CuSO4 induction (Fig. A.2 and 2.2).
2.3. Microbiological techniques
39
Figure 2.2 – Schematic view of the cloning site for the bait fragment in the pPCup1 -ubc9 CRU plasmid. After digestion of the plasmid with the restriction enzymes Sal I and NotI the yeast specific gene ubiquitin-conjugating enzyme 9 (ubc9 ) was replaced by the bait sequence. In red the copper dependent promoter PCup1 driving bait expression, in blue the C-terminal half of the ubiquitin moiety (Cub) and in green the Uracil complementation gene URA3 (R-ura). Cub and R-ura transcripts were in frame with the bait protein. The numbers close to the two restriction enzymes identify the site of the restriction sites in the plasmid (vertical black bars).
To test the correct expression of the bait, 1.5 ml YPD culture of JD53 were transformed with a mixture of bait plasmid (100-200 ng), carrier DNA, and 500 µl PEG/LiAc. After 30 minutes of incubation at 30◦ C the mixture was heat shocked for 30 minutes at 42◦ C and plated on Synthetic Dextrose minimum medium lacking Histidine (SD − HIS), SD medium −
lacking Histidine and Uracil (SD
−
HIS
−
URA), and SD medium lacking Histidine and
−
Uracil but enriched in CuSO4 (SD HIS URA + 100 µM CuSO4 ). Correct transformation was tested on −
URA
+
−
HIS plates and correct expression of the construct was tested on
CuSO4 . Plates with SD
−
HIS
−
−
HIS
URA served as negative controls. Once the
correct expression of the bait protein was confirmed, a culture of bait-transformed yeast was cotransformed with the prey cDNA library or with single prey clones to be tested. The screening to search new interactors of the U-box domain of HsCHIP was conducted using a cDNA library from human fetal brain fused N-terminally to the N-terminal part of Ubiquitin (Nub) (GPC Biotech AG, Munich, Germany) (Fig. A.3). The interaction of the prey with its bait generates a quasi native ubiquitin protein N-terminal to the URA3 protein that triggers the proteolytic cleavage of the URA3 reporter gene itself. Consequently the yeast cells lose their ability to grow on Uracil deficient plates and this allows a positive selection for the toxic URA3p-specific antimetabolite 5-fluoroorotic acid (5-FOA), enabling a more stringent selection of true positives. Moreover the pNubI-LEU prey vector bear the LEU gene that confers autotrophy for Leucine. It is therefore possible to select positively for the prey on yeast medium lacking Leucine (Fig. A.3). Positive clones were extracted from yeast with the QUIAGEN plasmid purification kit (QIAGEN, Hilden, Germany) according to manufacturer’s instructions. Yeast cells were lysated in 250 µl of P1 lysis buffer (QIAGEN, Hilden, Germany) and 2.5 µl Zymoliase. The mixture was incubated at 37◦ C for 1 hour and the rescued plasmid was retrans-
40
2. METHODS
formed into E. coli (Sec. 2.3.2). After verification of correct insert presence by colony PCR, the correct plasmids were purified and sequenced by GATC biotech, Konstanz, Germany. The sequences were analysed with the BLAST algorithm against the annotated EST of Homo sapiens. Once the human sequence of the prey was identified, each C. elegans prey homologue was identified using the InParanoid Eukaryotic Ortholog Groups database (http://inparanoid.sbc.su.se/cgi-bin/index.cgi). The human p53 protein as bait (p53 -CRU) and the human protein phosphatase 5 (pp5) as prey (NubI-pp5 ) served as positive controls for the split ubiquitin system.
2.4 2.4.1
Cell culture techniques Maintenance of HEK293 cell line
All expression experiments for biochemical analysis of protein tagged with the V5 tag were done in HEK293 cells (Tab 5.18). Maintenance and propagation of HEK293 was performed in 10 cm dishes (Greiner, Frickenhausen, Germany). Cells were grown in MEM+/+ medium supplemented with 50 U/ml penicillin G, 50 µg/ml streptomycin, and 10% FCS (Tab. 5.11), and transferred into a new plate with fresh medium every two to three days. First the old medium was removed with a vacuum pump followed by a brief wash with pre-warmed 1× PBS (Tab. 5.9) in order to eliminate cell debris and dead cells. The dish containing the HEK cells was then incubated for 2 minutes at 37◦ C with Trypsin/EDTA in order to detach the cells from the surface. Finally, the cells were resuspended in new medium and diluted 1:3 or 1:10 in a new 10 cm dish. All HEK293 handling was performed strictly under sterile conditions in laminar flow work benches (Heraeus, Hanau, Germany). HEK293 cells were kept for growing at 37◦ C and 5% CO2 in an incubator dedicated to HEK293 cells maintenance (Heraeus, Hanau, Germany).
2.4.2
Cryopreservation and storage of HEK293 cells
For long term conservation of HEK293 cells a 10 cm dish with 70% confluence (70% of the plate was covered by the cells) was washed off the medium and cleaned from debris with PBS 1× as previously described in section 2.4.1. Subsequently the cells were detached from the plates by incubating for 2 minutes at 37◦ C with Trypsin/EDTA and resuspended in freezing medium containing 70% MEM−/− medium, 20% FCS, and 10% DMSO at concentration of about 1 million cells ml-1 . Finally, 1 ml aliquots of the freezing medium containing the cells were pipetted into cryovials (Thermo Fisher Scientific, Langenselbold, Germany) and freezed at -80◦ C in Styrofoam containers to slow down the freezing process. After overnight incubation at -80◦ C the vials were permanently stored in liquid nitrogen. For recovery of frozen cells an aliquot of cryoconserved HEK cells was mixed with chilled plain MEM−/− medium in a 15 ml falcon tube. Once the aliquot melted the falcon
2.5. C. elegans methods
41
tube was centrifuged at 1,000 g for 5 minutes at 4◦ C. Upon removal of the supernatant the sedimented cells were resuspended in MEM+/+ and plated in a 10 cm petri dish.
2.4.3
Overexpression of target protein in HEK293 cells upon transient transfection
In order to overexpress target proteins in HEK293 cells the constructs were cloned in pcDNA3.1/nV5 expression vector (Tab. 5.15 and Fig. A.8). On the day before the transfection of the cells, an aliquot of HEK293 cells from a 10 cm mother plate that had reached 70% confluence was divided into 6 wells plates as follows. After removing the old medium the cells were treated with trypsin as described in section 2.4.1 and poured into a fresh well of a 6 wells plate after dilution 1:1 with new MEM+/+ medium. On the next day, for each single transfection 100 µl of serum free MEM−/− medium was mixed with 3 µl of GeneJuice Transfection Reagent (Novagen, Darmstadt, Germany), agitated vigorously and kept at room temperature for 5 minutes. Then, 1 µg of DNA of interest was added to the mixture, mixed by gently pipetting and kept at room temperature for 5-15 minutes. Finally the entire volume of GeneJuice reagent/DNA mixture was poured drop-wise over the surface of the cell-containing medium. Transfected HEK293T cells were incubated for 24 hours at 37◦ C and 5% CO2 , and finally harvested. As positive control for successful transfection a vector for expression of EGFP (pEGFP-N1) was used (Tab. 5.15). The qualitative observation of GFP glowing cells accounted for the efficiency of transfection.
2.4.4
Harvesting of overexpressed target proteins from HEK293 cells
Cells in each well of a 6 wells plate were washed once with 1× PBS to eliminate cells debris. 300 µl CytoBuster extraction buffer (Novagen, Darmstadt, Germany) was added to each well and incubated for 5 minutes on ice. The cell layer was scraped off from the bottom of each well and the resulting lysate transferred to a reaction tube. After centrifugation at 16,000 g for 15 minutes at 4◦ C to remove insoluble debris and chromosomal DNA the supernatant was stored at -20◦ C and used for pull-down analyses (Sec. 2.2.3).
2.5 2.5.1
C. elegans methods Maintenance of C. elegans strains
Animals were maintained on NGM agar plates (∅ 3.5 cm, 5 cm, or 9 cm) seeded with E. coli OP50 (Tab. 5.16) as previously described (Brenner, 1974). The basic culture methods were performed as described elsewhere (Lewis & Fleming, 1995; Stiernagle, 2005). NGM plates with worms were kept in cardboard boxes at 15, 20, 23, or 25◦ C.
42
2. METHODS For experiments involving worms bearing the hlh-1(cc561) allele, strains object to study
and the required controls were kept strictly at 15◦ C. All mutants and transgenic strains used in this study are listed in table 5.19.
2.5.2
Freezing of C. elegans strains
Slow freezing at -80◦ C allows storing of C. elegans strains for a long time period at cryogenic temperatures. When needed, a 9 cm plate was grown until fully covered by slightly starved L1 and L2 stage animals. After washing the worms off the plate with M9 medium (Tab. 5.6), they were placed in a 15 ml tube. Animals were washed two times by spinning at 1,000 g at 4◦ C for 2 minutes and changing the M9 buffer twice. Finally the worm suspension was mixed 1:1 with freezing buffer (Tab. 5.6) and aliquots of 1 ml were transferred into cryovials (Thermo Fisher Scientific, Langenselbold, Germany). Six cryovials were placed in Styrofoam boxes and placed in an ultra low temperature freezer at -80◦ C overnight. The following day a control vial was thawed and the content placed in a fresh NGM plate seeded with OP50. When the rescue of freezed worms exceeded hundred units the freezing procedure was considered successful. In this case four of the five remaining vials were kept at -80◦ C and 1 stored as backup in liquid nitrogen.
2.5.3
Crossing of C. elegans strains
To cross two C. elegans strains 1 hermaphrodite and 5 males at L4 stage were placed on a single small plate. Worms were transferred to a fresh plate every 24 hours. The eggs laid in the first 24 hours were discarded due to the high probability of self fertilisation. F1 progeny obtained by eggs laid in the following 24 hours was controlled for a ratio of hermaphrodites to males of 1:1, which was considered as a successful cross. About 20 young F1 hermaphrodite animals were singled out in separate plates at the L4 stage to test the F2 generation. The F2 progeny was analysed in detail to confirm the success of the cross. Depending on the features of the mutant involved, the genotype of the F2 generation was analysed by SW-PCR (Sec. 2.5.5), phenotype analysis, fluorescent marker, or the combination of these methods. Once the correct F2 worms were identified a final confirmation of F3 worms completed the crossing experiment. All crosses generated in this study are listed in table 5.19.
2.5.4
Outcrossing of C. elegans strains
All not outcrossed mutant strains obtained by external laboratory or produced from our lab were backcrossed at least five times with the C. elegans N2 wild type animals in order to eliminate possible unwanted secondary mutations from the genome. L4 stage hermaphrodites and males were placed in small plates in a ratio of 1 to 5, respectively and transferred into a fresh small plate after 24 hours. The whole experiment was conducted
2.5. C. elegans methods
43
according to the procedure described in section 2.5.3.
2.5.5
Worm lysis for single worm PCR (SW-PCR)
To determine the genotype of the worms via PCR, single worms were transferred into single PCR reaction tubes with 4 µl lysis buffer (Tab. 5.6). The tubes were incubated at -80◦ C for 40 minutes then heated to 65◦ C for 40 minutes and to 95◦ C for 10 minutes and kept on ice until the time of usage. The worm lysate was then used directly for the specific PCR reaction. PCR settings were optimized according to the characteristics of the primers and the length of the fragment of interest. The primers were designed according to the specific mutation to give a clear PCR product in case of homozygosity or heterozygosity either of the wild type or the mutant allele. All oligonuclotides used for SW-PCR are listed in section 5.5.
2.5.6
Decontamination and synchronization of C. elegans strains
For the decontamination or synchronization of C. elegans strains, worms were treated with alkaline hypochlorite. To obtain a synchronised population, well fed worms from one to two 10 cm plates were rinsed off with M9 buffer into a 15 ml falcon tube. The animals were washed by spinning at 1,000 g at 4◦ C for 2 minutes exchanging the M9 buffer once. After the second centrifugation the M9 buffer was eliminated and replaced by 9 ml of bleaching solution (Tab. 5.6). The falcon tube was shaken vigorously to destroy the cuticle of gravid animals and allow the release of eggs. After 15 minutes of vigorous shaking eggs and worm debris were pelleted by centrifugation at 1,000 g at 4◦ C for 2 minutes. The hypochlorite was then replaced by fresh M9 medium. After at least four washing steps the eggs were resuspended in fresh M9 medium and transferred into a new falcon tube. After 2 more washing steps eggs were incubated overnight in M9 medium. On the following morning synchronised L1 worms were ready to be used for subsequent analysis.
2.5.7
Generation of transgenic C. elegans strains
All extra chromosomal transgenic strains were generated as previously described (Mello et al., 1991). The method consisted of the co-injection of the expression plasmid of interest and a marker plasmid into the germ line of a young hermaphrodite adult worm and the observation of the following generations. Worms bearing the transgenic marker gene were kept for further analysis. The transgenic strains and the transgenic arrays generated were named individually so that the history of each transgenic line was clearly identified. All transgenic lines generated in this study are listed in table 5.19, all plasmids used for the generation of transgenic lines are listed in table 5.15.
44
2. METHODS
2.5.7.1
Neuronal
chn-1
rescue
strains
of
chn-1(by155)
dys-1(cx18);
hlh-1(cc561) A first transgenic line with neuron-specific expression of chn-1 from a construct bearing the genomic sequence of chn-1 under the aex-3 promoter was generated by injecting 1 ng/µl of plasmid pBY2330 (pBY2085-Paex-3 ::chn-1 ), 5 ng/µl pBY2085 (Paex-3 ::gfp), and 20 ng/µl pBY2550 (Pmyo-2 ::mCherry), into chn-1(by155) dys-1(cx18)I; hlh-1(cc561)II animals generating strain BR4294 chn-1(by155) dys-1(cx18)I; hlh-1(cc561)II byEx584[Paex-3 ::chn-1; Paex-3 ::gfp; Pmyo-2 ::mCherry]. A second transgenic line with neuron-specific expression of chn-1 from a construct bearing the genomic sequence of chn-1 under the aex-3 promoter was generated by injecting 1 ng/µl pBY2330 (pBY2085-Paex-3 ::chn-1 ), 10 ng/µl pBY2085 (Paex-3 ::gfp), 20 ng/µl pBY2550 (Pmyo-2 ::mCherry), and 40 ng/µl salmon sperm DNA into chn-1(by155) dys-1(cx18)I generating strain BR4312 chn-1(by155) dys-1(cx18)I byEx644. Strain BR4312 was
crossed
with
chn-1(by155) dys-1(cx18)I; hlh-1(cc561)II
to
generate
BR4315 chn-1(by155) dys-1(cx18)I; hlh-1(cc561)II byEx644[Paex-3 ::chn-1;
strain
Paex-3 ::gfp;
Pmyo-2 ::mCherry]. 2.5.7.2
Muscular
chn-1
rescue
strains
of
chn-1(by155)
dys-1(cx18);
hlh-1(cc561) A first transgenic line with muscle-specific expression of chn-1 from a construct bearing the genomic sequence of chn-1 under the unc-54 promoter was generated by injecting 1 ng/µl pBY2331 (pPD30.38-Punc-54 ::chn-1 ), 10 ng/µl pPD114.95 (Pmyo-3 ::gfp), 20 ng/µl pBY2550 (Pmyo-2 ::mCherry), and 70 ng/µl salmon sperm DNA in N2 animals to generate strain BR4287 byEx577. Strain BR4287 was crossed first with chn-1(by155) dys-1(cx18)I and then with hlh-1(cc561)II animals to generate strain BR4339 chn-1(by155) dys-1(cx18)I; hlh-1(cc561)II byEx577[Punc-54 ::chn-1; Pmyo-3 ::gfp; Pmyo-2 ::mCherry]. A second transgenic line with muscle-specific expression of chn-1 from a construct bearing the genomic sequence of chn-1 under the unc-54 promoter was generated by injecting 1 ng/µl pBY2331 (pPD30.38-Punc-54 ::chn-1 ), 10 ng/µl pPD114.95 (Pmyo-3 ::gfp), 20 ng/µl pBY2550 (Pmyo-2 ::mCherry), and 40 ng/µl salmon sperm DNA in chn-1(by155) dys-1(cx18)I to generate strain BR4309 chn-1(by155) dys-1(cx18)I byEx641. BR4309 was crossed with chn-1(by155) dys-1(cx18)I; hlh-1(cc561)II to generate BR4316 chn-1(by155) dys-1(cx18)I; hlh-1(cc561)II byEx641[Punc-54 ::chn-1; Pmyo-3 ::gfp; Pmyo-2 ::mCherry]. 2.5.7.3
Reporter strains of genomic chn-1 under aex-3 promoter
To verify the correct expression of the genomic sequence of chn-1 (Seq. A.1) under the aex-3 promoter, 50 ng/µl pBY2001 (pha-1(+)), 10 ng/µl pBY2330 (Paex-3 ::chn-1::gfp), and 40 ng/µl salmon sperm DNA were injected in pha-1(e2123)III worms to generate strain BR4481 pha-1(e2123)III byEx607[pha-1(+); Paex-3 ::chn-1::gfp].
2.5. C. elegans methods
45
A second independent reporter line was generated injecting 120 ng/µl pBY260 (rol-6(su1006)), 10 ng/µl pBY2330 (Paex-3 ::chn-1::gfp), and 40 ng/µl salmon sperm DNA to generate strain BR4529 byEx647[rol-6(su1006); Paex-3 ::chn-1::gfp]. 2.5.7.4
Reporter strains of genomic chn-1 under unc-54 promoter
To verify the correct expression of the genomic sequence of chn-1 (Seq. A.1) under the unc-54
promoter,
120 ng/µl pBY260 (rol-6(su1006)),
10 ng/µl pBY2502
(Punc-54 ::chn-1::gfp), and 40 ng/µl salmon sperm DNA were injected into N2 to generate the F1 strains BR4521 byEx609[rol-6(su1006);
Punc-54 ::chn-1::gfp]
and BR4522
byEx610[rol-6(su1006); Punc-54 :: chn-1::gfp]. A third independent reporter line was generated injecting 120 ng/µl pBY260 (rol-6(su1006)), 10 ng/µl pBY2502 (Punc-54 ::chn-1::gfp), and 40 ng/µl salmon sperm DNA to generate strain BR4523 byEx611[rol-6(su1006); Punc-54 ::chn-1::gfp].
2.5.8
RNA interference (RNAi) feeding experiments
For RNAi experiments the clones of interest were isolated from the Ahringer RNAi feeding library (Fraser et al., 2000; Kamath & Ahringer, 2003) or the open reading frame (ORFeome) RNAi feeding library (Rual et al., 2004). All RNAi clones were inserted into L4440 plasmid vector or its derivative. The L4440 plasmid has an IPTG inducible T7 promoter on both ends of the construct leading to the transcription of the sense and antisense strand of the inserted DNA under IPTG induction (Fig. A.1). The ingested dsRNA triggers the knock-down of the gene of interest in C. elegans. The bacterial strain of choice for the RNAi experiments was E. coli HT115 (DE3) (Tab. 5.16) (Fire et al., 1998). In practice, the RNAi experiments were conducted on NGM agar plates. Synchronised L1 larvae obtained by bleaching as described in section 2.5.6 were poured onto RNAi plates. For the preparation of RNAi plates, cultures of HT115 (DE3) bacteria containing the RNAi clone of interest were grown overnight in LB medium containing 12 µg/ml tetracycline and 50 µg/ml carbanecilin. After overnight incubation the bacterial culture was diluted 1:1 in fresh LB medium with the aforementioned antibiotics. The expression of the RNAi construct was induced in the 1:1 diluted culture for 6 hours with 1 mM IPTG. After the period of induction the bacteria were plated on NGM plates containing 1 mM IPTG and 100 µg/ml carbanecilin or ampicillin, and dried. As negative and positive controls bacteria transformed with the empty vector L4440 or the RNAi construct of pat-3 were chosen, respectively. The worm strain dys-1(cx18) ccIs4251; hlh-1(cc561) was used for the RNAi screening experiments performed with the Ahringer clones. Once the worms reached second day adulthood the motility and the general muscular integrity of RNAi treated worms was scored under the dissecting microscope and compared to the control fed with empty L4440. To define the motility amelioration, three arbitrary classes were used to classify pools of
46
2. METHODS
worms treated with the same RNAi clone: Class
−1
– worms moved similarly to the control and the general fluorescence of muscular
nuclei did not show any clear difference to the control fed with empty L4440. Class 0 – worms moved better and presented better nuclear-GFP distribution in body wall muscle cells than the control fed with empty L4440. Class +1 – worms moved clearly better and had a better GFP distribution in muscle nuclei than the control fed with empty L4440. Once the first generation was scored, 20 gravid worms for each RNAi clone were allowed to lay eggs for 2 hours on fresh RNAi plates. Once the second generation reached second day adulthood it was evaluated with the same parameters. For the RNAi screening experiments using the ORFeome RNAi library the dys-1(cx18); hlh-1(cc561) and dys-1(cx18); hlh-1(cc561); pdr-1(tm395) genotypes were tested. A synchronised L1 population was placed on RNAi plates and qualitatively assayed once grow to second day adult. Worms treated with the same RNAi clone were evaluated on an arbitrary scale similar to that used for the Ahringer RNAi screening and divided in three classes: Class
−1
– worms moved similarly to the control fed with empty L4440.
Class 0 – worms moved better than the control fed with empty L4440. Class +1 – worms moved clearly better than the control fed with empty L4440. All RNAi experiments were performed at 15◦ C.
2.5.9
20 -deoxy-5-fluorouridine (FUdR) treatment
FUdR was solved in ddH2 O to obtain a 100 mM stock solution. The stock solution was applied to the E. coli seeded RNAi NGM plates (L4440 or F33H1.4 RNAi) to obtain a final concentration of 100 µM. For the experiment late L4 worms were placed on the FUdR plates and examined once grown to second day adult.
2.5.10
Counts of eggs in uterus
Fertilized eggs in uterus of independent second day adult worms were counted by microscopic analysis at high magnification.
2.5.11
Food race assay
The food race assay, a modified chemosensory assay, was used to analyse locomotion. This experiment was carried out at 15◦ C.
2.5. C. elegans methods
47
50 µl fresh OP50 were dropped on one side of 5 cm NGM-agar plates and incubated at 37◦ C until drying. Before starting the experiment the plates were incubated for 12 hours at 15◦ C. Synchronised second-day adult animals were washed off the plates with pre-chilled M9 buffer. After 2 quick washes, the worms were suspended in M9 and dropped into 20 µl aliquotes on the NGM-agar plates, on the opposite side of the bacteria (OP50). The time measurement started when the M9 buffer was soaked into the agar. After defined time periods the percentage of the worms that had reached the food was calculated.
2.5.12
Motility assay
The motility assay to determine the speed of individual animals was conducted at 15◦ C. Synchronised second day adult wild type and mutant animals were washed off the plates with pre-chilled M9 buffer. After 2 quick washes, the worms were suspended in M9 and dropped into 20 µl aliquotes on NGM-agar plates without OP50 and pre chilled at 15◦ C. Once the M9 was soaked into the NGM; plates were incubated 60 minutes at 15◦ C before analysis for acclimatization of the worms. Movies of animals were recorded using QuickTimeVideo with a Dolphin F145B video camera (Allied Technology) mounted on a Leica MZ75 dissecting microscope. Videos were analysed with the DIAS 3.4.2 software (Solltech Inc., Iowa city, IA USA) using the “Centroid, Centre of Area” method (Fig. 2.3). Every Movie (15 frames/sec) consists of 151 frames (10.1 seconds duration). The speed value expressed in pixel/sec was converted in µm/sec by multiplying it by the conversion parameter 10.4 µm/pixel. Only video captures in which the worms moved unidirectionally and constantly for the entire period of 151 frames were considered for the analysis. For the analysis of transgenic strains generated as described in section 2.5.7, arbitrarily chosen second day adult transgenic worms with the brightest GFP expression were selected for the motility assay.
Figure 2.3 – Example of motility analysis of two wild type animals with the DIAS 3.4.2 software. In green the digitalized area of two N2 animals, heading to the left. The red dot indicates the starting position of the centroid or geometric center of the bidimensional area subtending the starting position of the worm. The blue dot indicates the final position of the centroid after 151 frames. The thick dark line connecting the red and the blue dot shows the actual path of the centroid in 10.1 seconds. The software ultimately renders the exact mean speed (pixel/sec) of each single worm calculated over 10.1 seconds.
48
2. METHODS
2.5.13
Treatment with proteasome inhibitor MG132
For the preparation of NGM plates containing the proteasome inhibitor MG132 (Calbiochem, Darmstadt, Germany), the chemical was first dissolve in pure DMSO and successively mixed in liquid NGM so that the final plate had an MG132 concentration of 200 µM and 500 µM as well as 1% v/v of diluents DMSO. An NGM plate with 1% v/v DMSO served as control. Once the NGM was solidified, E. coli strain OP50 was poured on top of the plate as described in section 2.5.1. L2 larvae were placed on the plates until second day adult stage and then the motility was analysed with the food race method described in section 2.5.11.
2.5.14
Phalloidin staining
Phalloidin staining was conducted as described elsewhere (Waterston et al., 1984). Synchronised second-day adult worms were fixed in 3% formaldehyde for 2 hours and washed twice with M9 buffer. After the second washing step, residual M9 was removed with a speed vacuum pump. To permeabilize the animals, the worms were incubated with pure acetone at -20◦ C for 2 minutes. Meanwhile, 2 U of Alexa Fluorr 488-phalloidin (Invitrogen, Karlsruhe, Germany) were precipitated from the methanol with a vacuum centrifuge and resuspended in 20 µl S-mix (Tab 5.6). Once the acetone was removed with the speed vacuum pump, the 20 µl S-mix with phalloidin was added to the dry worms and incubated overnight in complete darkness. The day after, the worms were washed once with M9 buffer and analysed under the microscope as described in section 2.7.
2.5.15
Lifespan analysis
Synchronised L1 worms were placed on NGM plates seeded with E. coli and kept at 15◦ C until young adult stage. Once the worms reached adulthood the plates were shifted to 25◦ C. The gravid hermaphrodite worms were transferred to a new plate every day until the egg-laying process ceased. Worms that were dying due to aging processes were scored each day.
2.6
Statistical analyses
One-way ANOVA was applied to test statistical differences between independent groups within the same experiment. Statistical significance was tested with Newman-Keuls, Tukey, and Bonferroni post tests. Two-tailed unpaired Student’s t-test was used to examine direct differences between independent groups and to validate ANOVA results. Statistical analysis of survival curves for the lifespan experiments were conducted by use of the Logrank (Mantel-Cox) test.
2.7. Microscopy and imaging
2.7
49
Microscopy and imaging
Routine observation of worms was done with the dissecting stereo microscope Olympus SZX7. For the microscopic analysis of worms, animals of interest were mounted on 2% agarose pad. Before mounting, the worms were anesthetized with a solution of 20 mM sodium azide (Tab. 5.6). Detailed anatomical analysis was performed as described elsewhere (Sulston & Horvitz, 1977). Image acquisition was done with Imager Z1 microscope, whereas images elaboration and documentation was performed with Axiovision software version 4.6 (both Carl Zeiss, G¨ ottingen, Germany) and Photoshop software (Adobe Systems GmbH, M¨ unchen, Germany).
2.8
Bioinformatics tools
Analysis of protein architecture was conducted using Simple Modular Architecture Research Tool (SMART) (http://smart.embl-heidelberg.de/). Pairwise dot plot analysis of proteins was performed with Windotter (Sonnhammer & Durbin, 1995). Specific set ups for each dot plot are indicated in the respective figure caption. Search for orthologous of C. elegans genes was conducted with the InParanoid Eukaryotic Ortholog Groups database (http://inparanoid.sbc.su.se). Multiple sequence alignments were performed with ClustalW2 and edited with Jalview. Generic information search for genes and literature was carried out through the NCBI website (http://www.ncbi.nlm.nih. gov/). Information on C. elegans genes and genetic sequences was obtained from WormBase (www.wormbase.org). The BLAST algorithm (Basic Local Alignment Search Tool) and its derivatives were used to search homologues DNA and proteins sequences in the aforementioned databases (Altschul et al., 1990, 1997).
3. Results 3.1
Characterisation of Pdys-1 ::gfp, dys-1(cx18), and dys-1(cx18); hlh-1(cc561)
3.1.1
T
Expression pattern of Pdys-1 ::gfp
o corroborate non-exclusive muscular expression of dys-1 (McKay et al., 2003), the BC20230 Pdys-1 ::gfp transgenic strain was analysed (Tab. 5.19). The Pdys-1 ::gfp
from the extra chromosomal array cEX20230 is a transcriptional fusion of the first 2,759 bp of dys-1 promoter 50 to gfp. The muscular expression of dystrophin first observed by Bessou et al. (1998) was confirmed, but in contrast to these results, a clear non muscular expression of dystrophin was found in the ventral nerve cord (Fig. 3.1).
Figure 3.1 – Expression analysis of Pdys-1 ::gfp. Transgenic expression of Pdys-1 ::gfp reporter in an adult worm. a, b, and c: body wall muscle expression of gfp. d, e, and f: neuronal expression of gfp in ventral nerve cord. g, h, and i: expression of gfp in vulva muscles and neurons of the nerve chord. a, d, and g: Differential Interference Contrast (DIC) images. b, e, and h: gfp expression. c, f, and i: merge of DIC and gfp images. a: scale bar 20 µm. d and g: scale bar 50 µm.
50
3.1. Characterisation of dys-1, dys-1(cx18), and dys-1(cx18); hlh-1(cc561)
3.1.2
51
Aging experiments
During routine handling of animals, dys-1(cx18) worms appeared to have a shorter life span than the wild type strain. In order to quantify this difference a survival rate assay was performed. To rule out possible epistasis effects with the insulin-like signalling pathway, shown to be one of the major pathway involved in life span regulation (Kenyon et al., 1993), the daf-2(e1370) and daf-2(e1370); dys-1(cx18) mutants were included in the analysis. The experiment confirmed the shorter lifespan of dys-1(cx18) compared to N2 as well as a shorter lifespan of daf-2(e1370); dys-1(cx18) compared to daf-2(e1370) (Fig. 3.2).
Figure 3.2 – Aging graph of dys-1(cx18). Survival curves of four different worm strains. The percentage of surviving worms at each day of adulthood is presented. Full strain genotypes: N2: wild type – dys-1 : dys-1(cx18) – daf-2 : daf-2(e1370) – daf-2; dys-1: daf-2(e1370); dys-1(cx18). n indicates the number of worms of the given strain used for the experiment. P values are as follow: ??? P
