kinesin-like proteins are involved in postmitotic ... - Science Direct

Cell Biology International 2002, Vol. 26, No. 8, 689–697 doi:10.1006/cbir.2002.0920, available online at http://www.idealibrary.com on

KINESIN-LIKE PROTEINS ARE INVOLVED IN POSTMITOTIC NUCLEAR MIGRATION OF THE UNICELLULAR GREEN ALGA MICRASTERIAS DENTICULATA ANDREAS HOLZINGER* and URSULA LU } TZ-MEINDL Institute of Plant Physiology, University of Salzburg, Austria Received 7 February 2002; accepted 18 April 2002

The unicellular green alga Micrasterias denticulata performs a two-directional postmitotic nuclear migration during development, a passive migration into the growing semicell, and a microtubule mediated backward migration towards the cell centre. The present study provides first evidence for force generation by motor proteins of the kinesin family in this process. The new kinesin specific inhibitor adociasulfate-2 causes abnormal nuclear displacement at 18 M. AMP-PNP, a non hydrolyseable ATP analogue or the general ATPase inhibitors calyculin A and sodium orthovanadate also disturb nuclear migration. In addition kinesin-like proteins are detected by means of immunoblotting using antibodies against brain kinesin, plant derived antibodies to kinesin-like proteins and a calmodulin binding kinesin-like protein. Immunoelectron microscopy suggests a correlation of conventional kinesinlike proteins, but not of the calmodulin binding kinesin-like protein to the microtubule 2002 Elsevier Science Ltd. All rights reserved. apparatus associated with the migrating nucleus. K: adociasulfate; kinesin; microtubules; motor protein; nuclear migration. A: AMP-PNP, 5 adenylylimidodippghosphate; AS-2, adociasulfate-2; BSA, bovine serum albumine; ECL, enhanced chemiluminescence; KCBP, kinesin-like calmodulin binding protein; KLP, kinesin-like protein; MT, microtubule; MTOC, microtubule organizing center; TBS, tris buffered saline.

INTRODUCTION Kinesins and kinesin-like proteins (KLPs) are ATP dependent motor proteins, involved in different cellular processes like chromosome segregation, translocation of vesicles and organelles and organization of the ER (for reviews see Bloom and Endow, 1994; Sack et al., 1999; Woehlke and Schliwa, 2000). Conventional kinesins and KLPs generate a plus end directed movement along microtubules (MTs), whereas C-type KLPs move towards the minus ends. Besides the classical function of moving tightly bound cargo along MTs, currently several functions of KLPs are recognized including movements of membrane associated rafts, zippering, crosslinking and organizing MTs, *To whom correspondence should be addressed: Institute of Plant Physiology, University of Salzburg, Hellbrunnerstr. 34, A-5020 Salzburg, Austria. Fax: 0043-662-8044-619; E-mail: Andreas. [email protected] 1065–6995/02/$-see front matter

catalysation of MT depolymerization, signalling and even functions independent form MTs are discussed (for summary see Goldstein and Philip, 1999). The molecular weight of the different kinesin families varies largely between 60–140 kD, and may even be as high as 250 kD (Whoelke and Schliwa, 2000). Plant cells possess conventional KLPs (Tiezzi et al., 1992; Cai et al., 1993; Liu et al., 1994; Bernstein, 1995; Liu and Palevitz, 1996; Liu et al., 1996; Asada et al., 1997; Lee and Liu, 2000; Cai et al., 2000; Liu and Lee, 2001; Lee et al., 2001; Matsui et al., 2001) and recently a C-type KLP with calmodulin binding capacities (KCBP) has been detected (Reddy et al., 1996; Wang et al., 1996; Bowser and Reddy, 1997; Song et al., 1997; Abdel-Ghany and Reddy, 2000; Abdel-Ghany et al., 2000; Vos et al., 2000). For a comprehensive overview on plant kinesins see Reddy (2001). 2002 Elsevier Science Ltd. All rights reserved.

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The unicellular green alga Micrasterias is an ideal object for investigation of both, morphogenesis (Meindl, 1993) and postmitotic nuclear migration (Meindl, 1983, for summary see Meindl, 1992). The morphogenetic process in Micrasterias is mostly actin-dependent (for summary see Lu¨tzMeindl and Menzel, 2000), whereas nuclear migration and chloroplast movement are MT mediated (Meindl, 1983). Micrasterias cells perform a special type of postmitotic nuclear migration, where the nucleus moves into the growing semicell, stops at a distinct point, and then migrates back to its interphase position in the cell centre (Kiermayer, 1964). Translocation of the nucleus into the growing semicell has been regarded as a passive process driven only by the pressure that moves the cytoplasm and chloroplast into the growing bulb, whereas the backward movement seems to be created by an active MT-dependent moving force (Meindl, 1992). Thus it is most interesting to elucidate which force generating motor proteins are involved in this process. The in vivo function of KLPs is investigated in Micrasterias by various inhibitors. Pharmacological inhibition of MT motors has been difficult to perform so far as general ATPase inhibitors like sodium orthovanadate or calyculin A as well as nucleotide analogues (AMP-PNP) may inhibit kinesin function (Menzel et al., 1995; Vale et al., 1985), but show general toxicity on ATP dependent processes. The discovery of adociasulfate-2 (AS-2; Sakowicz et al., 1998; Blackburn et al., 1999), a marine sponge product as specific inhibitor of kinesin function provides a new tool. AS-2 prevents binding of kinesin to MTs and inhibits MT stimulated kinesin ATPase, its structure does not resemble that of nucleotide triphosphates and the substance does not influence MT polymerization (Sakowicz et al., 1998). Additionally, in the present study KLPs are detected and localized by immunomethods employing antibodies against conventional KLPs and KCBP.

MATERIAL AND METHODS Unless otherwise stated, chemicals were obtained from Sigma Chemical Co. (St Louis, MO, U.S.A.). Algal cultures Micrasterias denticulata Bre´b. cells were grown in Desmidiacean medium (Schlo¨sser, 1994) diluted in

Cell Biology International, Vol. 26, No. 8, 2002

a ratio 1:1 with distilled water and kept at a light/dark regime of 14/10 h at 20C1C. Inhibitor studies AS-2 (kindly provided by Dr C. L. Blackburn, Scripps Inst. of Oceanography, University of California, San Diego, La Jolla, U.S.A.) was dissolved in DMSO to obtain a 10 mM stock solution, diluted in distilled water for working concentrations of 5, 10, 15, 16, 17, 18, 20, 25, 30, 50, 100 M. AMP-PNP was diluted with A. dest. to concentrations of 50, 100, 200, 500, 550, 600, 700, 800 M, 1, 5 mM. Sodium orthovanadate was dissolved in distilled water to obtain concentrations of 100 M, 1, 5, 10 mM. Calyculin A was diluted from a 100 M stock in DMSO with distilled water to working concentrations of 10, 100, 200, 500 nM, 1 M. For all inhibitor experiments young developmental stages of Micrasterias denticulata prior to pattern formation (45 min after mitosis, for AS-2 experiments also cells during mitosis) were treated in various concentrations for time periods of 1–5 h. For recovery experiments cells were treated for 0.5–1 h, subsequently the inhibitor was washed out by several rinses, and cells were allowed to develop for 2–6 h after treatment. Images were taken with a Reichert Univar microscope, 40 1.0 NA plan apo oil iris objective, (Reichert, Vienna, Austria), onto Kodak Tmax 100 Film (Kodak, Rochester, NY, U.S.A.). Western blotting Protein extraction. Micrasterias denticulata cells were homogenized in a Potter homogeniser and extracted at 4C in a buffer containing 50 mM Tris, pH 7.4, 5 mM EDTA, 5 mM DTT, 10 g/ml TAME, Complet protease inhibitor cocktail (Boehringer, Mannheim, Germany) and centrifuged at 10,000g for 10 min. The supernatant was collected and used for SDS-PAGE and Western blotting. SDS-PAGE and Western blotting were carried out as described in detail by Holzinger et al., 1999, 2000. Briefly, protein extracts were SDS-denatured (2.5% SDS, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 5% -mercaptoethanol, 0.01% bromphenol blue), separation was performed onto precasted 10–15% gradient gels (Pharmacia Biotech, Uppsala, Sweden) by the control of a Phast system (Pharmacia). Gels were Coomassie stained


(PhastGel Blue R) or semi dry blotted onto nitrocellulose (Satorius, Go¨ttingen, Germany) by control of Phast system at 15C for 8 Vh. Blots were blocked with 5% non-fat dry milk containing 0.1% Tween-20, antibody incubation steps were carried out in 1% BSA, 0.1% Tween-20, washing steps in 0.1% BSA, 0.1% Tween-20. Detection was achieved by ECL system (Amersham, Little Chalfont, U.K.) on hyperfilm (Amersham). Dot blot experiments To demonstrate that OsO4 and uranyl acetate used as fixatives during freeze-substitution for electron microscopy do not influence the immunoreactions, dot blot experiments were performed. Freshly extracted Micrasterias protein (see above) was mixed 1:1 with 4% OsO4, 4% OsO4 and 0.1% uranyl acetate and incubated for 20 min at 4C. This mimics the concentrations used for electron microscopic preparations (see below). 0.5 l samples were transferred onto nitrocellulose and allowed to dry. Antibody detection was carried out as described above. Immunoelectron microscopy Immunoelectron microscopy was essentially carried out as described by Holzinger et al. (1999, 2000). Micrasterias denticulata cells were high pressure frozen and freeze substituted according to the methods of Meindl et al. (1992), and embedded in LR white resin and UV polymerized according to Brosch-Salomon et al. (1998). Blocking was achieved by incubation of sections in a mixture of 1% BSA, 1% acetylated BSA and 0.2% Tween-20 for 1 h. Primary antibody was diluted 1:50 or 1:100 in TBS, containing 1% BSA, 0.1% acetylated BSA, 0.01% Tween-20 for 20 h at 4C. Sections were washed four times for 15 min in TBS, containing 1% BSA, incubated in the secondary antibody for 1.5 h at room temperature, washed and examined at a LEO 912 AB transmission electron microscope at 80 kV. Images were captured with a Gatan Slow Scan Camera under control of a Gatan Digital Micrograph software or onto Kodak SO-163 electron image film (Eastman Kodak Company, Rochester, NY, U.S.A.). Antibodies Different primary antibodies were used. A monoclonal antibody directed against bovine brain kinesin (Sigma K-1005), anti-AtPAKRP1-

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antibodies against Arabidopsis KLPs, antiRICS4872A-antibodies raised against rice KLPs (both kindly provided by Dr B. Liu; University of California Davis, Davis, U.S.A., for preparation see Lee and Liu, 2000) and anti-KCBP-antibodies (kindly provided by Prof. Dr A. S. N. Reddy; Colorado State University, Fort Collins, U.S.A., for preparation see Bowser and Reddy, 1997). As secondary antibodies for Western blotting horseradish peroxidase conjugated goat anti-rabbit IgG (Sigma A-4914), goat anti-mouse IgM (Sigma A-8786) and for immunoelectron microscopy a 10 nm gold conjugated goat anti-rabbit IgG (Sigma G-7402) or a goat anti-mouse IgM (Sigma G-5652) were used.

RESULTS Inhibitor studies The kinesin inhibitor AS-2 in concentrations higher than 50 M is lethal to Micrasterias denticulata, concentrations lower than 10 M show no light microscopically visible effect. Mitosis is inhibited by AS-2 in concentrations above 15 M, cells stop development and remain in the stage reached prior to treatment (Fig. 1a). AS-2, when applied to young developmental stages (45 min after mitosis) of Micrasterias denticulata for 4 h in concentrations above 18 M (Fig. 1b, c) results in growth retardation and abnormal displacements of the nucleus, when compared to an untreated control cell of the same age (Fig. 1e). However, even when abnormally dislocated, the nucleus still exhibits the typical remigration shape, i.e. a non-spherical appearance, being distorted towards the cell centre (Fig. 1c). When cells are treated for only 1 h and are allowed to recover from treatment for 4 h, the nucleus is able to migrate back to its interphase position, despite that the cell shape is malformed and the chloroplast is not spread properly (Fig. 1d). The nucleotide analogue AMP-PNP also leads to abnormal nuclear migration when applied at 500 M, the nucleus may be dislocated into the polar lobe (Fig. 2a) or to any other abnormal position in the young semicell. Concentrations of AMP-PNP exceeding 700 M are lethal, concentrations lower than 50 M have no effects. The ATPase inhibitor calyculin A markedly retards cell development and results in abnormal nuclear displacement when applied at concentrations of 100 nM and above. The nucleus does not stay on its track but can be abnormally

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Fig. 1. Developing semicells of Micrasterias denticulata under influence of AS-2 (a–d) and corresponding control (e): (a) treatment with 15 M during mitosis for 5 h, mitosis inhibited, (b) treatment with 18 M at young developmental stage for 4 h, abnormal displacement of the nucleus (N), (c) treatment with 30 M at young developmental stage for 4 h, abnormal displacement of the nucleus (N) which is distorted towards the cell centre, delay in development, (d) treatment for 1 h with 18 M, subsequent recovery for 4 h, nucleus (N) in normal position in the cell centre, chloroplast not spread properly, (e) untreated control cell, 4 h after mitosis. Bar 20 m.

displaced by cytoplasmic streaming to any position in the growing semicell (Fig. 2b). Sodium orthovanadate inhibits development of Micrasterias denticulata cells when applied in concentrations exceeding 5 mM (Fig. 2c). The nucleus appears to be fixed in the isthmus region, and only small lobes of the chloroplast are able to move into the young semicell. General ATPase inhibitors like calyculin A and sodium orthovanadate exhibit a narrow concentration range of activity, in higher concentrations the substances are lethal, in concentrations below the effective concentration, almost no light microscopically visible effect occurs. Western blotting The Coomassie stained total protein extract of Micrasterias denticulata (Fig. 3a) exhibits a similar

protein pattern as whole cell lysates (Holzinger et al., 1999, 2000). By use of monoclonal antibodies to bovine brain kinesin, bands at 135–140 kD and an additional band at 100 kD are stained in Micrasterias denticulata extract (Fig. 3b). Antibodies against KCBP detect a main band at 70 kD and additional bands at about 100 and 130 kD (Fig. 3c). Anti-AtPAKRP1-antibodies detect a band at 120 kD and an additional faint band at about 160 kD (Fig. 3d), anti-RICS4872Aantibodies detect a band at about 140 kD (Fig. 3e). Dot blot experiments Dot blot experiments demonstrate that the fixatives used for electron microscopy do not influence the immunoreaction. The intensity of staining of the different dots, either pure Micrasterias extract, or extract containing 2% OsO4 alone or containing a


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Fig. 2. Developing semicells of Micrasterias denticulata treated at young developmental stage with different inhibitors: (a) treatment with 500 M AMP-PNP for 3 h, abnormal displacement of the nucleus (N) towards the polar lobe, (b) treatment with 100 nM calyculin A for 5 h, nucleus (N) abnormally displaced, chloroplast migration into the young semicell inhibited, (c) treatment with 5 mM sodium orthovanadate for 4 h, development and migration of the nucleus (N) inhibited. Bar 20 m.

Fig. 3. Detection of kinesin-like proteins in Micrasterias denticulata: m, Molecular-weight marker: (a) Coomassie-stained gel, (b–e) Western blots; (b) detection with monoclonal antibody to bovine brain kinesin; (c) detection with antibody to KCBP; (d) detection with anti-AtPAKRP1-antibodies, (e) detection with anti-RICS4872A-antibodies; (f) Dot blot— immunodetection with antibody to brain kinesin (1) extract, (2) extract containing 2% OsO4, (3) extract containing 2% OsO4 and 0.05% uranyl acetate.

mixture of 2% OsO4 and 0.05% uranyl acetate is the same after antibody detection with monoclonal antibody against bovine brain kinesin (Fig. 3f). Immunoelectron microscopy Gold particles are present at the distinct MT apparatus associated with the nucleus of Micrasterias denticulata during its backward migration, when immunolabelled with monoclonal antibodies

against bovine brain kinesin (Fig. 4a–c), anti-RICS4872A-antibodies and anti-AtPAKRP1antibodies give similar results, but with weaker signal (data not shown). Especially in the area where the MTs are in close spatial correlation to the nuclear membrane, gold particles are detected (Fig. 4b). However, random staining is also located in the cytoplasm and the nucleus. Immunolocalization with an antibody to KCBP did not reveal any correlation to the MT apparatus in Micrasterias (Fig. 5a, b). Control experiments omitting the primary antibody or using various non-immunized control sera resulted in an almost complete abolishment of gold particles in Micrasterias (see Holzinger et al., 1999, 2000; Lu¨tz-Meindl and Brosch-Salomon, 2000).

DISCUSSION This study provides first evidence for the occurrence of KLPs in the green alga Micrasterias denticulata. Abnormal nuclear displacement caused by the specific kinesin inhibitor AS-2 (Sakowicz et al., 1998) in Micrasterias corroborates an involvement of KLPs in nuclear migration. Most interestingly, the dislocated nucleus still possesses its remigration shape, which points towards an intact MT system, that has lost its direction and moving force. The effects of the general ATPase inhibitors and of AMP-PNP additionally indicates an involvement of KLPs in MT dependent nuclear migration and chloroplast spreading.

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Fig. 4. Immunogold labelling of high pressure frozen Micrasterias denticulata cells during nuclear migration with antibody to brain kinesin. (a) Gold particles at longitudinally sectioned microtubules, (b) microtubules in close contact with the nucleus (N) are labelled, (c) accumulation of gold particles at microtubules. Bar 0.2 m.

KLPs are detected by means of immunoblotting and both, results from immunolocalization and inhibitor studies demonstrate their involvement in postmitotic nuclear and chloroplast migration. The latter processes have been demonstrated to occur under participation of MTs (e.g. Meindl, 1983, 1992). Monoclonal antibodies against bovine brain kinesin detect bands at 135–140 kD in Micrasterias which is at the same molecular weight as in enriched rat brain preparation with the same antibodies (Yang et al., 1990). Additionally in Micrasterias a band at about 100 kD most likely represents a breakdown product. The other plant derived antibodies against KLPs used in this study were able to detect bands at expected molecular

masses (Lee and Liu, 2000), an antibody against KCBP additionally detected putative breakdown products (Abdel-Ghany and Reddy, 2000). These results indicate that different epitopes of kinesin and KLPs are present in Micrasterias denticulata. Immunolocalization of kinesin and KLPs at the MT apparatus associated with the postmitotic nucleus in Micrasterias moreover demonstrates involvement in nuclear migration. Control experiments omitting the primary antibody, heat denaturing the primary antibody, using preimmunesera or a variety of non-immunized antisera (Holzinger et al., 1999, 2000; Lu¨tz-Meindl and BroschSalomon, 2000) have demonstrated that distinct staining of particular structures (e.g. the MT cytoskeleton) never occurs under these conditions.


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Fig. 5. Immunogold labelling of high pressure frozen Micrasterias denticulata cells with antibodies to KCBP. (a) Random distribution of staining in the cytoplasm, (b) no staining at the microtubules. Bars 0.2 m.

As conventional KLPs known to generate plus end directed movement (e.g. Woehlke and Schliwa, 2000) are detected at the MT cage in Micrasterias, indirect information on the orientation of MTs in the cage is obtained. Thus it seems that the minus ends point towards the nuclear envelope, which in plants can function as the principal MTOC (Staehelin, 1997). Evidence for kinesin involvement in unidirectional nuclear movement has been obtained also in Saccaromyces cervisiae, where destruction of the KIP3 function leads to oscillations in the nuclear position (DeZwaan et al., 1997). Nuclear migration and positioning in fungal hyphe is mostly dynein mediated, however functions of KLPs are considered (Fischer, 1999; Steinberg, 2000). As in Micrasterias MTs are generally not involved in the morphogenetic process (Schmid and Meindl, 1992), the effects of the different kinesin targeting drugs on morphogenesis is likely to be an indirect one. In MT-dependent fungal morphogenesis (Seiler et al., 1997; Steinberg, 2000) and trichome formation in higher plants

(Oppenheimer et al., 1997; Krishnakumar and Oppenheimer, 1999) kinesin is involved as molecular motor. In summary our results provide first evidence for the presence of KLPs in Micrasterias and suggest their function as force-generating motor in postmitotic nuclear migration. ACKNOWLEDGEMENTS Adociasulfate-2 was kindly provided by Dr C. L. Blackburn, Scripps Inst. of Oceanography, University of California, San Diego, La Jolla, U.S.A. We are thankful to Prof. Dr A. S. N. Reddy, Colorado State University, Fort Collins, U.S.A., for the kind gift of anti-KCBP antibody, for anti-AtAKRP1-antibodies and antiRICS4872A-antibodies we thank Dr B. Liu, University of California Davis, Davis, U.S.A. We are grateful to Prof. Dr A. Ellinger and Prof. Dr M. Pavelka, University of Vienna, for providing access to a Balzers hyperbaric freezer. Moreover we wish to thank Dr M. Ho¨ftberger for cultivating the

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