Generation of a Semi-Dominant Mutation with ...

J. Nrurogmelics, IS: 75-95. 2001

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GENERATION OF A SEMI-DOMINANT MUTATION WITH TEMPERATURE SENSITIVE EFFECTS ON BOTH LOCOMOTION AND PHOTOTRANSDUCTION IN DROSOPHILA MELANOGASTER BRENDON 0. WATSON, ILYA VILINSKY and DAVID L. DEITCHER Department of Neurobiology and Behavior, Cornell University, Ithaca, N Y (Received 29 November 2000: uccepred 12 June 2001).

A novel Drosophila mutant named Tripped-and-fell (Tad was isolated in a F1 screen for dominant temperature sensitive paralytics. Recombination mapping using multiply marked chromosomes and P elements have pinpointed the locus of Taf to polytene band 93 on the right arm of the third chromosome (3R). When exposed to restrictive temperatures, both Taj' heterozygotes and homozygotes paralyzed; however, homozygotes paralyzed at lower temperatures and took longer to recover than heterozygotes. There are also positive correlations between recovery time from paralysis and both duration and temperature of exposure. Electroretinograms (ERGs) revealed that both homozygotes and heterozygotes have a grossly normal light response at 22"C, but at 37"C, the ERGs from both homozygotes and heterozygotes are unable to maintain a normal sustained depolarization and have a This research was funded by American Heart Association Development Grant (D.L.D.) and NIH Cell and Molecular Neurobiology Training Grant (5T32GM07469 to I.V.). The authors would like to thank Dr. Patricia Rivlin, Sujata Rao, Dr. Bryan Stewart, Dr. R. S. Stowers and Dr. Cole Gilbert for helpful discussions, and Jennifer Bestman for statistical advice. We would especially like to thank Dr. Ron Hoy and Dr. Elke Buschbeck for generously allowing us to use their electrophysiological recording and analysis equipment. Address correspondence to: David L. Deitcher, Department of Neurobiology and Behavior, Cornell University, Ithaca, NY 14853. E-mail: [email protected]

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reduced off-transient potential. The severity of the ERG repolarization phenotype is greater in homozygotes than in heterozygotes.


Keywords: receptor potential; paralysis; electroretinogram (ERG); genetic mapping; neurotransmission; mutant screen

INTRODUCTION Drosophila melanogaster has proven to be an extremely valuable system for understanding the genetics and biochemistry of basic neuronal function. In particular, mutations which confer reversible temperature sensitive paralytic phenotypes have been very instructive. Flies with these phenotypes cease to locomote if their body temperature rises above a restrictive temperature but recover movement when they are cooled back to permissive temperatures. The first temperature sensitive paralytic mutant identified, para'", has an alteration in the sodium channel gene that is essential for normal excitability in Drosophila neurons (Suzuki et a/., 1971; Loughney et a/., 1989). Mutations of the gene encoding the endocytosis protein, dynamin, can cause the temperature-induced paralysis of the shibire phenotype (Chen et al., 1991). Several missense alleles in the gene encoding the N-ethylmaleimidesensitive fusion factor (NSF) cause the temperature sensitive paralytic mutant comatose (comt) phenotype (Siddiqi and Benzer, 1976; Pallanck et a/., 1995). Similarly, a single base pair change in the syntaxin ZA gene, which encodes a protein critical for synaptic vesicle fusion, causes temperature sensitive paralysis (Littleton et a/., 1998). Deletion of the gene for cysteine string protein (csp) causes a temperature-induced paralysis and a block of synaptic transmission (Zinsmaier et al., 1994). Finally, single amino acid changes in the sequence of the Rop gene cause abnormal light responses as measured by electroretinogram after exposure to restrictive temperature for multiple days (Harrison et al., 1994). The common feature of all of these genes is that they are all required for normal synaptic transmission. The phenotype of paralysis under restrictive conditions, then, appears to be one that is mediated by genes important in this basic neuronal function. Generation and isolation of more mutant flies with similar phenotypes might allow us to identify new genes involved in neurotransmission or other essential neuronal functions. One of the primary advantages of temperature sensitive paralytic mutations is that they display the mutant phenotype primarily at elevated temperatures. At room temperature they permit development to proceed normally enough to allow survival to adulthood. As a result, the acute


Novel TS Paralytic Mutant

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effects of a mutation can be examined in vivo at any developmental stage simply by raising the temperature. Therefore, temperature sensitive phenotypes represent a uniquely powerful tool for understanding the biochemistry of synaptically important proteins. Temperature sensitive paralytic mutants can be phenotypically characterized by a number of means. The most obvious way to characterize a temperature sensitive paralytic is to examine the kinetics of paralysis itself. Defining the temperature ranges that cause paralysis, affect recovery time, or prevent recovery altogether may give clues as to the type of protein involved. For instance, if the flies paralyze immediately, this would suggest that the protein’s activity is necessary for continued neural (or muscle) function. An analysis of the impact of the mutation on neural function can be carried out using the electroretinogram (ERG) (Hengstenberg and Gotz, 1964). This method allows one to observe the combined activity of multiple cell types within the eye to normal light stimuli. Consequently, this method allows one to explore many aspects of neuronal function in one procedure (Pak, 1995). When carried out at restrictive temperatures on temperature sensitive flies, the ERG can reveal defects in specific aspects of neurotransmission and can also be suggestive of the nature of the protein mutated. Hence, these methods of characterizing a paralytic mutant can point to the function of the protein involved, even before the gene has been cloned. In this paper, we describe a novel semi-dominant temperature sensitive mutation named Tripped-und-fell ( T a f ) . We have mapped the locus of Taf to polytene band 93 on the right arm of the third chromosome. Phenotypic characterization through behavioral mobility testing after exposure to elevated temperatures, as well as disturbed response to light stimuli as measured by ERG, reveal that this mutation is unique.

MATERIALS AND METHODS

Fly Stocks Balancer stocks, the ru h th st cu sr e ca stock for recombination mapping, and P element lines were obtained from the Bloomington Drosophilu Stock Center, Indiana University.

Generation and Isolation of Temperature Sensitive Mutation A red and ebony (red, e ) line was made isozygous for the third chromosome by crossing red, e males to T M 3 , SerlSb virgin females. T M 3 is a multiply inverted third chromosome, which contains the dominant marker Ser and the recessive marker ebony (e). Then, red, e l T M 3 , Ser males


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were selected from the progeny and individually backcrossed to TM3, S e r l S b virgins. The resulting red, e/TM3, Ser males and virgin females from individual vials were crossed to each other and red, e homozygotes were selected to establish six independent red, e isozygous stocks. One such red, e homozygous stock was then used for the mutagenesis. red, e males were fed a sucrose solution containing 25 mM ethylmethane sulfonate (EMS) overnight. These male flies were then mated en masse to red virgin females to produce approximately 60,000 F 1 progeny. F1 progeny were tested for temperature sensitivity at 38°C. Approximately 5000 flies were placed in a pre-warmed clear plastic box measuring approximately 45 cm x 30 cm x 23 cm and the whole box was placed into a 38°C incubator for 7 minutes. The box was tilted about 45 degrees and tapped so that any flies that were unconscious on the bottom of the box slid through an opening leading to a 5 cm x 4 cm chamber that ran the width of the box (Figure 1C). The box was then inverted to trap the temperature sensitive flies in the smaller compartment and carbon dioxide was released into the box through an uncorked hole, rendering all of the flies unconscious. The flies in the smaller compartment were collected and tested for the ability to regain consciousness or motion to ensure that they were not dead, and were retested for temperature sensitivity to eliminate any false positives. All males believed to be mutant were crossed to TM3, SerlSb females. From the above cross, stocks balanced with TM3 were generated. A line of flies containing an apparent dominant temperature sensitive allele was recovered during this screen, and a true breeding stock was generated. Segregation from third chromosome markers indicated the temperature sensitive allele was on the third chromosome.

Behavioral Phenotypic Characterization To analyze the temperature sensitivity of the Taf allele, a total of 55 heterozygotes (TafITM3, Ser), 55 homozygotes (TaflTaf) and 10 wild type (red, ebony) flies were tested for 10 minutes at each of 10 temperatures ranging from and including 27-36°C. Flies were tested in empty plastic vials, with no more than 20 flies in a vial at a time. Flies were transferred into room-temperature vials, and then the vials were put into a Hybaid hybridization oven (Hybaid Instruments, Holbrook, NY) at elevated temperatures for 10 minutes. Immediately after the 10 minute exposure to elevated temperature, flies were shifted to room-temperature vials and were tested for the ability to walk, once righted, as a result of tapping the vials. In order to examine the effect of temperature on recovery time after paralysis, two vials each containing 10 flies were exposed to temperatures between 35 and 39°C for 10 minutes. As above, flies were transferred into



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vials and then placed in the oven for 10 minutes. After the 10 minute exposure, flies were transferred to room-temperature vials and were allowed to recover. During the recovery, vials were tapped once every 30 seconds for the first 5 minutes after removal from elevated temperature then once every minute thereafter. The time point at which 5 out of the 10 flies in each vial were seen to walk in response to tapping was recorded. To determine whether time of exposure at restrictive temperature can change the recovery time from paralysis, two vials each containing 10 flies were exposed to 36°C for 4, 6, 8 or 10 minutes. In this case, however, flies were transferred to vials that had been pre-warmed to 36°C in the oven and were immediately placed into the oven. This was done so that time of exposure to restrictive temperature could be known precisely, avoiding the ambiguity of the time necessary for the vial and the flies to warm up after they are placed in a warm oven. After the exposure time was finished, flies were transferred to room-temperature vials and were allowed to recover. During the recovery, vials were tapped once every 30 seconds for the first 5 minutes after removal from elevated temperature then once every minute thereafter. The time point at which 5 out of the 10 flies in each vial were seen to walk in response to tapping was recorded.

Genetic Analysis of the Tuf Mutation Taf males (which were marked with red and e ) were crossed to rucuca virgins, containing eight recessive third chromosome marker genesroughoid, hairy, thread, scarlet, curled, stripe, ebony and claret-in order to carry out recombination mapping. Female progeny from this cross were collected and crossed to rucucu males. Those progeny of this second cross that became paralyzed when exposed to restrictive temperature were collected and were assumed to carry the Taf allele. They were then scored for six of the eight rucuca markers. roughoid was not scored since it was determined to be very far away from Tuf and ebony was not scored because all the progeny were ebony. Analysis of recombination frequencies between Tuf and each of these six markers involved initially placing Taf relative to the markers based on single cross-over events. Then, with knowledge of which markers the mutation was between, double cross-over events were figured and added into the recombination counts (see Table I). P elements between cytological regions 88-96 were then used to map Taf with somewhat finer resolution. w’; Taf/Taf females were crossed to males containing w f P elements between 88-96 (see Table 11). Female progeny of crosses between Tafand various P element lines were crossed to y w males. Temperature sensitive progeny of this second cross were collected and scored for the w f P element. The number of temperature sensitive flies that had wi was divided by the total number of tempera-

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TABLE I Recombination Frequency between Tuf and Recessive Markers Gene symbol


Number of recombinants Percent recombination Genetic location of gene Predicted genetic location of Tuf

h

th

st

cu

sr

ca

394 40.5 26.5 67

259 26.6 43.2 69.8

249 25.6 44 69.6

180 18.5 50 68.5

66 6.8 62 68.8

267 27.5 100.7 73.2

Recombination frequencies of Tuf with each of six recessive markers on the rucuca chromosome. 972 flies were collected and scored for temperature sensitive paralysis, the number that were found to be recombinant between each gene and Tuf are written below the symbol for that gene. The percent recombination was added to the known genetic location of each gene to calculate a predicted locus for the Tufgene. The estimates from recombination with each gene were averaged to get a mean predicted value of 69.5 on the third chromosome.

ture sensitive flies to obtain a recombination percentage for Taf with each P element insertion. The result of this mapping placed Taf between 92B and 93F13. Four additional P elements were used for recombination mapping between 93B1 and 93F13 to get a more exact localization of TaJ w ; Taf/Taf females were crossed to males containing w + P elements from the 93B1 to 93F13 region. The resulting females that were heterozygous for Taf and the P element were crossed to w; TM3, Ser, e / S b males. All w+/TM3, Ser progeny of this cross were scored for ebony (which is a closely linked recessive marker on the Taf chromosome) and were then temperature tested. Recombination was scored between Taf and the P element as well as between the P element and the recessive marker ebony (Table 111). The recombination data between ebony and the P elements was used to help resolve the position of the Taf locus.

Electroretinogram Measurements ERGS were performed by inserting a sharp ground electrode into the thorax and placing a blunt recording electrode on the eye surface of a fly immobilized on a cover slip. Both electrodes were filled with 0.85% NaC1. Signals were recorded using an intracellular amplifier (AM systems), digitized with an A/D converter (Tucker Davis Technologies), and analyzed using Matlab (Math Works, Inc.). Light pulses were applied using a fiber light fitted with a manual shutter, and a photovoltaic diode placed in the light path was used to ensure that all light pulses were of the same intensity. Flies were rapidly warmed with a heatable microscope stage controller (20/20 Technologies), and rapid cooling was accomplished by placing an ice pack on the stage until the thermometer readout dropped to 22°C.


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TABLE I1 Recombination Frequency between Tuf and P Elements P line

Cytological location

# of ts flies

# of w + flies

% recombinants

12135 12136 10281 12140 10286 11438 10821 10877 10300 10763 10301 10293 10305 10896 10932 10307 12142 10920 12146 12148 10311 10312 10761 12151 10315 10318 10320 10926 10331 10335 1I440 11442 10337 12312 10340 10342

88A4-5 88B1-2 88C9-11 88D5-6 88E3-4 88F 89A 89B 89B11-13 89C-D 89Dl-2 88F7-8 89E10-11 90D-E 90D-E 90F 1-2 90F6-7 92B 93B1 93C1-2 93D3-4 93D9-10 93F13 94A 1-2 94A5-7 94B4-5 94C 1-2 95F 95Fll-12 95F14-96A 1 95-96 96A 96B3-5 96B3-5 96B19-20 96C8-9

216 150 143 205 166 122 164 176 197 127 156 172 240 84 213 188 162 165 128 173 175 224 130 218 215 249 248 200 160 219 168 273 200 145 202 182

40 28 29 32

18.5 18.7 20.3 15.6 9.0 18.0 14.6 10.8 15.2 15.0 14.7 13.4 15.0 10.7 8.5 8.5 4.3 3.6 0.8 1.7 1.1 0.0 3.1 2.8 4.2 8.0 4.0 15.5 15.0 13.2 2.4 16.5 13.0 17.9 13.9 13.7

~

~ _ _ _ _

15

22 24 19 30 19 23 23 36 9 18 16 7 6 1

3 2 0 4 6 9 20 10

31 24 29 4 45 26 26 28 25 ~

___

Frequency of recombination between Tuf and w + marked P elements at various cytological locations. Temperature sensitive progeny ( ts ) from the cross Tuf/P element females to y M’ males were collected and the number of w + flies was recorded. The recombination frequency was calculated by dividing the number of M I + flies by the total temperature sensitive flies.

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TABLE 111 Frequency of Recombination between Tuf and P Elements between 93B1 and 93F13 Cytological location

# of segregants

% ebony,

P line

not ts

O/O ts, not ebony

YOebony, ts

12146 12148 10312 10761

93B1 93C1 93D9 93F13

1698 788 853 396

1.35 0.76 0 0

0 0.38 0.47 0

0 0 0.35 2.02

Tuf/Tuf virgin females were crossed to the w+ marked P elements indicated above. Progeny Tuj; e/P element females were crossed to w; TM3, Ser, ejSb males and all/'w TM3, Ser, e, progeny were collected, tested for temperature sensitivity, and scored for ebony. The recombination frequency was obtained by dividing the number of temperature sensitive or ebony flies by the total number of wf/TM3, Ser, e flies. N o recombinants (in this set of experiments) were obtained between the P element line 12146 located at 93B1 and Tufin 1698 segregants so it was not possible to determine whether Tufis proximal or distal to this P element. Though cytological information places 12146 at 93A4-5,the sequence of flanking DNA places it at 93B1 (Spradling et ul., 1999).

Initially, homozygous Tuf flies were examined: heating to 37°C nearly abolished the entire E R G signal. No on- or off- transients were present, and no sustained depolarization was observed during the light pulse. However, the Tufmutation was generated in an ebony background. ebony has an ERG phenotype of suppressing on- and off- transient spikes (Hotta and Benzer, 1969), though it does not affect the sustained depolarization of the photoreceptor. The lack of a sustained component appeared to be unique to the Tufmutation. In order to more readily observe this phenotype, we recombined ebony away from the Taf' allele by crossing it to y w flies, selecting Tuf/+ females and crossed them to TM6B, Tb, e / S b males. Single males that were paralytic and lacked ebony were used to establish three individual stocks. Two of the stocks were also tested for abnormal ERGs and both were found to have the same depolarization defect. One of these lines was then used for the subsequent ERG analysis. To perform ERGs of Tqfheterozygotes, Tuf homozygous males were crossed to Oregon R virgin females and Tuf/+ progeny were selected. Oregon R flies were used for control ERGs. At least three flies of each genotype were used and only w+ flies were used. Receptor potentials were measured 0.2 sec after the start of the light pulse, to ensure measurement was out of the range of the on- transient and all repolarization comparisons were made within the same fly. Similar ERGs were also obtained with a line produced by a recombination of Taf with the P


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element line 1031 1. In fact, all independent recombinant temperature sensitive paralytic stocks derived from the original Taf allele that were tested by light stimulation exhibited the mutant ERG phenotype. This suggests that these phenotypes are not genetically separable and are likely due to a mutation at a single locus.


RESULTS In order to isolate dominant temperature sensitive paralytic mutations in Drosophila melanogaster, we collected 60,000 F1 progeny of EMS-treated parental males and screened them for temperature sensitivity. Temperature sensitive paralytic flies were initially selected by heating the F1 flies to 38°C in batches of -5000 as shown (Figure 1).

load flies

A

B

cork

_ .. _... ._.... . _. .. ..

., .

gap

'

lid

Heated for 7 minutes at 38OC collect paralytic flies

C

D

tilt box to collect paralyticflies

discard nonparaiylicflies

FIGURE 1 Strategy for isolation of temperature sensitive paralytic flies. A) Flies were added to a prewarmed box. B) Box was heated for 7 minutes at 38"C, causing temperature sensitive flies to fall. C) Box was tilted and fallen flies slide into a separating compartment. D) Box was flipped over, temperature sensitive flies were trapped in separating compartment; the box could then be filled with C 0 2 so that the remaining flies could be immobilized and more easily sorted.


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Paralytic flies recovered from this large scale screen were retested in smaller batches in a water jacketed chamber as described by Ramaswami et ul. (1993). Initially, 30 flies were selected from the two paralytic tests. Stocks were established over balancer chromosomes from each of these flies. Each of these balanced lines were retested for paralysis and only a single line was found to retain the temperature sensitive paralytic phenotype. This dominant temperature sensitive paralytic fly line was selected for further study. This line will be referred to as Tripped-und:fell (Tuf), based upon its temperature sensitive locomotion defect.

Behavioral Phenotype The Tufmutation was initially identified by its paralysis at 38°C. In order to determine whether the mutant is sensitive to temperatures lower than 38°C and whether Tuf heterozygotes differ from homozygotes in their temperature sensitivity, Tuf heterozygotes, homozygotes, and control (red, ebony) flies were tested for 10 minutes a t each of 10 temperatures ranging from 27-36°C. After heating, the flies were transferred to a room-temperature vial, and the number of individual flies that were able to move in response to tapping the vial was recorded. The results of these experiments are presented in Figure 2. Both Tuf homozygotes and heterozygotes fell in larger proportions as temperature increased. However, homozygotes fell, on average, at about 3 degrees lower than heterozygotes. This difference is most dramatic at 32°C where 100% of homozygotes remain motionless after tapping whereas only 1 of 55 heterozygotes tested remained motionless. Since the duration of heating and the temperature of heating has been shown to affect recovery time in other temperature sensitive paralytics (Siddiqi and Benzer, 1976), we examined both of these parameters as they relate to the recovery time in the Tufmutant. Vials containing flies either heterozygous or homozygous for the Tufmutation were each exposed to paralyzing temperatures ranging from 3539°C. As seen in Figure 3, this experiment demonstrates a positive correlation between temperature of exposure and recovery time. Homozygotes apparently need a longer time to recover than heterozygotes. Similar experiments were conducted to explore whether the duration of exposure to restrictive temperatures affects the time to recovery. Both homozygotes and heterozygotes showed exposure time dependent increases in recovery time, with homozygotes remaining unable to respond for longer periods than heterozygotes (Figure 4). However, there may be an upper limit for recovery time of heterozygotes at 36°C since the 8 and 10 minute exposures led to essentially the same recovery time measurement.


Novel T S Paralytic Mutant

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29

30

31

32

33

85

34

35

36

Temperature ("C)

FIGURE 2 The dependence of temperature on paralysis of the Tafmutation. The percent of flies of indicated genotype able to respond to a tap stimulus at various temperatures is plotted. 5 5 Tuf homozygotes (diamond), 55 Taf heterozygotes (square) and 10 red ebony (wild type) flies (open triangle), in vials of 10 to 20 flies at a time were exposed to temperatures from 27 to 36°C for 10 minutes. After the exposure, vials were tapped on a bench to motivate flies that were capable of movement. Wild type flies showed no temperature sensitive paralysis. 100% of homozygotes were paralyzed at 32°C and 100% of heterozygotes were paralyzed at 35°C. At 3 2 T , whereas all homozygotes were down, only 2% of heterozygotes were down.

Electroretinogram Phenotype Electroretinograms (ERGs) have been used as a sensitive assay for identifying synaptic mutants and defining defects in synaptic transmission (Koenig and Merriam, 1977; Leung et al., 2000; Pak, 1995; Pearn et al., 1996; Stowers and Schwarz, 1999). Since a temperature sensitive paralytic phenotype often indicates a deficit in synaptic transmission, this mutant was assayed by ERG recordings. Stimuli and recordings were made at 22°C after heating to 37°C for five minutes, and then again after cooling back to 22°C for ten minutes (recovery). Control Oregon R flies were compared to Taf heterozygotes (Taf/+) and to homozygotes ( T a f / T q f ) , and the results are presented in Figure 5. Control flies showed normal ERGs under all circumstances. At 22"C, both Taf heterozygotes and homozygotes had robust on- and off- transients as well a sustained depolarization component. When the tem-

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FIGURE 3 Recovery from paralysis as a function of temperature. The time for 50% of flies to recover from temperature induced paralysis versus exposure temperature is plotted. Two vials of 10 Tufhomozygotes (open diamond) and two vials of 10 Taf heterozygotes (square) were put into a n oven at temperatures ranging from 35 to 39°C for 10 minutes. After the 10 minute exposure, vials were removed to room temperature and were quickly double tapped once every 30 seconds for 5 minutes and then once a minute thereafter. After each tapping, vials were examined in order to determine the time at which 5 out of the 10 flies in each vial were able to respond by either walking or moving. Both homozygotes and heterozygotes showed increased recovery times after exposure to increased temperatures. Homozygotes have longer recovery times than heterozygotes (error bars represent standard deviations).

perature was raised to 37°C for 5 minutes, both Taf homozygotes and heterozygotes still had on- and off-transients, though the size of the offtransient was substantially reduced to 38% of normal in heterozygotes and 17% of normal in homozygotes (Figures 5 and 6A). In addition, there was a dramatic decrease in the sustained depolarization component of their ERGs. In Tuf homozygotes, light induced receptor potential quickly (within 0.2 sec) repolarized to only 21% of its value at 22°C even while the light stimulus remained. Heterozygotes showed a similar, though less severe phenotype: photoreceptor potential repolarized to 49% of its value at stimulus onset (Figures 5 and 6B). After cooling the flies back to 22°C for 10 minutes, the sustained depolarization component of the ERGs in both homozygotes and heterozygotes was restored to preheating levels. Although full ERG recovery was observed after cooling to 22°C for 10 minutes, locomotion in homozygotes was restored more


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40

4

5

6

7

8

9

10

Exposure time (min)

FIGURE 4 Recovery from paralysis as a function of time at 36°C. The time for 50% of flies to recover from temperature induced paralysis versus time of exposure to restrictive temperature is plotted. Two vials of 10 Taf homozygotes (diamond) and two vials of 10 Tafheterozygotes (square) were put into an oven at 36°C for 4,6, 8 or 10 minutes. After the exposure to restrictive temperature, vials were removed to room temperature and were quickly double tapped once every 30 seconds for 5 minutes and then once a minute thereafter. After each tapping, vials were examined in order to determine the time at which 5 out of the 10 flies in each vial were able to respond by moving. Both homozygotes and heterozygotes showed increased recovery times after exposure for increased lengths of time. Homozygotes have longer recovery times than heterozygotes. Heterozygotes appear to show a plateau in recovery time after exposure of more than 8 minutes (error bars represent standard deviations).

slowly. Recovery of locomotion for 50% of homozygotes and heterozygotes was 46 minutes and 8.5 minutes respectively.

Genetic Mapping We determined that the Taf gene was on the third chromosome by segregation from the TM3, Ser balancer. To map the mutation of a putative single-gene etiology for these defects, we applied the markers described in Material and Methods. Also in Table I are the known genetic locations of each of these six marker genes. This led to estimates of the location of the mutation to 3-70 (Table I), corresponding to the cytological region 92-93 (Lindsley and Zimm, 1992). Finer mapping, using w +

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88 22O


wt

37 O

22' recovery

li 1

ZL +

Y

FIGURE 5 Temperature dependent changes in the electroretinograms of Taf mutants. Recombinant TaA which lacks the ERG- altering ebony allele, was used in lieu of Taf' (containing ebony), for the ERG experiments (see Methods). Representative ERGS of control (Oregon R) (top row), Taf'heterozygotes (Taf/+) (middle row) and Taf homozygotes (Tuf/Tuf) (bottom row) before, during, and after heating to 37°C are shown. Each genotype displayed a normal E R G a t 22°C. Incubation for five minutes at 37°C (middle column) resulted in a dramatic reduction in the sustained photoreceptor depolarization in both heterozygotes and homozygotes, and suppression of the off-transient potential (arrows), but no significant decrease in the on-transient potential (middle column). Homozygotes showed greater loss of sustained photoreceptor depolarization and a greater reduction in off-transient potential. Rapid cooling and a 10 minute recovery to 22°C showed a complete recovery of sustained photoreceptor depolarization in both heterozygotes and homozygotes (third column).


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A

wt

Taf/+

wt

Taf/+

Tafflaf

B 1

Tafflaf

FIGURE 6 Relative reductions in sustained photoreceptor depolarization and off-transient potentials in Tqf mutants after heating. A) Off-transient potentials are essentially unchanged in wt flies (96% of that at 22"C), but are reduced to 38% in Taf heterozygotes and to 17% in Tuf homozygotes. Differences were statistically significant between wt and Tufheterozygotes ( p = 0.005, single factor ANOVA) and between wt and Tufhomozygotes ( p = 0.001), but not between Tuf heterozygotes and Tuj homozygotes. B) Receptor potentials in control flies are essentially unchanged upon heating to 37°C (97% of that at 22°C). Tuf heterozygote receptor potentials dropped to 49% of that at 2 2 T , and homozygous Taf flies showed a receptor potential drop to 21 % of normal upon heating (differences between all genotypes are statistically significant, p < 0.001 by single factor ANOVA). Three wt, five Taf heterozygotes and eight Tuf homozygotes were tested. Error bars represent SEM.

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10761

93D2 9339

93F13

ebony 9;Bl

co;tectin 93C1


probable Taf locus

FIGURE 7 Predicted location of Tufand candidate genes. The location of three candidate genes Atpalpha (a subunit of the Na+/K+-ATPase gene), Cafx (Na+/Ca2+ exchanger), and cortuctin are shown. The P elements and the location of ebony which were used to map Tufare also shown.

carried in the transposons (Table 11) implied that the mutation is most likely within cytological region 92B-93F13. Four w + marked P elements from this region were used to further refine the map position of the mutation. The inability to generate recombinants between the P element insertion at 93B1 in nearly 1700 segregants indicates that this P element was very close to TuJ This location is further corroborated by finding that the P insertion at 93C1 lies between Taf and ebony (Table I11 and Figure 7). Temperature sensitive kinetics of flies heterozygous for Taf and the P element at 93Bl were identical to that of Taf/+ heterozygotes indicating that Tqf is likely to be an allele of a different gene (1. Vilinsky, unpublished results).

DISCUSSION The experiments presented in this paper show that the Tafmutation is a semi-dominant temperature sensitive allele of an as yet undetermined gene. It acts as a dominant mutation above the restrictive temperature of heterozygotes (above approximately 33°C) and it acts recessively between the restrictive temperatures of homozygotes and heterozygotes (between approximately 31" and 33°C). The ERG experiments allow us to more narrowly define the nature of the mutated protein. The ERG measures activity from two types of cells: the presynaptic photoreceptor cell and the post-synaptic laminar neuron. Two changes in the ERG were seen after heating to 37°C. The sustained depolarization of the photoreceptor was significantly reduced by the Taf mutation indicating that some aspect of phototransduction was affected. In addition, the off-transients of the ERGS were reduced suggesting that synaptic transmission is also affected (Pak, 1995, Stowers and Schwarz, 1999). The current model of the photoreceptor cell phototransduction cascade in Drosophila starts with light activation of rhodopsin into metarhodopsin, activating a G-protein (reviewed in Montell, 1999). This G



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protein is thought to activate phospholipase C (PLC), which then cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to yield inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. IP3 may open calcium channels in the sarcoplasmic reticulum leading to the release Ca2' into the cytosol. The increase in intracellular calcium may open the transient receptor potential (TRP) and transient receptor potential-like (TRPL) ion channels, which are known to open in response to light. Recent evidence suggests that the effect of intracellular Ca2+ may be to regulate PIP2 levels which in turn regulates TRP channel function (Hardie et ul., 2001). TRP is cation channel with high preference for Ca2+ over Na+ (Hardie and Minke, 1992), while TRPL is a non-selective cation channel (Kunze et al., 1997); combined these two appear to allow photoreceptor cells to maintain depolarization during the light stimulus. Most of the current flowing into the photoreceptors is due to Ca2+ through TRP and extremely high levels of intracellular Ca2+ (up to 600 pM) have been shown to accumulate in the rhabdomeres of blowfly photoreceptors following light stimulation (Oberwinkler and Stavenga, 2000). Ca2+ not only acts as a key activator of phototransduction but also likely plays a crucial role in phototransduction termination. Ca2+ can reduce the activity of rhodopsin, TRP and TRPL channels (Byk et al., 1993; Vinos et al., 1997), and PLC (Inoue et al., 1988). Ca2+, through the action of calmodulin, also affects the termination response through NINAC, a myosin 111-like protein (Li et al., 1998). Mutations in many of the above genes cause ERG phenotypes that are qualitatively different than those seen with Tuf at high temperatures (Pak, 1995; Pearn et al., 1996). However, the TRP and TRPL mutants yield electroretinograms that are similar to those seen with TaL though the time course of photoreceptor repolarization in TRP and TRPL mutant flies is slower (Cosens and Manning, 1969; Leung et d., 2000). TRP and TRPL genes do not map to the Taf locus, but their similar ERG phenotype suggests that the protein encoded by the Taf locus either directly or indirectly alters the flow of ions into the photoreceptor following light stimulation. The reduction in the off-transients in Taf mutants also indicates that synaptic transmission, in at least some synapses, between the photoreceptors and laminar neurons is reduced. Whichever genes are considered as candidates for the Tuf locus, it should be taken into account that they must be able to cause the paralysis phenotype as well as the photoreceptor phenotype at elevated temperatures. Therefore, unlike many ERG mutants which only have eye specific phenotypes, Tafmust be expressed elsewhere in the nervous system or in the muscle. Further restricting our search, we know that the Tqfgene is located very near cytological region 93B on the third chromosome. An examination of predicted genes from the region of 93B (Adams et al., 2000) revealed several potential candidates for the Taf locus (Figure 7). One candidate is the gene encoding the c1 subunit of the


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Na+/K+-ATPase. The Na+/K+-ATPase is critical for maintaining the ionic balance of Na+ and K + in epithelial and in excitable cells, such as muscle and neurons (reviewed in Therien and Blostein, 2000). The a subunit of the Na+/K+-ATPase is expressed in high levels in the nervous system, muscle, and malpighian tubules (Lebovitz et ul., 1989). A P insertion, that causes a reduction in expression of the a subunit of the Na+/K+-ATPase, has a cold sensitive locomotion defect (Feng et ul., 1997). A similar hypomorphic P allele is bang sensitive but does not appear to have an altered phototactic response (Schubiger et ul., 1994). If Tujis an allele of the a subunit of the Na+/K+-ATPase gene, it is unlikely to be a reduction of expression mutant like the cold sensitive allele. This is because Tujis a dominant, not a recessive mutation, and it is paralytic at elevated temperatures, not cold or bang sensitive. We crossed the Tujallele to a null allele of the a subunit of the Na+/K'-ATPase gene and tested recovery time from paralysis. We observed a mild slowing of recovery time as compared to Tufheterozygotes (I. Vilinsky, unpublished results). However, since Tuf is likely to alter ion channel function in some way, a reduction in Na+/K+-ATPase levels could enhance the paralytic phenotype without necessarily being allelic with Tuf: Although no phototactic phenotype was seen in the bang sensitive allele of the a subunit of the Na+/K+-ATPase gene, the protein has been localized in photoreceptors. The protein was detected on the peripheral surface but not in the rhabdomere (Yasuhara et ul., 2000). Its localization makes it less likely to be directly involved in the initial events in phototransduction but it is possible that it could influence the ERG phenotype by altering the membrane potential of the photoreceptors. Another candidate for Taf is the Na+/Ca2+ exchanger protein gene, Culx (Schwarz and Benzer, 1997). The Na+/Ca2+ exchanger protein along with the Ca2+ ATPase is responsible for maintaining the low basal level of Ca2+ within cells (reviewed in Blaustein and Lederer, 1999). The Na+/ Ca2+ exchanger gene is expressed in many Drosophilu tissues including the photoreceptors (Schwarz and Benzer, 1997; Haug-Collet et ul., 1999) but subcellular localization of this protein in the photoreceptors has yet to be determined. A mutation in the Na+/Ca2+ exchanger protein gene could alter Ca2+ clearance from the rhabdomere following light stimulation and could affect numerous aspects of the phototransduction pathway. In addition, the exchanger could alter the excitability of muscle cells and neurons, leading to paralysis. There are currently no mutations in this gene. It is possible the Tuf gene is not directly involved in ion transport but instead, it may be involved in signal transduction, which in turn affects ion transport or movement. One candidate gene involved in signal transduction in the 93B region is cortactin. Cortactin is a protein that associates with actin (Wu and Parsons, 1993) and with scaffolding proteins that interact with the PDZ domain proteins (Naisbitt et ul., 1999). The phototransduction pathway proteins in Drosophilu has been shown



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to be highly concentrated in supramolecular complexes known as signalplexes (reviewed in Montell, 1998). Such signalplexes are dependent on a PDZ containing protein, INAD. In photoreceptors, mutations in inaD have been shown to affect the localization of PKC, TRP, and PLC. INAD also binds NINAC, an actin binding protein. It is possible that cortactin may also interact with INAD and tether the signalplex to the actin cytoskeleton in the microvilli of the rhabdomeres. The novel temperature sensitive paralytic mutation, Taf; alters the function of a protein that is certainly critical for normal Drosophila visual perception. Most of the mutants affecting fly vision have only an eye specific phenotype. We have demonstrated that Taf affects phototransduction, synaptic transmission between the photoreceptors and the laminar neurons, and locomotion. Identification of the gene responsible for the Tafphenotype will likely play an important role in better defining the phototransduction cascade. Since Tuf function affects the activity of laminar neurons, it is likely to be an important protein regulating the activity of neurons throughout the nervous system.

REFERENCES Adams, M. D., Celniker, S. E., Holt, R. A.. Evans, C . A., Gocayne, J. D. et ul. (2000) The Genome of Drosophilu melrmoguster. Science, 287, 21 85-2 195. Blaustein, M . P., and Lederer, W. J. (1999) Sodium/calcium exchange: its physiological implications. Physiol. Rev., 79, 763-854. Byk, T., Bar-Yaacov, M., Doza, Y. N., Minke, B., and Selinger, Z. (1993) Regulatory arrestin cycle secures the fidelity and maintenance of the fly photoreceptor cell. Proc. Nutl. Acud. Sci. U S A , 90, 1907-191 1. Chen, M. S., Obar, R. A,, Schroeder, C. C . , Austin, T. W., Poodry, C. A., Wadsworth, S. C., and Vallee, R. B. (1991) Multiple forms of dynamin are encoded by shihire, a Drosophilu gene involved in endocytosis. Nuture, 351, 583-586. Cosens, D. J.. and Manning, A. (1969) Abnormal electroretinogram from a Drosophilu mutant. Nuture, 224, 285-287. Feng, Y., Huynh. L., Takeyasu, K.. and Fambrough, D. M. (1997) The Drosophilu Na+/K+-ATPdse alpha-subunit gene: gene structure, promoter function and analysis of a cold-sensitive recessive-lethal mutation. Genes Funct., 1, 99-1 17. Hardie, R. C., and Minke, B. (1992) The rrp gene is essential for a light-activated C a Z t channel in Drosophilu photoreceptors. Neuron, 8. 643-65 I . Hardie, R. C., Raghu, P., Moore, S., Juusola, M., Baines, R. A,, and Sweeney, S. T. (2001) Calcium Influx via T R P Channels Is Required to Maintain PIPz Levels in Drosc~philrr Photoreceptors. Neuron, 30, 149-159. Harrison, S. D., Broadie, K., van de Goor, J., and Rubin, G. M. (1994) Mutations in the Drosophilu Rop gene suggest a function in general secretion and synaptic transmission. Neuron, 13, 555-566. Haug-Collet, K., Pearson, B.. Webel, R., Szerencsei, R. T., Winkfein, R. J., Schnetkamp, P. P., and Colley. N. J. (1999) Cloning and characterization of a potassium-dependent sodium/calcium exchanger in Drosophilu. J . Cell. Eiol., 147, 659-670.


94

Watson et al.

Hengstenberg, R., and Gotz, K. G. (1964) [Effect of facet-separating pigments on the perception of light and contrast in eye mutants of Drosophila]. Kyberkinerik, 3, 276. Hotta, Y., and Benzer, S. (1969) Abnormal electroretinograms in visual mutants of Drosophila. Nature, 222, 354356. Inoue, H., Yoshioka, T., and Hotta, Y. (1988) Membrane-associated phospholipase C of Drosophila retina. J . Biochem. ( T o k y o ) , 103, 91-94. Koenig, J . , and Merriam, J. R. (1977) Autosomal ERG mutants. Drosophila Information Service, 52, 50-51. Kunze, D. L., Sinkins, W. G., Vaca, L., and Schilling, W. P. (1997) Properties of single Drosophila Trpl channels expressed in Sf9 insect cells. Am. J . Physiol., 272, C27-34. Lebovitz, R. M., Takeyasu, K., and Fambrough, D. M. (1989) Molecular characterization and expression of the Na+/K+-ATPase alpha-subunit in Drosophila melanogaster. EMBO J . , 8, 193-202. Leung. H -T., Geng, C., and Pak, W. L. (2000) Phenotypes of trpl Mutants and Interactions between the Transient Receptor Potential (TRP) and TRP-Like Channels in Drosophila. J . Neurosci., 20, 6797-6803. Li, H. S., Porter, J. A,, and Montell, C. (1998) Requirement for the NINAC kinase/myosin for stable termination of the visual cascade. J . Neurosci., 18, 9601-9606. Lindsley, D. L., and Zimm, G. G. (1992) The Genome of Drosophila melanogaster. San Diego: Academic Press. p. 1132. Littleton, J. T., Chapman, E. R., Kreber, R., Garment, M. B., Carlson, S. D., and Ganetzky, B. (1998) Temperature-sensitive paralytic mutations demonstrate that synaptic exocytosis requires SNARE complex assembly and disassembly. Neuron, 21, 401413. Loughney, K., Kreber, R., and Ganetzky, B. (1989) Molecular analysis of the para locus, a sodium channel in Drosophila. Cell, 58, 1143-1 154. Montell, C. (1998) TRP trapped in fly signaling web. Curr. Opin. Neurobiol., 8, 389-397. Montell, C. (1999) Visual transduction in Drosophila. Annu. Rev. Cell. Dev. Biol., 15, 231-268. Naisbitt, S., Kim, E., Tu, J. C., Xiao, B., Sala, C., ValtschanoK, J., Weinberg, R. J., Worley, P. F., and Sheng, M. (1999) Shank, a novel family of postsynaptic density proteins that binds to the NMDA receptor/PSD-95/GKAP complex and cortactin. Neuron, 23, 569-582. Oberwinkler, J., and Stavenga, D. G. (2000) Calcium transients in the rhabdomeres of darkand light-adapted fly photoreceptor cells. J . Neurosci., 20, 1701-1709. Pdk, W. (1995) Drosophila in vision research. Invesl. Opthamol. and Visual Sci., 36, 23402357. Pallanck, L., Ordway, R. W., Ramaswami, M., Chi, W. Y., Krishnan, K. S., and Ganetsky, B. (1 995) Distinct roles for N-ethylmaleimide-sensitive fusion protein (NSF) suggested by the identification of a second Drosophila NSF homologue. J . Biol. Chem., 270, 1874218744. Pearn, M. T., Randall, L. L., Shortridge, R. D., Burg, M. G., and Pak, W. L. (1996) Molecular, biochemical, and electrophysiological characterization of Drosophila norpA mutants. J . Biol. Chem., 271, 49374945. Ramaswami, M., Rao, S., van der Bliek, A., Kelly, R. B., and Krishnan, K. S. (1993) Genetic studies on dynamin function in Drosophila. J . Neurogen., 9, 73-87. Schubiger, M., Feng, Y., Fambrough, D. M., and Palka, J. (1994) A mutation of the Drosophila sodium pump alpha subunit gene results in bang-sensitive paralysis. Neuron, 12, 373-381.



95

Schwarz, E. M., and Benzer, S. (1997) Cul.~,a N a + / C a + exchanger gene of Drosophilu melanogaster. Proc. Natl. Acud. Sci. USA, 94, 10249-10254. Siddiqi, O., and Benzer. S. (1976) Neurophysiological defects in temperature-sensitive paralytic mutants of Drosophilu rnelunoguster. Proc. Natl. Acud. Sci. USA. 73,3253-3257. Spradling, A. C., Stern, D., Beaton, A,, Rhem, E. J., Laverty, T., Mozden, N., Misra, S., and Rubin, G . M. (1999) The Berkeley Drosophilu Genome Project gene disruption project: Single P-element insertions mutating 25% of vital Drosophilu genes. Genetics, 153, 135-177. Stowers, R. S., and Schwarz, T. L. (1999) A genetic method for generating Drosophilu eyes composed exclusively of mitotic clones of a single genotype. Genetics, 152, 1631-1639. Suzuki, D. T., Grigliatti, T.. and Williamson, R. (1971) Temperature-sensitive mutations in Drosophilu melunoguster, VII. A mutation (para") causing reversible adult paralysis. Proc. Nutl. Acud. Sci. USA, 68, 890-893. Therien, A. G., and Blostein, R. (2000) Mechanisms of sodium pump regulation. Am. J . Physiol. Cell. Physiol., 279, C541-566. Vinos, J., Jalink, K., Hardy, R. W., Britt, S. G., and Zuker, C. S. (1997) A G proteincoupled receptor phosphatase required for rhodopsin function. Science, 277, 687-690. Wu, H., and Parsons, J. T. (1993) Cortactin, an 80/85-kilodalton pp60src substrate, is a filamentous actin-binding protein enriched in the cell cortex. J . Cell. Biol., 120, 14171426. Yasuhara, J. C., Baumann, O., and Takeyasu, K. (2000) Localization of Na+/K+-ATPase in developing and adult Drosophilu melanogaster photoreceptors. Cell Tissue Res., 300, 239-249. Zinsmaier, K. E., Eberle, K. K., Buchner, E., Walter, N., and Benzer, S. (1994) Paralysis and early death in cysteine string protein mutants of Drosophila. Science, 263, 977-980.