Nucleoside Diphosphate Kinase, a Source of GTP, Is Required for Dynamin-Dependent Synaptic Vesicle Recycling

Neuron, Vol. 30, 197–210, April, 2001, Copyright 2001 by Cell Press

Nucleoside Diphosphate Kinase, a Source of GTP, Is Required for Dynamin-Dependent Synaptic Vesicle Recycling K. S. Krishnan,† Richa Rikhy,† Sujata Rao,† Madhuri Shivalkar,† Michael Mosko,* Radhakrishnan Narayanan,* Paul Etter,* Patricia S. Estes,* and Mani Ramaswami*‡ * Department of Molecular and Cellular Biology and ARL, Division of Neurobiology University of Arizona Tucson, Arizona 85721 † Department of Biological Sciences Tata Institute for Fundamental Research Homi Bhabha Road Colaba, Bombay 400005 India

Summary Nucleoside diphosphate kinase (NDK), an enzyme encoded by the Drosophila abnormal wing discs (awd) or human nm23 tumor suppressor genes, generates nucleoside triphosphates from respective diphosphates. We demonstrate that NDK regulates synaptic vesicle internalization at the stage where function of the dynamin GTPase is required. awd mutations lower the temperature at which behavioral paralysis, synaptic failure, and blocked membrane internalization occur at dynamin-deficient, shits, mutant nerve terminals. Hypomorphic awd alleles display shits-like defects. NDK is present at synapses and its enzymatic activity is essential for normal presynaptic function. We suggest a model in which dynamin activity in nerve terminals is highly dependent on NDK-mediated supply of GTP. This connection between NDK and membrane internalization further strengthens an emerging hypothesis that endocytosis, probably of activated growth factor receptors, is an important tumor suppressor activity in vivo. Introduction Dynamin is a GTPase required for fission of clathrincoated endocytic vesicles from the plasma membrane (Kosaka and Ikeda, 1983; Chen et al., 1991; van der Bliek and Meyerowitz, 1991; Damke et al., 1994; Takei et al., 1995). Dynamin functions at the rate-limiting step of endocytosis; thus, increasing the stability of the active, GTP bound form of dynamin results in accelerated membrane internalization in cultured fibroblasts (Sever et al., 1999, 2000b). Dynamin-mediated endocytosis is required in several cellular processes such as synaptic vesicle recycling, nutrient uptake, or downregulation of activated growth factor receptors (Kramer et al., 1991; De Camilli and Takei, 1996; Futter et al., 1996; Vieira et al., 1996). This probably accounts for the pleiotropic phenotypes of Drosophila shibirets (shits) mutants that have a conditional block in dynamin function (Kelly and Suzuki, 1974; Poodry, 1990; Ramaswami et al., 1993). ‡ To whom correspondence should be addressed (e-mail: mani@

u.arizona.edu).

The most striking phenotype of shits mutants is rapid paralysis upon exposure to nonpermissive temperature, a behavior that reflects synaptic failure due to blocked vesicle recycling at presynaptic terminals (Poodry and Edgar, 1979; Kosaka and Ikeda, 1983; Koenig and Ikeda, 1989). In addition to dynamin, several other proteins involved in coated vesicle formation at synapses and other cellular contexts have been biochemically purified and analyzed (Cremona and De Camilli, 1997; Marsh and McMahon, 1999). However, many “endocytosis proteins” and their regulators probably remain unknown. To identify potentially novel molecules required for synaptic vesicle recycling in vivo, we performed a classical genetic screen designed to identify mutations that modify the conditional paralysis phenotype of Drosophila shibirets mutants; our expectation being that these mutations will identify other genes in the same cellular pathway (Simon et al., 1991). Mutant shits flies show remarkably tight and penetrant temperature-dependent paralysis (Kim and Wu, 1990; Ramaswami et al., 1993). The tightness of this t.s. phenotype does not reflect a sudden, step-like decline in dynamin activity. Rather, in shits animals, dynamin activity is likely to be steadily reduced as temperature is raised. Thus, the restrictive temperature for a given allele reflects the point at which dynamin activity drops below a level required for sustained activity of critical synapses in the fly (Ramaswami et al., 1993, 1994; Krishnan et al., 1996; Grant et al., 1998). The tightness of the shits paralytic phenotype allowed us to identify second-site “enhancer” mutations that cause shits mutants to be paralyzed at lower temperatures. In a screen of about 190,000 mutagenized shits flies, we isolated three enhancer of shibire, e(shi), mutations. Our analysis indicates that all three are alleles of abnormal wing discs (awd), the Drosophila ortholog of a human tumor suppressor gene, nm23 (Steeg et al., 1988a, 1988b; Rosengard et al., 1989; Biggs et al., 1990). awd/nm23 encodes cytoplasmic nucleoside diphosphate kinase (NDP kinase, or NDK), an enzyme required for ATP dependent synthesis of nucleoside triphosphates from the respective nucleoside diphosphates. Several lines of genetic and cell biological evidence presented here indicate that Awd/Nm23 functions to provide GTP required for dynamin function at nerve terminals. Because levels of dynamin:GTP determine the rate of endocytosis (Sever et al., 1999), our data suggest that NDK-dependent supply of GTP helps determine the rate of endocytosis in vivo. In humans, the tumor/metastasis suppressor nm23 is a small gene family comprising at least three different homologous genes H1, H2, and H3. Reduced expression of nm23 appears to be a primary determinant of tumor progression, invasiveness, and serum-stimulated motility of several metastatic tumors (Leone et al., 1991, 1993; Kantor et al., 1993; Baba et al., 1995; Parhar et al., 1995; Fukuda et al., 1996). Our data suggest that an early effect of nm23 inhibition may be to slow down dynamindependent membrane internalization. In light of recent links between putative tumor suppressors and other components of endocytosis, our observations strengthen the

Neuron 198

Table 1. Temperature at which 100% of Flies Are Paralyzed in Three Minutes Alleles

shits2/shits2

shiCK2/shiCK2

parats1

comttp7

⫹/⫹

⫹/⫹ MSM95/MSM95 MSF15/MF15 CM47/CM47

27 22 22 26

35 29 29 33

27 27 27 27

35 35 35 35

⬎42 40 40 ⬎42

The MSM95, MSF15, and CM47 mutations specifically reduce the temperature required for shits paralysis; the mutations have no effect on the paralytic behavior of parats and comtts alleles tested.

hypothesis that endocytosis is an important tumor suppressor function in vivo (Floyd and De Camilli, 1998; Levkowitz et al., 1998; Babst et al., 2000). Results Isolation of Three Allelic Mutations that Enhance shits Paralysis In a behavioral genetic screen (Experimental Procedures), we isolated three independent, EMS-induced, e(shi) mutations, MSM95, MSF15, and CM47, that reduced the temperature at which shits mutants paralyze. We subsequently discovered (see below) that these three mutations are alleles of a single gene that we termed e(shi)A. When e(shi)A mutations were heterozygous, their slight dominance was indicated by a marginal reduction in the temperature for paralysis of shi; e(shi)A/⫹ flies when compared to shi; ⫹/⫹ controls (Table 2). However, when homozygous, these mutations dramatically reduced the restrictive temperature for paralysis of shits flies (Figure 1; Tables 1 and 2). For instance, w shiCK2; ⫹/⫹ flies require about 2 min to be paralyzed at 35⬚C and are not affected at all at temperatures below 33⬚C; in contrast, w shiCK2; MSM95/MSM95 animals are rapidly (within 2 min) paralyzed at 29⬚C, an “enhancement” of about 6⬚C. The effect of e(shi)A mutations on paralytic behavior was specific to shits mutants: e(shi) mutations had no effect on paralysis of Drosophila, comatosets, and parats mutants (Table 1) that affect synaptic vesicle exocytosis (Pallanck et al., 1995) and axonal conduction (Loughney et al., 1989), respectively. The specificity of these genetic interactions argued a specific effect of e(shi)A mutations on synaptic vesicle recycling, the function affected in shi mutant animals. All experiments to genetically map the mutations were performed in a shits background because e(shi)A mutations do not show robust dominant or recessive pheno-

Table 2. Temperatures for Paralysis of shiShy Flies Carrying Heteroalleleic Combinations of e(shi)A Allele/Allele

⫹

MSM95

MSF15

CM47

⫹ MSM95 MSF15 CM47

35

33 29

33 30 29

34 32.5 33 33

The data indicate noncomplementation among MSM95, MSF15, and CM47 for the enhancer of shi phenotype. Recombination analyses first indicated close genetic linkage of the MSM95, CM47, and MSF15 mutations (Experimental Procedures). Allelism was further indicated by results from the complementation analysis shown above.

types in a wild-type background (Table 1). We determined that MSM95, MSF15, and CM47 are alleles of the same gene by segregation analysis, chromosome mapping and genetic complementation. All three mutations mapped to the vicinity of claret, a genetic marker close to the tip of the right arm of the third chromosome. The mutations did not complement one another: for instance shits; MSM95/MSF15 flies were paralyzed at much lower temperatures than shits; MSM95/⫹ or shits; MSF15/⫹. The suggestion of allelism was further strengthened by finer recombination analysis. From more than 1000 progeny of shits/shits; MSM95/MSF15 or shits/shits; MSM95/CM47 transheterozygous females, we failed to obtain any recombinants between the different e(shi) alleles; thus, MSM95, MSF15, and CM47 are very tightly linked (⬍0.1 cM). We tentatively concluded (and subsequently confirmed by transgene rescue experiments) that the three mutations were allelic and performed more detailed genetic studies on MSM95, the strongest allele of e(shi)A.

e(shi)A Is Allelic to awd and e(shi)A Mutations Affect NDP Kinase Activity Recombination and genetic deficiency analysis (Experimental Procedures) localized e(shi)A to near cytological map position 100E, within a centimorgan of a P insertion in strain 2166, in a region deleted in the deficiency strain Df(3R) faf-BP (missing 100D–100F). A recessive lethal line, 2167, that carries a P insertion at 100E did not complement MSM95. To confirm that this noncomplementation resulted from the transposon insertion, rather than an independent mutation on the chromosome, we mobilized the P element in 2167 and obtained several P element excision lines. Of 11 excision lines obtained, 4 were wild-type for e(shi)A; excision chromosomes that did not complement e(shi)A were presumed to carry imprecise excisions of the P element typically obtained in such P element mobilization experiments (Experimental Procedures). These observations confirmed that the P insertion in line 2167 disrupts e(shi)A gene function. The Berkeley Drosophila Genome Project (BDGP; www.fruitfly.org) has sequenced genomic DNA flanking the P insertion in line 2167. A BLAST search using this genomic sequence showed that it derives from the 5⬘ untranslated sequence of the mRNA for the awd gene that also maps to the same region of chromosome 3R. Interestingly, awd, the Drosophila ortholog of the human tumor suppresor nm23, encodes NDK, a protein not previously associated with endocytosis. We confirmed the accuracy of the BDGP report on the insertion site for 2167 by PCR, using oligonucleo-

NDK Regulates Dynamin Function In Vivo 199

Figure 1. enhancer of shibire Mutations Alter Temperature Dependence of shits Paralysis (A) MSM95 (B) MSF15 (C) CM47

tides from P element and awd DNA sequences, as well as by isolating and sequencing genomic DNA flanking the P insertion (Figure 2A). Three lines of evidence demonstrated that e(shi)A mutations MSM95, MSF15, and CM47 are alleles of awd. First, two lethal alleles of awd, awdb3, a severe hypomorph (Dearolf et al., 1988a, 1988b), and awdKRs6, a null mutation (Timmons et al., 1995), did not complement any of the e(shi)A mutations: for instance, w shiCK2; MSM95/ awdKRs6 flies are rapidly debilitated at 28⬚C, compared to 34⬚C for w shiCK2; MSM95/⫹ controls (Figure 2B and Table 3 show representative data). Second, an awd transgene (PAS7), which includes native transcriptional control sequences fused to the awd cDNA, rescues the enhancer of shi phenotype of MSM95. Thus, w shiCK2; PAS7/⫹; MSM95/MSM95 flies are paralyzed at 34⬚C, almost identical to w shiCK2 (Figure 2D). Remarkably, we could also effect unambiguous (though incomplete) phenotypic rescue with either of two human nm23 transgenes H1 (line 1) or H2 (line 4) driven by the native awd promoter (Xu et al., 1996), an observation that emphasizes phylogenetic conservation of Awd/Nm23 functions in vivo (Table 3). An awd transgene, H119A3, identical to PAS7 but for a point mutation at histidine 119, the residue essential for the kinase activity of NDK, was unable to rescue or alleviate paralysis phenotypes of awd. As the H119A3 transgene produces a stable Awd protein (Xu et al., 1996), this result indicates that the enzymatic activity of NDK is required for its synaptic functions. Finally, when we measured NDK activity in extracts from control and mutant fly heads, we found a roughly 90% reduction of activity in MSM95/MSM95 heads when compared to appropriate controls (Figure 2C). This reduction in enzyme activity is associated with reduced protein levels probably deriving from altered gene regulation (Figure 2E). We examined the behavior of a variety of awd mutant combinations (Table 3). The most striking observation was that specific allelic combinations of awd showed rapid and reversible temperature-dependent paralysis in a wild-type background, a behavior remarkably similar to that observed in shits mutants. For instance, a temperature of 38⬚C rapidly paralyzed awdMSM95/awdKRs6 flies, a phenotype completely rescued by the presence of a single copy of the rescuing awd⫹ transgene, PAS7, or either human nm23 transgene H1 or H2 (Table 3). In general, awd alleles that enhance or mimic shits phenotypes are hypomorphs, and their severity corresponds roughly with allelic strength (Table 3). An unusual allele called killer of prune (awdKpn) only slightly reduces intrin-

sic NDK activity, but severely affects a protein phosphotransferase activity of the protein (Lascu et al., 1992; Freije et al., 1997). This viable allele shows a dominant lethal interaction with prune (pn) mutations that affect a potential GTPase activating protein (GAP) required for normal eye color (Sturtevant, 1956; Biggs et al., 1988; Teng et al., 1991, Lascu et al., 1992). We found that awdKpn had no effect on shits paralysis. Conversely, the shi-enhancer allele awdMSM95 had no effect on the viability of pn1 (Table 3). That pn-interacting alleles (or allelic combinations) show no overlap with the shi-interacting awd alleles indicates that the two genetic interactions occur through different underlying mechanisms. Our observation that reduction in NDK activity causes shits flies to paralyze at lower temperatures was consistent with synaptic vesicle endocytosis being regulated by levels of Awd activity in vivo. We directly addressed this issue via a series of experiments designed to determine the effect of awd mutations on synaptic physiology, specifically on synaptic vesicle recycling. The awd Alleles Increase the Temperature Sensitivity of Synaptic Failure in shits The temperature sensitivity of synaptic responses in shits, shits; awd, or awd mutants exactly corresponded with observations made on the paralytic behavior of whole flies. The compound response of the visual system to a light flash stimulus may be measured in a simple extracellular recording called an electroretinogram (ERG). A typical ERG includes different components: a “slow” receptor potential corresponds to photoreceptor depolarization; two transient spikes, the on and off transients (arrowheads on Figure 3), that flank the receptor potential represent compound synaptic responses of the optic lobe (Alawi and Pak, 1971; Wu and Wong, 1977; Zuker, 1996). In shits mutants, the on and off transients are lost at elevated temperature in a use-dependent manner. In shiCK2, for instance (n ⫽ 15), the synaptic transients clearly visible at room temperature and even at 34⬚C are lost at 36⬚C (Figure 3A, trace 1). However, in an awdMSM95/awdKRs6 mutant background, synaptic responses of shiCK2 flies disappear at 30⬚C (Figure 3A, trace 2; n ⫽ 18). That this “enhancement” of the shi synaptic transmission defect is caused by altered awd function, rather than genetic background effects, is shown by the greatly attenuated effect of awdMSM95/awdKRs6 when the wild-type awd transgene, PAS7, is present in the same genetic background (Figure 3A, trace 3; n ⫽ 14). We further discovered that electroretinograms recorded from ⫹/Y; awdMSM95/awdKRs6 animals (a background wild-

Neuron 200

Figure 2. Multiple Lines of Evidence to Show that e(shi)A is awd (A) A P element insertion in line 2167 that causes noncomplementation of the MSM95 phenotype is inserted into the 5⬘ untranslated region of the awd mRNA encoding NDP kinase. (B) An awd null allele awdKRs6 does not complement the enhancer of shi phenotype of MSM95. (C) Whole fly lysates from MSM95/MSM95 mutants show reduced NDP kinase activity relative to controls. Enzyme activity is represented in arbitrary units defined in terms of the slope of substrate utilization (OD units) as a function of time (see Experimental Procedures). (D) A single copy of a wild-type awd transgene (PAS7) rescues the enhancer of shi phenotype of MSM95; further transgene rescue data are shown in Table 3. (E) MSM95/MSM95 flies show severely reduced Awd protein in Western blots of head lysates relative to shi and wild-type controls. This figure shows data for alleles shiCK2, MSM95, and awdKRs6; they are representative of similar results obtained for various different combinations of shits, awd, and e(shi) alleles (Table 3 and data not shown).

type for shi) also lost their synaptic transients at 39⬚C in a use-dependent manner (n ⫽ 10). Again, this abnormality was rescued by the PAS7 transgene (n ⫽ 9),

indicating that it derives from a genetic reduction of awd function (Figure 3B). In all of these mutant combinations, loss of synaptic transmission at elevated temperatures

Table 3. Paralytic Temperature or Viability of awd Alleles in Combination with shi, prune, and awd/nm23 Transgenes awdMSM95

⫹/Y ⫹/Y; PAS7/⫹ ⫹/Y; H1/⫹ ⫹/Y; H2/⫹ shiCK2/Y shiCK2/Y; PAS7/⫹ shiCK2/Y; H119A3/⫹ shiCK2/Y; H1/⫹ shiCK2/Y; H2/⫹ shiCK2/Y; H1/H1 shiCK2/Y; H2/H2 pn1/Y

awdMSM95

awdKpn

MSM95

Kpn

awdMSM95

awdKpn

⫹/⫹

awd

awd

awd

awd

awdKRs6

⬎42 ⬎42 ⬎42 ⬎42 35 35 35 35 35 35 35 ⬎42

38 ⬎42 ⬎42 ⬎42 27 33 27 30 31 ND ND ND

40 ⬎42 ⬎42 ⬎42 29 34 ND 30 31 30.5 31.5 viable 40

⬎42 ND ND ND 35 ND ND ND ND ND ND lethal

⬎42 ND ND ND 34.5 ND ND ND ND ND ND lethal

⬎42 ND ND ND 34.5 ND ND ND ND ND ND lethal

KRs6

Kpn

The awd allelic combination, awdMSM95/awdKRs6, shows rapid, reversible t.s. paralysis at 39⬚C, a phenotype rescued by an awd⫹ transgene PAS7. The enhancement of shits paralysis by awd is rescued by the awd⫹ transgene PAS7, but not by an identically expressed transgene H119A3 that carries a point mutation in the active site histidine required for phosphate transfer in NTP synthesis from NDP. The awdKpn (Killer of prune) allele does not interact with shits. The enhancer of shi allele, awdMSM95, does not interact with pn1 mutant in a potential GTPase activating protein (GAP) (Teng et al., 1991). Human transgenes H1 and H2 partially rescue the enhancer of shi phenotype. Entries in bold font indicate the most important observations; ND ⫽ not done.

NDK Regulates Dynamin Function In Vivo 201

Figure 3. Hypomorphic awd Alleles Not Only Reduce the Temperature for Synaptic Failure in shits Synapses (A), but also Show Synaptic Failure in Isolation (B) (A) Numbers in parentheses beside the genotypes indicate restrictive temperature for adult paralysis. In the electroretinogram recordings shown, transient spikes that precede (arrows) and follow (arrowheads) compound responses of photoreceptors to light stimulation derive from synaptic transmission between photoreceptor cells and the optic lobes. These “on” and “off” transients are selectively lost in shiCK2 flies at 36⬚C ([A], trace 1); however, in shiCK2; awdMSM95/awdKRs6 double mutants, synaptic failure is evident at 30⬚C ([A], trace 2). In the presence of a wild-type awd transgene (PAS7), the effect of awd mutations is alleviated: thus, ERGs of shiCK2; PAS7/⫹; awdMSM95/awdKRs6 flies do not lose their on and off transients until 34⬚C ([A], trace 3). (B) Shows loss of synaptic on and off transients in ERG recordings from y w/Y; awdMSM95/awdKRs6 mutants at 39⬚C ([B], trace 1) and controls to show that this temperature dependent synaptic failure is not shown in the presence of a rescuing PAS7 transgene ([B], trace 2) or in wild-type flies ([B], trace 3).

is reversible: that is, synaptic transients reappear if the flies are restored to permissive temperatures. Thus, Awd is essential for normal synaptic transmission, and conditional synaptic defects are seen in mutant animals that otherwise appear completely normal. awd Function Is Required for Synaptic Vesicle Recycling To test whether the synaptic transmission block in shi; awd or awd mutants derive from blocked dynamin-

dependent, synaptic vesicle recycling, we studied the presynaptic effects of awd mutations. We specifically examined effects of awd mutations on nerve terminal ultrastructure, viewed by electron microscopy (EM), and on membrane internalization, assayed by observing endocytic uptake of a fluorescent dye into recycling synaptic vesicles. When shits mutants are exposed to bright light at their restrictive temperature, their photoreceptor terminals accumulate deep, flat invaginations emanating from the plasma membrane (Kosaka and Ikeda, 1983; Koenig and Ikeda, 1996). At temperatures permissive for dynamin function, such structures are not seen. Although the origins of these invaginations remains poorly understood, it is clear that they are vesicle recycling intermediates that accumulate when dynamin function is inhibited (Koenig and Ikeda, 1996, 1999). If our awd mutations altered synaptic transmission by reducing dynamin activity, then we expected to observe these recycling intermediates at lower temperatures in shits; awd double mutants than in shits mutants alone. Furthermore, we anticipated observing such membrane invaginations in ⫹/Y; awdMSM95/awdKRs6 mutants at 39⬚C, a temperature at which they show a synaptic transmission defect in our electroretinogram recordings. In cross-sectional view, the membrane invaginations appear as thin, elongated electron-dense structures (arrow in Figure 4B). After establishing conditions for observing these structures in our shits mutants, we analyzed various experimental and control photoreceptor terminals by EM. For each animal, we selected single cross-sections through the lamina and counted the number of structures visible in individual cartridges, modules containing synaptic terminals of the photoreceptor cells (R1–R6). As predicted, we observed these characteristic structures at lower temperatures in a shits; awd mutant background than in shits or shits; PAS7; awd controls (Figures 4A–4C and 4F). At 31⬚C, shiCK2; awdMSM95/awdKRs6 nerve terminals show 5.3 ⫾ 0.2 structures per section through a laminar cartridge (19 cartridges, 4 animals), compared to 0.9 ⫾ 0.1 structures per section/cartridge in shiCK2; ⫹/⫹ flies under identical conditions (31 cartridges, 6 animals). This increase (p ⬍ 10⫺9) in the observed number of recycling intermediates is temperature and awd dependent as evidenced by two control observations. First, at 19⬚C, shiCK2; awdMSM95/ awdKRs6 show almost no evidence of abnormal recycling (0.1 ⫾ 0.1 structures per section/cartridge based on 15 cartridges in 3 animals examined); second, the presence of the PAS7 transgene greatly reduces (p ⬍ 10⫺4) the number of tubular structures observed in shiCK2; awdMSM95/awdKRs6 nerve terminals at 31⬚C (1.8 ⫾ 0.4 structures per section/cartridge; 18 cartridges, 5 animals). Structures similar to those observed in shits mutant photoreceptor terminals were also observed at low levels in ⫹/Y; awdMSM95/awdKRs6 terminals exposed to light at 39⬚C (Figure 4D; 23 cartridges, 5 animals). This number, 0.7 ⫾ 0.3 structures per section/cartridge, was significantly reduced (p ⫽ 0.03) to 0.05 ⫾ 0.05 by the presence of the PAS7 transgene (Figure 4E and 4F; 17 cartridges, 4 animals). Our observation that awd mutants cause accumulation of recycling intermediates indistinguishable from those observed in shits mutant nerve terminals strongly

Neuron 202

Figure 4. Similar Membrane Intermediates Are Arrested in awd and shits Mutant Nerve Terminals Characteristic recycling intermediates (white arrows) arrested in photoreceptor terminals of shits flies at nonpermissive temperatures are observed in shi; awd double mutants at much lower temperatures; remarkably, similar structures are also visible in awdMSM95/ awdKRs6 animals at elevated temperatures. Abbreviations: shi ⫽ shiCK2; awd ⫽ awdMSM95/ awdKRs6; P(awd⫹) ⫽ PAS7. (A)–(C) show representative control (w), w shiCK2; awdMSM95/ awdKRs6 and control w shiCK2; PAS7/⫹; awdMSM95/awdKRs6 terminals under EM at 31⬚C. (D) and (E) show y w/Y; awdMSM95/awdKRs6 and y w/Y; PAS7/⫹; awdMSM95/awdKRs6 terminals at 39⬚C. The temperatures were selected based on behavioral and ERG analysis to most effectively explore potential differences between shi; awd and shi; P(awd⫹); awd mutants (31⬚C) or awd and P(awd⫹); awd (39⬚C), respectively. The histogram in (F) shows number of invaginations apparent per section through a laminar cartridge in the various genotypes. Note that these structures are absent at 19⬚C in shi; awd double mutants that show large numbers of these structures at 31⬚C.

indicates that awd mutations affect the internalization step in vesicle recycling, at, or very close to, the stage at which dynamin is required. We looked to see whether an internalization block could be directly observed by studying the temperature dependence of fluorescent dye (FM1-43) uptake into recycling synaptic vesicles at motor synapses on bodywall muscles of third instar larvae (Betz and Bewick, 1992; Ramaswami et al., 1994). When stimulated at 38⬚C with high-potassium (60 mM) saline containing the endocytic tracer FM1-43, wshiCK2/Y; awdMSM95/awdKRs6 motor terminals (n ⫽ 8) on larval bodywall muscles were labeled poorly or not at all; in contrast, motor terminals on wshiCK2/Y; PAS7/⫹; awdMSM95/awdKRs6 muscles were robustly labeled (Figure 5; n ⫽ 8). At room temperature, terminals in animals of either genotype were strongly labeled (data not shown). Although we did observe some variability in the uptake into a single identified synapse (see Experimental Procedures), the data unequivocally indicated a role for awd function in stimulation-dependent dye loading. To roughly monitor the efficiency of exocytosis at these synapses, we tested whether terminals loaded with dye at permissive temperatures would unload their contents if stimulated at 38⬚C. Consistent with a specific effect of awd, exocytosis, monitored by such dye-unloading experiments, was not detectably different between experimental and control larvae (n ⫽ 4). Taken together with our physiological and ultrastructural studies on synaptic transmission at photoreceptor synapses, these results establish that synaptic vesicle endocytosis is severely compromised by hypomorphic awd mutations.

awd Mutations Do Not Alter Presynaptic Organization or Dynamin Levels Before considering how a relatively specific effect on dynamin function may derive from a reduced Awd activity, we tested whether any of the awd mutant phenotypes could derive from defects in synapse architecture or altered levels or localization of dynamin. The general morphology of the motor nerve endings in an awd background, visualized by synaptotagmin staining and viewed by confocal microscopy, was indistinguishable from controls (Figures 6A and 6B). In wild-type (data not shown), w shiCK2; awdMSM95/awdKRs6 (Figure 6A), and w shiCK2; PAS7/⫹; awdMSM95/awdKRs6 animals (Figure 7B), the characteristic arborization pattern of the two motor axons was clearly visible: synaptotagmin was tightly associated with presynaptic varicosities (boutons), where the transmitter release machinery is localized. Viewed at even higher resolution, a more detailed organization of dynamin, in a “honeycomb” pattern that surrounds active zones for vesicle release, exists within individual wild-type varicosities (Figure 6C; Estes et al., 1996; Gonzalez-Gaitan and Jackle, 1997; Roos and Kelly, 1999; Wan et al., 2000). This organization is preserved in w shiCK2; awdMSM95/awdKRs6 (Figure 6D) as well as w shiCK2; PAS7/⫹; awdMSM95/awdKRs6 (Figure 6E) animals. Finally, as judged by Western blotting of head lysates (Figure 6F), dynamin levels are unchanged in awdMSM95/awdKRs6 mutants analyzed in both wild-type and w shiCK2 backgrounds. Thus, the effect of awd mutations on vesicle recycling occurs downstream of mechanisms that regulate presynaptic organization and levels of dynamin, yet at a stage very close to that requiring dynamin.

NDK Regulates Dynamin Function In Vivo 203

Figure 5. awd Mutations Affect Synaptic Vesicle Internalization Reduced uptake of a fluorescent endocytosis tracer, FM1-43, into nerve terminals of w shiCK2; awdMSM95/awdKRs6 larvae (A) compared to w shiCK2; PAS7/⫹; awdMSM95/awdKRs6 controls (B) identically stimulated at 38⬚C with high-potassium saline in the presence of FM1-43. Terminals on muscles 6 and 7, imaged in both preparations after washing at 38⬚C to remove surface-bound dye, are clearly visible in (B). Terminals of both these genotypes take up dye at lower temperatures; control shiCK2/Y; ⫹/⫹ and ⫹/Y; awdMSM95/awdKRs6 preparations efficiently internalize FM1-43 under the conditions of the experiment (data not shown). The temperatures required for observing a complete block in membrane internalization at shits larval motor terminals are typically about 6⬚C higher than the temperature at which behavioral paralysis is observed (Ramaswami et al., 1993, 1994; Krishnan et al., 1996).

Most Cellular Dynamin and NDK Are Not Physically Associated In Vivo While experiments described above demonstrate that Awd is essential for internalization of synaptic vesicle membrane, they do not address the mechanism of Awd function in endocytosis. Several lines of data suggested a weak physical association between dynamin and NDK. First, the original report on purification of dynamin described isolation of NDK as a dynamin cofactor required for observing a microtubule-stimulated ATPase activity (Shpetner and Vallee, 1989). Second, there are intriguing observations to suggest that NDP kinase can act as a local GDP-GTP exchange factor by directly associating with specific G proteins, thereby providing a local supply of GTP (Zhu et al., 1999). We found little direct support for physical interactions

between dynamin and NDK using coimmunolocalization, cell fractionation, coimmunopreciptation, and GST pulldown experiments. Although Awd/NDK is highly expressed in the CNS and present in synaptic regions of the brain, it is not selectively enriched at presynaptic terminals as might be predicted if it existed predominantly in a dynamin-bound form (Figure 7). In biochemical fractionation experiments, we, like Shpetner and Vallee (1992), found Drosophila dynamin to be substantially present in pellet fractions P2 and P3, while the Awd protein is highly enriched in the final supernatent S3 (data not shown). Beads conjugated to affinity-purified dynamin antibodies (2074) or a GST fusion to a domain of DAP160/Intersectin (Roos and Kelly, 1998) brought down dynamin but not Awd/NDK from an S2 fraction containing both proteins. Attempts to trap transient inFigure 6. The Effect of awd Mutations on Vesicle Recycling Is Downstream of Mechanisms that Regulate Synapse Morphology, Dynamin Organization, and Dynamin Levels (A and B) In w shiCK2; awdMSM95/awdKRs6 double mutants (A), nerve terminal arborization viewed by synaptotagmin immunoreactivity appears normal compared to PAS7 transgene carrying controls (B). (C) Dynamin (green) is organized as a presynaptic meshwork (Estes et al., 1996) that surrounds active zones for vesicle fusion (red) (Roos and Kelly, 1999). Active zones for fusion are labeled by anti-myc antibodies in MHC-GluRIIb-myc flies expressing a myc epitope–tagged glutamate receptor in muscle; immunoelectron microscopy has shown that these receptors cluster at postsynaptic sites directly opposed to presynaptic, electron-dense bodies (Petersen et al., 1997). (D and E) This honeycomb organization of dynamin can be observed in w shiCK2; awdMSM95/awdKRs6 terminals (D) as well as w shiCK2; PAS7/⫹; awdMSM95/awdKRs6 controls (E). (F) Western blot data to show that dynamin (Dyn) levels are not altered by awd mutations in a wild-type or shits background. ␤-tubulin (␤-tub) serves as a loading control.

Neuron 204

Figure 7. Awd Protein Is Present in the Neuronal Cell Bodies, Axons, and Synaptic Regions of the Brain Immunocytochemical staining of sections through adult head sections show that Awd (A) is expressed in high levels in the brain being present in somatic as well as neuropile regions that are highly enriched in cysteine string protein Csp (B). In awdMSM95/awdMSM95 homozygotes, Awd immunoreactivity is severely reduced. Analysis of the larval nmj reveals that, unlike dynamin or the synaptic vesicle protein Csp, Awd present in nerve and muscle (G) is not selectively enriched at nerve terminals (H) where dynamin and synaptic vesicle protein csp are enriched (I). (H) and (I) are a confocal image pair from a single preparation double stained for Awd (H) and csp (I).

teractions between NDK and dynamin, using high concentrations of GDP and ATP or ATP␥-s for instance, were also unsuccessful (data not shown). These admittedly negative data indicate that dynamin and NDK are predominantly not associated in vivo. However, they do not rule out weak or transient associations between dynamin and NDK molecules at nerve terminals that are suggested by the following genetic data. An elegant genetic analysis of awd indicates that NDK function in vivo requires association with specific intracellular proteins (Xu et al., 1996). Human H1 or H2 transgenes that provide levels of global NDP kinase activity greater than, or similar to, that provided by the Drosophila PAS7 transgene only partially rescue organismal phenotypes of awd null mutants (Xu et al., 1996). This suggested that enzyme activity required for viability needs to be provided in the local context of specific, yet unidentified, protein partners (Xu et al., 1996). Our analysis of the relative efficiency of awd rescue by the human and Drosophila transgenes provides support for the notion that Awd function in endocytosis requires association with dynamin or a molecule in the proximity of dynamin (Table 3). Xu et al. (1996) have demonstrated that two copies of the PAS7, H1 (line 1) and H2 (line 4) transgenes, respectively, provide 46%, 21%, and 45% of wild-type NDK activity measured under identical conditions in adult fly homogenates. Single copies of the transgenes provide half as much activity. We observe (Table 3) that human transgenes providing more global NDK activity than a Drosophila transgene are yet less effective in providing NDK function required for endocytosis. For instance (Table 3), two copies of the H2 transgene that provide 46% wild-type NDK activity result in a 2.5⬚C alleviation (40% rescue) of shi; awd t.s. paralysis compared to a 5⬚C alleviation (80% rescue) by a Drosophila transgene that provides only half as much (23%) NDK activity. This indicates that Awd/Nm23 activity required for synaptic vesicle endocytosis is not predicted simply

by NDK activity measured in fly homogenates. It further suggests that Awd and H1/H2 may need to bind a specific partner protein for appropriate function in endocytosis: presumably, this binding involves protein domains whose sequences have diverged through evolution. Discussion The major conclusion of this study, that dynamin function at nerve terminals is particularly sensitive to inhibition of Awd function, is significant for three reasons. First, because dynamin appears to serve as a “master regulator” of endocytosis (Sever et al., 1999), the rate of synaptic vesicle recycling in vivo could be acutely regulated by NDK-dependent supply of GTP. Second, our results imply that NDK, as the major facilitator of dynamin’s GDP-GTP exchange reaction (or simply GTP loading) essentially acts as an unconventional guanosine nucleotide exchange factor (GEF) for presynaptic dynamin. Finally, because awd and nm23 are tightly conserved functional homologs, the in vivo link between Awd and the dynamin may be relevant to understanding mechanisms that underlie nm23-mediated tumor suppression. Awd Regulates Dynamin Function In Vivo The GTPase dynamin occupies a unique position in the process of membrane internalization. Its activity, specifically the lifetime of the GTP-bound form of dynamin (dynamin:GTP), controls the rate of membrane internalization in non-neuronal cells where this issue has been analyzed (Sever et al., 1999, 2000b). Thus, the entire process of membrane internalization occurs faster in cells expressing a mutant dynamin in which its assembly-stimulated GTP hydrolysis is inhibited. Because dynamin:GTP participates in the rate-determining step of membrane internalization, mechanisms that regulate the lifetime of dynamin:GTP are particularly important from the context of synaptic vesicle recycling. In principle,

NDK Regulates Dynamin Function In Vivo 205

altered rates of synaptic vesicle recycling could be achieved by regulation of dynamin activity. Multiple lines of evidence presented here indicate that reducing Awd activity in vivo specifically reduces the efficiency of dynamin, a GTPase essential for endocytosis. In a search for genes encoding regulators of synaptic vesicle endocytosis, we identified three viable, hypomorphic alleles of awd, the Drosophila homolog of human tumor suppressor nm23, that encodes a cytoplasmic NDP kinase activity (Rosengard et al., 1989; Biggs et al., 1990; Figures 1–2). The specificity of the effects of awd on the temperature sensitivity of shi paralysis suggested a significant requirement for Awd in synaptic vesicle recycling (Table 1). We confirmed by electrophysiology (Figure 3), tracer uptake experiments (Figure 5), and electron microscopy (Figure 4) that shits; awd double mutant synapses show all the characteristic cellular phenotypes of dynamin inhibition at lower temperatures than shits mutants alone. No new phenotypes were seen. Importantly, EM analysis shows that characteristic recycling intermediates may be arrested at lower temperatures in shits; awd double mutant nerve terminals than in shits mutant terminals alone. This localizes the effect of Awd inhibition to a stage indistinguishable from the dynamin-requiring step. Also significant, genetic reduction of awd function alone caused synaptic failure at high temperature, in a manner similar to shits mutants that carry missense mutations in the GTPase domain of dynamin (van der Bliek and Meyerowitz, 1991; Grant et al., 1998; Figure 3–4). Thus, our experiments demonstrate that relatively high levels of Awd activity are required for normal synaptic vesicle recycling. We show, using an active site mutant transgene (Xu et al., 1996; Table 3), that NDK activity rather than other potential activities of the protein is essential for Awd function in synaptic vesicle recycling; thus, the effect of Awd in vesicle recycling is likely mediated by a nucleotide triphosphate (NTP). Given that dynamin is a GTPase and that Awd/NDK synthesizes GTP from GDP, it is likely that this effect of awd mutations occurs via altered dynamin function. Furthermore, as Awd levels appear limiting for dynamin function at nerve terminals, our observations indicate that NDK can regulate dynamin activity in vivo. The import of this is that levels and stability of dynamin:GTP, which controls the rate limiting step in endocytosis, may be regulated not only by GAP activity triggered by dynamin assembly, but also by the efficiency of GTP loading, a step dependent on NDK-generated GTP (Figure 8A). The Specificity of NDK–Dynamin Interactions Phenotypes we observe in our viable awd mutants are remarkably specific: the mutations either enhance or independently display shits phenotypes. Defects in development or general behavior are not obvious. This suggests a rather specific connection between NDK and dynamin functions in vivo. One straightforward explanation for this specificity is that dynamin, in vivo, is unusually sensitive to levels of cytoplasmic GTP that, in turn, depend on levels of NDK. Experiments with purified GTPases indicate that most bind GTP in a range of concentrations (10⫺7–10⫺11) much lower than the resting cytoplasmic GTP concentration (100 ␮M; Bourne et al.,

Figure 8. Models for Awd/NDK Function (A) Model indicating how Awd/NDK may facilitate formation of active dynamin:GTP. In this model, NDK acts like an unconventional GEF that facilitates dynamins’s GTP loading or GDP-GTP exchange reaction. This could occur either via regulation of global GTP levels to which presynaptic dynamin is particularly sensitive or via regulation of local GTP levels at nerve terminals or in the immediate proximity of dynamin. Dynamin exists as an oligomer in vivo, probably as a tetramer. Although shown as a single step, the process of dynamin activation is likely to involve multiple, NDK-sensitive, reversible steps, in which individual dynamin monomers exchange GDP for GTP. It is not yet clear whether a dynamin multimer associated with both GTP (on some subunits) and GDP (on others) is partially active. Adapted from Sever et al. (1999). (B) The pathway for receptor downregulation with postulated stages at which different tumor suppressor functions c-Cbl, Tsg101, and Nm23 (Levkowitz et al., 1998; Babst et al., 2000; and this report) are required. This diagram is adapted from Babst et al. (2000).

1991). For these GTPases, the availability of GTP- or GDP-bound forms are modulated by respective groups of accessory proteins such as GAPs and GEFs/GDIs that regulate GTP hydrolysis and GDP-GTP exchange, respectively (Bourne et al., 1990, 1991). In contrast, GTP loading of dynamin may be determined substantially by local concentrations of cellular GTP: the Km of purified dynamin is between 10 ␮M and 30 ␮M, and accessory GDP-GTP exchange factors have never been identified (Baba et al., 1995; Warnock and Schmid, 1996; Sever et al., 2000a). Dynamin’s unusual sensitivity to a general reduction in GTP concentration may be easily explained if the affinities for dynamin and other GTPases, determined using purified proteins in vitro, closely reflect their

Neuron 206

Table 4. Temperature-Sensitive Paralytic Mutations Used and Their Restrictive Temperatures Mutant Line

Restrictive Temp (⬚C)

shits2 shits4 shiShy shiCK2 parats1 comttp7

27 28.5 34.5 35 27 35

properties in vivo. As most biochemical properties of dynamin have been obtained in vitro, supporting in vivo data are both important and informative. Thus, in this model, NDK provides GTP required for generating dynamin:GTP either by facilitating GTP loading or by acting like a local or remote GEF (Figure 8A). It is also feasible to consider an alternative model in which NDK transiently associates with dynamin and is optimally positioned to provide a very high local concentration of soluble GTP. This would promote GDP for GTP exchange and increase the availability of GTP-bound dynamin. Several previous analyses of NDP kinase interactions in vitro have suggested that NDKs can associate with GTPases and activate them in situ, via direct phosphorylation of protein-associated GDP (Zhu et al., 1999; but see Randazzo et al., 1992). Although we found no physical evidence for association of Awd/NDK with dynamin, genetic data (Table 3) nevertheless support the idea that NDK activity required for dynamin function is provided close to the site of dynamin action. The two models we propose to explain the specific effect of awd mutations on dynamin function are not mutually exclusive. Model 1 suggests that dynamin is most sensitive to reductions in global GTP concentrations, model 2 that NDP kinase provides a local supply of GTP to dynamin. It is possible to imagine that weak associations between NDP kinase and dynamin (or a proximal molecule) have evolved specifically to accommodate the low Kd of dynamin for GTP. Finally, it is important to appreciate that the effect of NDK inhibition on GTP levels depends not only on NDK activity but also on the rate at which GTP is depleted; this could vary substantially under different physiological conditions and in different subcellular regions. Little is currently known about how GTP metabolism varies within cells. It is easy to conceive that GTP in a nerve terminal undergoing rapid bursts of exocytosis, or in a rapidly dividing cell, may be more rapidly turned over than, for instance, GTP in a quiescent nondividing cell like an osteoclast. Thus, dynamin in these contexts may be particularly sensitive to levels of NDK activity. Insight into nm23-Deficient, Invasive Tumors Our analysis directly addresses NDK regulation of dynamin in the context of synaptic vesicle recycling. However, the discovery that awd mutations affect dynamindependent endocytosis may also have implications for understanding the role of the mammalian ortholog, nm23, in tumor/metastasis suppression. Nm23 was identified based on its reduced expression in several metastatic tumors (Steeg et al., 1988a, 1988b). Its potential role as a “metastasis suppressor” has been con-

firmed in elegant experiments analyzing effects of constitutively expressing nm23 in several murine and human nm23-deficent invasive tumor cell lines (Leone et al., 1991, 1993; Baba et al., 1995; Parhar et al., 1995; Fukuda et al., 1996). Although NDP kinase is essential for a wide range of biological functions such as microtubule polymerization and signal transduction, it is not yet understood which of these varied processes are involved in tumor suppression. Our observations demonstrate that nm23 inhibition has the capacity to reduce the rate of dynamin-dependent endocytosis in vivo. It is conceivable that this is irrelevant to the tumor suppressor function of nm23. For instance, nm23 inhibition could affect dynamin activities unrelated to membrane internalization (Whistler and von Zastrow, 1999; Fish et al., 2000; McNiven et al., 2000), nm23 activities unrelated to GTP synthesis (Engel et al., 1995, 1998; MacDonald et al., 1996; Freije et al., 1997; Wagner et al., 1997) or activities of other dynamin family members similarly sensitive to GTP levels (Richter et al., 1995; van der Bliek, 1999). However, it is interesting to consider the possible connections between endocytosis and tumor suppression. Figure 8B suggests a model in which Nm23 functions as a tumor suppressor by facilitating the downregulation of activated growth factor receptors via endocytosis (Futter et al., 1996; Figure 8B). Such a model is supported by recent demonstrations that two putative tumor suppressor proteins, Tsg101 and c-Cbl function at deeper stages of the endocytic pathway to lysosomes (Jongeward et al., 1995; Yoon et al., 1995; Levkowitz et al., 1998, 1999; Babst et al., 2000; Figure 8B). In addition, several other tumor suppressor genes have been mapped to small chromosomal intervals that encode, among others, proteins known to be involved in endocytosis (Floyd and De Camilli, 1998). Data we present make a strong case that an early effect of nm23 inhibition may be to compromise dynamin activity in vivo. This association of Nm23 with a GTPase involved in endocytosis may be important for understanding how nm23 functions in tumor suppression. Interpreted in the context of recent links between cancer and endocytosis, our results support an emerging idea that endocytosis and receptor downregulation are critical for tumor suppression in vivo. Experimental Procedures Drosophila Stocks and Culture Conditions Drosophila cultures were maintained between 18⬚ and 25⬚. The shits4 strain was provided by Dr. Chun-Fang Wu; parats1 was obtained from Dr. Barry Ganetzky. A list of t.s. paralytic mutants used and their temperatures of paralysis is shown in Table 4. The awd lethal alleles, the awd transgene stocks (PAS7 and H119A3), and the two human nm23 transgenes H1-1 and H2-4 were from Dr. Allen Shearn; the awdKpn; prune alleles were provided by Dr. Tadmiri Venkatesh; and transgenic MHC-DGluRIIb-myc flies were from Dr. Aaron Di Antonio. All other strains, duplications, deficiencies, conventional, and GFP balancer chromosomes were either from our lab stocks or from the Bloomington stock center (GFP balancers, Df(3R) faf-BP). Measurements of Paralysis To study paralysis, we used a previously described, temperaturecontrolled glass chamber called the “sushicooker” (Ramaswami et al., 1993; Grant et al., 1998). Paralysis was empirically defined as the condition where the animal lies on its back with little effective movement of legs and wings. About 10–30 flies were loaded into

NDK Regulates Dynamin Function In Vivo 207

a glass chamber held at different temperatures. The number of paralyzed flies was noted at varying time points 0–5 min after introduction into the chamber. Between experiments, flies were allowed to recover for at least 10 min at 18⬚C–20⬚C. The flies tested were between 2 and 4 days of age; there is no significant variation in the paralytic behavior of shits flies within this age group. Screen for enhancers of shibire We used the temperature-controlled sushicooker to screen for enhancers of shibire paralysis (See Ramaswami et al., 1993). Two- to four-day-old adult male shiShy or shiCK2 flies were subjected to EMS mutagenesis and mated with appropriate shits/shits virgin females (15–20 males and 30 females per bottle). About 190,000 progeny from these crosses were screened for enhanced paralysis. Candidate mutant flies were crossed with appropriate shits/shits mates and their progeny rescreened. We identified at least three different loci that enhanced shi paralysis. Here, we focus our discussion on three mutations, MSM95, MSF15, and CM47 that, when homozygous, strongly enhance the t.s. paralysis of shits flies. For these e(shi) mutations, when shits/Y; e(shi)/⫹ males were mated to shits/shits; e(shi)/⫹ females, a quarter of the progeny showed very strongly enhanced paralysis. When these flies were crossed to each other, a stable stock of homozygous shits/shits; e(shi)/e(shi) flies that showed very strongly enhanced paralysis could be generated. Mapping of e(shi)A Mutations to awd Recombination Mapping of MSM95 We mapped MSM95 finely to the 100E region of chromosome III using recombination and deficiency mapping. We obtained a series of w⫹-marked transgenes [P(w⫹) stocks: 2140 (88D5–D6), 2144 (91F10–11), 2159 (97B8–9), 2160 (98E), 2161 (99D1–2), 2162 (99D1–2), 2163 (99E1–2), 2164 (100B2–4), and 2166 (100E1-E2)] that had been mapped by the Berkeley Drosophila Genome Project (BDGP) to specific regions of chromosome 3R. We plotted the fraction of recombinants obtained between MSM95 and P(w⫹) as a function of P element map position. The points fell into a roughly straight line that intersects the ordinate at 100E, the position of the P insertion in line 2166 (0 recombinants obtained from 500 test progeny). This map position was strengthened by deficiency mapping—the deficiency Df(3R) faf-BP that deletes 100D–F does not complement MSM95. Isolation of a Transposon-Induced Allele of e(shi)A; Establishing Identity between awd and e(shi)A When we tested 352 and 2167, two other chromosomes carrying P insertions in 100E, we discovered that 2167, but not 352, failed to complement MSM95. This result was surprising, given a BDGP database report that both insertions occur within a few base pairs of each other. PCR and sequence analysis confirmed that while the 2167 insertion is present in awd as reported, the P insertion in 352 is either lost or present elsewhere on 3R. To confirm that the enhancer of shi phenotype resulted from the P element insertion, we mobilized the P element by crossing a transposase source (⌬2–3 ) into the 2167 background and determined that several excision lines, from progeny in which the whiteexpressing P element was lost by transposition, were now wild-type for e(shi)A function. For final proof that MSM95, MSF15, and CM47 phenotypes are caused by mutations in awd, we performed genetic complemention and rescue analyses using awd lethal alleles (awdb3 and awdKRs6) and transgenic strains expressing awd, or either of two human nm23 genes, under awd promoter control (PAS7, H1, and H2, respectively). These strains were generously provided by Allen Shearn (Johns Hopkins University). Assaying NDP Kinase Activity in Drosophila Lysates We used a previously described procedure for assaying NDP kinase from Drosophila lysates (Biggs et al., 1990; Timmons et al., 1995). In brief, 20 adult flies were homogenized in 100 ␮l of ice-cold buffer containing 100 mM Tris-HCl, 10 mM MgCl2, and 100 mM KCl. The homogenate was centrifuged, supernatant extracted, and diluted 1:20 in the same homogenization buffer. Ten microliters of diluted lysate was added to 990 ␮l of reaction mixture containing 10 mM MgCl2, 100 mM KCl, 0.4 mM NADH, 6 mM ATP, 0.7 mM TDP, 4 mM PEP, and 10 units of pyruvate kinase and lactate dehydrogenase each (Timmons et al., 1993). The absorbence of NADH at 340 nm

was recorded in a spectrophotometer and read at intervals of 10 s for 10 min. The slope of a linear plot of OD versus time was used as a measure of enzymatic activity. We compared enzymatic activity in lysates of w1118shiCK2/w1118shiCK2; MSM95/MSM95 flies to those of control w1118shiCK2/w1118shiCK2; ⫹/⫹; ⫹/⫹. Electroretinogram (ERG) Recording Extracellular recordings of light-evoked visual responses were made from eyes of 2- or 3-day-old flies grown at 22⬚C. Flies, lightly anesthetized by cooling on ice, were mounted upright on modeling clay with the right eye facing the light. Electrodes were heat-pulled glass capillaries with a tip resistance of 3–5 M⍀ filled with 3 M KCl. When placed in contact with the eye, the recording electrode sensed membrane potential of photoreceptor and laminar cells. A reference ground electrode was inserted into the back of the fly’s head (in shits mutants, this avoids signals from spontaneous thoracic ganglion activity at elevated temperatures). Light flashes were generated using a custom-made pulsing circuit to control a 60 watt bulb, connected to an optic fiber output placed 3–4 cm from the eye of the fixed fly. A plate connected to a DC power supply heated the preparation; a digital probe monitored temperature very close to the fly. The ramp time for the temperature to change from 20⬚C to 39⬚C was ⵑ5 min. Signals were amplified using an intracellular preamplifier (WPI, Waltham, MA) and data acquired directly from the oscilloscope display using a Polaroid camera. Recordings shown represent the steady state ERG achieved after a series of light flashes at the indicated temperature. Electron Microscopy of Retinular Cell Synapses Three-day-old male flies were used in all experiments. Flies were placed into P 2 or P 20 pipette tips cut at the end such that fly heads, but not thoraces, protruded from the tip. In some instances, the flies were secured with a small drop of superglue. They were dark adapted in a moist dark chamber for at least 15 min. The fly’s head was then immersed in saline held at the appropriate temperature by a Peltier system (Physitemp, NJ). The fly was then exposed to 1 min of light stimulation from a Leica Lux 1000 fiber optic illuminator placed 1 cm from the retina before fixation at the same temperature as the saline. Either of two fixatives used (Koenig and Ikeda, 1996; Stimson et al., 1998) yielded tissue of similar quality. For fixation, the proboscis was quickly removed, and fixative was then perfused into the resulting hole in the anterior portion of the head for five minutes using a drawn out Pasteur pipette. The head was fixed at elevated temperature for an additional 30 min, removed from the body, and kept in 4% glutaraldehyde or 4% paraformaldehyde, 1% glutaraldehyde overnight at 4⬚C. The next day, the heads were rinsed in 0.1 M cacodylate buffer with 264 mM sucrose, post-fixed in 1% or 2% osmium tetroxide, and stained “en bloc” during ethanol dehydration with 1% uranyl acetate. Heads embedded in Epon/Araldite were thin sectioned at 60 nm. Sections stained with 2% uranyl acetate and 1% lead citrate were examined with a Jeol 1200EX electron microscope. A single section through the middle of the lamina was analyzed per individual fly. We counted and analyzed the number of thin, elongated, electrondense structures in a single cross-section through a given cartridge (Figure 4F). A detailed serial analysis on a small number of these structures confirmed that they are flat membranous cisternae emanating from near the active zone as previously reported by Koenig and Ikeda, 1996. The two lipid bilayers of invaginated membrane are tightly apposed (within 30 nm and form complex folds in the cytosol (data not shown). Analysis of FM1-43 Uptake and Release at Larval Motor Terminals Larval dissections, salines, and FM1-43 concentrations were as previously described (Ramaswami et al., 1994; Estes et al., 1996). In brief, wandering third instar larvae were filleted to reveal entire musculature and nervous system; for loading and unloading procedures, the CNS was left attached. Because complete block in endocytosis at shits larval motor terminals requires temperatures about 6⬚C higher than the restrictive temperature for adult paralysis (Ramaswami et al., 1994), we were restricted in the range of useful temperatures for analysis of awd phenotypes. After empirical testing, we chose

Neuron 208

38⬚C as the most convenient temperature at which to study potential differences in dye uptake between w shiCK2; PAS7/⫹; awdMSM95/ awdKRs6 and w shiCK2; awdMSM95/; awdKRs6 larval synapses. We initially focused our attention on the synapses innervating muscles 6 and 7 of abdominal segments A-2 and A-3; however, we observed more consistent results when we considered all of the visible muscle fibers. The preparations were examined with a Zeiss Axioskop through a water immersion lens (40⫻, NA 0.75 or 63⫻, NA 0.9). Images were captured using a chilled CCD camera (Princeton Instruments). After background subtraction (muscle surface close to bright boutons) digitized images were identically stretched and processed to maximize contrast in the brighter image, while preserving the relative intensities between control and experimental samples. Loading and unloading experiments were carried out in pairs—one control and one experimental animal were treated together. For the unloading experiments, preparations were loaded with FM1-43 at room temperature, and images were acquired. The preparations were prewarmed, stimulated with high K⫹ saline for 5 min, and then viewed again under the fluorescence microscope in Ca2⫹-free saline. Immunohistochemistry and Confocal Microscopy Dissected larvae were fixed in 3.5% paraformaldehyde and processed for antibody staining as previously described (Estes et al., 1996; Stimson et al., 1998). A Bio-Rad 600 confocal microscope running COMOS (Bio-Rad, Richmond, CA) or a PCM-2000 laserscanning confocal microscope (Nikon, Melville, NY) and Simple32 software (C Imaging, Cranberry Township, PA) were used for image acquisition. Rabbit anti-synaptotagmin antibody DSYT2 was used at a 1:500 dilution, mouse anti-CSP antibody mab49 at 1:20, rabbit anti-dynamin antibody 2074 at 1:200, and anti-Awd antibody HA1 was used at 1:100 dilution. Anti-myc epitope monoclonal antibody 9E10 (Sigma) was used at 1:100 dilution; secondary antibodies (Cappel) were used at 1:200. Biochemical Fractionation, Immunoprecipitation, GST Pulldowns, and Western Blotting Fractionation procedures were largely as described by Van de Goor et al. (1995) and Roos and Kelly (1998). For immunoprecipitations or GST pulldowns, the samples were diluted to 1 mg/ml of protein in IP buffer (50 mM Tris 7.4, 0.1 mM EDTA, 150 mM Nacl, and 0.5%Tween-20). To 1 ml of sample, affinity-purified 2074 (at 1:25 final dilution) or 10 ␮g of GST-DAP SH3-B fusion protein (Roos and Kelly, 1998) was added, and the mixture incubated for 3 hr at 4⬚C. In some instances, the buffer also contained 100 ␮M of either GDP, GDP-␤-S, GTP, or GTP-␥-S in the presence of ATP or ATP-␥-S. The samples were then mixed with protein A sepharose (Pharmacia) slurry (for IPs) or 30 ␮l of glutathione agarose resin (for GST pulldowns) for 30 min. Beads were washed with IP buffer at least 6 times, spun down, and then boiled in sample buffer. Under these conditions, a substantial fraction of dynamin was recovered with the beads, but NDK was found exclusively in the unbound fraction. No effect of varying nucleotides was observed on the absence of NDK from the bound fraction. For Western blotting, samples were subjected to SDS–PAGE and gels were electroblotted onto Hybond-PVDF (Amersham, Arlington Heights, IL), blocked with 5% nonfat dry milk powder in PBS/0.05% Tween 20, and incubated with 1:1000 dilution of anti-dynamin Ab2074 or a 1:2000 dilution of anti-Awd HA1 (Timmons et al., 1995). A 1:1000 dilution of mouse monoclonal anti-␤-tubulin was used as loading control. Detection was done with an HRP-conjugated secondary antibody (Cappel) and developed with an ECL detection system (Amersham, Arlington Heights, IL) according to the manufacturer’s directions. For comparing relative amounts of dynamin, we selected a dilution of lysate (1/2 head for dynamin) at which the ECL signal was maximally sensitive, and corresponded roughly linearly, to protein concentration. Acknowledgments We are particularly grateful to Allen Shearn for generous supply of awd reagents and for several useful discussions. We thank Patricia Steeg, Jitu Mayor, Veronica Rodrigues, Tadmiri Venkatesh, Charles Dearolf, Subhabrata Sanyal, Ted Weinert, and members of the Ra-

maswami lab for useful discussions and/or comments on the manuscript. We acknowledge Subhabrata Sanyal for help with ERG recordings, Chuck Hoeffer for expert help with adult head sections, and Narmada Khare for help in identifying a transposon-induced awd allele. We are grateful to Gina Zhang, ARL Division of Biotechnology Imaging Facility, for EM sectioning, and to Patty Jansma for essential assistance with confocal and electron microscopy, performed using instruments belonging to the ARL Division of Neurobiology. We thank Troy Littleton and Hugo Bellen for anti-syt antibodies, Erich Buchner and Konrad Zinsmaier for anti-csp antibodies, Aaron Di Antonio for MHC-DGluRIIb-myc flies, and Jack Roos for anti-dynamin antibodies, GST-DAP160 constructs, and advice on biochemistry. The work was funded by NIH grants to M. R. (NS34889 and KO2-NS02001), an HSFP grant to M. R. (and five others), funds from the McKnight and Alfred P Sloan Foundations (M. R.), as well as grants from the Government of India, Departments of Science and Technology and Biotechnology to K. S. K. Received August 21, 2000; revised January 23, 2001. References Alawi, A.A., and Pak, W.L. (1971). On-transient of insect electroretinogram: its cellular origin. Science 172, 1055–1057. Baba, H., Urano, T., Okada, K., Furukawa, K., Nakayama, E., Tanaka, H., Iwasaki, K., and Shiku, H. (1995a). Two isotypes of murine nm23/ nucleoside diphosphate kinase, nm23-M1 and nm23-M2, are involved in metastatic suppression of a murine melanoma line. Cancer Res. 55, 1977–1981. Baba, T., Damke, H., Hinshaw, J.E., Ikeda, K., Schmid, S.L., and Warnock, D.E. (1995b). Role of dynamin in clathrin-coated vesicle formation. Cold Spring Harb. Symp. Quant. Biol. 60, 235–242. Babst, M., Odorizzi, G., Estepa, E., and Emr, S. (2000). Mammalian TSG101 and the yeast homologue Vps23p, both function in late endosomal trafficking. Traffic 1, 248–258. Betz, W.J., and Bewick, G.S. (1992). Optical analysis of synaptic vesicle cycling at the frog neuromuscular junction. Science 255, 200–203. Biggs, J., Tripoulas, N., Hersperger, E., Dearolf, C., and Shearn, A. (1988). Analysis of the lethal interaction between the prune and Killer of prune mutations of Drosophila. Genes Dev. 2, 1333–1343. Biggs, J., Hersperger, E., Steeg, P.S., Liotta, L.A., and Shearn, A. (1990). A Drosophila gene that is homologous to a mammalian gene associated with tumor metastasis codes for a nucleoside diphosphate kinase. Cell 63, 933–940. Bourne, H.R., Sanders, D.A., and McCormick, F. (1990). The GTPase superfamily: a conserved switch for diverse cell functions. Nature 348, 125–132. Bourne, H.R., Sanders, D.A., and McCormick, F. (1991). The GTPase superfamily: conserved structure and molecular mechanism. Nature 349, 117–127. Chen, Y.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 the shibire, a Drosophila gene involved in endocytosis. Nature 351, 583–586. Cremona, O., and De Camilli, P. (1997). Synaptic vesicle endocytosis. Curr. Opin. Neurobiol. 7, 323–330. Damke, H., Baba, T., Warnock, D.E., and Schmid, S.L. (1994). Induction of mutant dynamin specifically blocks endocytic coated vesicle formation. J. Cell Biol. 127, 915–934. De Camilli, P., and Takei, K. (1996). Molecular mechanisms in synaptic vesicle endocytosis and recycling. Neuron 16, 481–486. Dearolf, C.R., Hersperger, E., and Shearn, A. (1988a). Developmental consequences of awdb3, a cell-autonomous lethal mutation of Drosophila induced by hybrid dysgenesis. Dev. Biol. 129, 159–168. Dearolf, C.R., Tripoulas, N., Biggs, J., and Shearn, A. (1988b). Molecular consequences of awdb3, a cell-autonomous lethal mutation of Drosophila induced by hybrid dysgenesis. Dev. Biol. 129, 169–178. Engel, M., Veron, M., Theisinger, B., Lacombe, M.L., Seib, T., Dooley, S., and Welter, C. (1995). A novel serine/threonine-specific protein

NDK Regulates Dynamin Function In Vivo 209

phosphotransferase activity of Nm23/nucleoside-diphosphate kinase. Eur. J. Biochem. 234, 200–207. Engel, M., Seifert, M., Theisinger, B., Seyfert, U., and Welter, C. (1998). Glyceraldehyde-3-phosphate dehydrogenase and Nm23-H1/ nucleoside diphosphate kinase A. Two old enzymes combine for the novel Nm23 protein phosphotransferase function. J. Biol. Chem. 273, 20058–20065. Estes, P.S., Roos, J., van der Bliek, A., Kelly, R.B., Krishnan, K.S., and Ramaswami, M. (1996). Traffic of dynamin within individual Drosophila synaptic boutons relative to compartment-specific markers. J. Neurosci. 16, 5443–5456. Fish, K.N., Schmid, S.L., and Damke, H. (2000). Evidence that dynamin-2 functions as a signal-transducing GTPase. J. Cell. Biol. 150, 145–154.

A Pro/Ser substitution in nucleoside diphosphate kinase of Drosophila melanogaster (mutation killer of prune) affects stability but not catalytic efficiency of the enzyme. J. Biol. Chem. 267, 12775– 12781. Leone, A., Flatow, U., King, C.R., Sandeen, M.A., Margulies, I.M., Liotta, L.A., and Steeg, P.S. (1991). Reduced tumor incidence, metastatic potential, and cytokine responsiveness of nm23-transfected melanoma cells. Cell 65, 25–35. Leone, A., Flatow, U., VanHoutte, K., and Steeg, P.S. (1993). Transfection of human nm23–H1 into the human MDA-MB-435 breast carcinoma cell line: effects on tumor metastatic potential, colonization and enzymatic activity. Oncogene 8, 2325–2353.

Floyd, S., and De Camilli, P. (1998). Endocytosis proteins and cancer: a potential link. Trends Cell Biol. 8, 299–301.

Levkowitz, G., Waterman, H., Zamir, E., Kam, Z., Oved, S., Langdon, W.Y., Beguinot, L., Geiger, B., and Yarden, Y. (1998). c-Cbl/Sli-1 regulates endocytic sorting and ubiquitination of the epidermal growth factor receptor. Genes Dev. 12, 3663–3674.

Freije, J.M., Blay, P., MacDonald, N.J., Manrow, R.E., and Steeg, P.S. (1997). Site-directed mutation of Nm23–H1. Mutations lacking motility suppressive capacity upon transfection are deficient in histidine-dependent protein phosphotransferase pathways in vitro. J. Biol. Chem. 272, 5525–5532.

Levkowitz, G., Waterman, H., Ettenberg, S.A., Katz, M., Tsygankov, A.Y., Alroy, I., Lavi, S., Iwai, K., Reiss, Y., Ciechanover, A., et al. (1999). Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1. Mol Cell 4, 1029–1040.

Fukuda, M., Ishii, A., Yasutomo, Y., Shimada, N., Ishikawa, N., Hanai, N., Nagata, N., Irimura, T., Nicolson, G.L., and Kimura, N. (1996). Decreased expression of nucleoside diphosphate kinase alpha isoform, an nm23-H2 gene homolog, is associated with metastatic potential of rat mammary-adenocarcinoma cells. Int. J. Cancer 65, 531–537.

Loughney, K., Kreber, R., and Ganetzky, B. (1989). Molecular analysis of the para locus, a sodium channel gene in Drosophila. Cell 58, 1143–1154.

Futter, C.E., Pearse, A., Hewlett, L.J., and Hopkins, C.R. (1996). Multivesicular endosomes containing internalized EGF-EGF receptor complexes mature and then fuse directly with lysosomes. J. Cell Biol. 132, 1011–1023. Gonzalez-Gaitan, M., and Jackle, H. (1997). Role of Drosophila alpha-adaptin in presynaptic vesicle recycling. Cell 88, 767–776. Grant, D., Unadkat, S., Katzen, A., Krishnan, K.S., and Ramaswami, M. (1998). Probable mechanisms underlying interallelic complementation and temperature-sensitivity of mutations at the shibire locus of Drosophila melanogaster. Genetics 149, 1019–1030. Jongeward, G.D., Clandinin, T.R., and Sternberg, P.W. (1995). sli-1, a negative regulator of let-23-mediated signaling in C. elegans. Genetics 139, 1553–1566. Kantor, J.D., McCormick, B., Steeg, P.S., and Zetter, B.R. (1993). Inhibition of cell motility after nm23 transfection of human and murine tumor cells. Cancer Res. 53, 1971–1973. Kelly, L.E., and Suzuki, D.T. (1974). The effects of increased temperature on electroretinograms of temperature-sensitive paralysis mutants of Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 71, 4906–4909. Kim, Y.-T., and Wu, C.-F. (1990). Allelic interactions at the shibire locus of Drosophila: effects on behaviour. J. Neurogenet. 7, 1–14. Koenig, J., and Ikeda, K. (1989). Disappearance and reappearance of synaptic vesicle membrane upon transmitter release observed under reversible blockage of membrane retrieval. J. Neurosci. 9, 3844–3860. Koenig, J.H., and Ikeda, K. (1996). Synaptic vesicles have two distinct recycling pathways. J. Cell Biol. 135, 797–808. Koenig, J.H., and Ikeda, K. (1999). Contribution of active zone subpopulation of vesicles to evoked and spontaneous release. J. Neurophysiol. 81, 1495–1505. Kosaka, T., and Ikeda, K. (1983). Possible temperature-dependent blockage of synaptic vesicle recycling induced by a single gene mutation in Drosophila. J. Neurobiol. 14, 207–225. Kramer, H., Cagan, R.L., and Zipursky, S.L. (1991). Interaction of bride of sevenless membrane-bound ligand and the sevenless tyrosine-kinase receptor. Nature 352, 207–212. Krishnan, K.S., Chakravarti, S., Raghuram, V., and Ramaswami, M. (1996). Specific alleviation of the temperature-sensitive paralytic phenotype of shibirets mutants in Drosophila by subanesthetic concentrations of carbon dioxide. J. Neurogenet. 10, 221–238. Lascu, I., Chaffotte, A., Limbourg-Bouchon, B., and Veron, M. (1992).

MacDonald, N.J., Freije, J.M.P., Stracke, M.L., Manrow, R.E., and Steeg, P.S. (1996). Site-directed mutagenesis of nm23–H1. Mutation of proline 96 or serine 120 abrogates its motility inhibitory activity upon transfection into human breast carcinoma cells. J. Biol. Chem. 271, 25107–25116. Marsh, M., and McMahon, H.T. (1999). The structural era of endocytosis. Science 285, 215–220. McNiven, M.A., Cao, H., Pitts, K.R., and Yoon, Y. (2000). The dynamin family of mechanozymes: pinching in new places. Trends Biochem. Sci. 25, 115–120. Pallanck, L., Ordway, R.W., and Ganetzky, B. (1995). A Drosophila NSF mutant. Nature 376, 25. Parhar, R.S., Shi, Y., Zou, M., Farid, N.R., Ernst, P., and al-Sedairy, S.T. (1995). Effects of cytokine-mediated modulation of nm23 expression on the invasion and metastatic behavior of B16F10 melanoma cells. Int. J. Cancer 60, 204–210. Petersen, S.A., Fetter, R.D., Noordermeer, J.N., Goodman, C.S., and DiAntonio, A. (1997). Genetic analysis of glutamate receptors in Drosophila reveals a retrograde signal regulating presynaptic transmitter release. Neuron 19, 1237–1248. Poodry, C.A. (1990). shibire: a neurogenic mutant of Drosophila. Dev. Biol. 138, 464–472. Poodry, C.A., and Edgar, L. (1979). Reversible alterations in the neuromuscular junctions of Drosophila melanogaster bearing a temperature-sensitive mutation, shibire. J. Cell. Biol 81, 520–527. Ramaswami, M., Rao, S., van der Bliek, A., Kelly, R.B., and Krishnan, K.S. (1993). Genetic studies on dynamin function in Drosophila. J. Neurogenet. 9, 73–87. Ramaswami, M., Krishnan, K.S., and Kelly, R.B. (1994). Intermediates in synaptic vesicle recycling revealed by optical imaging of Drosophila neuromuscular junctions. Neuron 13, 363–375. Randazzo, P.A., Kahn, R.A., and Northup, J.K. (1992). Nucleoside diphosphate kinase: conclusions withdrawn. Science 257, 862. Richter, M.F., Schwemmle, M., Herrmann, C., Wittinghofer, A., and Staeheli, P. (1995). Interferon-induced MxA protein. GTP binding and GTP hydrolysis properties. J. Biol. Chem. 270, 13512–13517. Roos, J., and Kelly, R.B. (1998). Dap160, a neural-specific Eps15 homology and multiple SH3 domain-containing protein that interacts with Drosophila dynamin. J. Biol. Chem. 273, 19108–19119. Roos, J., and Kelly, R.B. (1999). The endocytic machinery in nerve terminals surrounds sites of exocytosis. Curr. Biol. 9, 1411–1414. Rosengard, A.M., Krutzsch, H.C., Shearn, A., Biggs, J.R., Barker, E., Margulies, I.M., King, C.R., Liotta, L.A., and Steeg, P.S. (1989). Reduced Nm23/Awd protein in tumour metastasis and aberrant Drosophila development. Nature 342, 177–180.

Neuron 210

Sever, S., Muhlberg, A.B., and Schmid, S.L. (1999). Impairment of dynamin’s GAP domain stimulates receptor-mediated endocytosis. Nature 398, 481–486.

visual system of Drosophila: genetic dissection of electroretinogram components. J. Gen. Physiol. 69, 705–724.

Sever, S., Damke, H., and Schmid, S.L. (2000a). Garrotes, springs, ratchets and whips: putting dynamin models to the test. Traffic 1, 385–392.

Xu, J., Liu, L.Z., Deng, X.F., Timmons, L., Hersperger, E., Steeg, P.S., Veron, M., and Shearn, A. (1996). The enzymatic activity of Drosophila AWD/NDP kinase is necessary but not sufficient for its biological function. Dev. Biol. 177, 544–557.

Sever, S., Damke, H., and Schmid, S.L. (2000b). Dynamin:GTP controls the formation of constricted coated pits, the rate limiting step in clathrin-mediated endocytosis. J. Cell Biol. 50, 1137–1148.

Yoon, C.H., Lee, J., Jongeward, G.D., and Sternberg, P.W. (1995). Similarity of sli-1, a regulator of vulval development in C. elegans, to the mammalian proto-oncogene c-cbl. Science 269, 1102–1105.

Shpetner, H., and Vallee, R. (1989). Identification of dynamin, a novel mechanochemical enzyme that mediates interactions between microtubules. Cell 59, 421–432.

Zhu, J., Tseng, Y.H., Kantor, J.D., Rhodes, C.J., Zetter, B.R., Moyers, J.S., and Kahn, C.R. (1999). Interaction of the Ras-related protein associated with diabetes rad and the putative tumor metastasis suppressor NM23 provides a novel mechanism of GTPase regulation. Proc. Natl. Acad. Sci. USA 96, 14911–14918.

Shpetner, H.S., and Vallee, R.B. (1992). Dynamin is a GTPase stimulated to high levels of activity by microtubules. Nature 355, 733–735. Simon, M.A., Bowtell, D.D., Dodson, G.S., Laverty, T.R., and Rubin, G.M. (1991). Ras1 and a putative guanine nucleotide exchange factor perform crucial steps in signaling by the sevenless protein tyrosine kinase. Cell 67, 701–716. Steeg, P.S., Bevilacqua, G., Kopper, L., Thorgeirsson, U.P., Talmadge, J.E., Liotta, L.A., and Sobel, M.E. (1988a). Evidence for a novel gene associated with low tumor metastatic potential. J. Natl. Cancer Inst. 80, 200–204. Steeg, P.S., Bevilacqua, G., Pozzatti, R., Liotta, L.A., and Sobel, M.E. (1988b). Altered expression of NM23, a gene associated with low tumor metastatic potential, during adenovirus 2 Ela inhibition of experimental metastasis. Cancer Res. 48, 6550–6554. Stimson, D.T., Estes, P.S., Smith, M., Kelly, L.E., and Ramaswami, M. (1998). A product of the Drosophila stoned locus regulates neurotransmitter release. J. Neurosci. 18, 9638–9649. Sturtevant, A.H. (1956). Genetics 41, 118–123. Takei, K., McPherson, P.S., Schmid, S., and De Camilli, P. (1995). Tubular membrane invaginations coated by dynamin rings are induced by GTPgS in nerve terminals. Nature 374, 186–190. Teng, D.H., Engele, C.M., and Venkatesh, T.R. (1991). A product of the prune locus of Drosophila is similar to mammalian GTPaseactivating protein. Nature 353, 437–440. Timmons, L., Hersperger, E., Woodhouse, E., Xu, J., Liu, L.Z., and Shearn, A. (1993). The expression of the Drosophila awd gene during normal development and in neoplastic brain tumors caused by lgl mutations. Dev. Biol. 158, 364–379. Timmons, L., Xu, J., Hersperger, G., Deng, X.F., and Shearn, A. (1995). Point mutations in awdKpn which revert the prune/Killer of prune lethal interaction affect conserved residues that are involved in nucleoside diphosphate kinase substrate binding and catalysis. J. Biol. Chem. 270, 23021–23030. van der Bliek, A. (1999). Functional diversity in the dynamin family. Trends Cell Biol. 9, 96–102. van der Bliek, A., and Meyerowitz, E. (1991). Dynamin like protein encoded by the Drosophila shibire gene associated with membrane traffic. Nature 351, 411–414. van de Goor, J., Ramaswami, M., and Kelly, R.B. (1995). Redistribution of synaptic vesicles and their proteins in shibirets1 mutant Drosophila. Proc. Natl. Acad. Sci. USA 92, 5739–5743. Vieira, A.V., Lamaze, C., and Schmid, S.L. (1996). Control of EGF receptor signaling by clathrin-mediated endocytosis. Science 274, 2086–2089. Wagner, P.D., Steeg, P.S., and Vu, N.D. (1997). Two-component kinase-like activity of nm23 correlates with its motility-suppressing activity. Proc. Natl. Acad. Sci. USA 94, 9000–9005. Wan, H.I., DiAntonio, A., Fetter, R.D., Bergstrom, K., Strauss, R., and Goodman, C.S. (2000). Highwire regulates synaptic growth in Drosophila. Neuron 26, 313–329. Warnock, D.E., and Schmid, S.L. (1996). Dynamin GTPase, a forcegenerating molecular switch. Bioessays 18, 885–893. Whistler, J.L., and von Zastrow, M. (1999). Dissociation of funtional roles of dynamin in receptor-mediated endocytosis and mitogen signal transduction. J. Biol. Chem. 274, 24575–24578. Wu, C.F., and Wong, F. (1977). Frequency characteristics in the

Zuker, C.S. (1996). The biology of vision of Drosophila. Proc. Natl. Acad. Sci. USA 93, 571–576.