1 a Conserved Role for Vezatin Proteins in Cargo-Specific Regulation of Retrograde

Genetics: Early Online, published on August 11, 2020 as 10.1534/genetics.120.303499

1 A conserved role for Vezatin proteins in cargo-specific regulation of retrograde

2 axonal transport

6 Michael A. Spinner*, Katherine Pinter†, Catherine M. Drerup†, and Tory G. Herman*1

7 * Institute of Molecular Biology, University of Oregon, Eugene, OR 97403

8 † Unit on Neuronal Cell Biology, NICHD, NIH, Bethesda, MD 20892

9 1. Corresponding author: [email protected]

13 Running title: Vezatin is required for axonal transport

14 Keywords: Vezatin, Diamond, axonal transport, retrograde, endosome

17 7 Figures + 4 Supplemental Figures + 1 Supplemental Table + 1 Supplemental Movie

18 The authors declare no competing financial interests.

21 Active transport of organelles within axons is critical for neuronal health. Retrograde axonal

22 transport, in particular, relays neurotrophic signals received by axon terminals to the

23 nucleus and circulates new material among en passant synapses. A single motor protein

24 complex, cytoplasmic dynein, is responsible for nearly all retrograde transport within axons:

25 its linkage to and transport of diverse cargos is achieved by cargo-specific regulators. Here

26 we identify Vezatin as a conserved regulator of retrograde axonal transport. Vertebrate

27 Vezatin (Vezt) is required for the maturation and maintenance of cell-cell junctions and has

28 not previously been implicated in axonal transport. However, a related fungal protein, VezA,

29 has been shown to regulate retrograde transport of endosomes in hyphae. In a forward

30 genetic screen, we identified a loss-of-function mutation in the Drosophila vezatin-like (vezl)

31 gene. We here show that vezl loss prevents a subset of endosomes, including signaling

32 endosomes containing activated BMP receptors, from initiating transport out of motor

33 neuron terminal boutons. vezl loss also decreases the transport of endosomes and dense

34 core vesicles (DCVs) but not mitochondria within axon shafts. We disrupted vezt in

35 zebrafish and found that vezt loss specifically impairs the retrograde axonal transport of

36 late endosomes, causing their accumulation in axon terminals. Our work establishes a

37 conserved, cargo-specific role for Vezatin proteins in retrograde axonal transport.

2 39 INTRODUCTION

40 Neurons mediate cognition and behavior by forming networks that can have complex

41 connectivity and span considerable distances. As a consequence, neurons have elongated

42 processes along which passive diffusion is inefficient and materials must be actively

43 transported (recently reviewed in Guedes-Dias and Holzbaur 2019). Within axons, cargoes

44 move along uniformly polarized microtubules by binding to active kinesin or cytoplasmic

45 dynein motors: the former move associated cargo in the anterograde direction, toward

46 microtubule plus ends and the axon terminal, and the latter move cargo in the retrograde

47 direction, back to the soma (Guedes-Dias and Holzbaur 2019). Bidirectional axonal

48 transport is essential for the function and survival of neurons, and abnormalities in this

49 process are an early hallmark of neurodegenerative diseases such as Huntington's,

50 Alzheimer's, and Parkinson's diseases (Guo et al. 2020).

52 How axonal transport is regulated remains a central question. While multiple kinesin family

53 members transport the cargos that move toward axon terminals, the majority of retrograde

54 axonal transport is accomplished by a single motor protein complex, cytoplasmic dynein.

55 Dynein's ability to bind cargo and microtubules is enhanced by its cofactor, dynactin.

56 However, a variety of cargo-specific regulators are additionally required: some link cargoes

57 to dynein-dynactin complexes, some promote the motility of cargo-bound dynein-dynactin,

58 and some do both (Reck-Peterson et al. 2018). While many such regulators have been

59 identified, others undoubtedly remain to be discovered.

61 The Drosophila larval neuromuscular junction (NMJ) is a powerful system in which to

62 identify and analyze genes required for neural development and function, including genes

3 63 required for axonal transport (Neisch et al. 2016). Each motor neuron forms a stereotyped

64 pattern of branched chains of synaptic boutons on its target muscle and adds new boutons

65 in response to a muscle-secreted Bone Morphogenetic Protein (BMP) signal during larval

66 growth (reviewed in Deshpande and Rodal 2016). Within larval motor neuron axons, two

67 broad types of cargoes are retrogradely transported: (1) those, such as signaling

68 endosomes containing activated BMP receptors, that originate in the boutons themselves

69 and are transported through the NMJ and axon shaft to the soma (Lloyd et al. 2012; Smith

70 et al. 2012); and (2) those, such as dense core vesicles (DCVs), that originate in the soma,

71 are anterogradely transported to the terminal boutons of each NMJ, but require retrograde

72 transport within the NMJ itself in order to be distributed among the other, en passant,

73 boutons (Wong et al. 2012). Both types of cargo accumulate in terminal boutons when

74 dynactin is disrupted by loss of its core subunit p150Glued (Lloyd et al. 2012).

76 Forward genetic screens in invertebrates continue to uncover new relationships between

77 molecules and phenotypes. In such a screen, we identified a loss-of-function mutation in

78 the Drosophila gene, diamond (Graziadio et al. 2018), that results in enlarged terminal

79 boutons containing accumulated retrograde cargos. We found that the predicted Diamond

80 protein is most closely related to vertebrate Vezatin (Vezt) in amino acid sequence and on

81 that basis will refer to the Drosophila gene as vezatin-like (vezl). Vertebrate Vezt was

82 originally identified as an adherens junction-associated protein that links cadherin-catenin

83 complexes to the FERM-domain-containing proteins, myosin VIIa (Küssel-Andermann et al.

84 2000) and radixin (Bahloul et al. 2009). Vezt has subsequently been found postsynaptically

85 in both muscle and dendrites and is required for the maturation and maintenance of

86 adherens junctions (Hyenne et al. 2005; Hyenne et al. 2007), dendrites (Sanda et al. 2010;

4 87 Danglot et al. 2012), and NMJ synapses (Koppel et al. 2019). A forward genetic screen in

88 Aspergillus identified a Vezatin-like Aspergillus protein, VezA, as being required for dynein-

89 dependent transport of endosomes in hyphae (Yao et al. 2015). Here we show that both fly

90 vezl and zebrafish vezt are required for the retrograde transport of late endosomes in

91 axons. Our data identify a conserved role for Vezatin proteins as cargo-specific regulators

92 of retrograde axonal transport.

5 93 MATERIALS AND METHODS

95 Drosophila genetics

96 Flies were grown on standard food at 25°C. The 721 mutation was the result of exposing

97 wild-type FRT82-containing flies (Bloomington Drosophila Stock Center (BDSC) stock

98 #1359) to ethylmethane sulfonate (EMS) using standard methods (Ashburner 1989). The

99 vezl13.1 deletion was generated by imprecise excision of a white+ (w+) P element within

100 exon 1 (BDSC#31820). Briefly, w- lines generated by mobilizing the P element were tested

101 for their ability to complement 721 for lethality. DNA from those that failed to complement

102 721 was sequenced with primers flanking the CG7705 gene region. The 13.1 deletion

103 removed most of CG7705 without extending beyond it. Homozygous wild-type (FRT82),

104 FRT82 vezl721, or FRT82 vezl13.1 R7s were created by GMR-FLP-induced mitotic

105 recombination (Lee et al. 2001) and labeled by the MARCM technique (Lee and Luo 1999)

106 using actin-Gal4 to drive UAS-synaptotagmin-GFP. Df(3R)Exel6180 (BDSC#7659) is a

107 molecularly-defined deletion that completely removes vezl along with 23 other genes;

108 vezl13.1/Df(3R)Exel6180 homozygous larvae are viable as late third instar larvae but die

109 either shortly before or after pupariation. All NMJ analyses were performed using motile

110 third instar larvae responsive to touch.

111

112 The UAS-Vezlwt:Venus and UAS-hVez:Venus transgenes were generated by cloning a full-

113 length vezl cDNA (LP22035; obtained from the Berkeley Drosophila Genome Project) or a

114 full-length human vezatin cDNA (BC064939, obtained from Dharmacon) into pUAST

115 vectors containing a C-terminal Venus open reading frame (Wang et al. 2012, Addgene

6 116 plasmid 35204). Transgenes were sequenced and then injected into embryos containing an

117 attP8 site on the X chromosome (BDSC#32233, BestGene).

118

119 Flies containing UAS-Tkv:mCherry were a generous gift from Thomas Kornberg (Roy et al.

120 2014). Stocks containing all other transgenes used were obtained from BDSC: actin-Gal4

121 (#4414), UAS-synaptotagmin:GFP (#6525), OK371-Gal4 (#26160); UAS-ANF:GFP

122 (#7001); UAS-YFP:Rab5 (#50788); UAS-Rab7:GFP (#42705); UAS-Rab11:GFP (#8506);

123 and UAS-mito:GFP (#8442). Because many transgenes used were located on the X

124 chromosome, we analyzed females only for consistency.

125

126 Sequence alignment and domain prediction

127 The third iteration of PSI-BLAST (https://blast.ncbi.nlm.nih.gov/) identified vertebrate

128 Vezatins as having sequence similarity to Vezl . Sequence alignments in Figure 1H were

129 performed with ClustalW2 (Geneious). Figure S1 shows the sequence alignment between

130 Vezl and human Vezatin that was generated by PSI-BLAST, as well as the alignment with

131 the pfam12632 "Vezatin domain" consensus sequence. Domains indicated in Figure 1H

132 were predicted using Interpro (https://www.ebi.ac.uk/interpro/), SMART (http://smart.embl-

133 heidelberg.de/), or PrDOS (http://prdos.hgc.jp/cgi-bin/top.cgi).

134

135 Drosophila dissections, fixation, and immunostaining

136 Adult brains were dissected and fixed in 4% paraformaldehyde at room temperature for 20

137 minutes. Late third-instar larval NMJs were dissected in Schneider’s insect medium

138 (Sigma-Aldrich) and fixed with Bouin’s solution (Sigma-Aldrich) for 5 min. All samples were

139 then stained by standard methods: washed 3x in PBS containing 0.3% Triton-X 100 (PBT),

7 140 blocked for 30 min in 5% normal goat serum, incubated with primary antibodies at 4°C

141 overnight, washed 3x with PBT, incubated with secondary antibodies at 4°C overnight,

142 washed 3x with PBT, and mounted in Vectashield (Vector Laboratories). We used the

143 following primary antibodies from the Developmental Studies Hybridoma Bank (DSHB):

144 anti-Chp (24B10, at dilution 1:200); anti-Dlg (4F3, 1:250); anti-CSP (6D6, 1:250); and anti-

145 Brp (nc82, 1:100). Other primary antibodies used were: rabbit anti-GFP (1:1000; Abcam);

146 fluorescence-conjugated anti-HRP (1:250; Jackson Immuno Labs); rabbit anti-GFP

147 (1:1000; Abcam); rabbit anti-pMAD (1:500, a gift from Carl Heldin; Persson et al. 1998);

148 and rabbit anti-GluRIIC (1:2500, a gift from Aaron DiAntonio; Marrus et al. 2004). All

149 secondary antibodies were goat IgG coupled to Alexa Fluor 488, 555, or 633 (1:500;

150 Thermo Fisher). As noted in the Figure 6A legend, Vezl:Venus was imaged by dissecting

151 but not fixing late third-instar larvae; fluorescence-conjugated anti-HRP was added to the

152 dissection media.

153

154 Quantification of Drosophila R7 and NMJ morphology in fixed samples

155 For all comparative experiments, animals from each genotype were grown, dissected, and

156 stained in parallel and imaged on the same day under identical microscope settings. Z-

157 sections were collected on a Leica SP2 microscope (with a 63x/1.4 oil objective). For

158 Figure S2C-E, structured illumination microscopy (SIM) images were collected using a

159 Zeiss ELYRA S.1/LSM 880 confocal microscope with a Plan-Apochromat 63x/1.4 oil

160 objective. Within each experiment, the images were randomized, and quantifications were

161 performed blind to genotype. For Figure 1E, ~100 R7 axons were scored within each

162 animal to determine, for that animal, the percentage of R7s with terminal blobs. For Figures

163 2-4 and Figures S2 and S3, maximum-intensity projections of z-stacks were generated and

8 164 analyzed with ImageJ software (Schindelin et al. 2012; http://fiji.sc/). As is standard (e.g.

165 Ball et al. 2010), we counted boutons based on shape, as visualized by anti-HRP and anti-

166 Dlg staining on muscle 4 on both sides of segment A3 and calculated the mean for each

167 animal. Satellite boutons were identified as smaller, non-chain-forming, individual boutons

168 that emerged from larger boutons on the main NMJ axis (e.g. Marie et al. 2004); they were

169 counted separately and not included in total NMJ bouton counts. The relative sizes and

170 staining intensities of terminal boutons were measured in ImageJ by manually tracing each

171 terminal bouton and its immediate neighbors. Each z-stack in which intensities were

172 quantified consisted of only 3 to 5 z-sections (boutons are shallow) with little variation in

173 intensity at a given xy position within the stack except at positions that were outside the

174 bouton in a subset of slices; maximum-intensity projections therefore provided an accurate

175 assessment of the accumulation of signal within terminal relative to non-terminal boutons.

176

177 Drosophila live imaging and analysis

178 Wandering late third-instar larvae were dissected to expose the area of interest in Ca2+-free

179 HL3 media on a custom made Sylgard mount, secured with a coverslip, and immediately

180 imaged in a temperature-controlled room during similar times of day. Live imaging within

181 single planes was conducted using a Zeiss ELYRA S.1/LSM 880 (with a Plan-Apochromat

182 63x/1.4 oil objective) with the pinhole set at >1 Airy Unit to optimize the ability to monitor

183 individual particles. Videos were recorded with a minimum of two frames per second over

184 the course of 6-10 minute timespans to ensure the larvae did not die during imaging.

185 Movement of individual Tkv:mCherry puncta within NMJ boutons (Figure 5) was trackable

186 without the use of FRAP. But we did use FRAP to track individual ANF:GFP puncta

187 movement within NMJ boutons: we bleached a region of interest adjacent to the terminal

9 188 bouton with a 488nm laser for 60s at high power followed by regular image acquisition. In

189 all cases, the number of puncta that entered or left the terminal bouton over the movie's

190 timespan was counted manually. Imaging of ANF:GFP, Tkv:mCherry, and mito:GFP

191 movement within axons was performed in individual axon bundles, near the ventral nerve

192 cord. Velocities were calculated based on the slopes of kymographs generated by the Multi

193 kymograph tool in ImageJ. Puncta were counted and then watched for directional

194 movement over a fixed timeframe of 3 to 5 minutes in order to categorize them as

195 stationary, anterograde-moving, or retrograde-moving. These numbers were normalized to

196 the area of the region being imaged.

197

198 Zebrafish husbandry and generation of the vezt mutant line

199 Adult zebrafish were housed at 28°C and bred using established protocols (Westerfield

200 1993) in accordance with NICHD/IACUC protocol Drerup 18.008. Embryos and larvae were

201 housed at 28°C in embryo media and staged by established methods (Kimmel et al. 1995).

202 The TgBAC(neurod:egfp)nl1 transgenic line was used to label neurons with cytoplasmic

203 GFP (Obholzer et al. 2008).

204

205 The vezty624 mutant line was generated using CRISPR/Cas9 mutagenesis (Burger et al.

206 2016). Briefly, 500ng Cas9 protein (Integrated DNA Technologies) was coinjected with

207 ~200pg gRNA targeted against exon 3 of the zebrafish vezt ortholog

208 (GTGGCGCTGTCTCGACGCAG). Resulting F0 animals were raised and crossed to wild-

209 type animals to generate F1s. Sequencing of the F1 larvae revealed an F0 carrier of a 5

210 base pair insertion in exon 3 of vezt, predicted to cause the insertion of 40 amino acids

211 followed by a premature stop site. Fragment length analysis of PCR products flanking this

10 212 region were used to genotype (Carrington et al. 2015). The vezty624 mutation is

213 homozygous lethal, and the axon terminal swelling phenotype it causes is recessive.

214

215 Immunolabeling and fluorescence intensity quantification in zebrafish

216 Homozygous embryos were generated by vezty624 heterozygous crosses. Larvae were

217 raised to 4 dpf, fixed in 4% paraformaldehyde, and stained as in Drerup and Nechiporuk

218 (2013). Antibodies used were: Anti-Dync1h1 (Proteintech #12345), Anti-p150 (BD

219 transduction laboratories #610473), Anti-Cytochrome C (BD Pharma #556432), Anti-GFP

220 (Aves #gfp-1020), and Alexa Fluor 488/568 secondaries (ThermoFisher).

221

222 After immunolabeling, larvae were imaged on a Zeiss LSM 800 confocal microscope with a

223 63X/NA1.4 oil objective. Optical sections were obtained at ~0.5µm intervals spanning the

224 full depth of the axon terminal. For quantification of immunofluorescence, a custom ImageJ

225 macro was used to isolate the fluorescence signal from the GFP-labeled axons. A summed

226 projection was generated and then the mean fluorescence intensity quantified. Mean

227 fluorescence was normalized to background. Statistical analyses were done in JMP.

228

229 In vivo analysis of axon terminal volume and endosome localization and transport in

230 zebrafish

231 To determine axon terminal volumes, axon terminals were imaged using an LSM800

232 confocal microscope with a 63X/NA1.2 water immersion objective and identified using the

233 GFP axon fill from the TgBAC(neurod:egfp)nl1 transgene. Axon terminal volumes were

234 calculated in ImageJ using the 3D object counter plugin.

235

11 236 Larvae mosaically expressing endosome markers were generated as previously described

237 (Drerup and Nechiporuk 2013). Briefly, zygotes were microinjected with DNA plasmids

238 encoding a neuron-specific promotor (5kbneurod) driving expression of the particular Rab

239 fused to mRFP. Larvae expressing the construct of interest in pLL sensory neurons were

240 isolated using an AxioZoom fluorescence dissecting microscope, mounted in 1.5% low melt

241 agarose, and imaged on a LSM800 confocal microscope using a 63X/NA1.2 water

242 immersion objective (Zeiss). For analyses of Rab5/7/11 vesicle localization, z-sections

243 were obtained through each expressing pLL axon terminal. A standard deviation projection

244 was then done in ImageJ and the total particle area quantified. The axon terminal area was

245 also measured and the number of axon terminals recorded so that the endosome area

246 could be normalized to axon terminal area or to axon number in order to account for the

247 mosaicism of the Rab labeling and the increased axon terminal volume in vezty624 mutants.

248

249 For analyses of vesicle transport, larvae were mounted similarly and imaged with the

250 LSM800 confocal using the 63X/NA1.2 water immersion objective (Zeiss) as previously

251 described (Drerup and Nechiporuk 2016). Images were taken 3 times per second for 150

252 seconds over a region of pLL axon in a single plane to track the location of moving puncta.

253 Imaging sessions were analyzed using Kymograph analysis in Metamorph (Molecular

254 Devices). Transport distance was quantified and statistical significance analyzed using

255 JMP.

256

257 Experimental design, statistical analyses, and data availability

258 All experiments were scored blind to genotype. For comparisons in Figures 1 to 6 and

259 Figures S2 and S3, one-way ANOVA was performed, followed by select post hoc

12 260 comparisons using two-tailed t-tests; for multiple comparisons (Figures 1E and Figure 2I-L),

261 Bonferroni-corrected p values were used. For comparisons in Figure 7 and Figure S4, two-

262 way ANOVA was performed. For all figures: ns, not significant; *, p<0.05; **, p<0.01; ***,

263 p<0.001. The means, n values, SEMs, p values, and Bonferroni-corrected p values

264 presented in the bar graphs are available in Table S1. The Supplemental Figures S1-S4,

265 Movie S1, and Table S1 are available on the GSA FigShare Portal. All specifics of the data

266 are available upon request, as are DNA constructs and fly and fish stocks.

267

13 268 RESULTS

269

270 Loss of a Vezatin-like (Vezl) protein alters the morphology of terminal synaptic

271 boutons in adult eye and at the larval NMJ

272 Drosophila R7 photoreceptor neurons are a convenient cell type with which to identify

273 essential genes that are required cell-autonomously for neuronal function. Photoreceptors

274 are not required for viability, and the GMR-FLP/MARCM technique can be used to generate

275 mosaic animals in which Green Fluorescent Protein (GFP)-labeled R7s are homozygous

276 for randomly mutagenized chromosome arms within otherwise heterozygous animals (Lee

277 and Luo 1999; Lee et al. 2001). In a screen of EMS-induced mutations, we identified a

278 mutation, 721, that alters R7 synaptic bouton morphology. Both wild-type and 721 mutant

279 R7 axons terminate in ellipsoid boutons within the M6 layer of the optic lobe (Figure 1A,B).

280 However, 721 mutant R7 terminals also frequently extend short, thin projections that end in

281 distinctive blobs beyond M6 (Figure 1A,B).

282

283 Animals homozygous for the 721-containing chromosome do not survive to adulthood.

284 Using recombination mapping and complementation tests between the 721-containing

285 chromosome and molecularly-defined deletions, we found that both the lethality and the R7

286 defect are linked to a gene, CG7705, recently renamed "diamond" (Graziadio et al. 2018).

287 The predicted CG7705/Diamond protein has no obvious sequence motifs, and simple

288 BLAST searches identify homologs only in other Drosophilid species. However, Position-

289 Specific Iterative (PSI)-BLAST identifies a similarity between CG7705/Diamond and

290 vertebrate Vezt proteins (Figure 1F,H; Figure S1; Küssel-Andermann et al. 2000). For this

291 reason, and because of the shared function we describe later in this paper, we will refer to

14 292 CG7705/Diamond as Vezatin-like (Vezl). Despite a low percentage of amino acid sequence

293 identity in any pairwise comparison (Figure 1F), Vezl, Vezt, and the related fungal protein

294 VezA share a predicted overall structure, consisting of central alpha-helical transmembrane

295 domains and N- and C-terminal disordered regions (Figure 1H). We note that a so-called

296 "Vezatin domain," which, in vertebrate Vezt, binds the FERM domains of myosin VIIa and

297 radixin (Küssel-Andermann et al. 2000; Bahloul et al. 2009), is not detected by an InterPro

298 scan of Vezl (Figure 1H, S1).

299

300 To verify that vezl loss is responsible for the R7 blobs, we used imprecise excision of a

301 previously identified P element insertion within the first exon of vezl to generate a deletion

302 allele, vezl13.1, that is missing most of the predicted vezl open reading frame (Figure 1G).

303 vezl13.1 homozygotes die either shortly before or after pupariation, and homozygous vezl13.1

304 mutant R7 terminal boutons within heterozygous animals have 721-like short extensions

305 that end in blobs (Figure 1C,E). R7-specific expression of a UAS-vezl transgene

306 constructed from the vezl cDNA with the largest open reading frame fully rescues the R7

307 defect (Figure 1D,E). We conclude that vezl is required for normal R7 synaptic bouton

308 morphology.

309

310 The observation that vezl loss causes late larval/early pupal lethality led us to wonder

311 whether vezl might be generally required for synaptic bouton development. To test this, we

312 turned to the larval NMJ. vezl loss was previously reported to disrupt mitotic spindles and

313 chromosome segregation (Graziadio et al. 2018). However, we never observed missing

314 NMJs or other gross abnormalities in axon morphology or targeting, indicating that vezl loss

315 does not affect motor neuron survival or muscle target choice. We were therefore able to

15 316 analyze Vezl's role in NMJ bouton development. We found that vezl mutants have

317 significantly fewer boutons per NMJ than wild-type animals, a defect that is fully rescued by

318 expression of a UAS-vezl transgene in motor neurons (Figure 2A-C,I). In addition, Vezl loss

319 specifically alters the morphology of NMJ terminal boutons, leaving all other, i.e. en

320 passant, boutons apparently unaffected. vezl mutant terminal boutons are abnormally large

321 (Figure 2E,F,J) and stain more intensely with the neuronal membrane marker anti-HRP

322 (Figure 2E,F,K). In addition, vezl mutant terminal boutons are frequently surrounded by

323 smaller "satellite" boutons that stain intensely with anti-HRP (Figure 2G,L). We conclude

324 that Vezl is required in both R7s and motor neurons for normal terminal bouton

325 morphology. We note that expressing Vezl in wild-type motor neurons has gain-of-function

326 effects that are superficially similar to defects caused by Vezl loss: an increase in terminal

327 bouton size and an increase in satellite bouton number (Figure 2H,J,L). However, the Vezl

328 gain and loss effects are likely distinct: Vezl gain induces satellite boutons along the whole

329 length of the NMJ instead of specifically from the terminal bouton and also does not cause

330 an accumulation of HRP in the terminal bouton. Finally, we note that expressing Vezl

331 specifically in motor neurons does not rescue the lethality caused by Vezl loss, consistent

332 with its previously described broader effects on development (Grazadio et al. 2018).

333

334 Vezl loss results in excessive DCVs and endosomes in terminal boutons and

335 disrupts retrograde BMP signaling

336 Many different manipulations can cause a decrease in bouton number or increase in

337 satellite boutons in fly larval NMJs. However, the only previously reported instance of

338 enlarged terminal boutons with increased anti-HRP staining is that caused by disrupting

339 essential components of the dynein-dynactin complex in motor neurons: knockdown of

16 340 dynactin or dynein subunits traps dynein-dependent cargoes such as DCVs and

341 endosomes in terminal boutons, causing the latter to expand in size (Lloyd et al. 2012). To

342 test whether Vezl might also be involved in this process, we first examined the localization

343 of retrograde cargoes in fixed samples. When expressed in fly neurons, ANF:GFP, a fusion

344 of GFP to a rat neuropeptide, is packaged and transported in DCVs in the same way as

345 endogenous neuropeptides (Rao et al. 2001; Levitan et al. 2007; Wong et al. 2012). We

346 found that Vezl loss, like loss of the essential dynactin subunit p150Glued, increases

347 ANF:GFP specifically in terminal boutons (Figure 3A,B,I; Lloyd et al. 2012). The early

348 endosome marker YFP:Rab5 and the late endosome marker Rab7:GFP also accumulate in

349 terminal boutons in the absence of p150Glued (Lloyd et al. 2012). We found that both of

350 these endosome markers are likewise specifically increased in terminal boutons in vezl

351 mutants (Figure 3C-F,I). By contrast, the recycling endosome marker Rab11:GFP, which

352 does not normally undergo retrograde transport, and the synaptic vesicle protein Cysteine

353 String Protein (CSP) do not show such an increase in vezl mutants (Figure 3G-I; Figure

354 S2A,B). Similarly, the major active zone (AZ) protein Bruchpilot (Brp) is not increased:

355 instead, the density of Brp puncta in terminal boutons is decreased by vezl loss, although

356 these puncta remain apposed to postsynaptic glutamate receptors, as in wild type (Figure

357 S2C-E).

358

359 The increase in ANF:GFP, YFP:Rab5 and Rab7:GFP in NMJ terminal boutons implicate

360 Vezl in the initiation of their retrograde transport and suggest a potential explanation for the

361 reduced NMJ size in vezl mutants. Normal NMJ growth requires motor neurons to

362 transduce a muscle-released BMP signal through the axon to the nucleus (McCabe et al.

363 2003; reviewed in Deshpande and Rodal 2016). The BMP signal binds the receptors

17 364 Wishful thinking (Wit), Saxophone (Sax) and Thick veins (Tkv), which phosphorylate the

365 transcription factor Mothers Against Dpp (MAD). Phosphorylated MAD (pMAD) can then

366 enter the motor neuron nucleus and activate transcription of growth-promoting factors

367 (Marqués et al. 2002; Aberle et al. 2002; Rawson et al. 2003; McCabe et al. 2003; McCabe

368 et al. 2004). Previous work has shown that pMAD is not retrogradely transported (Smith et

369 al. 2012). Instead, entry of pMad into the nucleus depends on the retrograde transport of

370 activated BMP receptors that have been endocytosed (Smith et al. 2012). To determine

371 Vezl's effect on this pathway, we examined the localization of a tagged form of the BMP

372 receptor Tkv. In fixed samples, Tkv:mCherry normally localizes to puncta distributed

373 throughout the motor neuron axon and boutons (Figure 4A). We found that Vezl loss

374 causes an increase in Tkv:mCherry specifically in terminal boutons (Figure 4D,E),

375 consistent with a defect in its retrograde transport. Previous work has shown that BMP

376 signaling also acts locally, phosphorylating MAD within synaptic boutons by a pathway that

377 does not require retrograde transport (O'Connor-Giles et al. 2008; Higashi-Kovtun et al.

378 2010; Sulkowski et al. 2016). This presynaptic pMAD participates with postsynaptic

379 glutamate receptors in a positive feedback that regulates the distribution of glutamate

380 receptor subtypes (Sulkowski et al. 2016). We were unable to detect nuclear pMAD

381 staining above background in either wild type or vezl mutants. However, we did find that

382 pMAD levels are significantly increased in vezl mutant terminal boutons (Figure 4C-E),

383 suggesting that Vezl loss traps activated Tkv in terminal boutons, preventing the increase in

384 NMJ bouton number that depends on retrograde BMP signaling.

385

386 Vezl is required for the retrograde movement of Tkv out of terminal boutons

18 387 We next directly tested the effect of Vezl loss on the movement of individual vesicles in live

388 animals. We first tracked the movement of Tkv:mCherry. In wild-type animals, significantly

389 more Tkv:mCherry puncta move out of the terminal bouton, i.e. retrogradely, than in the

390 opposite direction (Figure 5A,C). By contrast, in vezl mutant animals, very few Tkv:mCherry

391 puncta move out of terminal boutons (Figure 5B,C). Instead, most Tkv:mCherry puncta

392 within vezl mutant terminal boutons move sporadically and non-directionally (Figure 5B),

393 suggesting that they are not moving along microtubule tracks. We conclude that vezl is

394 required for the initiation of Tkv:mCherry retrograde transport out of terminal boutons.

395

396 The possibility remained that vezl loss might only affect Tkv:mCherry transport indirectly -

397 perhaps by causing the accumulation of excess HRP-positive membrane. If so, one would

398 expect that most or all retrogradely-transported cargoes would be similarly affected. We

399 therefore next tracked the movement of individual ANF:GFP puncta. Because these puncta

400 are more numerous than Tkv:mCherry puncta and therefore difficult to follow individually,

401 we used fluorescence recovery after photobleaching (FRAP). We photobleached the

402 boutons adjacent to the terminal bouton and measured the number of vesicles that entered

403 the bleached area either by retrograde transport from the terminal bouton or by

404 anterograde transport from more proximal boutons. As expected of cargoes that use

405 retrograde transport to circulate among en passant boutons, we found that ANF:GFP

406 puncta do not display the bias toward retrograde movement that we observed for

407 Tkv:mCherry puncta: in wild-type animals, roughly equal numbers of ANF:GFP puncta

408 move out of and toward the terminal bouton (Figure 5D,F). We found that vezl loss does

409 not significantly alter anterograde or retrograde flux of ANF:GFP at terminal boutons

410 (Figure 5E,F). The mild increase in ANF:GFP levels in terminal boutons (Figure 3B,I)

19 411 suggests that a subtle defect may nonetheless exist and manifest as a slow accumulation

412 over time. However, our live imaging indicates that, despite its strong effect on

413 Tkv:mCherry, Vezl loss does not uniformly prevent retrograde cargoes from exiting the

414 terminal boutons.

415

416 Vezl is enriched in terminal boutons but is also required for normal transport of

417 DCVs and Tkv along axons

418 Retrograde transport out of terminal boutons is initiated when dynein-cargo complexes are

419 loaded onto microtubules by the dynactin subunit p150Glued (Lloyd et al. 2012; Moughamian

420 and Holzbaur 2012; Moughamian et al. 2013). Presumably to facilitate this, p150Glued is

421 highly enriched in terminal boutons, unlike dynein itself (Lloyd et al. 2012; Moughamian and

422 Holzbaur 2012). To test whether Vezl might specifically regulate transport out of terminal

423 boutons, we examined Vezl localization by driving expression of Venus-tagged full-length

424 Vezl in motor neurons of wild-type animals. Detecting Vezl:Venus signal requires two

425 copies each of OK371-Gal4 and UAS-Vezl:Venus, resulting, presumably, in considerable

426 overexpression. Under these conditions, Vezl:Venus is highly enriched in terminal boutons,

427 consistent with its role in transport initiation (Figure 6A). However, Vezl:Venus is also

428 present at lower levels in non-terminal boutons and along axon shafts. While most

429 Vezl:Venus signal appears to be stationary, we did observe occasional movement of

430 Vezl:Venus puncta in both anterograde and retrograde directions (Movie S1).

431

432 To test whether Vezl might be required for normal transport within axons, we tracked the

433 movement of ANF:GFP, Tkv:mCherry, and the mitochondrial marker Mito:GFP in nerve

434 bundles emerging from the ventral nerve cord. We found that Vezl loss decreases the

20 435 density of ANF:GFP and Tkv:mCherry puncta within axons and increases the proportion of

436 these puncta that are stationary (Figure 6B-G). As a consequence, both anterograde and

437 retrograde flux of Tkv:mCherry is decreased, as is retrograde flux of ANF:GFP (Figure 6I).

438 The effect on retrograde flux is particularly striking: Vezl loss significantly decreases the

439 proportion of motile ANF:GFP and Tkv:mCherry puncta that move in the retrograde

440 direction (Figure 6H). By contrast, Vezl loss does not alter the number of Mito:GFP puncta

441 present within axons (Figure 6F) and does not have a specific effect on retrograde

442 Mito:GFP transport, although there is a slight increase in the proportion of Mito:GFP that

443 are stationary (Figure 6G). Together, these results indicate that Vezl is strongly required for

444 the retrograde transport of DCVs and signaling endosomes in axons but is largely

445 dispensable for mitochondrial transport. We note that Vezl loss decreases the velocities of

446 retrograde ANF:GFP and anterograde Tkv:mCherry movement (Figure 6J), but how this

447 might relate to the other axon transport defects is unclear.

448

449 Loss of Vezt in zebrafish impairs retrograde transport of Rab7-positive endosomes

450 Vertebrate Vezt has not previously been implicated in axonal transport. To test its potential

451 role in this process, we first examined whether expression of tagged human Vezt (hVez)

452 might rescue fly vezl mutant NMJ defects, despite the low amino acid sequence identity

453 between the fly and human proteins. We found that, unlike fly Vezl, hVezt expressed in fly

454 motor neurons is not enriched at terminal boutons and does not restore either NMJ size or

455 terminal bouton morphology in vezl mutants (Figure S3). We therefore turned to a

456 vertebrate model system, zebrafish, to directly test whether vertebrate Vezt plays a role in

457 retrograde axonal transport. The posterior lateral line (pLL) sensory axons of the larval

458 zebrafish are ideal for analyses of cargo localization and transport in axons, in part

21 459 because of their considerable length (Drerup and Nechiporuk 2016). We used CRISPR

460 technology to create a zebrafish line, vezty624, in which the single vezt ortholog within the

461 zebrafish genome is disrupted. In this line, a 5 base pair insertion in exon 3 disrupts the

462 open reading frame, leading to a 40 amino acid insertion and premature stop site. We

463 found that vezty624 homozygotes are grossly normal but die by late larval stages.

464

465 As in fly, loss of dynein or dynactin in zebrafish causes retrogradely-transported cargoes to

466 accumulate in axon terminals, resulting in characteristic axon terminal swellings (Drerup

467 and Nechiporuk 2013; Drerup et al. 2017). We found that at 4 days post-fertilization (dpf),

468 pLL axon terminals in vezt mutant zebrafish are noticeably swollen: quantification of axon

469 terminal volume revealed a significant increase in vezt mutants compared to their wild-type

470 siblings (638±49 µm3 vs 417±63 µm3; Figure S4A-F). To determine whether loss of

471 retrograde cargo movement correlated with this defect, we first analyzed the localization of

472 dynein and dynactin components in pLL axons by immunofluorescence. We found that

473 excess p150 accumulates in vezt mutant terminals as compared to wild-type siblings at 4

474 dpf (Figure S4A,B,G), suggestive of a defect in retrograde transport. Dynein heavy chain

475 (DHC) and mitochondria are localized normally (Figure S4C-G).

476

477 To directly assess the localization and transport of endosome subtypes, we mosaically

478 expressed mRFP-tagged Rab5, Rab7, and Rab11 in pLL neurons of live larvae. We found

479 that mRFP:Rab7-positive late endosomes significantly accumulate in pLL axon terminals in

480 vezt mutants at 4 dpf, while Rab5- and Rab11-labeled endosomes localize normally (Figure

481 7A-C,E-G,I-K). We then analyzed the movement of these markers in axons and found that

482 retrograde transport of mRFP:Rab7-positive endosomes is specifically impaired in vezt

22 483 mutants, as assessed by distance traveled (Figure 7H); Rab5-positive early endosome

484 anterograde transport showed a modest decrease that failed to reach significance (Figure

485 7D), and recycling endosomes moved normally (Figure 7L). We conclude that Vezt is

486 required for normal retrograde transport of Rab7-labeled endosomes in vertebrate axons.

487

488

23 489 DISCUSSION

490 Here, we describe the identification of a Vezt-like fly protein, Vezl, that is enriched at NMJ

491 terminal boutons and required for normal retrograde transport of a subset of cargos. In the

492 absence of Vezl, Rab5 and Rab7-positive endosomes, including signaling endosomes, and

493 DCVs accumulate within NMJ terminal boutons. The retrograde movement of signaling

494 endosomes and DCVs but not of mitochondria is also impaired within axon shafts. We find

495 that loss of Vezt from zebrafish impairs the retrograde movement of Rab7-labeled

496 endosomes in sensory axons. This work identifies a new, conserved regulator of dynein-

497 dependent endosomal transport in neurons.

498

499 While neither fly Vezl nor vertebrate Vezt have previously been implicated in axonal

500 transport, a distantly related Aspergillus protein, VezA, is required for the dynein-dependent

501 retrograde transport of endosomes in hyphae (Yao et al. 2015). Tagged VezA is enriched

502 at microtubule plus ends within hyphal tips as Vezl is at terminal boutons, and loss of VezA

503 decreases the frequency of retrograde endosome movement, causing endosomes to

504 accumulate at microtubule plus ends (Yao et al. 2015). VezA is thought to act by promoting

505 binding between the endosome-specific cargo adaptor HookA and the dynein-dynactin

506 complex (Yao et al. 2015). Might Vezl and Vezt act similarly?

507

508 While the Hook family of cargo adaptors is conserved across species (Xiang et al. 2015;

509 Reck-Peterson et al. 2018), the single Hook protein in fly does not seem to be required for

510 retrograde axonal transport of endosomes. Fly Hook can associate with Rab5- and Rab7-

511 positive endosomes and has some effects on endosome trafficking (Krämer and Phistry

512 1999). However, hook null mutants are viable (Krämer and Phistry 1999; Sunio et al. 1999;

24 513 Szatmári et al. 2014), their NMJs do not have the terminal bouton swelling that is

514 characteristic of vezl mutants (Narayanan et al. 2000), and their NMJs have more boutons

515 rather than fewer (Narayanan et al. 2000). The cargo specificity of Vezl toward endosomes

516 and, to a lesser extent, DCVs, but not mitochondria, indicates that, like VezA, Vezl does not

517 alter the general properties of the dynein-dynactin motor or its microtubule substrate.

518 Instead, Vezl likely promotes interactions between cargoes and dynein-dynactin that are

519 required for the initiation of retrograde transport both out of terminal boutons and within

520 axon shafts. Such interactions could be direct or could instead involve another cargo

521 adaptor such as Bicaudal, which shares a similar structure and interaction space on the

522 dynein motor with Hook proteins (McKenney et al. 2014; Schlager et al. 2014; Urnavicius et

523 al. 2018).

524

525 Unlike fly Hook, vertebrate Hooks are well-established as activating adaptors for cargo-

526 bound dynein-dynactin (Reck-Peterson et al. 2018). Zebrafish Vezt may therefore act

527 analogously to VezA and promote binding between the Rab7-specific Hook protein Hook3

528 and Rab7-endosome-bound dynein-dynactin. Vertebrate Vezt was originally identified

529 based on its association with cell-cell junction proteins, including the cadherin-catenin

530 complex within epithelial cells (Küssel-Andermann et al. 2000; Hyenne et al. 2005), PSD95

531 within hippocampal dendrites (Danglot et al. 2012), and, more recently, muscle

532 acetylcholine receptor at NMJs (Koppel et al. 2019). Consistent with this localization

533 pattern, Vezt is required for the maturation and maintenance of adherens junctions

534 (Hyenne et al. 2005; Hyenne et al. 2007), dendrites (Sanda et al. 2010; Danglot et al.

535 2012), and NMJ synapses (Koppel et al. 2019). The precise mechanism by which Vezt has

536 these effects is unclear, but its binding partners include the small GTPase Arf6 (Sanda et

25 537 al. 2010), which regulates dynein-dependent endosome movements during cytokinesis,

538 exocytosis, and macropinocytosis (Montagnac et al. 2009 ; Marchesin et al. 2015;

539 Williamson and Donaldson 2019). It is therefore possible that Vezt's ability to interact with

540 Arf6 contributes to its regulation of endosome movement in axons.

541

542 A significant consequence of Vezl loss at fly NMJ is its disruption of neurotrophic BMP

543 signaling. The failure of Tkv to undergo retrograde transport out of terminal boutons

544 provides a simple explanation for the reduced number of boutons at vezl mutant NMJs. The

545 observation that pMAD is increased in vezl mutant terminal boutons suggests that Vezl loss

546 does not prevent the trafficking of activated receptors into endosomes but instead

547 specifically prevents their retrograde transport. Like p150Glued, Vezl is normally enriched in

548 NMJ terminal boutons, suggesting that, like p150Glued, Vezl is particularly important in

549 initiating this process.

550

551 vezl loss was previously found to disrupt the behavior of mitotic and meiotic spindles and

552 their attachment to chromosomes (Graziadio et al. 2018). Despite this defect, we never

553 observed missing NMJs, and vezl mutants do not die until they are late larvae or early

554 pupae; this may be because wild-type vezl mRNA is present in the maternal germline

555 (Fisher et al. 2012). Graziadio et al. (2018) did not recognize the similarity of Vezl to

556 vertebrate Vezt or identify the mechanism by which Vezl affects spindle behavior. One

557 possibility is that Vezl regulates dynein-dynactin-dependent movements during cell division

558 as well as in axons. In support of this, Graziadio et al. (2018) observed that Vezl loss

559 causes the failure of a dynein-dependent event during mitosis: in vezl mutants, the dynein-

560 associated protein Zw10 fails to spread along spindle microtubules and instead

26 561 accumulates at kinetochores. Alternatively, like vertebrate Vezt, Vezl may have functions

562 that are independent of its effects on dynein-dependent transport.

563

564 ACKNOWLEDGMENTS

565 This work was supported by National Institutes of Health (NIH) grants R03NS100027 (to

566 T.G.H.), T32GM007759 (M.A.S.), and 1ZIAHD008964-02 (to C.M.D). Stocks obtained from

567 the Bloomington Drosophila Stock Center (supported by NIH grant P40OD018537) were

568 used in this study, as were DNA constructs obtained from the Drosophila Genomics

569 Resource Center (supported by NIH grant 2P40OD010949). The 24B10 and 6D6

570 antibodies developed by S. Benzer, the 4F3 antibody developed by C. Goodman, the 3H2

571 antibody developed by K. Zinn, and the nc82 antibody developed by E. Buchner were

572 obtained from the Developmental Studies Hybridoma Bank, which was created by the

573 NICHD of the NIH and is maintained at The University of Iowa.

574

27 575 FIGURE LEGENDS

576

577 Figure 1. Vezl is required in R7 photoreceptors for normal axon terminal

578 morphology.

579 (A-D) Adult medullas in which homozygous R7s generated by GMR-FLP/MARCM express

580 Synaptotagmin:GFP (green). All R7 and R8 axons are labeled with anti-Chaoptin (Chp;

581 red). The M6 layer of the medulla is indicated by a dashed yellow line. Scale bar is 10 µm.

582 (A) Wild-type (homozygous FRT82) R7 axons terminate in ellipsoid synaptic boutons in M6.

583 (B) The axon terminals of R7s homozygous for the 721 mutation are ellipsoid but frequently

584 have short extensions that end in blobs (arrows) beyond M6.

585 (C) R7s homozygous for 13.1, which deletes most of the CG7705/vezl open reading frame

586 (see panel G), have the same axon terminal defect as 721 homozygous R7s (arrows point

587 to terminal blobs).

588 (D) Expression of a vezl cDNA in 721 homozygous R7s fully rescues their axon terminal

589 defect.

590 (E) Quantification of ectopic terminal blobs in (A-D) genotypes. The percentage of R7s with

591 terminal blobs was calculated for each animal. n = animals, indicated on each bar. Error

592 bars indicate standard error of the mean (SEM). ***p<0.001, based on two-tailed t-tests

593 with Bonferroni correction. Means, SEMs, and p values are available in Table S1.

594 (F) Amino acid sequence comparisons among fly Vezl, human Vezatin (hVez), A. nidulans

595 VezA, and zebrafish Vezatin (zVez). Each value in orange indicate the percentage of amino

596 acid identity between a given pair.

597 (G) Diagram showing the approximate breakpoints of the vezl13.1 deletion relative to the

598 locations of the vezl exons found in the two types of vezl mRNA identified, "CG7705-RA"

28 599 and "CG7705-RB". The first exon is at the left. The large deficiency Df(3R)Exel6180

600 completely removes vezl together with 23 additional genes. Amino acid coding sequences

601 (CDS) are in orange and untranslated regions (UTR) are in gray.

602 (H) Sequence alignment of Vezatin family proteins. Black regions indicate conserved

603 sequence and dark grey indicates >80% conserved sequence (ClustalW2). Interpro detects

604 a "Vezatin domain" (purple bars) in vertebrate Vezatins and Aspergillus VezA but not in fly

605 Vezl. Helical transmembrane domains predicted by two different algorithms are indicated

606 by blue (Interpro) and red (SMART) bars. Green bars indicate low complexity regions

607 predicted by PrDOS. A detailed amino acid alignment among fly Vezl, human Vezt, and the

608 pfam12632 "Vezatin domain" consensus sequence is provided in Figure S1.

609

610 Figure 2. Vezl is required in larval motor neurons for normal NMJ size and terminal

611 bouton morphology

612 (A-D) Third-instar larval NMJs at muscle 4 in abdominal segment 3 (A3) stained with anti-

613 HRP (white). Scale bar is 10 µm.

614 (A) Wild type.

615 (B) vezl13.1/Df(3R)Exel6180 mutant NMJs have a reduced number of boutons.

616 (C) Expression of a vezl cDNA in motor neurons of vezl13.1/Df(3R)Exel6180 mutants

617 restores NMJ size to that of wild type (quantified in I).

618 (D) Expression of a vezl cDNA in motor neurons of wild-type animals increases the number

619 of small, "satellite" boutons throughout the NMJ.

620 (E-H) Larger magnifications of NMJs (different from those in panels A-D), stained with anti-

621 HRP (white). Scale bar is 10 µm.

29 622 (E) In wild type, the terminal bouton (top) and en passant boutons (below) have similar

623 levels of anti-HRP staining.

624 (F) In vezl13.1/Df(3R)Exel6180 mutants, the terminal bouton (top) is often enlarged and

625 contains significantly increased anti-HRP staining. Both defects are fully rescued by

626 neuronal expression of a wild-type vezl transgene (quantified in panels J and K).

627 (G) In vezl13.1/Df(3R)Exel6180 mutants, the terminal bouton is often surrounded by satellite

628 boutons containing increased anti-HRP staining.

629 (H) Expression of a vezl cDNA in motor neurons of wild-type animals causes an increase in

630 satellite boutons, including those that branch off the terminal bouton (as in this example).

631 (I-L) Quantifications of the phenotypes mentioned in (A-H). n = animals, indicated on each

632 bar. Error bars indicate SEM. ***p<0.001, **p<0.01, *p<0.05, ns = not significant, based on

633 two-tailed t-tests with Bonferroni correction. Means, SEMs, and p values are available in

634 Table S1.

635

636 Figure 3. Loss of vezl causes an increase in DCVs and endosomes specifically in

637 terminal boutons

638 (A-H) Third-instar larval NMJs stained with anti-HRP (blue) and anti-Dlg (red). In each

639 panel, dashed boxes indicate the regions containing en passant boutons (top) and terminal

640 boutons (bottom) that are displayed with increased magnification in adjacent panels to the

641 right. Scale bar is 10 µm.

642 (A) In wild-type motor neurons, similar levels of the DCV marker ANF:GFP (green) localize

643 to terminal and en passant boutons.

644 (B) ANF:GFP is increased specifically in terminal boutons in vezl13.1/Df(3R)Exel6180

645 mutants.

30 646 (C) In wild-type motor neurons, similar levels of the early endosome marker YFP:Rab5

647 (green) localize to terminal and en passant boutons.

648 (D) YFP:Rab5 is increased specifically in terminal boutons in vezl13.1/Df(3R)Exel6180

649 mutants.

650 (E) In wild-type motor neurons, similar levels of the early-late endosome marker Rab7:GFP

651 (green) localize to terminal and en passant boutons.

652 (F) Rab7:GFP is increased specifically in terminal boutons in vezl13.1/Df(3R)Exel6180

653 mutants.

654 (G) In wild-type motor neurons, similar levels of the recycling endosome marker

655 Rab11:GFP (green) localize to terminal and en passant boutons.

656 (H) The localization of Rab11:GFP in vezl13.1/Df(3R)Exel6180 mutants resembles that in

657 wild type.

658 (I) Quantification of fluorescence intensities in (A-H) genotypes. n = animals, indicated on

659 each bar. Error bars indicate SEM. ***p<0.001, **p<0.01, ns = not significant, based on

660 two-tailed t-tests. Means, SEMs, and p values are available in Table S1.

661

662 Figure 4. Loss of vezl disrupts retrograde BMP signaling

663 (A,B) Third-instar larval NMJs stained with anti-HRP (blue) in which motor neurons express

664 Tkv:mCherry (red). Each terminal bouton is on the right. Scale bar is 10 µm.

665 (A) In wild-type motor neurons, similar levels of the BMP type I receptor marker

666 Tkv:mCherry (red) localize to terminal and en passant boutons.

667 (B) Tkv:mCherry is increased specifically in terminal boutons in vezl13.1/Df(3R)Exel6180

668 mutants.

669 (C,D) Third-instar larval NMJs stained with anti-HRP (green) and anti-pMAD (white).

31 670 (C) In wild type, similar levels of phosphorylated Mad (pMad), the downstream target of

671 BMP signaling in motor neurons, localize to terminal and en passant boutons.

672 (D) Anti-pMad staining is increased specifically in terminal boutons in

673 vezl13.1/Df(3R)Exel6180 mutants.

674 (E) Quantification of staining intensities in (A-D) genotypes. n = animals, indicated on each

675 bar. Error bars indicate SEM. ***p<0.001, **p<0.01, based on two-tailed t-tests. Means,

676 SEMs, and p values are available in Table S1.

677

678 Figure 5. Vezl is required for the retrograde movement of Tkv out of terminal boutons

679 (A,B,D,E) Timecourses of vesicle movements in live third-instar larvae. Each terminal

680 bouton is on the left (dashed circles) and retrograde transport occurs toward the right.

681 Scale bars are 10 µm.

682 (A) In wild type, Tkv:mCherry puncta (red; arrows) frequently move retrogradely out of the

683 terminal bouton.

684 (B) Tkv:mCherry puncta (red; arrows) in vezl13.1/Df(3R)Exel6180 mutants often fail to exit

685 the terminal bouton and instead circulate in an unorganized fashion. Note the extended

686 recording time compared to that in (A). And note that panel B is shown with reduced

687 brightness relative to panel A so that individual puncta are more evident.

688 (C) Quantification of Tkv:mCherry movement. Retrograde movement out of the terminal

689 bouton is significantly decreased in vezl mutants. n = animals, indicated on each bar. Error

690 bars indicate SEM. **p<0.01, based on two-tailed t-tests. Means, SEMs, and p values are

691 available in Table S1.

692 (D,E) Regions adjacent to the terminal bouton were photobleached, allowing the

693 movements of individual ANF:GFP puncta into the bleached region to be monitored.

32 694 (D) In wild type, ANF:GFP puncta (green; arrow) frequently move retrogradely out of the

695 terminal bouton. (Note that the angle of the single z-section in this example creates an

696 illusion that, prior to photobleaching, ANF:GFP is higher in the non-terminal boutons than in

697 the terminal bouton; there is no such difference (see Figure 3A,I)).

698 (E) The movement of ANF:GFP puncta (green; arrow) in vezl13.1/Df(3R)Exel6180 mutants is

699 similar to that in wild type.

700 (F) Quantification of ANF:GFP movement. n = animals, indicated on each bar. Error bars

701 indicate SEM. ns = not significant based on two-tailed t-tests. Means, SEMs, and p values

702 are available in Table S1.

703

704 Figure 6. Vezl is enriched in terminal boutons but is also required for normal

705 transport of DCVs and Tkv along axons

706 (A) NMJ from a wild-type third-instar larva containing two copies each of the motor neuron

707 driver OK371-Gal4 and the UAS-Vezlwt:Venus transgene. Larvae were dissected but not

708 fixed, and fluorescence-conjugated anti-HRP (blue) was added to the dissection media.

709 Vezl:Venus (green) is highest in terminal boutons but also present in en passant boutons

710 and the axon shaft. (Vezl:Venus was difficult to detect in fixed samples or in animals

711 containing fewer transgenes.) Scale bar is 10 µm. Most Vezl:Venus signal appears to be

712 stationary, but we did observe movement of some Vezl:Venus puncta within axon bundles

713 (Movie S1).

714 (B,C,D,E) Representative kymographs of ANF:GFP (B,D) or Tkv:mCherry (C,E) in third-

715 instar larval axons; for experiments in (B-J), one bundle of axons was imaged per animal,

716 near the ventral nerve cord. The x-axes represent distance, and y-axes represent time.

717 Lines that slope downward from left to right are puncta moving in the anterograde direction;

33 718 lines that slope downward from right to left are puncta moving in the retrograde direction.

719 Stationary puncta result in vertical lines. In wild type, ANF:GFP (B) and Tkv:mCherry (C)

720 puncta move in both directions. In vezl13.1/Df(3R)Exel6180 mutants, retrograde movement

721 of ANF:GFP (D) and Tkv:mCherry (E) puncta is considerably reduced.

722 (F-J) Quantification of ANF:GFP, Tkv:mCherry, and Mito:GFP movement in wild type and

723 vezl13.1/Df(3R)Exel6180 mutants. Loss of vezl decreases the density of ANF:GFP and

724 Tkv:mCherry puncta within axons (F), increases the fraction of those puncta that are

725 stationary (G), and decreases the proportion of motile ANF:GFP and Tkv:mCherry puncta

726 that are moving in the retrograde direction (H), resulting in a severe decrease in their

727 retrograde flux (I). vezl loss also affects the velocities of ANF:GFP and Tkv:mCherry puncta

728 (J). By contrast, we observed only a mild effect of vezl loss on Mito:GFP movement (F-J).

729 In all bar graphs, n = animals, indicated on each bar. Error bars indicate SEM. *p<0.05,

730 **p<0.01, ***p<0.001, ns = not significant, based on two-tailed t-tests. Means, SEMs, and p

731 values are available in Table S1.

732

733 Figure 7. Zebrafish vezt mutants have decreased retrograde transport of late

734 endosomes, which accumulate in axon terminals

735 (A,B,E,F,I,J) pLL axon terminals marked by cytoplasmic GFP (green) in the

736 TgBAC(neurod:egfp)nl1 transgenic line. Axon terminal swellings caused by vezt loss are

737 particularly evident in panel B (see also Figure S4 and the quantification of wild-type and

738 vezt mutant axon terminal volumes in Results). Animals were mosaic for transgenes driving

739 neuronal expression of Rab5:mRFP (A-D), Rab7:mRFP (E-H), or Rab11:RFP (I-L).

740 Because of this mosaicism and the axon terminal swelling caused by vezt loss, Rab

741 accumulation was assessed in (C), (G), and (K) by normalizing Rab area to axon terminal

34 742 area. Normalizing instead to axon terminal number yielded similar results (see Table S1).

743 Scale bar is 10 µm.

744 (A,B) The accumulation of Rab5:mRFP (magenta) in axon terminals is similar in wild-type

745 (A) and vezty624 mutant animals (B). Quantified in (C,D), which also shows largely normal

746 movement of Rab5:mRFP.

747 (E,F) By contrast, Rab7:mRFP (magenta) accumulates significantly more in vezty624 mutant

748 axon terminals (F) than in wild-type terminals (E). Quantified in (G,H), which also shows a

749 decreased retrograde transport distance in vezty624 mutants.

750 (I-L) Rab11:mRFP accumulation and movement are unaffected by vezt loss.

751 In all bar graphs, n = animals, indicated on each bar. Error bars indicate SEM. *p<0.05,

752 **p<0.01, ns = not significant, based on two-way ANOVA. Means, SEMs, and p values are

753 available in Table S1.

754

755

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44 ABwt 721 E F *** *** Vezl 40 (592aa) 16% 13% 30 hVezt 15% VezA (748aa) (615aa) CDvezl13.1 721 + 20 vezlwt 15% 43% 15% 10 zVezt (783aa) 10 10 10 12

%R7s with terminal blobs 0

wt 13.1 wt 721 vezl

721 + vezl G P-element CDS UTR

CG7705-RA vezl (CG7705) CG7705-RB

vezl 13.1

Df(3R)Exel6180

hVezt

zVezt

Vezl

VezA

hVezt Vezatin domain (Interpro)

zVezt helical transmembrane (Interpro)

Vezl helical transmembrane (SMART)

VezA disordered regions (PrDOS)

Figure 1 wt vezl13.1 vezl13.1 + wt + A B C vezlwt D vezlwt

HRP

wt vezl13.1 vezl13.1 wt + E F G H vezlwt

HRP

I J K L ns ns ** ) ** 10 50 2 30 2 *** *** * m *** *** 9 ns 40 *** 25 8 1.5 *** 7 20 30 6 15 1 5 20 4 10 3 0.5 10 2

5 # Satellite boutons 14 12 12 10 14 12 12 10 14 12 12 10 1 14 12 12 10

0 bouton size ( Terminal 0 0 0

wt wt wt wt wt wt wt wt Total # of non-satellite boutons Total relative to non-terminal boutons wt /Df wt /Df intensity in terminal bouton HRP wt /Df wt /Df 13.1 13.1 13.1 13.1

vezl vezl vezl vezl /Df + vezlwt + vezl /Df + vezlwt + vezl /Df + vezlwt + vezl /Df + vezlwt + vezl 13.1 13.1 13.1 13.1

vezl vezl vezl vezl

Figure 2 A wt + ANF C wt + Rab5 E wt + Rab7

proximal

distal

vezl13.1 + vezl13.1 + vezl13.1 + B ANF D Rab5 F Rab7

HRP HRP HRP DLG DLG DLG ANF Rab5 Rab7

Rab7:GFP wt + Rab11 2.5 G I *** YFP:Rab5 ANF:GFP 2 *** ** Rab11:GFP ns

1.5

1 vezl13.1 + H Rab11

0.5 ﬂuorescence intensity in terminal

15 10 16 10 14 12 16 14 GFP

bouton relative to non-terminal boutons 0

HRP wt /Df wt /Df wt /Df wt /Df DLG 13.1 13.1 13.1 13.1 RAB11 vezl vezl vezl vezl

Figure 3 A wt + Tkv E 2.5 HRP Tkv:mCherry Tkv Tkv *** 2 13.1 B vezl + Tkv -pMAD **

1.5 HRP Tkv Tkv

1 C wt

0.5

HRP pMAD relative to non-terminal boutons 14 15 12 15 13.1 vezl Fluorescence intensity in terminal bouton 0

D wt /Df wt /Df 13.1 13.1

vezl vezl HRP pMAD

Figure 4 A wt + Tkv C 1.75 Tkv:mCherry 1.5 ** Out 1.25 In 1 Tkv t=0 t=5s t=30s t=60s retrograde 0.75 transport 0.5 7 13.1 vezl + Tkv 0.25 erminal bouton ﬂux

B (# vesicles/minute) 5 T 0

wt /Df 13.1

Tkv t=0 t=5s t=60s t=120s vezl

wt + ANF D F ANF:GFP 4 ns Out 3 In ANF t=0 t=5s t=15s t=30s retrograde 2 transport vezl13.1 + ANF 1

E erminal bouton ﬂux 9 6 (# vesicles/minute) T 0

wt /Df 13.1

ANF t=0 t=5s t=15s t=30s vezl

Figure 5 A wt +Vezl Anterograde movement Retrograde movement

B ANF:GFP in wt C Tkv:mCherry in wt

D ANF:GFP in vezl 13.1 E Tkv:mCherry in vezl 13.1

HRP Vezl Vezl

F G H retrograde anterograde 1.2 ANF:GFP 1 Mito:GFP ANF:GFP Tkv:mCherry Mito:GFP ns *** ANF:GFP *** *** 1 Tkv:mCherry * 100 0.8 *** ** Tkv:mCherry 2 0.8 80 m *** 0.6 Mito:GFP 0.6 ns 60 0.4

Puncta/ 0.4 40 (% of total) 0.2 0.2 20 6 7 6 6 7 5 6 7 6 6 7 5

0 Fraction of stationary puncta 0 6 7 6 6 7 5

Directionality of motile puncta Directionality of motile 0 wt 13.1 wt 13.1 wt 13.1 wt 13.1 wt 13.1 wt 13.1 wt 13.1 wt 13.1 wt 13.1 vezl vezl vezl vezl vezl vezl vezl vezl vezl I J 2 Tkv:mCherry 1.75 retrograde ANF:GFP ** ns 1 ANF:GFP anterograde 1.5 ns ** *** 0.8 Tkv:mCherry 1.25 *** 1 Mito:GFP 0.6 ns ns 0.75

0.4 Mito:GFP (um/s) Velocity 0.5 ns 6

Flux (puncta/min) 0.2 7 5 0.25 6 7 6 12 10 12 10 10 11 10 11 10 14 10 14 0 0

wt 13.1 wt 13.1 wt 13.1 wt 13.1 wt 13.1 wt 13.1 wt 13.1 wt 13.1 wt 13.1

vezl vezl vezl vezl vezl vezl vezl vezl vezl anterograde retrograde anterograde retrograde anterograde retrograde

Figure 6 wt A C D ns 0.5 ns 8

0.4 6 ns 0.3

merge Rab5 4 0.2 B vezty624 Distance (µm) 2 0.1 Rab5 vesicle area/axon area 11 9 9 8 10 8 0.0 0 wt y624 wt y624 wt y624 vezt vezt vezt Rab5 merge anterograde retrograde

E wt G H 0.5 8 ** ns

0.4 6

0.3 * merge Rab7 4 y624 F vezt 0.2 Distance (µm) 2 0.1 Rab7 vesicle area/axon area 13 14 13 13 12 13 0.0 0 wt y624 wt y624 wt y624 merge Rab7 vezt vezt vezt anterograde retrograde

I wt K L 0.4 ns 8 ns

0.3 6 ns

merge Rab11 0.2 4 J vezty624 Distance (µm) 0.1 2

Rab11 vesicle area/axon area Rab11 10 14 11 11 11 11 0.0 0

wt y624 wt y624 wt y624 merge Rab11 vezt vezt vezt anterograde retrograde

Figure 7

fly 1 MQELKQRFERRQLADDLKQKQVIQDHL--LQKILY 33 F + DDL K I L L IL human 1 MTPEFDEEVVFENSPLYQYLQDLGHTDFEICSSLSPKTEKCTTEGQQKPPTRVLPKQGILLKVAETIKSWIFFSQCNKKDDLLHKLDIGFRLDSLHTILQ 100

fly 34 SKRLLVDHVEALEH------CMQELQTASSASSGRQLLTTRNA--CLILGGTLLEHLITPRGIRYTMVPSILISGTFAALF----AVKSVFVCRNKIVE 120 + LL + VE +E Q Q + L T N +L + ++ P +V S L+ G ++ ++++ + R ++ human 101 QEVLLQEDVELIELLDPSILSAGQSQQQENGHLPTLCSLATPNIWDLSMLFAFISLLVMLPTWW---IVSSWLVWGVILFVYL---VIRALRLWRTAKLQ 194 L A I L+ W + SS L G L + + +IR+L WR +LQ vez dom LIALIIFLLKQAFYW---lSSSSLFKGFKLLLLIstkLIRVlfywRRQRLQ

fly 121 RK------LEQLVKTIDEFGNCIRRNMTYFQEIIIMKQAELIES---RQIER-AWDCITAAKEVTEILYD------ATRKLETDYPLPTKYSAY 198 LE + F N +R+ + QE ++ + + S + + + +Y AT + +YPL ++ human 195 VTLKKYSVHLEDMATNSRAFTNLVRKALRLIQETEVISRGFTLVSAACPFNKA-GQHPSQHLIGLRKAVYRTLRANFQAARLATLYMLKNYPLNSESDNV 293 + K LE+ NS+AF LVRKAL LIQE E++SRG+ L PF++ Q S+ IGLRKA+Y TL F R A K PL++ DN+ vez dom IIRHKALNQLEEFVANSQAFDKLVRKALILIQEVELVSRGyrlslplppfsrlEDQSQSRRCIGLRKALYSTLSFLFLNLRQAIS---KLLPLSN-GDNL

fly 199 -----YAPMEEL------RECEYFKNNVTD-YSPKHIKDFHNIFAYVQSQYLLRLALTITT--RPSISQLSEDLV--KIDSLVRQ-LVQEEEQHFSNLA 280 P +EL + E +N TD +S +K + ++ S++ RLAL ++T P L+ L+ +I S V Q L L human 294 TNYICVVPFKELGLGLSEEQISEEEAHNFTDGFSLPALKVWFQLWVAQSSEFFRRLALLLSTANSPPGPLLTPALLPHRILSDVTQGLPHAHSACLEELK 393 Y + EL GLSEE +S+EEA TD SLPALK FQ + EF +L++LLS + P A vez dom EKYCDIYNINEL--GLSEESlsdeeAEELTDSESLPALKFLFQRFNGLRKEFLCQLLALLSdGSKpNFFRWKVAdqfltpdrr------

fly 281 LALQ----NKKQLELAELNATRAEQQQSGPIAVLQHSSLKLSACMVAVAAECQALDVTLQKLTVTEAAKKNNSKELVAVANNMHGIENALAVCCDDFQRL 376 + + + Q + ++ QQ+S + + + L + A+ E L+ L+KL T+ ++ S+ + + I+ + + ++ human 394 RSYEFYRYFETQHQSVPQCLSKT-QQKSRELNNVHTAVRTLQLHLKALLNEVIILEDELEKLVCTKETQELVSEAYPILEQKLKLIQPHVQASNNCWEEA 492 + + +SR+LN++ T +R vez dom ------eraraqsrKLNSLSTLIR

fly 377 MLVYNKFLHSQLDLEGELPKPKQDL------DDEFPESILRVEFSQNV---DAPQQRDDFYAYMYDENEEQHFEAEQNAPFPSPEKELLN 457 + +K L D +G+ ++ D PE +E + D+ ++DDFY ++ E Q E E++ K +L human 493 ISQVDKLLRRNTDKKGKPEIACENPHCTVVPLKQPTLHIADKDPIPEE-QELEAYVDDIDIDSDFRKDDFYYLSQEDKERQKREHEESKRVLQELKSVLG 591

fly 456 F-EKRITKGRFK------PVLKQL--KDRIDPIRQVMLEKEREVLASKGINVDELFGKMERVEQQQEDNRLADAAPLPVYESSSNSDSDSADEEAFRRR 547 F + ++K VLK L D ++PI E + + G V + + E + DN ++ S + D++ E F + human 592 FKASEAERQKWKQLLFSDHAVLKSLFPVDPVEPISNS----EPSMNSDMG-KVSKNDTEEESNKSATTDNEIS-RTEYLCENSLEGKNKDNSSNEVFPQG 685

fly 548 SKQH--KERDNFAEMRQFLAQKQAINLFKLPPPPVAAGGEELLESEC 592 +++ + ++ E + human 686 AEERMCYQCESEDEPQADGSGLTTAPPTPRDSLQPSIKQRLARLQLSPDFTFTAGLAAEVAARSLSFTTMQEQTFGGEEEEQIIEENKNEIEEK 779

Figure S1. Detailed sequence alignment of fly Vezl, human Vezt, and the "Vezatin domain" consensus sequence. The alignment between fly Vezl (NCBI reference sequence NP_650741.1 (isoform A); "fly") and human Vezt (NCBI reference sequence NP_060069.3 (isoform b); "human") was generated by PSI-BLAST. The alignment between human Vezt and the pfam12632 Vezatin domain consensus sequence ("vez dom") was generated from the consensus sequence alignment at https://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=372233. Amino acid identities and similarities are indicated between each pair of aligned proteins. 13.1 A wt B vezl

HRP

CSP

13.1 C wt D vezl E *** 2.5

2 in terminal

2 1.5

HRP Brp HRP Brp m

1 boutons 0.5 13 14 0

wt /Df # Brp puncta / 13.1 GluRIIC merge GluRIIC merge vezl

Figure S2. The synaptic vesicle marker CSP and active zone marker Brp do not accumulate in NMJ terminal boutons in vezl mutants. (A,B) Third-instar larval NMJs stained with anti-HRP (white) and anti-CSP (red). Terminal boutons are on the right (because of branching there are two terminal boutons in panel A). Scale bar is 10 µm. Unlike HRP, the SV protein CSP is not enriched in terminal boutons in vezl13.1/Df(3R)Exel6180 mutants (the mean anti-CSP fluorescence intensity in the terminal bouton relative to non-terminal boutons is 1.01 (n = 7 animals, SEM = 0.11) in wild type and 1.06 (n = 7 animals, SEM = 0.04) in vezl mutants; p = 0.739 based on a two-tailed t-test). (C,D) Structured illumination microscopy (SIM) images of terminal boutons stained with antibodies against HRP (blue), the major AZ protein Brp (green) and the post-synaptic glutamate receptor subunit GluIIC (red). Scale bar is 10 µm. In wild-type (C), Brp and GluIIC are in apposition. In vezl13.1/Df(3R)Exel6180 mutants (D), Brp and GluIIC remain in apposition, but are at decreased density. Barren areas appear to correlate with areas having increased anti-HRP staining (merge panel has reduced HRP intensity to show detail). (E) Quantification of anti-Brp puncta density in terminal boutons. n = animals, indicated on each bar. Error bars indicate SEM. ***p<0.001, based on a two-tailed t-test. Means, SEMs, and p values are available in Table S1. 45 A wt + hVez B ns 40 *** 35

20 HRP 15

5 11 7 8 3 Total # of non-satellite boutons Total 0

wt /Df 13.1

vezl wt + hVez /Df + hVez 13.1

vezl hVez

Figure S3. Human vezatin expressed in fly motor neurons is not enriched in terminal boutons and does not rescue the decreased NMJ size caused by vezl loss. (A) A wild-type third-instar larval NMJ at which human vezatin (hVez) tagged with Venus is expressed in the motor neuron and detected with anti-GFP (green). Anti-HRP is in blue. Unlike Vezl:Venus, hVez:Venus is not enriched in the terminal bouton (top). Like Vezl:Venus, hVez:Venus was difficult to detect. Unlike Vezl:Venus, hVez:Venus was best imaged in fixed samples stained with anti-GFP antibodies (the signal in muscle is non-specific background). Scale bar is 10 µm. (B) Quantification of NMJ bouton numbers. Expression of hVez:Venus in motor neurons does not restore NMJ size in vezl13.1/Df(3R)Exel6180 mutants or alter NMJ size in wild type. n = animals, indicated on each bar. Error bars indicate SEM. ***p<0.001, ns = not significant, based on two-tailed t-tests. Means, SEMs, and p values are available in Table S1. A wt C wt

merge p150 merge Dync1h1

y624 B vezt D vezty624

merge p150 merge Dync1h1

wt E G 1.5

Dync1h1 CytoC p150 ns ns

1.0 *

merge CytoC F vezty624 mean Fl(A.U.) 0.5

4040 28 28 11 1311 13 1413 14 13 0.0

merge CytoC wt y624 wt y624 wt y624

vezt vezt vezt

Figure S4. p150 accumulates in vezt mutant axon terminals (A-F) Immunofluorescence analysis of the dynactin component p150, Dynein Heavy Chain (Dync1h1), and mitochondria (CytoC) in zebrafish pLL axon terminals marked by cytoplasmic GFP (green) in the TgBAC(neurod:egfp)nl1 transgenic line. Arrows point to axon terminal swellings in vezty624 mutants. Scale bar is 10 µm. (G) Quantification of (A-F). Values were normalized to background. p150 but not Dync1h1 or CytoC accumulates significantly in vezty624 mutant terminals. n = animals, indicated on each bar. Error bars indicate SEM. *p<0.05, ns = not significant, based on two-way ANOVA. Means, SEMs, and p values are available in Table S1. Table S1. Numerical values for bar graphs in Figures 1-7 and Figures S2-S4. Figure Panel Mean SEM +/- n= comparison p-value Bonferroni-corrected p Figure 1 E wt 10.34 2.1 10 wt to vezl721 4.75E-08 9.50E-08 vezl721 39.25 1.7 10 vezl721 to vezl721 + vezlwt (rescue) 4.11E-10 8.22E-10 vezl13.1 33.93 1.4 10 721 WT vezl + vezl 10.48 1.6 12 Figure 2 I wt 35 1.9 14 wt to vezl13.1 0.0001727 0.000691 vezl13.1 23.16 1.8 12 wt to vezl13.1 + vezlwt (rescue) 0.7638 3.06 vezl13.1 + vezlwt 35.92 1.9 12 vezl13.1 to vezl13.1 + vezlwt (rescue) 0.0035 0.014 wt wt wt vezl + vezl 42.1 3.1 10 wt to wt + vezl (OE) 0.0656 0.2624 J wt 10.57 1.12 14 wt to vezl13.1 0.000031 0.000124 vezl13.1 23.87 2.5 12 wt to vezl13.1 + vezlwt (rescue) 0.5925 2.37 vezl13.1 + vezlwt 11.49 0.76 12 vezl13.1 to vezl13.1 + vezlwt (rescue) 0.00018 0.00072 wt wt wt vezl + vezl 18.93 2.1 10 wt to wt + vezl (OE) 0.002121 0.00848 K wt 0.96 0.04 14 wt to vezl13.1 2.03E-08 8.12E-08 vezl13.1 1.59 0.075 12 wt to vezl13.1 + vezlWT (rescue) 0.5499 2.2 vezl13.1 + vezlwt 0.94 0.076 12 vezl13.1 to vezl13.1 + vezlwt (rescue) 8.68E-06 3.47E-05 wt wt wt vezl + vezl 0.95 0.064 10 wt to wt + vezl (OE) 0.6124 2.45 L wt 1.28 0.32 14 wt to vezl13.1 7.36E-06 2.94E-05 vezl13.1 5.42 0.69 12 wt to vezl13.1 + vezlWT (rescue) 0.1812 0.7248 vezl13.1 + vezlwt 2.67 0.88 12 vezl13.1 to vezl13.1 + vezlwt (rescue) 0.029 0.116 wt wt wt vezl + vezl 7.8 1.64 10 wt to wt + vezl (OE) 0.000381 0.001524 Figure 3 I wt ANF:GFP 1.24 0.11 15 wt to vezl13.1 (ANF:GFP) 0.007953 vezl13.1 ANF:GFP 1.62 0.09 10 wt YFP:Rab5 1.1 0.04 16 wt to vezl13.1 (YFP:Rab5) 0.0001801 vezl13.1 YFP:Rab5 1.72 0.04 10 wt Rab7:GFP 0.92 0.09 14 wt to vezl13.1 (Rab7:GFP) 0.0001445 vezl13.1 Rab7:GFP 1.98 0.27 12 wt Rab11:GFP 1.21 0.06 16 wt to vezl13.1 (Rab11:GFP) 0.5895 13.1 vezl Rab11:GFP 1.27 0.26 14 Figure 4 E wt Tkv:mCherry 1.02 0.04 14 wt to vezl13.1 (Tkv:mCherry) 0.0004521 vezl13.1 Tkv:mCherry 1.92 0.14 15 wt anti-pMAD 1.12 0.08 12 wt to vezl13.1 (anti-pMAD) 0.007105 13.1 vezl anti-pMAD 1.52 0.103 15 Figure 5 C wt Tkv:mCherry In 0.25 0.075 5 wt to vezl13.1 (Tkv:mCherry In) 0.4253 wt Tkv:mCherry Out 1.05 0.063 5 vezl13.1 Tvk:mCherry In 0.11 0.086 7 wt to vezl13.1 (Tkv:mCherry Out) 0.009173 13.1 vezl Tvk:mCherry Out 0.048 0.024 7 F wt ANF:GFP In 1.23 0.2 9 wt to vezl13.1 (ANF:GFP In) 0.7126 wt ANF:GFP Out 1.79 0.39 9 vezl13.1 ANF:GFP In 1.37 0.32 6 wt to vezl13.1 (ANF:GFP Out) 0.7807 13.1 vezl ANF:GFP Out 1.48 0.33 6

Figure Panel Mean SEM +/- n= comparison p-value Figure 6 F wt ANF:GFP 0.96 0.06 6 wt to vezl13.1 (ANF:GFP) 0.0005902 vezl13.1 ANF:GFP 0.57 0.06 7 wt Tkv:mCherry 0.72 0.09 6 wt to vezl13.1 (Tkv:mCherry) 0.00825 vezl13.1 Tvk:mCherry 0.28 0.03 6 wt mito:GFP 0.25 0.025 7 wt to vezl13.1 (mito:GFP) 0.3479 13.1 vezl mito:GFP 0.234 0.013 5 G wt ANF:GFP 0.12 0.03 6 wt to vezl13.1 (ANF:GFP) 0.0007503 vezl13.1 ANF:GFP 0.39 0.072 7 wt Tkv:mCherry 0.12 0.048 6 wt to vezl13.1 (Tkv:mCherry) 0.0009128 vezl13.1 Tvk:mCherry 0.55 0.067 6 wt mito:GFP 0.66 0.012 7 wt to vezl13.1 (mito:GFP) 0.01058 13.1 vezl mito:GFP 0.25 0.025 5 wt ANF:GFP H (anterograde) 55.4 2.89 6 wt to vezl13.1 (ANF:GFP) 8.64E-09 vezl13.1 ANF:GFP (anterograde) 93.6 1.13 7 wt Tkv:mCherry (anterograde) 65 3.42 6 wt to vezl13.1 (Tkv:mCherry) 0.0009573 vezl13.1 Tvk:mCherry (anterograde) 90.9 1.91 6 wt mito:GFP (anterograde) 48 5.07 7 wt to vezl13.1 (mito:GFP) 0.3479 vezl13.1 mito:GFP (anterograde) 45.2 4.7 5 wt ANF:GFP I (anterograde) 0.47 0.05 6 wt to vezl13.1 (anterograde ANF:GFP) 0.1706 wt ANF:GFP (retrograde) 0.37 0.03 6 wt to vezl13.1 (retrograde ANF:GFP)) 0.00001796 vezl13.1 ANF:GFP (anterograde) 0.34 0.063 7 vezl13.1 ANF:GFP (retrograde) 0.02 0.0048 7 wt Tkv:mCherry wt to vezl13.1 (anterograde) 0.4 0.055 6 (anterograde Tkv:mCherry) 0.000166 wt Tkv:mCherry wt to vezl13.1 (retrograde) 0.24 0.08 6 (retrograde Tkv:mCherry) 0.0007431 vezl13.1 Tvk:mCherry (anterograde) 0.11 0.041 6 vezl13.1 Tvk:mCherry (retrograde) 0.024 0.0075 6 wt mito:GFP (anterograde) 0.022 0.0046 7 wt to vezl13.1 (anterograde mito:GFP) 0.117 wt mito:GFP (retrograde) 0.024 0.0061 7 wt to vezl13.1 (retrograde mito:GFP) 0.166 vezl13.1 mito:GFP (anterograde) 0.015 0.0021 5 vezl13.1 mito:GFP (retrograde) 0.017 0.0044 5 wt ANF:GFP J (anterograde) 1.03 0.03 12 wt to vezl13.1 (anterograde ANF:GFP) 0.2475 wt ANF:GFP (retrograde) 0.77 0.03 12 wt to vezl13.1 (retrograde ANF-GFP) 0.002424 vezl13.1 ANF:GFP (anterograde) 1.16 0.08 10 vezl13.1 ANF:GFP (retrograde) 0.57 0.06 10 wt Tkv:mCherry wt to vezl13.1 (anterograde) 1.55 0.07 10 (anterograde Tkv:mCherry) 0.009273 wt Tkv:mCherry wt to vezl13.1 (retrograde) 0.8 0.11 10 (retrograde Tkv:mCherry) 0.7723 vezl13.1 Tvk:mCherry (anterograde) 1.13 0.04 11 vezl13.1 Tvk:mCherry (retrograde) 0.73 0.08 11 wt mito:GFP (anterograde) 0.26 0.006 10 wt to vezl13.1 (anterograde mito:GFP) 0.2008 wt mito:GFP (retrograde) 0.49 0.02 10 wt to vezl13.1 (retrograde mito:GFP) 0.613 vezl13.1 mito:GFP (anterograde) 0.32 0.09 10 vezl13.1 mito:GFP (retrograde) 0.51 0.05 14 Figure Panel Mean SEM +/- n= comparison p-value Figure 7 C wt Rab5:mRFP 0.377 0.055 11 wt to vezty624 (Rab5:mRFP) 0.3999 y624 vezt Rab5:mRFP 0.306 0.061 9 wt Rab5:mRFP wt to vezty624 D (anterograde) 6.91 0.51 9 (anterograde Rab5:mRFP) 0.0562 vezty624 Rab5:mRFP (anterograde) 5.37 0.54 8 wt Rab5:mRFP wt to vezty624 (retrograde) 3.667 0.37 10 (retrograde Rab5:mRFP) 0.4655 vezty624 Rab5:mRFP (retrograde) 4.08 0.41 8 G wt Rab7:mRFP 0.25 0.03 13 wt to vezty624 (Rab7:mRFP) 0.0084 y624 vezt Rab7:mRFP 0.374 0.03 14 wt Rab7:mRFP wt to vezty624 H (anterograde) 5.55 0.32 13 (anterograde Rab7:mRFP) 0.9762 vezty624 Rab7:mRFP (anterograde) 5.56 0.32 13 wt Rab7:mRFP wt to vezty624 (retrograde) 3.83 0.24 12 (retrograde Rab7:mRFP) 0.0472 vezty624 Rab7:mRFP (retrograde) 3.12 0.23 13 wt to vezty624 K wt Rab11:mRFP 0.251 0.045 10 (anterograde Rab11:mRFP) 0.2256 y624 vezt Rab11:mRFP 0.324 0.038 14 wt Rab11:mRFP wt to vezty624 L (anterograde) 6.45 0.57 11 (anterograde Rab11:mRFP) 0.9091 vezty624 Rab11:mRFP (anterograde) 6.36 0.57 11 wt Rab11:mRFP wt to vezty624 (retrograde) 4.4 0.4 11 (retrograde Rab11:mRFP) 0.9618 vezty624 Rab11:mRFP (retrograde) 4.42 0.36 11 Figure 7 wt Rab5:mRFP 19.46 3.79 11 wt to vezty624 (Rab5:mRFP) 0.156 Rab area (µm2) vezty624 Rab5:mRFP 27.8 4.18 9 per axon wt Rab7:mRFP 13.73 3.21 11 wt to vezty624 (Rab7:mRFP) 0.0181 (alternative vezty624 Rab7:mRFP 24.65 2.85 14 normalization of wt Rab11:mRFP 14.4 2.57 8 wt to vezty624 (Rab11:mRFP) 0.7501 y624 data in C, G, K) vezt Rab11:mRFP 13.35 2.01 13 Supp Fig 2 E wt 2.25 0.11 13 wt to vezl13.1 3.25E-08 13.1 vezl 1.15 0.09 14 Supp Fig 3 B wt 32 1.3 11 wt to vezl13.1 0.0000137 vezl13.1 18.17 1.8 7 wt + hVezt 29.25 1.9 8 13.1 13.1 vezl + hVezt 18.3 1.1 13 wt to vezl + hVezt 5.27E-08 Supp Fig 4 G wt (anti-p150) 0.737 0.048 40 wt to vezty624 (anti-p150) 0.0224 vezty624 (anti-p150) 0.911 0.057 28 wt (anti-Dync1h1) 0.841 0.089 11 wt to vezty624 (anti-Dync1h1) 0.3055 vezty624 (anti-Dync1h1) 0.968 0.082 13 wt (anti-CytoC) 0.856 0.057 14 wt to vezty624 (anti-CytoC) 0.6339 y624 vezt (anti-CytoC) 0.895 0.059 13