DOI: 10.5061/dryad.pg4f4qs03
title: Data from: Kif1a and intact microtubules maintain synaptic-vesicle populations at ribbon synapses in zebrafish hair cells

description: Sensory hair cells of the inner ear utilize specialized ribbon synapses to
 transmit sensory stimuli to the central nervous system. This transmission
 necessitates rapid and sustained neurotransmitter release, which depends
 on a large pool of synaptic vesicles at the hair-cell presynapse. While
 previous work in neurons has shown that kinesin motor proteins traffic
 synaptic material along microtubules to the presynapse, the mechanisms of
 this process in hair cells remain unclear. Our study demonstrates that the
 kinesin motor protein Kif1a, along with an intact microtubule network, is
 essential for enriching synaptic vesicles at the presynapse in hair cells.
 Through genetic and pharmacological approaches, we disrupt Kif1a function
 and impair microtubule networks in hair cells of the zebrafish
 lateral-line system. These manipulations led to a significant reduction in
 synaptic-vesicle populations at the presynapse in hair cells. Using
 electron microscopy, in vivo calcium imaging, and electrophysiology, we
 show that a diminished supply of synaptic vesicles adversely affects
 ribbon-synapse function. Kif1a mutants exhibit dramatic reductions in
 spontaneous vesicle release and evoked postsynaptic calcium responses.
 Furthermore, kif1a mutants exhibit impaired rheotaxis, a behavior reliant
 on the ability of hair cells in the lateral line to respond to sustained
 flow stimuli. Overall, our results demonstrate that Kif1a-mediated
 microtubule transport is critical to enrich synaptic vesicles at the
 active zone, a process that is vital for proper ribbon-synapse function in
 hair cells.
description: <strong>Nocodazole treatment</strong>
 To destabilize microtubules,
 <em>Tg(myo6b:YFP-Hsa.TUBA)<sup>idc16Tg
 </sup></em>larvae at 5 dpf were incubated in 250 nM nocodazole
 (Sigma-Aldrich, SML1665) for 2 hours. Nocodazole was diluted in E3 media
 for a final concentration of 250 nM nocodazole and 0.1 % DMSO. After
 effective nocodazole treatment <em>YFP-Hsa.TUBA</em> labeling
 was visually disrupted. For controls, larvae were incubated in media
 containing 0.1 % DMSO. After 2 hours, larvae were fixed for
 immunohistochemistry or prepared for LysoTracker labeling (see
 below). <strong>Lysotracker labeling, and
 imaging</strong> After 2 hours of nocodazole
 treatment, 100 nM LysoTracker Red DND-99 (ThermoFisher, L7528) was added
 to the media for 15 min. Larvae were then embedded in 1 % low melt agarose
 prepared in E3 media containing 0.03 % tricaine (Sigma-Aldrich, A5040,
 ethyl 3-aminobenzoate methanesulfonate salt), 100 nM LysoTracker and
 either 250 nM nocodazole or 0.1 % DMSO. A similar labeling approach was
 used for Lysotracker labeling in <em>kif1aa</em> mutants.
 LysoTracker Red DND-99 was used to label
 <em>Tg(myo6b:YFP-Hsa.TUBA)<sup>idc16Tg
 </sup></em>larvae while LysoTracker Green DND-26<em>
 </em>(ThermoFisher, L7526) was used to label
 <em>Tg(myo6b:ctbp2a-TagRFP)<sup>idc11Tg</sup></em>
 larvae. Transgenic larvae were incubated in 100 nM LysoTracker dye in E3
 media for 15 min and mounted in 1 % low melt agarose prepared in E3 media
 containing 0.03 % tricaine and 100 nM Lysotracker dye.
 To image LysoTracker label, samples were imaged live on a Nikon
 A1R upright confocal microscope using a 60x 1 NA water objective lens.
 Denoised images were acquired using NIS Elements AR 5.20.02 with an 0.425
 µm z-interval. Z-stacks of whole or partial neuromasts were acquired in a
 top-down configuration using 488 and 561 nm lasers.
 <strong>Immunohistochemistry </strong>
 Immunohistochemistry was performed on whole larvae. Zebrafish
 larvae were fixed with 4 % paraformaldehyde (Thermo Scientific, 28906) in
 PBS for 3-4 hrs at 4 °C. After fixation samples were washed 5 × 5 min in
 PBS + 0.01 % Tween (PBST), followed by a 5-min wash in
 H<sub>2</sub>O. Larvae were then permeabilized with ice cold
 acetone (at -20 °C) for 5 min. Larvae were then washed again in
 H<sub>2</sub>O for 5 min, followed by a 5 × 5-min washes in
 PBST, and then blocked overnight at 4 °C with PBST containing 2 % goat
 serum, 2 % fish skin gelatin and 1 % bovine serum albumin (BSA). Primary
 antibodies were diluted in PBST containing 1 % BSA. Larvae were incubated
 in primary antibodies overnight at 4 °C. After 5 × 5 min washes in PBST to
 remove the primary antibodies, larvae were incubated in diluted secondary
 antibodies (1:1000) in PBST containing 1 % BSA for 3 hrs at room
 temperature. After 5 × 5 min washes in PBST to remove the secondary
 antibodies, larvae were rinsed in H<sub>2</sub>O and mounted
 in ProLong Gold Antifade (ThermoFisher, P36930).
 <strong>RNA-FISH to detect <em>kif1aa</em> and
 <em>kif1ab</em> mRNA in hair cells</strong>
 To detect mRNA for <em>kif1aa and
 kif1ab</em><strong><em> </em></strong>we
 followed the Molecular Instrument RNA-FISH Zebrafish protocol, Revision
 Number 10 (https://files.molecularinstruments.com/MI-Protocol-RNAFISH-Zebrafish-Rev10.pdf), with a few minor changes to the preparation of fixed whole-mount larvae as follows. For our dehydration steps we dehydrated using the following methanol series: 25, 50, 75, 100, 100 % methanol, with 5 min for each step in the series. To permeabilize we treated larvae with 10 µg/mL proteinase K for 20 min. RNA-FISH probes were designed to bind after the motor domain of zebrafish <em>kif1aa and kif1ab<strong> </strong></em>(Figure 1F, Molecular Instrument Probe lot # RTD364, RTD365, using B2 and B3 amplifiers respectively). <em>Tg(myo6b:Cr.ChR2-EYFP)<sup>ahc1Tg </sup></em>larvae were labeled using these probes; the strong EYFP label is retained after  RNA-FISH and allows for delineation of hair cells within the whole-mount larvae. After the RNA-FISH protocol the larvae were mounted in ProLong Gold Antifade (ThermoFisher, P36930). <strong>Confocal imaging of RNA-FISH and immunolabels</strong> Fixed immunostained and RNA-FISH samples were imaged on an inverted LSM 780 laser-scanning confocal microscope with an Airyscan attachment using Zen Blue 3.4 (Carl Zeiss) and an 63x 1.4 NA Plan Apo oil immersion objective lens. Neuromast and inner ear z-stacks were acquired every 0.15 µm. The Airyscan z-stacks were auto processed in 3D. Experiments were imaged with the same acquisition settings to maintain consistency between comparisons. For presentation in figures, images were further processed using Fiji (RRID:SCR_002285). <strong>Quantification of RNA-FISH and synaptic components</strong> Z-stack image acquisitions from zebrafish confocal images were processed in FIJI. Researchers were blinded to genotype during analyses. Hair-cell numbers were counted manually based on Myo7a, Cr.ChR2-EYFP, YFP-Hsa.TUBA or Acetylated tubulin label. Each channel was background subtracted using the rolling-ball radius method. Then each z-stack was max-intensity projected. A mask was generated by manually outlining the region or interest in the reference channel (ex: hair cells via Myo7a, Acetylated tubulin or <em>Tg(myo6b:Cr.ChR2-EYFP)</em>). This mask was then applied to the z-projection of each synaptic component or RNA-FISH channel.                 We then used automated quantification to quantify puncta using a customized Fiji-based macro. In this macro, each masked image was thresholded using an adaptive thresholding plugin by Qingzong TSENG  (https://sites.google.com/site/qingzongtseng/adaptivethreshold) to generate a binary image of the puncta (presynaptic, postsynaptic, Ca<sub>V</sub>1.3 cluster or RNA-FISH puncta). Individual synaptic or RNA-FISH puncta were then segmented using the particles analysis function in Fiji. A watershed was applied to the particle analysis result to break apart overlapping puncta. After the watershed, the particle analysis was rerun with size and circularity thresholds to generate ROIs and measurements of each punctum. For particle analysis, the minimum size thresholds of 0.025 μm<sup>2 </sup>(Rib b, Ca<sub>V</sub>1.3, <em>kif1aa</em> and <em>kif1ab</em> RNA-FISH particles) and 0.04 μm<sup>2</sup> (Maguk) were applied. A circularity factor of 0.1 was also used for the particle analysis. The new ROIs were applied to the original z-projection to get the average intensity and area of each punctum. To identify paired synaptic components, images were further processed. Here, the overlap and proximity of ROIs from different channels (ex: pre- and post-synaptic puncta) were calculated. ROIs with positive overlap or ROIs within 2 pixels were counted as paired components. The ROIs and synaptic component measurements (average intensity, area) and pairing results were then saved as Fiji ROIs, jpg images, and csv files. <strong>Synaptic vesicle quantification of live and fixed labels</strong> Z-stack image acquisitions from live or fixed confocal images were processed and quantified in FIJI. A minimum of 3-6 hair cells per neuromast analyzed. Hair cells with a clear side view were used for base to apex analyses. For base to apex, two rectangular ROIs with an area of 2.25 x 0.65 µm (L x W), were placed above and below the nucleus of each hair cell, in 1 z-slice. Nuclei were identified based on Acetylated tubulin or YFP-Hsa.TUBA labeling. The mean values were measured in all ROIs. The ratio of the base to apex was measured per hair cell to assess the enrichment of synaptic vesicles. Ratio values for hair cells were then averaged for a single neuromast base to apex fluorescence value. To quantify Lysotracker fluorescence at ribbons, a circular ROI was drawn around the ribbon indicated by Ctbp2a-tagRFP fluorescence, and the mean value was measured in FIJI. In the cristae, Vglut3 label enrichment at the cell base was measured the same manner as for neuromasts, but Vglut3 enrichment was only quantified in tall cells. To quantify the Vglut3 label in the anterior macula, a maximum projection of the stack imaged was generated in FIJI. An ROI was drawn outlining the macula, and the mean values were measured. <strong>Startle behavior</strong> A Zantiks MWP behavioral system (https://zantiks.com)  was used to assess acoustic startle responses in larvae at 5 dpf. The Zantiks system tracks and monitors behavioral responses using an infrared camera at 30 frames per second. During the tracking and stimulation, a Cisco router connected to the Zantiks system was used to relay <em>x</em>, <em>y</em> coordinates of each larva in every frame. A 12-well plate was used for behavioral analyses. Each well was filled with E3 and 1 larva. All fish were acclimated in the plate within the Zantiks chamber in the dark for 15 min before each test. A vibrational stimulus that triggered a maximal proportion of animals startling in control animals without any tracking artifacts (due to the vibration), was used for our strongest stimuli. We used 4 different levels of intensity (1-4, increasing in intensity), with level 4 as the highest intensity stimulus. To deliver the acoustic vibrational stimulus, the solenoid motor in the Zantiks system was set to move by 7.2° (level 4: 4 full steps), 3.6° (level 3: 2 full steps), 1.8° (level 2: 1 full step), 0.9° (level 1: 1/2 step) with a 4 x 4.25 ms motor speed moving in clockwise and anticlockwise movements. For our initial startle assay, each larva was presented with stimuli from intensity levels 1-3, 5 times, with 100 s between trials to avoid habituation. For each animal, the proportion of startle responses out of the 5 trials was plotted. For our habituation and recovery assay, a non-habituating stimulus, followed by a habituating stimulus train, and lastly recovery stimulus was performed. Our non-habituating stimulus was presented 3 times (intensity level 4), with 100 s between trials. This was followed by a habituating train of 30 stimuli (same stimulus intensity), presented with 5 s in between each stimulus. We then presented recovery stimuli once each at 20 s, 40 s, 1 min, and 2 min after the last stimulus in the habituating train. The proportion of startle responses out of the initial 3 non-habituating stimuli and the proportion of responses at each habituation block and recovery stimulus were plotted. To qualify as a startle response, a distance above 4 pixels or ∼1.9 mm was required within 2 frames after stimulus onset. Larvae were excluded from our analysis if no tracking data was recorded. Startle behavior was performed on at least three independent days. <strong>Rheotaxis behavior</strong> A custom microflume was used as the experimental apparatus for rheotaxis behavior. Laminar water flow of a constant velocity was provided by a 6V bow thruster motor (#108-01, Raboesch) inserted into the flume. An Arduino (UNO R3, Osepp) was programmed with custom scripts to coordinate the timing of the flow and video recording. An array of 196 LEDs emitting infrared light (850 nm) provided illumination for video capture through a layer of diffusion material (several Kimwipes sealed in plastic) and the translucent bottom of the flume. A monochromatic high-speed camera (SC1 without IR filter, Edgertronic.com) with a 60 mm manual focus macro lens (Nikon) was used to record behavioral trials at 60 fps. The flume was filled with EM media (28 °C) and the arena placed within the flume. Due to the heat generated by the IR lights, the temperature was monitored and miniature ice packs (2 × 2 cm; −20 °C) were used to maintain a consistent temperature range of 27–29 °C. For each rheotaxis behavior test, individual 6 dpf larval zebrafish were transferred by pipette to the arena within the flume and their swimming activity was monitored for ~10 s to ensure that it exhibited burst-and-glide swimming behavior; larva that did not exhibit normal swimming behavior during the pre-trial period were not included in the analysis. Each larva was genotyped after behavior acquisition and analysis. A total of 43 wild-type siblings and 30 <em>kif1aa </em>mutant larvae were analyzed. Rheotaxis behavior was assessed blind, prior to genotyping. Data was collected from 6 experimental sessions on separate days. Larval fish were tracked using DeepLabCut. In brief, video files were downsampled to 1000 x 1000 px and cropped. A previously created and trained single animal maDLC project was used to annotate seven unique body parts (left and right eyes, swim bladder, four points along the tail) on each larva. Videos were analyzed with the maDLC project, and the detections were assembled into tracklets using the box method. The original videos were overlaid with the newly labeled body parts to check for trackelet accuracy. Misaligned tracklets were manually adjusted. Rheotaxis behavior was annotated and analyzed using a previously created custom Python feature extraction script <em>(SimBA)</em> that defined positive rheotaxis events as when the larvae swam into the oncoming water flow at an angle of 0 degrees ± 45 degrees for at least 100 ms.  Videos processed through DeepLabCut analysis were converted to AVI format using the SimBA video editor function and imported into SimBA as previously described. <strong>Calcium imaging and electrophysiology</strong> Larvae for electrophysiology recordings were either in a <em>Tg(myo6b:Cr.ChR2-EYFP)</em> transgenic background or a nontransgenic background. For calcium imaging either <em>Tg(myo6b:memGCaMP6s)<sup>idc1Tg</sup> </em>or <em>Tg(en.sill,hsp70l:GCaMP6s)</em><em><sup>idc8Tg </sup></em> transgenic larvae were used. To prepare larvae for calcium imaging and electrophysiology, 3-6 dpf larvae were anesthetized in 0.03 % Tricaine-S (SYNCAINE/MS-222, Syndel), pinned to a Sylgard-filled perfusion chamber at the head and tail. Then larvae were paralyzed by injection of 125 µM a-bungarotoxin (Tocris, 2133) into the heart cavity, as previously described (Lukasz & Kindt, 2018). Larvae were then rinsed once in E3 embryo media to remove the tricaine. Next, larvae were rinsed three times with extracellular imaging solution (in mM: 140 NaCl, 2 KCl, 2 CaCl<sub>2</sub>, 1 MgCl<sub>2</sub>, and 10 HEPES, pH 7.3, OSM 310±10) and allowed to recover prior to calcium imaging or electrophysiology. Researchers were blind to genotype during the acquisition. Calcium responses in the hair cells and afferent process were acquired on a Swept-field confocal system built on a Nikon FN1 upright microscope (Bruker) with a 60x 1.0 NA CFI Fluor water-immersion objective. The microscope was equipped with a Rolera EM-C2 EMCCD camera (QImaging), controlled using Prairie view 5.4 (Bruker). GCaMP6s was excited using a 488 nm solid state laser. We used a dual band-pass 488/561 nm filter set (59904-ET, Chroma). For evoked measurements, stimulation was achieved by a fluid jet, which consisted of a pressure clamp (HSPC-1, ALA Scientific) and a glass pipette (pulled and broken to achieve an inner diameter of ~50 µm) filled with extracellular imaging solution. A 500-ms pulse of positive or negative pressure was used to deflect the hair bundles of mechanosensitive hair cells along the anterior-posterior axis of the fish.  For GCaMP6s imaging in hair bundles or at the presynapse the <em>Tg(myo6b:memGCaMP6s)<sup>idc1Tg</sup> </em>line was used. For GCaMP6s imaging in the afferent terminal beneath lateral line hair cell, the <em>Tg(en.sill,hsp70l:GCaMP6s)</em><em><sup>idc8Tg </sup></em> line was used. GCaMP6s measurements were performed on larvae at 4 and 5 dpf. Each neuromast (L1-L4) was stimulated four times with an inter-stimulus interval of ~2 min. To acquire GCaMP6s evoked responses, 5 z-slices (0.5 µm step for mechanosensation, 1.5 µm step for presynaptic and 2.0 µm step for the afferent process) were collected per timepoint for 80 timepoints at a frame rate of 20 ms for a total of ~100 ms per z-stack and a total acquisition time of ~8 sec. Stimulation began at timepoint 31; timing of the stimulus was triggered by an outgoing voltage signal from Prairie view. GCaMP6s z-stacks were average projected, registered, and spatially smoothed with a Gaussian filter (size = 3, sigma = 2) in custom-written MatLab software as described previously (Zhang <em>et al.</em>, 2018). The first 10 timepoints (~1 sec) were removed to reduce the effect of initial photobleaching. Registered average projections analyzed in Fiji to make intensity measurements using the Time Series Analyzer V3 plugin. Here circular ROIs were placed on hair bundles or synaptic sites; average intensity measurements over time were measured for each ROI. GCaMP6s data was excluded in the case of excessive motion artifacts. Presynaptic responses were defined as &gt;20% ∆F/F0. Hair-bundle responses were defined as &gt;20% ∆F/F0. Postsynaptic responses were defined as &gt;5% ∆F/F0 and a minimum duration of 500 ms. Calcium imaging data then plotted in Prism 10 (Graphpad). The first 20 timepoints were averaged to generate an F0 value, and all responses were calculated as ∆F/F0. The Area under the curve (AUC) function of Prism was used to determine the peak value for each response. Responses presented in figures represent average responses within a neuromast. The max ∆F/F0 was compared between sibling and <em>kif1aa</em> mutants. Extracellular postsynaptic current recordings from afferent cell bodies of the posterior lateral-line ganglion (pLLg) of zebrafish at 3-6 dpf were performed. Briefly, borosilicate glass pipettes (Sutter Instruments, BF150-86-10 glass with filament) were pulled with a long taper, with resistances between 5 and 15 MW. The pLLg was visualized using an Olympus BX51WI fixed stage microscope equipped with a LumPlanFl/IR 60x 1.4 NA water dipping objective (N2667800, Olympus). An Axopatch 200B amplifier, a Digidata 1400A data acquisition system, and pClamp 10 software (Molecular Devices, LLC) were used to collect signals. To record spontaneous extracellular currents, afferent cell bodies were recorded using a loose-patch configuration with seal resistances ranging from 20 to 80 MW. Recordings were done in voltage-clamp mode, and signals were sampled at 50 μs/point and filtered at 1 kHz.  The number of spontaneous events from one neuron per min was quantified from a 3-5-min recording window using Igor Pro (Wavemetrics). <strong>Transmission electron microscopy </strong> Larvae were genotyped at 2 dpf using a larval fin clip method to identify <em>kif1aa</em> and wild-type siblings to prepare for TEM. Larval tail DNA was genotyped. At 5 dpf <em>kif1aa</em> and wild-type siblings were fixed in freshly prepared solution containing 1.6 % paraformaldehyde and 2.5 % glutaraldehyde in 0.1 M cacodylate buffer supplemented with 3.4% sucrose and 2 µM CaCl<sub>2</sub> for 2 h at room temperature, followed by a 24-h incubation at 4 °C in a fresh portion of the same fixative. After fixation, larvae were washed with 0.1 M cacodylate buffer with supplements, and post-fixed in 1 % osmium tetroxide for 30 min, and then washed with distilled water. Larvae were, dehydrated in 30 – 100 % ethanol series, which included overnight incubation in 70 % ethanol containing 2 % uranyl acetate, and in propylene oxide, and then embedded in Epon. Transverse serial sections (60-70 nm thin sections) were used to section through neuromasts. Sections were placed on single slot grids coated with carbon and formvar, and then sections were stained with uranyl acetate and lead citrate. All reagents and supplies for TEM were from (Electron Microscopy Sciences). Samples were imaged on a JEOL JEM-2100 electron microscope (JEOL Inc.). Whenever possible, serial sections were used to restrict our analysis to central sections of ribbons adjacent to the plasma membrane and a well-defined postsynaptic density.             To quantify ribbon area, ROIs were drawn in FIJI outlining the electron-dense ribbon, excluding the filamentous “halo” surrounding the ribbon. Vesicles with a diameter of 30–50 nm and adjacent (within 60 nm of the ribbon) to the “halo” were counted as tethered vesicles. Readily releasable vesicles were defined as tethered vesicles between the ribbon and the plasma membrane. To quantify reserve vesicles, we counted vesicles that were not tethered to the ribbon but were within 200 nm of the edge of the ribbon. All distances and perimeters were measured in FIJI.
description: # Kif1a and intact microtubules maintain synaptic-vesicle populations at
 ribbon synapses in zebrafish hair cells
 [https://doi.org/10.5061/dryad.pg4f4qs03](https://doi.org/10.5061/dryad.pg4f4qs03) ## Description of the data and file structure Paper associated with dataset: The Journal of Physiology (2024) [https://doi.org/10.1113/JP286263](https://doi.org/10.1113/JP286263) This dataset includes raw data from the following data collected at the NIH/NIDCD: Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 This dataset uses RNA-FISH to show that kif1aa but not kif1ab mRNA are highly expressed in zebrafish hair cells (Figure 2). This dataset includes live imaging of lysotracker, immunolabeling to stain synaptic vesicles or TEM to visualize individual synaptic vesicles in zebrafish lateral-line hair cells (L1-L4 at 5 dpf) in wild type, kif1aa mutants and after nocodazole treatment (Figure 4,6,7) or immunolabeling of synaptic vesicles in hair cells of the zebrafish inner ear (Figure 5). This data shows that synaptic vesicle enrichment at the hair cell base is reduced in kif1aa mutants or after nocodazole treatment. This dataset using immunolabeling to quantify ribbons synapses in wildtype and kif1aa mutants (Figure 3,8). This data shows that there are fewer synapses per hair cell in kif1aa mutant and each synapse has more calcium channels. In zebrafish, pre- and post-synaptic calcium imaging in the lateral-line, spontaneous spikes from posterior lateral line neurons, and acoustic startle behavior for control and kif1aa zebrafish mutants are also included (Figure 9,10,11). This data shows normal presynaptic responses, but a reduction in post-synaptic calcium responses and spontaneous spike rate, but no change in acoustic startle behavior. Folders included in this submission have the following names: RAWDATA_Figure 1 RAWDATA_Figure 2 RAWDATA_Figure 3 RAWDATA_Figure 4 RAWDATA_Figure 5 RAWDATA_Figure 6 RAWDATA_Figure 7 RAWDATA_Figure 8 RAWDATA_Figure 9 RAWDATA_Figure 10 RAWDATA_Figure 11 ### Files and variables #### File: RAWDATA\_FIgure\_1.zip **Description:**  Summary of Figure 1 folder contents: This folder contains 2 Zeiss Airyscan processed images. There are 2 Zeiss Airyscan processed images that represent the raw data in Figure 1B and C. These files end in .czi. This is data acquired from 2 wildtype neuromasts. These data are fixed samples that label hair cell microtubules, synaptic vesicle and ribbons in neuromasts (D1 or L2) of the posterior lateral line. Zebrafish were fixed and stained at day 5. Information on immunostaining: Larvae were fixed with 4 % paraformaldehyde in PBS for 3.5 hr at 4C. After fixation, larvae were washed 5 x 5 min in 0.1% Tween in PBS (PBST). Prior to block, larvae were permeabilized with acetone. For this permeabilization, larvae were washed for 5 min with H2O. The H2O was removed and replaced with ice-cold acetone and placed at -20C for 5 min, followed by a 5 min H2O wash. The larvae were then washed for 5 x 5 min in PBST. Larvae were then blocked overnight at 4C in blocking solution (2% goat serum, 1% bovine serum albumin, 2% fish skin gelatin in PBST). Larvae were then incubated in primary antibodies in antibody solution (1% bovine serum albumin in PBST) overnight, nutating at 4C. The next day, the larvae were washed for 5 x 5 min in PBST to remove the primary antibodies. Secondary antibodies in antibody solution were added and larvae were incubated for 2 hrs at room temperature. After 5 x 5 min washes min in PBST to remove the secondary antibodies, larvae were rinsed in H2O and mounted in Prolong Gold (ThermoFisher Scientific). The following primary antibodies were used: mouse anti-Acetylated Tubulin (IgG2b) (Sigma T7451; 1:5000); rabbit anti-Vglut3 (Obholzer *et al.*, 2008; 1:1000) and mouse anti-CTBP (IgG2a) (Santa Cruz 55502; 1:1000) With the following secondary antibodies were used at 1:1000: #A-11008, # A-21141, # A-11035 and # A-21136 (ThermoFisher Scientific) Or mouse anti-Acetylated Tubulin (IgG1)(Sigma T7451; 1:5000); mouse anti-rab3a (IgG1) (Synaptic System 107 011; 1:1000); and rabbit anti-Vglut3 (Obholzer *et al.*, 2008; 1:1000) With the following secondary antibodies were used at 1:1000: #A-11008, # A-21141, # A-21123 # A-21070, (ThermoFisher Scientific) Metadata for Zeiss Airyscan acquisition: Fixed zebrafish samples were imaged on an inverted Zeiss LSM 780 laser-scanning confocal microscope with Airyscan (Carl Zeiss AG) using a 63x 1.4 NA oil objective lens and the following lasers lines: 488, 546 and 647. Gain = 800 all laser lines 588x588 or 704x704 pixels 0.185 microns per slice Processed in Zen with an Airyscan processing factor of 6.5 (2D). The 2 Airyscan files generated are as follows: 040824_kif1aa_d5_acetub_488_vglut3_546_CTBP_647_r3_F3_D1_Out.czi Channel 1 = acetylated tubulin; Channel 2 = vglut3; Channel 3 = CTBP 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS1_R1_F2_L2-Airyscan Processing-03.czi Channel 1 = acetylated tubulin; Channel 2 = rab3a; Channel 3 = vglut3 Analysis in FIJI Airyscan images were processed in FIJI to max project 5 slices of each z-stack image. 040824_kif1aa_d5_acetub_488_vglut3_546_CTBP_647_r3_F3_headNM_Out.czi (Channel 2 and 3) 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS1_R1_F2_L2-Airyscan Processing-03.czi (Channel 1) These images correspond to the images in Figure 1 B and C. #### File: RAWDATA\_Figure\_2.zip **Description:**  Summary of Figure 2 folder contents: This folder contains 38 Zeiss Airyscan processed images and one excel file. There are 35 Zeiss Airyscan processed images that represent the quantified raw data in the results, and 3 more Zeiss Airyscan processed images that are used as example images in Figure 2. These files end in .czi. This is data acquired from 18 sibling and 16 kif1aa neuromasts. These data are samples that label hair cells and kif1aa and kif1ab mRNA in neuromasts (L1-L4) of the posterior lateral line and in the medial crista and anterior macula of the inner ear. Zebrafish were fixed and stained at day 5. The data extracted from these files is summarized in the excel file. Information on RNA-FISH: To detect mRNA for kif1aa and kif1ab we followed the Molecular Instrument RNA-FISH Zebrafish protocol, Revision Number 10 ([https://files.molecularinstruments.com/MI-Protocol-RNAFISH-Zebrafish-Rev10.pdf](https://files.molecularinstruments.com/MI-Protocol-RNAFISH-Zebrafish-Rev10.pdf)), with a few minor changes to the preparation of fixed whole-mount larvae as follows. For our dehydration steps we dehydrated using the following methanol series: 25, 50, 75, 100, 100 % methanol, with 5 min for each step in the series. To permeabilize we treated larvae with 10 µg/mL proteinase K for 20 min. RNA-FISH probes were designed to bind after the motor domain of zebrafish kif1aa and kif1ab (Figure 1F, Molecular Instrument Probe lot # RTD364, RTD365, using B2 and B3 amplifiers respectively). Tg(myo6b:Cr.ChR2-EYFP)ahc1Tg larvae were labeled using these probes; the strong EYFP label is retained after RNA-FISH and allows for delineation of hair cells within the whole-mount larvae. After the RNA-FISH protocol the larvae were mounted in ProLong Gold Antifade (ThermoFisher, P36930). Information on RNA-FISH quantification: Z-stack image acquisitions from zebrafish confocal images were processed in FIJI. Researchers were blinded to genotype during analyses. Hair-cell numbers were counted manually based on Myo7a, Cr.ChR2-EYFP, YFP-Hsa.TUBA or Acetylated tubulin label. Each channel was background subtracted using the rolling-ball radius method. Then each z-stack was max-intensity projected. A mask was generated by manually outlining the region or interest in the reference channel (ex: hair cells via Myo7a, Acetylated tubulin or Tg(myo6b:Cr.ChR2-EYFP)). This mask was then applied to the z-projection of each synaptic component or RNA-FISH channel.                  We then used automated quantification to quantify puncta using a customized Fiji-based macro. In this macro, each masked image was thresholded using an adaptive thresholding plugin by Qingzong TSENG  ([https://sites.google.com/site/qingzongtseng/adaptivethreshold](https://sites.google.com/site/qingzongtseng/adaptivethreshold)) to generate a binary image of the puncta (presynaptic, postsynaptic, CaV1.3 cluster or RNA-FISH puncta). Individual synaptic or RNA-FISH puncta were then segmented using the particles analysis function in Fiji. A watershed was applied to the particle analysis result to break apart overlapping puncta. After the watershed, the particle analysis was rerun with size and circularity thresholds to generate ROIs and measurements of each punctum. For particle analysis, the minimum size thresholds of 0.025 μm2 (Rib b, CaV1.3, kif1aa and kif1ab RNA-FISH particles) and 0.04 μm2 (Maguk) were applied. A circularity factor of 0.1 was also used for the particle analysis. The new ROIs were applied to the original z-projection to get the average intensity and area of each punctum. To identify paired synaptic components, images were further processed. Here, the overlap and proximity of ROIs from different channels (ex: pre- and post-synaptic puncta) were calculated. ROIs with positive overlap or ROIs within 2 pixels were counted as paired components. The ROIs and synaptic component measurements (average intensity, area) and pairing results were then saved as Fiji ROIs, jpg images, and csv files. Metadata for Zeiss Airyscan acquisition: Fixed zebrafish samples were imaged on an inverted Zeiss LSM 780 laser-scanning confocal microscope with Airyscan (Carl Zeiss AG) using a 63x 1.4 NA oil objective lens and the following lasers lines: 488, 543 and 633. Gain = 800 all laser lines 512x512 pixels 0.18 microns per slice Processed in Zen with an Airyscan processing factor of 3D. The 38 Zeiss Airyscan files generated are as follows: Channel 1: ChR2-EYFP             Channel 2: kif1aa         Channel 3: kif1ab 022824_HCR_kif1aa_ab_from_040523_S2_CS2_r1_F1_L1_Out.czi 022824_HCR_kif1aa_ab_from_040523_S2_CS2_r1_F1_L2_Out.czi 022824_HCR_kif1aa_ab_from_040523_S2_CS2_r1_F2_L1_Out.czi 022824_HCR_kif1aa_ab_from_040523_S2_CS2_r1_F2_L2_Out.czi 022824_HCR_kif1aa_ab_from_040523_S2_CS2_r1_F2_L3_Out.czi 022824_HCR_kif1aa_ab_from_040523_S2_CS2_r1_F3_L1_Out.czi 022824_HCR_kif1aa_ab_from_040523_S2_CS2_r1_F3_L2_Out.czi 022824_HCR_kif1aa_ab_from_040523_S2_CS2_r1_F3_L3_Out.czi 022824_HCR_kif1aa_ab_from_040523_S2_CS2_r1_F4_L1_Out.czi 022824_HCR_kif1aa_ab_from_040523_S2_CS2_r1_F4_L2_Out.czi 022824_HCR_kif1aa_ab_from_040523_S2_CS2_r1_F5_L1_Out.czi 022824_HCR_kif1aa_ab_from_040523_S2_CS2_r1_F5_L2_Out.czi 022824_HCR_kif1aa_ab_from_040523_S2_CS2_r1_F6_L1_Out.czi 022824_HCR_kif1aa_ab_from_040523_S2_CS2_r1_F6_L3_Out.czi 031824_HCR_kif1aa_ab_from_040523_S2_CS2_r2_F7_L3_Out.czi 031824_HCR_kif1aa_ab_from_040523_S2_CS2_r2_F7_L4_Out.czi 031824_HCR_kif1aa_ab_from_040523_S2_CS2_r2_F8_L1_Out.czi 031824_HCR_kif1aa_ab_from_040523_S2_CS2_r2_F8_L2_Out.czi 031824_HCR_kif1aa_ab_from_040523_S2_CS2_r2_F8_L4_Out.czi 040824_HCR_kif1aa_ab_from_040523_S1_CS1_r1_F1_L3_Out.czi 040824_HCR_kif1aa_ab_from_040523_S1_CS1_r1_F1_L4_Out.czi 040824_HCR_kif1aa_ab_from_040523_S2_CS2_r2_F9_L1_Out.czi 040824_HCR_kif1aa_ab_from_040523_S2_CS2_r2_F9_L2_Out.czi 040824_HCR_kif1aa_ab_from_040523_S2_CS2_r2_F9_L3_Out.czi 040824_HCR_kif1aa_ab_from_040523_S2_CS2_r2_F9_L4_Out.czi 040824_HCR_kif1aa_ab_from_040523_S2_CS2_r2_F10_L1_Out.czi 040824_HCR_kif1aa_ab_from_040523_S2_CS2_r2_F10_L4_Out.czi 040824_HCR_kif1aa_ab_from_040523_S2_CS2_r2_F11_L1_Out.czi 040824_HCR_kif1aa_ab_from_040523_S2_CS2_r2_F11_L2_Out.czi 040824_HCR_kif1aa_ab_from_040523_S2_CS2_r2_F11_L3_Out.czi 040824_HCR_kif1aa_ab_from_040523_S2_CS2_r2_F11_L4_Out.czi 040824_HCR_kif1aa_ab_from_040523_S2_CS2_r2_F12_L1_Out.czi 040824_HCR_kif1aa_ab_from_040523_S2_CS2_r2_F12_L2_Out.czi 040824_HCR_kif1aa_ab_from_040523_S2_CS2_r2_F12_L3_Out.czi 040824_HCR_kif1aa_ab_from_040523_S2_CS2_r2_F12_L4_Out.czi 031824_HCR_kif1aa_ab_from_040523_S2_CS2_r2_F8_AM_Out.czi 031824_HCR_kif1aa_ab_from_040523_S2_CS2_r2_F8_MC_Out.czi 042224_HCR_kif1aa_ab_from_040523_S1_CS2_r1_F4_PM_Out.czi After imaging genotyping was done using standard PCR followed by a Bsl1 restriction enzyme digest. *Kif1aa* genotyping primers used were as follows: *kif1aa*_FWD 5’-AACACCAAGCTGACCAGTGC-3’ and *kif1aa*_REV 5’-TGCGGTCCTAGGCTTACAAT-3’. *Kif1aa* mutants were compared to either heterozygous and wild-type siblings (sibling controls) or to wild-type siblings. The excel file “kif1aa_HCR results_dryad.xlsx” contains the HCR puncta counting results derived from the *CompleteSynpaseCounter2Dv5.2.ijm* Fiji macro ([https://github.com/KindtLab/CompleteSynpaseCounter2D](https://github.com/KindtLab/CompleteSynpaseCounter2D)). This data has one sheet separated into two sections by genotype and includes the filename and kif1aa mRNA puncta per neuromast. This data is explained in the results section. The following files: HCR_CS1_r1_f5_nm1_Out.czi 031824_HCR_kif1aa_ab_from_040523_S2_CS2_r2_F8_AM_Out.czi 031824_HCR_kif1aa_ab_from_040523_S2_CS2_r2_F8_MC_Out.czi 042224_HCR_kif1aa_ab_from_040523_S1_CS2_r1_F4_PM_Out.czi Were processed in FIJI (partial max projections) and used as example images in the figure. #### File: RAWDATA\_Figure\_3.zip **Description:**  Summary of folder contents: This folder contains 33 Zeiss Airyscan processed images and 1 excel file. The 33 Zeiss Airyscan processed images that represent the raw data in Figure 3A-F. These files end in .czi. This is data acquired from wild type and kif1aa mutant neuromasts. These data are fixed samples that label hair cells (Myosin7a), ribbons (Ribeyeb) and postsynapses (Maguk) in neuromasts (L1-L4) of the posterior lateral line. Zebrafish were fixed and stained at day 5. The data extracted from these files is summarized in the .xlsx excel files. Information on immunostaining: Whole larvae were fixed with paraformaldehyde (PFA 4%; Thermoscientific; 28906) in PBS at 4°C for 3.5 hr. Antibody solutions were prepared with PBS + 0.1% Tween (PBST). After fixation, larvae were washed 4 × 5 min in PBST. Prior to block, larvae were permeabilized with acetone. For this permeabilization, larvae were washed for 5 min with H2O in glass vials. The H2O was removed and replaced with ice-cold acetone and larvae placed at −20°C for 5 min, followed by a 5 min H2O wash. The larvae were then washed for 4 × 5 min in PBST. Larvae then were blocked overnight at 4°C in blocking solution (2% goat serum, 1% bovine serum albumin, 2% fish skin gelatin in PBST). After block, larvae were incubated in primary antibodies in antibody solution (1% bovine serum albumin in PBST) overnight, nutating at 4°C. The next day, the larvae were washed for 4 × 5 min in PBST to remove the primary antibodies. Secondary antibodies in antibody solution were added, and larvae were incubated for 2 hrs at room temperature, with minimal exposure to light. Secondary antibodies were removed by washing with PBST for 4 × 5 min. Larvae were mounted on glass slides with Prolong Gold (ThermoFisher Scientific) using No. 1.5 coverslips.  The following primary antibodies were used: rabbit anti-Myosin7a (Proteus 25-6790; 1:1000), mouse anti-Ribeyeb (IgG2a) (Sheets et al., 2011); 1:10,000) and mouse anti-Pan Maguk (IgG1) (Millipore MABN7; 1:500). With the following secondary antibodies at 1:1000: # A-11008, # A-21143, # A-21240 (ThermoFisher Scientific).  Metadata for Zeiss Airyscan acquisition: Fixed zebrafish samples were imaged on an inverted Zeiss LSM 780 laser-scanning confocal microscope with Airyscan (Carl Zeiss AG Zen Black) using a 63x 1.4 NA oil objective lens and the following lasers lines: 488, 543 and 633.\ Laser powers: 0.1% (Myosin7a) for 488 and 0.3% (Ribeyeb) for 543 and 0.2% (Maguk) for 633 Gain = 800 all laser lines Zoom: 4.5x Pixel size 0.04 um 704x704 0.185 microns per slice Autoprocessed in 2D in Zen Black Channel 1 (488)= Myosin7a, Channel 2 (543)= Ribeyeb, Channel 3 (647) = Maguk The 27 Zeiss Airyscan files generated are as follows: Wild type: 021221_kif1aa_F1_L1-Airyscan Processing-01.czi 021221_kif1aa_F1_L2-Airyscan Processing-02.czi 021221_kif1aa_F2_L1-Airyscan Processing-03.czi 021221_kif1aa_F2_L2-Airyscan Processing-04.czi 021221_kif1aa_F3_L1-Airyscan Processing-05.czi 021221_kif1aa_F3_L3-Airyscan Processing-06.czi 021221_kif1aa_F6_L2-Airyscan Processing-11.czi 021221_kif1aa_F6_L3-Airyscan Processing-12.czi 021221_kif1aa_F8_L2-Airyscan Processing-16.czi 021221_kif1aa_F8_L3-Airyscan Processing-17.czi 021221_kif1aa_F10_L2-Airyscan Processing-21.czi 021221_kif1aa_F10_L3-Airyscan Processing-01.czi 021221_kif1aa_F11_L1-Airyscan Processing-01.czi 021221_kif1aa_F11_L3-Airyscan Processing-02.czi 021221_kif1aa_F12_L1-Airyscan Processing-03.czi 021221_kif1aa_F12_L2-Airyscan Processing-04.czi Kif1aa 021221_kif1aa_F4_L1-Airyscan Processing-07.czi 021221_kif1aa_F4_L2-Airyscan Processing-08.czi 021221_kif1aa_F5_L1-Airyscan Processing-09.czi 021221_kif1aa_F5_L2-Airyscan Processing-10.czi 021221_kif1aa_F7_L1-Airyscan Processing-13.czi 021221_kif1aa_F7_L2-Airyscan Processing-14.czi 021221_kif1aa_F7_L3-Airyscan Processing-15.czi 021221_kif1aa_F9_L1-Airyscan Processing-18.czi 021221_kif1aa_F9_L3-Airyscan Processing-19.czi 021221_kif1aa_F9_L4-Airyscan Processing-20.czi 021221_kif1aa_F13_L1-Airyscan Processing-05.czi 021221_kif1aa_F13_L2-Airyscan Processing-06.czi 021221_kif1aa_F14_L1-Airyscan Processing-07.czi 021221_kif1aa_F14_L2-Airyscan Processing-08.czi 021221_kif1aa_F14_L3-Airyscan Processing-09.czi 021221_kif1aa_F15_L2-Airyscan Processing-10.czi 021221_kif1aa_F15_L3-Airyscan Processing-11.czi After imaging genotyping was done using standard PCR followed by a Bsl1 restriction enzyme digest. *Kif1aa* genotyping primers used were as follows: *kif1aa*_FWD 5’-AACACCAAGCTGACCAGTGC-3’ and *kif1aa*_REV 5’-TGCGGTCCTAGGCTTACAAT-3’. *Kif1aa* mutants were compared to heterozygous and wild-type siblings (sibling controls). The [https://github.com/KindtLab/CompleteSynpaseCounter5.2](https://github.com/KindtLab/CompleteSynpaseCounter5.2) Fiji macro script was used to batch process the .czi images in the folder and* *to generate the data presented in Figure 3C-F. Hair cells were counted manually. The 021221_kif1aa_CSC5.2results_dryad.xlsx file contain the synapse quantification results derived from the CompleteSynpaseCounter2Dv5.2. 021221_kif1aa_CSC5.2results_dryad.xlsx contains a data table called synapses analysis that include the filename, genotype, hair cells per neuromast, Synapses per hair cell, area of paired ribbons, area of paired postsynpases. The following files: 021221_kif1aa_F2_L1-Airyscan Processing-03.czi 021221_kif1aa_F5_L2-Airyscan Processing-10.czi Were processed in FIJI (partial max projections) and used as example images in the Figure 3A-B. #### File: RAWDATA\_Figure\_4.zip **Description:**  Summary of Figure 4 folder contents: This folder contains 4 subfolders containing 20 NIS-Elements denoised images and 63 Zeiss Airyscan processed images, and one excel file. There are 20 NIS-Elements denoised images that represent the live raw data in Figure 4. These files end in .nd2. This is data acquired from 10 sibling and 10 kif1aa neuromasts (Figure 4A-B, C-D). There are 63 Zeiss Airyscan processed images that represent the fixed raw data in Figure 4. These files end in .czi. This is data acquired from 31 sibling and 32 kif1aa neuromasts (Figure 4E-F, G-L). These data are both fixed and live samples that label hair cells, ribbons and synaptic vesicles in neuromasts (L1-L4) of the posterior lateral line. Zebrafish were fixed and stained at day 5. The data extracted from these files is summarized in the excel file. Information on live dye: Larvae were incubated for 15 min in 100nM Lysotracker Green or Red and then embedded in 1% low-melt agarose prepared in E3 media containing 0.03% tricaine and 100nM Lysotracker. Information on immunostaining: Larvae were fixed with 4 % paraformaldehyde in PBS for 3.5 hr at 4C. After fixation, larvae were washed 5 x 5 min in 0.1% Tween in PBS (PBST). Prior to block, larvae were permeabilized with acetone. For this permeabilization, larvae were washed for 5 min with H2O. The H2O was removed and replaced with ice-cold acetone and placed at -20C for 5 min, followed by a 5 min H2O wash. The larvae were then washed for 5 x 5 min in PBST. Larvae were then blocked overnight at 4C in blocking solution (2% goat serum, 1% bovine serum albumin, 2% fish skin gelatin in PBST). Larvae were then incubated in primary antibodies in antibody solution (1% bovine serum albumin in PBST) overnight, nutating at 4C. The next day, the larvae were washed for 5 x 5 min in PBST to remove the primary antibodies. Secondary antibodies in antibody solution were added and larvae were incubated for 2 hrs at room temperature. After 5 x 5 min washes in PBST to remove the secondary antibodies, larvae were rinsed in H2O and mounted in Prolong Gold (ThermoFisher Scientific). The following primary antibodies were used: mouse anti-acetylated tubulin (IgG2b) (Sigma Aldrich T7451; 1:5000); chicken anti-GFP (Thermofisher A10262); rabbit anti-CSP (Millipore AB1576; 1:1000), mouse anti-CTBP (IgG2a) (Santa Cruz Biotechnology SC-55502; 1:1000); mouse anti-Ribeye b (IgG2a) (Sheets et al., 2011); mouse anti-Rab3 (IgG1) (Synaptic Systems 107011; 1:1000), and rabbit anti-Vglut3 c-terminus (Obholzer *et al.*, 2008; 1:1000). With the following secondary antibodies were used at 1:1000: # A-11008, # A-21137, # A-21240 (ThermoFisher Scientific). Metadata for Zeiss Airyscan acquisition: Fixed zebrafish samples were imaged on an inverted Zeiss LSM 780 laser-scanning confocal microscope with Airyscan (Carl Zeiss AG) using a 63x 1.4 NA oil objective lens and the following lasers lines: 488, 543 and 633. Gain = 800 all laser lines 672x672 pixels 0.18 microns per slice Processed in Zen with an Airyscan processing factor of 6.5 (2D). Metadata for Nikon A1R acquisition: Live zebrafish samples were imaged on a Nikon A1R upright confocal microscope using a 60x 1 NA water objective lens and the following lasers lines: 488 and 561. Denoised images acquired using NIS Elements AR 5.20.02 1024x512 pixels 0.5 microns per slice The 20 NIS-Elements and 63 Zeiss Airyscan files generated are as follows: Channel 1 = Lysotracker; Channel 2 = DIC Siblings: F1_L1_102821_zoom6_004.nd2 F1_L2_102821_zoom6_003.nd2 F3_L1_110321_zoom6_005.nd2 F3_L2_110321_zoom6_006.nd2 F3_L4_110321_zoom6_009.nd2 F4_L1_110321_zoom6_010.nd2 F4_L2_110321_zoom6_011.nd2 F4_L3_110321_zoom6_012.nd2 F5_L1_110321_zoom6_014.nd2 F5_L2_110321_zoom6_015.nd2 *kif1aa* mutants: F3_L1_102821_zoom6_010.nd2 F3_L2_102821_zoom6_012.nd2 F3_L3_102821_zoom6_013.nd2 F3_L4_102821_zoom6_014.nd2 F4_L1_102821_zoom6_015.nd2 F4_L2_102821_zoom6_017.nd2 F4_L3_102821_zoom6_018.nd2 F6_L1_102821_zoom6_026.nd2 F6_L2_102821_zoom6_027.nd2 F6_L4_102821_zoom6_030.nd2 Channel 1 = yfp-tubulin; Channel 2 = Vglut3; Channel 3 = Ribeye Siblings: p3_CS1_r1_f1_L2_kif1aa_d5_072921_Out.czi p3_CS1_r1_f1_L3_kif1aa_d5_072921_Out.czi p3_CS1_r1_f4_L1_kif1aa_d5_072921_Out.czi p3_CS1_r2_f1_L1_kif1aa_d5_072921_Out.czi p3_CS1_r2_f1_L2_kif1aa_d5_072921_Out.czi p3_CS2_r1_f1_L2_kif1aa_d5_080921_Out.czi p3_CS2_r1_f1_L3_kif1aa_d5_080921_Out.czi p3_CS2_r1_f3_L2_kif1aa_d5_080921_Out.czi p3_CS2_r1_f3_L3_kif1aa_d5_080921_Out.czi p3_CS2_r1_f4_L2_kif1aa_d5_080921_Out.czi p3_CS2_r1_f4_L3_kif1aa_d5_080921_Out.czi p3_CS2_r1_f4_L4_kif1aa_d5_080921_Out.czi Kif1aa mutants: p1_CS1_r1_f3_L2_kif1aa_d5_082521_Out.czi p1_CS1_r1_f3_L3_kif1aa_d5_082521_Out.czi p1_CS1_r1_f3_L4_kif1aa_d5_082521_Out.czi p3_CS1_r1_f3_L1_kif1aa_d5_072921_Out.czi p3_CS1_r1_f7_L1_kif1aa_d5_072921_Out.czi p3_CS1_r1_f7_L2_kif1aa_d5_072921_Out.czi p3_CS1_r1_f7_L3_kif1aa_d5_072921_Out.czi p3_CS1_r2_f6_L1_kif1aa_d5_080621_Out.czi p3_CS1_r2_f6_L2_kif1aa_d5_080621_Out.czi p3_CS1_r2_f6_L3_kif1aa_d5_080621_Out.czi p3_CS2_r2_f2_L1_kif1aa_d5_081021_Out.czi p3_CS2_r2_f2_L2_kif1aa_d5_081021_Out.czi p3_CS2_r2_f2_L3_kif1aa_d5_081021_Out.czi Channel 1 = acetylated tubulin; Channel 2 = CSP; Channel 3 = CTBP, rab3a Siblings: 040824_kif1aa_d5_acetub_488_CSP_546_CTBP_rab3a_r1_F3_L2_Out.czi 040824_kif1aa_d5_acetub_488_CSP_546_CTBP_rab3a_r1_F3_L3_Out.czi 040824_kif1aa_d5_acetub_488_CSP_546_CTBP_rab3a_r1_F3_L4_Out.czi 040824_kif1aa_d5_acetub_488_CSP_546_CTBP_rab3a_r1_F4_L2_Out.czi 040824_kif1aa_d5_acetub_488_CSP_546_CTBP_rab3a_r1_F4_L3_Out.czi 040824_kif1aa_d5_acetub_488_CSP_546_CTBP_rab3a_r2_F8_L2_Out.czi 040824_kif1aa_d5_acetub_488_CSP_546_CTBP_rab3a_r2_F8_L3_Out.czi 040824_kif1aa_d5_acetub_488_CSP_546_CTBP_rab3a_r2_F9_L3_Out.czi 040824_kif1aa_d5_acetub_488_CSP_546_CTBP_rab3a_r2_F16_L2_Out.czi 040824_kif1aa_d5_acetub_488_CSP_546_CTBP_rab3a_r2_F16_L3_Out.czi Kif1aa mutants: 040824_kif1aa_d5_acetub_488_CSP_546_CTBP_rab3a_r1_F2_L2_Out.czi 040824_kif1aa_d5_acetub_488_CSP_546_CTBP_rab3a_r1_F2_L3_Out.czi 040824_kif1aa_d5_acetub_488_CSP_546_CTBP_rab3a_r1_F5_L3_Out.czi 040824_kif1aa_d5_acetub_488_CSP_546_CTBP_rab3a_r1_F6_L3_Out.czi 040824_kif1aa_d5_acetub_488_CSP_546_CTBP_rab3a_r1_F7_L2_Out-1.czi 040824_kif1aa_d5_acetub_488_CSP_546_CTBP_rab3a_r1_F7_L4-1_Out.czi 040824_kif1aa_d5_acetub_488_CSP_546_CTBP_rab3a_r2_F11_L2_Out.czi 040824_kif1aa_d5_acetub_488_CSP_546_CTBP_rab3a_r2_F12_L2_Out.czi 040824_kif1aa_d5_acetub_488_CSP_546_CTBP_rab3a_r2_F13_L2_Out.czi 040824_kif1aa_d5_acetub_488_CSP_546_CTBP_rab3a_r2_F14_L2_Out.czi Channel 1 = Acetylated tubulin; Channel 2 = Rab3a; Channel 3 = Vglut3 Siblings: 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS1_R1_F5_L1-Airyscan Processing-08.czi 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS1_R1_F5_L2-Airyscan Processing-09.czi 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS1_R1_F5_L3-Airyscan Processing-10.czi 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS1_R2_F6_L1-Airyscan Processing-11.czi 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS1_R2_F6_L2-Airyscan Processing-12.czi 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS1_R2_F11_L1-Airyscan Processing-10.czi 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS1_R2_F11_L2-Airyscan Processing-09.czi 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS1_R2_F11_L4-Airyscan Processing-08.czi 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS2_R1_F4_L2-Airyscan Processing-02.czi Kif1aa mutants: 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS1_R1_F2_L1-Airyscan Processing-02.czi 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS1_R1_F2_L2-Airyscan Processing-03.czi 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS1_R1_F2_L3-Airyscan Processing-04.czi 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS1_R1_F2_L4-Airyscan Processing-05.czi 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS1_R2_F9_L2-Airyscan Processing-14.czi 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS1_R2_F9_L3-Airyscan Processing-15.czi 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS1_R2_F9_L4-Airyscan Processing-16.czi 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS2_R1_F1_L1-Airyscan Processing-13.czi 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS2_R1_F1_L2-Airyscan Processing-12.czi After imaging genotyping was done using standard PCR followed by a Bsl1 restriction enzyme digest. *Kif1aa* genotyping primers used were as follows: *kif1aa*_FWD 5’-AACACCAAGCTGACCAGTGC-3’ and *kif1aa*_REV 5’-TGCGGTCCTAGGCTTACAAT-3’. *Kif1aa* mutants were compared to either heterozygous and wild-type siblings (sibling controls) or to wild-type siblings. Zeiss Airyscan and Nikon NIS-Elements images were quantified in Fiji to measure base:apex fluorescence in hair cells. For this analysis ROIs of with an area of 2.25 x 0.65 μm (L x W) were used. The excel file “kif1aa_lyso and immuno neuromast results_dryad.xlsx” contains four sheets corresponding to each dye or stain (lysotracker, vglut3, rab3aa, csp), and each with data extracted from the raw data files (filename, genotype, and base:apex fluorescence ratio). The following files: 040824_kif1aa_d5_acetub_488_CSP_546_CTBP_rab3a_r1_F2_L3_Out.czi 040824_kif1aa_d5_acetub_488_CSP_546_CTBP_rab3a_r1_F3_L4_Out.czi 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS1_R1_F2_L1-Airyscan Processing-02.czi 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS1_R1_F5_L2-Airyscan Processing-09.czi F1_L1_102821_zoom6_004.nd2 F4_L3_102821_zoom6_018.nd2 p3_CS1_r2_f1_L2_kif1aa_d5_072921_Out.czi p3_CS1_r2_f6_L1_kif1aa_d5_080621_Out.czi Were processed in FIJI (partial max projections) and used as example images in the figure. #### File: RAWDATA\_Figure\_5\_.zip **Description:**  Summary of Figure 5 folder contents: This folder contains 29 Zeiss Airyscan processed images and one excel file. There are 29 Zeiss Airyscan processed images that represent the raw data in Figure 5. These files end in .czi. This data is contained in 2 subfolders, according to genotype (sibling or kif1aa) and was acquired from 8 sibling cristae, 6 sibling maculae, 9 *kif1aa-/- *mutant cristae, and 6 *kif1aa-/-* mutant maculae. These data are fixed samples that label hair cells and synaptic vesicles in inner ear hair cells. Zebrafish were fixed and stained at day 5. The data extracted from these files is summarized in the excel file. Information on immunostaining: Larvae were fixed with 4 % paraformaldehyde in PBS for 3.5 hr at 4C. After fixation, larvae were washed 5 x 5 min in 0.1% Tween in PBS (PBST). Prior to block, larvae were permeabilized with acetone. For this permeabilization, larvae were washed for 5 min with H2O. The H2O was removed and replaced with ice-cold acetone and placed at -20C for 5 min, followed by a 5 min H2O wash. The larvae were then washed for 5 x 5 min in PBST. Larvae were then blocked overnight at 4C in blocking solution (2% goat serum, 1% bovine serum albumin, 2% fish skin gelatin in PBST). Larvae were then incubated in primary antibodies in antibody solution (1% bovine serum albumin in PBST) overnight, nutating at 4C. The next day, the larvae were washed for 5 x 5 min in PBST to remove the primary antibodies. Secondary antibodies in antibody solution were added and larvae were incubated for 2 hrs at room temperature. After 5 x 5 min washes in PBST to remove the secondary antibodies, larvae were rinsed in H2O and mounted in Prolong Gold (ThermoFisher Scientific). The following primary antibodies were used: mouse anti-acetylated tubulin (IgG2b) (Sigma Aldrich T7451; 1:5000), mouse anti-Rab3a (Synaptic systems 107 011; 1:1000), and rabbit anti-Vglut3 c-terminus (Obholzer *et al.*, 2008; 1:1000). Or Chicken anti-GFP (Millipore AB1576) and rabbit anti-Vglut3 c-terminus (Obholzer *et al.*, 2008; 1:1000).   With the following secondary antibodies were used at 1:1000: # A-21131, # A-21127, # A-21071 (ThermoFisher Scientific). Or With the following secondary antibodies were used at 1:1000: # A-11039, #**A-21428** (ThermoFisher Scientific). Metadata for Zeiss Airyscan acquisition: Fixed zebrafish samples were imaged on an inverted Zeiss LSM 780 laser-scanning confocal microscope with Airyscan (Carl Zeiss AG) using a 63x 1.4 NA oil objective lens and the following lasers lines: 488 and 543 and 647. Gain = 800 all laser lines 800x800 to 1000x1000 pixels 0.18 microns per slice (Medial Crista); 0.20 microns per slice (Anterior Macula) Processed in Zen with an Airyscan processing factor of 6.5 (2D). The 29 Zeiss Airyscan files generated are as follows: Channel 1 = YFP-tubulin or Acetylated tubulin; Channel 2 = Vglut3 or rab3a; Channel 3: null or Vglut3 Siblings: 011824_kif1aa_YFPtub_vglut3_d5_CS1_r1_F1_AM_Out.czi 011824_kif1aa_YFPtub_vglut3_d5_CS1_r1_F1_MC_Out.czi 011824_kif1aa_YFPtub_vglut3_d5_CS1_r1_F2_MC_Out.czi 011824_kif1aa_YFPtub_vglut3_d5_CS1_r1_F4_AM_Out.czi 011824_kif1aa_YFPtub_vglut3_d5_CS1_r1_F4_MC_Out.czi 011824_kif1aa_YFPtub_vglut3_d5_CS1_r2_F10_AM_lf_Out.czi 011824_kif1aa_YFPtub_vglut3_d5_CS1_r2_F10_AM_rt_Out.czi 011824_kif1aa_YFPtub_vglut3_d5_CS1_r2_F10_MC_rt_Out.czi 011824_kif1aa_YFPtub_vglut3_d5_CS1_r2_F11_AM_lf_Out.czi 011824_kif1aa_YFPtub_vglut3_d5_CS1_r2_F11_AM_rt_Out.czi 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS2_R1_F4_MC_Out.czi 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS2_R1_F8_MC_Out.czi 071624_kif1aa\_ YFPtub_vglut3_d5_CS2_r2_F3_MC_lf_Out.czi 071624_kif1aa\_ YFPtub_vglut3_d5_CS2_r2_F7_MC_rt_Out.czi *kif1aa* mutants: 011824_kif1aa_YFPtub_vglut3_d5_CS1_r1_F3_AM\_ Out.czi 011824_kif1aa_YFPtub_vglut3_d5_CS1_r1_F3_MC_Out.czi 011824_kif1aa_YFPtub_vglut3_d5_CS1_r1_F9_MC_Out.czi 011824_kif1aa_YFPtub_vglut3_d5_CS1_r2_F9_AM_lf_Out.czi 011824_kif1aa_YFPtub_vglut3_d5_CS1_r2_F9_AM_rt2_Out.czi 011824_kif1aa_YFPtub_vglut3_d5_CS1_r2_F9_MC_rt_Out.czi 011824_kif1aa_YFPtub_vglut3_d5_CS1_r2_F14_MC_rt_Out.czi 011824_kif1aa_YFPtub_vglut3_d5_CS1_r2_F15_AM_rt_Out.czi 011824_kif1aa_YFPtub_vglut3_d5_CS1_r2_F15_MC_rt_Out.czi 071624_kif1aa_YFPtub_vglut3_d5_CS2_r2_F1_AM_rt_Out.czi 071624_kif1aa_YFPtub_vglut3_d5_CS2_r2_F1_MC_Out.czi 071624_kif1aa_YFPtub_vglut3_d5_CS2_r2_F4_MC_Out.czi 071624_kif1aa_YFPtub_vglut3_d5_CS2_r2_F5_AM_rt_Out.czi 071624_kif1aa_YFPtub_vglut3_d5_CS2_r2_F5_MC_rt_Out.czi 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS2_R1_F2_MC_Out.czi After imaging genotyping was done using standard PCR followed by a Bsl1 restriction enzyme digest. *Kif1aa* genotyping primers used were as follows: *kif1aa*_FWD 5’-AACACCAAGCTGACCAGTGC-3’ and *kif1aa*_REV 5’-TGCGGTCCTAGGCTTACAAT-3’. *Kif1aa* mutants were compared to heterozygous and wild-type siblings (sibling controls). Zeiss Airyscan images were quantified in Fiji to measure base:apex fluorescence Vglut3 label in tall  hair cells in the medial crista. For this analysis ROIs of with an area of 2.25 x 0.65 μm (L x W) were used. In addition, to quantify the Vglut3 label in the anterior macula, a maximum projection of the stack imaged was generated in FIJI. An ROI was drawn outlining the macula, and the mean intensity values were measured. The excel file “kif1aa_inner ear vglut3 results_dryad.xlsx” contains two sheets separated by genotype (kif1aa mutants, siblings) and each contain data extracted from the raw data. They are separated by inner ear structure (anterior macula, medial crista) and contain the filename and the mean intensity Vglut3 measurements for the anterior macula and the base to apex Vglut3 intensity for the medial crista. The following files: 011824_kif1aa_YFPtub_vglut3_d5_CS1_r1_F3_AM_Out.czi 011824_kif1aa_YFPtub_vglut3_d5_CS1_r2_F11_AM_rt_Out.czi 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS2_R1_F2_MC_Out.czi 060221_kif1aa_acetub_rab3_vglut3_Slide1_CS2_R1_F4_MC_Out.czi Were processed in FIJI (partial max projections) and used as example images in the figure. #### File: RAWDATA\_Figure\_6.zip **Description:**  Summary of Figure 6 folder contents: This folder contains 16 Zeiss Airyscan processed images, 20 Nikon NIS-Elements images, and one excel file. There are 16 Zeiss Airyscan processed images and 20 Nikon NIS-Elements images that represent the raw data in Figure 6. These files end in .czi or .nd2. This is data acquired from DMSO-treated or nocodazole-treated neuromasts (Figure 6A-D). These data are both fixed and live samples that label hair cells and synaptic vesicles in neuromasts (L1-L4) of the posterior lateral line. The data is contained in 2 subfolders, live imaging (Lysotracker), fixed imaging (Vglut3 immunostain) (Figure 6E-H). Within each of these subfolders, raw data files are sorted into subfolders by condition (DMSO or noco). Zebrafish examined at day 5. The data extracted from these files is summarized in the excel file. Information on live Lysotracker dye labeling: Larvae were incubated for 15 min in 100nM Lysotracker Green and then embedded in 1% low-melt agarose prepared in E3 media containing 0.03% tricaine, 100nM Lysotracker, and either 250 nM nocodazole or 0.1% DMSO. Metadata for Nikon A1R acquisition: Live zebrafish samples were imaged on a Nikon A1R upright confocal microscope using a 60x 1 NA water objective lens and the following lasers lines: 488 and laser scanning transmitted light. Denoised images acquired using NIS Elements AR 5.20.02 1024x512 or 512x256 pixels 0.4-0.5 microns per slice Information on fixed immunostaining: Larvae were fixed with 4 % paraformaldehyde in PBS for 3.5 hr at 4C. After fixation, larvae were washed 5 x 5 min in 0.1% Tween in PBS (PBST). Prior to block, larvae were permeabilized with acetone. For this permeabilization, larvae were washed for 5 min with H2O. The H2O was removed and replaced with ice-cold acetone and placed at -20C for 5 min, followed by a 5 min H2O wash. The larvae were then washed for 5 x 5 min in PBST. Larvae were then blocked overnight at 4C in blocking solution (2% goat serum, 1% bovine serum albumin, 2% fish skin gelatin in PBST). Larvae were then incubated in primary antibodies in antibody solution (1% bovine serum albumin in PBST) overnight, nutating at 4C. The next day, the larvae were washed for 5 x 5 min in PBST to remove the primary antibodies. Secondary antibodies in antibody solution were added and larvae were incubated for 2 hrs at room temperature. After 5 x 5 min washes in PBST to remove the secondary antibodies, larvae were rinsed in H2O and mounted in Prolong Gold (ThermoFisher Scientific). The following primary antibodies were used: mouse anti-acetylated tubulin (IgG2b) (Sigma Aldrich T7451; 1:5000); mouse anti-Rab3 (IgG1) (Synaptic Systems 107011; 1:1000); and rabbit anti-Vglut3 c-terminus (Obholzer *et al.*, 2008; 1:1000). The following secondary antibodies were used at 1:1000: # A-21131, # A-21127, # A-21071 (ThermoFisher Scientific). Metadata for Zeiss Airyscan acquisition: Fixed zebrafish samples were imaged on an inverted Zeiss LSM 780 laser-scanning confocal microscope with Airyscan (Carl Zeiss AG) using a 63x 1.4 NA oil objective lens and the following lasers lines: 488, 543 and 633. Gain = 800 all laser lines 672x672 pixels 0.18 microns per slice Processed in Zen with an Airyscan processing factor of 6.5 (2D). The 20 Nikon NIS_Elements files generated are as follows: Channel 1 = Lysotracker; Channel 2 = DIC DMSO: 051822_control_F1_L2_zoom4_001.nd2 051822_control_F2_L1_zoom4_016.nd2 111721_F1_L2_control002.nd2 111721_F1_L3_control003.nd2 111721_F1_L4_control004.nd2 111721_F1_L5_control005.nd2 111721_F2_L1_control.nd2 111721_F2_L2_control001.nd2 111721_F2_L4_control002.nd2 111721_F2_L5_control001.nd2 Nocodazole: 051822_noco_F2_L1_zoom4_008.nd2 051822_noco_F2_L3_zoom4_012.nd2 111721_F1_L1_250nM_noco004.nd2 111721_F1_L2_250nM_noco.nd2 111721_F1_L3_250nM_noco001.nd2 111721_F1_L4_250nM_noco003.nd2 111721_F2_L1_250nM_noco.nd2 111721_F2_L2_250nM_noco001.nd2 111721_F2_L4_250nM_noco002.nd2 111721_F2_L5_250nM_noco001.nd2 The 16 Zeiss Airyscan processed files generated are as follows: Channel 1 = Acetylated tubulin; Channel 2 = Vglut3; Channel 3 = Rab3aa DMSO: dmso_CS1_r1_f1_L1_d5_Out.czi dmso_CS1_r1_f2_L2_d5_Out.czi dmso_CS1_r1_f2_L3_d5_Out.czi dmso_CS1_r1_f3_L1_d5_Out.czi dmso_CS1_r1_f3_L2_d5_Out.czi dmso_CS1_r1_f3_L3_d5_Out.czi dmso_CS1_r1_f4_L2_d5_Out.czi dmso_CS1_r1_f4_L3_d5_Out.czi Nocodazole: noco_CS2_r1_f1_L3_d5_Out.czi noco_CS2_r1_f2_L1_d5_Out.czi noco_CS2_r1_f2_L3_d5_Out.czi noco_CS2_r1_f2_L4_d5_Out.czi noco_CS2_r1_f3_L2_d5_Out.czi noco_CS2_r1_f3_L3_d5_Out.czi noco_CS2_r1_f3_L4_d5_Out.czi noco_CS2_r1_f4_L1_d5_Out.czi Both Nikon NIS-Elements and Zeiss Airyscan images were quantified in Fiji to measure base:apex fluorescence of Lysotracker or Vglut3 in hair cells. For this analysis ROIs of with an area of 2.25 x 0.65 μm (L x W) were used. The excel file “noco_lyso and vglut3 neuromast results_dryad.xlsx” contains two sheets: data from live lysotracker labeling and data from a vglut3 immunostain. The data extracted from the raw data files are sorted by treatment condition, filename and base:apex fluorescence ratios of each neuromast measured. The following files: 111721_F2_L2_250nM_noco001.nd2 111721_F2_L2_control001.nd2 dmso_CS1_r1_f2_L2_d5_Out.czi noco_CS2_r2_f2_L1_d5_Out.czi Were processed in FIJI (partial max projections) and used as example images in the figure. #### File: RAWDATA\_Figure\_7.zip **Description:**  Summary of Figure 7 folder contents: This folder contains 25 .tif transmission electron microscopy (TEM) images, 22 Nikon NIS-Elements images, and one excel file. There are 2 subfolders that contain live Lysotracker images and fixed TEM images. The 22 Nikon NIS_Elements files represent the raw data in Figure 7A-C. These files end in .nd2. This is data acquired from 10 control neuromasts and 10 *kif1aa *mutant neuromasts that also express the following transgenic line: myo6b:ctbp2a-tagRFP. These data are from live samples acquired at day 5 or day 6. There also are 25 TEM images that represent the raw data in Figure 7E-M. These files end in .tif. This is data acquired from 4 wild-type siblings and 3 *kif1aa *mutants. The TEM data are from zebrafish that were fixed at day 5. The data extracted from these files is summarized in the excel file. Information on live Lysotracker dye: Larvae were incubated for 15 min in 100nM Lysotracker Green and then embedded in 1% low-melt agarose prepared in E3 media containing 0.03% tricaine and 100nM Lysotracker. Information on TEM preparation: Larvae were genotyped at day 2 using a larval fin clip method to identify *kif1aa* and wild-type siblings to prepare for TEM. At day 5 *kif1aa* and wild-type siblings were fixed in freshly prepared solution containing 1.6 % paraformaldehyde and 2.5 % glutaraldehyde in 0.1 M cacodylate buffer supplemented with 3.4% sucrose and 2 µM CaCl2 for 2 h at room temperature, followed by a 24-h incubation at 4 °C in a fresh portion of the same fixative. After fixation, larvae were washed with 0.1 M cacodylate buffer with supplements, and post-fixed in 1 % osmium tetroxide for 30 min, and then washed with distilled water. Larvae were dehydrated in 30 – 100 % ethanol series, which included overnight incubation in 70 % ethanol containing 2 % uranyl acetate, and in propylene oxide, and then embedded in Epon. Transverse serial sections (60-70 nm thin sections) were used to section through neuromasts. Sections were placed on single slot grids coated with carbon and formvar, and then sections were stained with uranyl acetate and lead citrate. All reagents and supplies for TEM were from Electron Microscopy Sciences. Metadata for EM: Samples were imaged on a JEOL JEM-2100 electron microscope (JEOL Inc.). Whenever possible, serial sections were used to restrict our analysis to central sections of ribbons adjacent to the plasma membrane and a well-defined postsynaptic density.           Metadata for Nikon A1R acquisition: Live zebrafish samples were imaged on a Nikon A1R upright confocal microscope using a 60x 1 NA water objective lens and the following lasers lines: 488 and 561. Denoised images acquired using NIS Elements AR 5.20.02 1024x512 pixels 0.5 microns per slice Zoom4x or 6x The 22 Nikon NIS_Elements files generated are as follows: Channel 1 = Lysotracker; Channel 2 = ctbp2a-tagRFP; Channel 3 = DIC Siblings: F1_L1_102821_zoom6_004.nd2 F1_L2_102821_zoom6_003.nd2 F3_L1_110321_zoom6_005.nd2 F3_L2_110321_zoom6_006.nd2 F3_L2_110321_zoom4_007.nd2 F3_L4_110321_zoom6_009.nd2 F4_L1_110321_zoom6_010.nd2 F4_L2_110321_zoom6_011.nd2 F4_L3_110321_zoom6_012.nd2 F5_L1_110321_zoom6_014.nd2 F5_L2_110321_zoom6_015.nd2 *kif1aa* mutants: F3_L1_102821_zoom6_010.nd2 F3_L2_102821_zoom6_012.nd2 F3_L3_102821_zoom6_013.nd2 F3_L4_102821_zoom6_014.nd2 F4_L1_102821_zoom6_015.nd2 F4_L2_102821_zoom6_017.nd2 F4_L3_102821_zoom6_018.nd2 F6_L1_102821_zoom6_026.nd2 F6_L2_102821_zoom6_027.nd2 F6_L2_102821_zoom4_028.nd2 F6_L4_102821_zoom6_030.nd2 The 24 .tif TEM image files generated are as follows: Siblings: 102221_ZF-wt2_0016-1.tif 112221_wt2_0016.tif 112221_wt2_0030.tif 112221_wt2_0042.tif 112221_ZF wt2_0093.tif 112321_ZF wt1_0008.tif 112321_ZF wt1_0014.tif NM1_0029_rib2ok.tif NM2_0054_rib3.tif NM3_0029.tif wt4_0046.tif wt4_0049.tif *kif1aa* mutants: 101821_ZF_mut6__0014_vesicles.tif 101821_ZF_mut6__0031_vesicles.tif 102221_ZF_mu6_0047.tif 111021_ZFmu4_2_0001.tif 111021_ZFmu4_2_0032.tif 111521_zf-mu6_0015.tif 111521_zf-mu6_0063.tif 111521_zf-mu6_0064.tif 111521_zf-mu6_0069.tif 111521_zf-mu6_0076.tif mu2_0046.tif mu2_NM3_0031_20.tif ZFmut6_0008.tif After live lysotracker imaging genotyping was done using standard PCR followed by a Bsl1 restriction enzyme digest. *Kif1aa* genotyping primers used were as follows: *kif1aa*_FWD 5’-AACACCAAGCTGACCAGTGC-3’ and *kif1aa*_REV 5’-TGCGGTCCTAGGCTTACAAT-3’. *Kif1aa* mutants were compared to heterozygous and wild-type siblings (sibling controls). Live Nikon NIS-Elements images were quantified in Fiji to quantify Lysotracker fluorescence at ribbons, a circular ROI was drawn around the ribbon indicated by Ctbp2a-tagRFP fluorescence, and the mean value was measured in FIJI. TEM images were quantified in FIJI. To quantify ribbon area, ROIs were drawn in FIJI outlining the electron-dense ribbon, excluding the filamentous “halo” surrounding the ribbon. Vesicles with a diameter of 30–50 nm and adjacent (within 60 nm of the ribbon) to the “halo” were counted as tethered vesicles. Readily releasable pool (RRP) of vesicles were defined as tethered vesicles between the ribbon and the plasma membrane. To quantify reserve vesicles, we counted vesicles that were not tethered to the ribbon but were within 200 nm of the edge of the ribbon. All distances and perimeters were measured in FIJI. The excel file “kif1aa_TEM and ribbon results_dryad.xlsx” contains three sheets: TEM vesicle counts, TEM ribbon area, and Lysotracker. The TEM vesicle counts sheet includes filename and vesicle counts from the RRP, tethered, and reserve vesicles.  TEM ribbon area includes filename and ribbon areas. Lyotracker includes the filename and the the base:apex Lysotracker fluorescence ratios of each neuromast measured. The following files: F3_L2_110321_zoom4_007.nd2 F3_L2_110321_zoom6_006.nd2 F6_L2_102821_zoom4_028.nd2 F6_L2_102821_zoom6_027.nd2 Were processed in FIJI (partial max projections) and used as example images in the figure. 111521_zf-mut6_0076.tif ZFmut6_0008.tif 112321_ZF wt1_0014.tif 112221_wt2_0016.tif Were also cropped in FIJI and used as example images in the figure. #### File: RAWDATA\_Figure\_8.zip **Description:**  Summary of Figure 8 folder contents: This folder contains 22 Zeiss Airyscan processed images and one excel file. There are 22 Zeiss Airyscan processed images that represent the raw data in Figure 8. These files end in .czi. This is data acquired from 13 sibling and 9 kif1aa neuromasts. These data are fixed samples that label calcium channels, ribbons and acetylated tubulin in neuromasts (O1, D1/earNM3 and L1-L4 ) of the lateral line. Zebrafish were fixed and stained at day 5. The data extracted from these files is summarized in the excel file. Information on immunostaining: Larvae were fixed with 4 % paraformaldehyde in PBS for 3.5 hr at 4C. After fixation, larvae were washed 5 x 5 min in 0.1% Tween in PBS (PBST). Prior to block, larvae were permeabilized with acetone. For this permeabilization, larvae were washed for 5 min with H2O. The H2O was removed and replaced with ice-cold acetone and placed at -20C for 5 min, followed by a 5 min H2O wash. The larvae were then washed for 5 x 5 min in PBST. Larvae were then blocked overnight at 4C in blocking solution (2% goat serum, 1% bovine serum albumin, 2% fish skin gelatin in PBST). Larvae were then incubated in primary antibodies in antibody solution (1% bovine serum albumin in PBST) overnight, nutating at 4C. The next day, the larvae were washed for 5 x 5 min in PBST to remove the primary antibodies. Secondary antibodies in antibody solution were added and larvae were incubated for 2 hrs at room temperature. After 5 x 5 min washes min in PBST to remove the secondary antibodies, larvae were rinsed in H2O and mounted in Prolong Gold (ThermoFisher Scientific). The following primary antibodies were used: rabbit anti-Cav1.3 ((Sheets *et al.*, 2011); 1:1000); mouse anti-ribeyeb (IgG2a) ((Sheets *et al.*, 2011); 1:10,000)  and mouse anti-Acetylated tubulin (IgG1) (Sigma T7451; 1:500). With the following secondary antibodies were used at 1:1000: #A-11008, # A-21137, # A-21240 (ThermoFisher Scientific). Metadata for Zeiss Airyscan acquisition: Fixed zebrafish samples were imaged on an inverted Zeiss LSM 780 laser-scanning confocal microscope with Airyscan (Carl Zeiss AG) using a 63x 1.4 NA oil objective lens and the following lasers lines: 488, 543 and 647. Gain = 800 all laser lines 608x608 pixels 0.185 microns per slice Processed in Zen with an Airyscan processing factor of 6.5 (2D). The 22 Zeiss Airyscan files generated are as follows: Sibling: 072121_kif1aa_5_cav_ribB_tub_CS1_R1_F1L2_Out 072121_kif1aa_5_cav_ribB_tub_CS1_R1_F4L2_Out 072121_kif1aa_5_cav_ribB_tub_CS1_R2_F6L2_Out 072121_kif1aa_5_cav_ribB_tub_CS1_R2_F8L2_Out 072121_kif1aa_5_cav_ribB_tub_CS1_R2_F8L3_Out 072121_kif1aa_5_cav_ribB_tub_CS1_R2_F9L2_Out 072121_kif1aa_5_cav_ribB_tub_CS1_R2_F12L2_Out 072121_kif1aa_5_cav_ribB_tub_CS2_R1_F2L1_Out 072121_kif1aa_5_cav_ribB_tub_CS2_R1_F2L2_Out 072121_kif1aa_5_cav_ribB_tub_CS2_R1_F6L2_Out 072121_kif1aa_5_cav_ribB_tub_CS2_R2_F10_L3_Out 072121_kif1aa_5_cav_ribB_tub_CS2_R2_F8L1_Out 072121_kif1aa_5_cav_ribB_tub_CS2_R2_F9earNM3_Out Kif1aa mutants 072121_kif1aa_5_cav_ribB_tub_CS1_R1_F3O1_Out 072121_kif1aa_5_cav_ribB_tub_CS1_R2_F10L1_Out 072121_kif1aa_5_cav_ribB_tub_CS1_R2_F10earNM3_Out 072121_kif1aa_5_cav_ribB_tub_CS1_R2_F11L2_Out 072121_kif1aa_5_cav_ribB_tub_CS1_R2_F11L3_Out 072121_kif1aa_5_cav_ribB_tub_CS1_R2_F13L3_Out 072121_kif1aa_5_cav_ribB_tub_CS1_R2_F13L4_Out 072121_kif1aa_5_cav_ribB_tub_CS2_R1_F3L1_Out 072121_kif1aa_5_cav_ribB_tub_CS2_R1_F5L2_Out Channel 1 = Cav1.3; Channel 2 = Ribeyeb Channel 3 = Acetylated tubulin After imaging genotyping was done using standard PCR followed by a Bsl1 restriction enzyme digest. *Kif1aa* genotyping primers used were as follows: *kif1aa*_FWD 5’-AACACCAAGCTGACCAGTGC-3’ and *kif1aa*_REV 5’-TGCGGTCCTAGGCTTACAAT-3’. *Kif1aa* mutants were compared to heterozygous and wild-type siblings (sibling controls). Zeiss Airyscan images were automatically quantified using a customized Fiji-based macro “Complete Synapse Counter v5.2” ([https://github.com/KindtLab/CompleteSynpaseCounter5.2](https://github.com/KindtLab/CompleteSynpaseCounter5.2)) to measure mean cav1.3 area mean integrated cav1.3 intensity and mean cav1.3 intensity and paired cav1.3-ribeyeb puncta. For this analysis the minimum size thresholds of 0.025 μm2 (Cav1.3 and Ribeyeb) were used. The excel file 072121_kif1aa-cav-rbb_CSC5.2results_Dryad contains data extracted from the raw data files including: filename, genotype, paired cav1.3-ribeye puncta per hair cell, mean area of paired cav1.3 puncta, mean integrated intensity of paired cav1.3 puncta, mean intensity of paired cav1.3 puncta. The following files: 072121_kif1aa_5_cav_ribB_tub_CS2_R1_F5L2_Out 072121_kif1aa_5_cav_ribB_tub_CS2_R1_F6L2_Out These files were processed in FIJI (partial max projections) and used as example images in the figure. #### File: RAWDATA\_Figure\_9.zip **Description:**  Summary of Figure RAW_DATA_Figure 9 folder contents: This folder contains 32 Prairieview files and 1 Excel file. The .xml files are the raw calcium imaging data from kif1aa mutants and sibling animals represented in Figure 9. This data is sorted into 2 subfolders: 500ms MET and 500ms presyn that separate the mechanosensitive and presynaptic measurements respectively for each sample. The data extracted from these files is summarized in the excel file .xlsx. This is data acquired from kif1aa mutants and sibling zebrafish at 5 dpf. The .xml files are measurements of mechanosensitive and presynaptic responses from 8 sibling and 8 kif1aa mutant neuromasts (L1-L4) in the posterior lateral line. Responses were made using the following transgenic line: myo6b:GCaMP6scaax. To stimulate hair cells a fluidjet was used. A 500ms saturating fluid flow stimulus was used, towards the anterior or posterior of the fish. The .xml data files are coded with the date acquired, Fish number (F) for each experimental day, the neuromast assayed (L1, L2, L3, L4) and the direction of the stimulus (ANT= towards the anterior, POST= towards the posterior). Each neuromast was stimulated in 2 directions, anterior or posterior; this stimulation was applied to measurements taken at the hair cell apex/MET and hair cell presynapse/base. Metadata for GCaMP6s hair cell calcium imaging acquisition: Acquired on a Swept field confocal microscope using Prairie-view (Bruker) software Nikon 60x water objective 20ms per image, 1.0 microns (presynaptic) 0.5 micron (apex/MET) per slice, 5 planes per Z-stack (each Z-stack or timepoint is ~0.1s) 400 tif images in total per acquisition (~8s), fluidjet stimulation at frame 150 (~3s) 70 micron slit EM gain 3900 2x2 binning Imaging ROI 128x128 Laser setting 125 for apex and 100 for base The following samples were acquired: Sibling m6bGCaMP6scaax_101123_F1_L2 m6bGCaMP6scaax_101123_F1_L3 m6bGCaMP6scaax_101123_F6_L1 m6bGCaMP6scaax_101123_F6_L3 m6b_memGCaMP6s_d5_101823_F3_L3 m6b_memGCaMP6s_d5_101823_F5_L2 m6b_memGCaMP6s_d5_101823_F5_L4 m6b_memGCaMP6s_d5_101823_F7_L2 kif1aa mutants: m6bGCaMP6scaax_101123_F2_L3 m6bGCaMP6scaax_101123_F3_L2 m6bGCaMP6scaax_101123_F4_L3 m6bGCaMP6scaax_101123_F5_L1 m6bGCaMP6scaax_101123_F5_L2 m6b_memGCaMP6s_d5_101823_F1_L3 m6b_memGCaMP6s_d5_101823_F2_L3 m6b_memGCaMP6s_d5_101823_F2_L4 Each sample has 2 acquisitions at the apex and 2 at the base. These .xml files are in the folders 500ms_MET and 500ms_presyn After imaging genotyping was done using standard PCR followed by a Bsl1 restriction enzyme digest. *Kif1aa* genotyping primers used were as follows: *kif1aa*_FWD 5’-AACACCAAGCTGACCAGTGC-3’ and *kif1aa*_REV 5’-TGCGGTCCTAGGCTTACAAT-3’. *Kif1aa* mutants were compared to heterozygous and wild-type siblings (sibling controls). Each .xml was processed using the GUI-based Matlab 2014Rb program IOS_Software.m This program average projects each z-stack. Then registers the Z-stack. The first second (10 frames) of the recording was removed for baseline stability. Then the resulting 70 frames per recording was then opened in FIJI/ImageJ. The Time Series Analyzer V3 plugin was used to create size circular ROIs. ROIs were placed on each responding hair bundle at the apex or presynaptic site at the cell base. The multi-measure feature in the ROI manager was used to obtain the mean gray value from each ROI and each image in the series. Background subtracted plots were created. The background was the first 20 rows (F0). The calculation to subtract background was Percentage different 100*(Value-Baseline)/Baseline. From the background subtracted plots (deltaF/F0), for each neuromast the anterior and posterior MET and presynpatic responses were determined. For each genotype, the average response magnitude (Max deltaF/F0) of the was determined for each neuromast. These analyses are summarized in 4 tables in the 011123_011823_kif1aa_Hair cell calcium IMG results_dryad.xlxs file. The table Summary MET lists base file name(column a), the genotype (Column B), and the Max deltaF/F0 (Column c) for each neuromast. The table MET AVG traces and SEM summarizes the average mutant and sibling MET response at each time point, along with the SEM. The table Summary presynaptic lists base file name (column a), the genotype (Column B), and the Max deltaF/F0 (Column c) for each neuromast. The table Presynaptic AVG traces and SEM summarizes the average mutant and sibling presynaptic response at each time point, along with the SEM. These tables represent the data shown in Figure 5F-I. The examples shown in Figure 9B-E were derived from the following raw data files: m6bGCaMP6scaax_101123_F4_L3_500ms m6bGCaMP6scaax_101123_F6_L1_500ms #### File: RAWDATA\_Figure\_10.zip **Description:**  Summary of Figure RAW_DATA_Figure 10 folder contents: This folder contains 38 Prairieview .xml files and 19 Igor Pro .abf files, and 2 excel files There are 2 subfolders, 500ms Afferent GCaMP6s and Electrophysiology Traces that contain the .xml and .abf files respectively. The .xml files are the raw calcium imaging data and the .abf files are the raw electrophysiology data from kif1aa mutants and sibling animals represented in Figure 10. The data extracted from these files is summarized in the 2 excel files. This is data acquired from kif1aa mutants and sibling zebrafish at 3-6 dpf. The .xml files are measurements of calcium responses in the afferent terminals beneath neuromasts from 10 sibling and 9 kif1aa mutant neuromasts (L1-L4) in the posterior lateral line. Responses were made using the following transgenic line: en.sill,hsp70l:GCaMP6s. To stimulate hair cells a fluidjet was used. A 500ms saturating fluid flow stimulus was used, towards the anterior or posterior of the fish. The .xml data files are coded with the date acquired, Fish number (F) for each experimental day, the neuromast assayed (L1, L2, L3, L4) and the direction of the stimulus (ANT= towards the anterior, POST= towards the posterior). Each neuromast was stimulated in 2 directions, anterior or posterior. The .abf files are spontaneous extracellular recordings from the afferent cell bodies in the posterior lateral line ganglion innervating lateral-line neuromasts. The .abf files are coded with the date acquired (first 5 digits) and number of recording on the day (last 3 digits). Metadata for GCaMP6s afferent calcium imaging acquisition: Acquired on a Swept field confocal microscope using Prairie-view (Bruker) software Nikon 60x water objective 20ms per image, 2.0 microns (presynaptic) 0.5 micron (apex/MET) per slice, 5 planes per Z-stack (each Z-stack or timepoint is ~0.1s) 400 tif images in total per acquisition (~8s), fluidjet stimulation at frame 150 (~3s) 70 micron slit EM gain 3900 2x2 binning Imaging ROI 128x128 Laser setting 125 Metadata for electrophysiology recording acquisition: Borosilicate glass pipettes (Sutter Instruments, BF150-86-10 glass with filament) were pulled with a long taper, resistances between 5 and 15 MW. pLLg was visualized using an Olympus BX51WI fixed stage microscope equipped with a LumPlanFl/IR 60x 1.4 NA water dipping objective (N2667800, Olympus). Axopatch 200B amplifier, a Digidata 1400A data acquisition system, and pClamp 10 software (Molecular Devices, LLC) were used to collect signals. Recordings were done in voltage-clamp mode, and signals were sampled at 50 μs/point and filtered at 1 kHz.  The number of spontaneous events from one neuron per min was quantified from a 5-min recording window using Igor Pro (Wavemetrics). The following samples were acquired: Calcium Imaging: Sibling: SILLGC6s_d4_050223_F1_L2_500ms SILLGC6s_d4_050223_F2_L2_500ms SILLGC6s_d4_050223_F3_L2_500ms SILLGC6s_d4_050223_F8_L2_500ms 050323_SILLGC6s_d5_F1_L2_500ms 050323_SILLGC6s_d5_F3_L2_500ms SILLGCa6s_050923_d4_F1_L3_500ms SILLGCa6s_050923_d4_F1_L2_500ms SILLGCa6s_050923_d4_F2_L2_500ms SILLGC6s_050923_d4_F6_L2_500ms kif1aa mutants: SILLGC6s_d4_050223_F6_L2_500ms SILLGC6s_d4_050223_F6_L3_500ms SILLGC6s_d4_050223_F7_L2_500ms 050323_SILLGC6s_d5_F5_L3_500ms 050323_SILLGC6s_d5_F5_L2_500ms SILLGCa6s_050923_d4_F3_L2_500ms SILLGCa6s_050923_d4_F3_L3_500ms SILLGCa6s_050923_d4_F3_L4_500ms SILLGCa6s_050923_d4_F4_L2_500ms Each sample has 2 acquisitions, ANT and POST. Electrophysiology Recordings: Sibling: ABF1_24227002 ABF1_24227003 ABF1_24227004 ABF1_24228002 ABF1_24228008 ABF1_24228011 ABF1_24401000 ABF1_24402001 kif1aa mutants: ABF1_24226003 ABF1_24228001 ABF1_24229000 ABF1_24325000 ABF1_24326000 ABF1_24326001 ABF1_24326002 ABF1_24327004 ABF1_24329001 ABF1_24401001 ABF1_24402004 After imaging or recording spikes genotyping was done using standard PCR followed by a Bsl1 restriction enzyme digest. *Kif1aa* genotyping primers used were as follows: *kif1aa*_FWD 5’-AACACCAAGCTGACCAGTGC-3’ and *kif1aa*_REV 5’-TGCGGTCCTAGGCTTACAAT-3’. *Kif1aa* mutants were compared to heterozygous and wild-type siblings (sibling controls). Each .xml was processed using the GUI-based Matlab 2014Rb program IOS_Software.m This program average projects each z-stack. Then registers the Z-stack. The first second (10 frames) of the recording was removed for baseline stability. Then the resulting 70 frames per recording was then opened in FIJI/ImageJ. The Time Series Analyzer V3 plugin was used to create size circular ROIs. ROIs were placed on each responding afferent site. The multi-measure feature in the ROI manager was used to obtain the mean gray value from each ROI and each image in the series. Background subtracted plots were created. The background was the first 20 rows (F0). The calculation to subtract background was Percentage different 100*(Value-Baseline)/Baseline. From the background subtracted plots (deltaF/F0), for each neuromast the anterior and posterior responsive sites were determined. For each genotype, the average response magnitude (Max deltaF/F0) of the was determined for each neuromast. These analyses are summarized in kif1aa_Afferent calcium IMG results_dryad.xlxs file. The table Summary Afferent lists base file name, (column a), the age of the animal (column b), the genotype (column c), and the Max deltaF/F0 (Column d) for each neuromast. The table Afferent AVG traces and SEM summarizes the average mutant and sibling afferent terminals response at each time point, along with the SEM. These tables represent the data shown in Figure 10G-H. Each .abf file was processed in Igor Pro (Wavemetrics) using the zPhys Panel plugin from Josef Trapani ([https://gitlab.amherst.edu/jtrapani/zphys_panel](https://gitlab.amherst.edu/jtrapani/zphys_panel)). Alternatively these files can be opened with R studio using the "readABF" package. Spikes were counted in a 3-5 min time window, and spikes/minute were calculated. These analyses are summarized in kif1aa_Ephys results_dryad.xlsx file. This file includes filename, genotype, the time window used for analysis, and the spikes/minute calculation. The examples shown in Figure 10B and Figure 10E-F were derived from the following raw data files: SILLGC6s_d4_050223_F2_L2_500ms SILLGC6s_d4_050223_F6_L3_500ms ABF1_24227003 ABF1_24228001 #### File: RAWDATA\_Figure\_11.zip **Description:**  Summary of Figure 11 folder contents: This folder contains 2 subfolders named by experimental paradigm (startle or habituation recovery), each with subfolders named by plate ran on the day. In total there are 224 videos of plates ran and 22 excel files. There are 21 excel files with extension .csv and 224 videos with extension .avi generated by the Zantiks system that represent the data in figure 11A-B. There is one excel file with extension .xlsx denoting all data points and which experiment and date they are derived from, along with genotypes. This is live behavioral data acquired from 103 fish aged 5 dpf on 4 separate days. Video .avi files in the startle folder beginning with “S1-VIB1” correspond to the highest intensity stimulus, “S2-VIB2” the medium intensity stimulus, “S3-VIB3” the low intensity stimulus, and “S4-VIB4” the control with no stimulus. Video .avi files in the habituation recovery folder beginning with “NON-HAB-PRE” correspond to the pre-habituation stimuli, “HAB” the train of habituation stimuli, and “NON-HAB-POST” the recovery stimuli following the habituation stimuli. Information on behavioral assay: A Zantiks MWP behavioral system ([https://zantiks.com](https://zantiks.com)) was used to assess acoustic startle responses in larvae at 5 dpf. The Zantiks system tracks and monitors behavioral responses using an infrared camera at 30 frames per second. During the tracking and stimulation, a Cisco router connected to the Zantiks system was used to relay x and y coordinates of each larva in every frame. The Zanzik systems creates video files (.avi) during each stimulus. In addition, from the x,y, coordinates a .cvs (vib-stim prefix) containing the distance traveled in pixels for each time point for each well of the plate (column I-T). This data is contained in the .csv files for each plate. Behavioral trials were performed at 5 dpf, on three independent days. For this behavioral analysis, we compared *kif1aa+/+ *wild-type siblings and* kif1aa+/- *heterozygotes to *kif1aa-/-* mutants for an in-clutch, sibling comparison. A 12-well plate was used for behavioral analyses. Each well was filled with E3 and 1 larva. All fish were acclimated in the plate within the Zantiks chamber in the dark for 15 min before each test. A vibrational stimulus that triggered a maximal proportion of animals startling in control animals without any tracking artifacts (due to the vibration) was used for our strongest stimuli. We used 4 different levels of intensity (1-4, increasing in intensity), with level 4 as the highest intensity stimulus. To deliver the acoustic-vibrational stimulus, the solenoid motor in the Zantiks system was set to move by 7.2° (level 4: 4 full steps), 3.6° (level 3: 2 full steps), 1.8° (level 2: 1 full step), and 0.9° (level 1: 1/2 step), with a 4 x 4.25 ms motor speed moving in clockwise and anticlockwise movements. We used an Optimus+ Red Sound Level Meter (Cirrus Research) to measure the intensity (dB) of each stimulus in the Zantiks chamber. The meter recorded the following sound intensities: 26.4 dB (Level 4), 857 23.3 dB (Level 3), 17.8 dB (Level 2), and 11.9 dB (Level 1). For our initial startle assay, each larva was presented with stimuli from intensity levels 1-3, 5 times, with 100 s between trials to avoid habituation. For each animal, the proportion of startle responses out of the 5 trials was plotted. For our habituation and recovery assay, a non-habituating stimulus, followed by a habituating stimulus train, and lastly a recovery stimulus train was performed. Similar to previous studies, our non-habituating stimulus was presented 3 times (intensity level 4), with 100 s between trials. This was followed by a habituating train of 30 stimuli (same stimulus intensity), presented with a 5 s interstimulus interval (ISI), an ISI shown to result in habituation (Marsden & Granato, 2015). We then presented recovery stimuli once each at 20 s, 40 s, 1 min, and 2 min after the last stimulus in the habituating train. The proportion of startle responses out of the initial 3 non-habituating stimuli and the proportion of responses at each habituation block and recovery stimulus were plotted. To qualify as a startle response, a distance above 4 pixels, or ∼1.9 mm, was required within 2 frames after stimulus onset. Larvae were excluded from our analysis if no tracking data was recorded. Startle behavior was performed on at least three independent days. The folders by date are as follows: Startle: startle 011922 startle 020222 startle 021522 Habituation Recovery: recovery 022223 recovery 030823 recovery 040523 recovery 042623 recovery 062823 Each plate has 12 wells. Well position is designated by three rows A-C and 4 column 1-4; for example, the upper left well is A1 and the lower right well is C4. The data from the .csv files in the startle folder was extracted using Python code ‘zantiks processing-0.3s_eachtmpt.ipynb’. This code detects whether each animal travels more than 4 pixels or ~1.9 mm within 2 frames after stimulus onset. This is counted as a startle response. Then the number of times (out of 5 trials) the animal startles is determined. This data is summarized in the excel file. The table Summary data outlines the date, plate and well for each animal. In addition, this table lists the proportion of times (out of 5 trials) that the animal startled in response to the each vibrational stimulus (HI, MED, LO and control (no stimulus)). This data is sorted by genotype and summarized in the table data plotted. This is the data plotted in Figure 11A. The data from the .csv files in the habituation recovery folder was extracted in excel by removing all lines in the spreadsheet that contained “QUIET” and calculating the proportion of times a fish startled in a given condition (out of 3 prehabituation stimuli, out of every 5 habituation stimuli, each individual recovery stimuli). The same criteria of travel distance greater than 4 pixels was used to qualify as a startle response. This is the data plotted in Figure 11B. The data from both the startle and habituation recovery conditions is summarized in the excel file "082024 Zantiks behavior linking sheet.xlsx". This file contains two sheets: startle and recovery, for each condition. The summarized data in this file includes folder, plate number, filename, well, genotype, and value for stimulus administered.  After testing, genotyping was done using standard PCR followed by a Bsl1 restriction enzyme digest. *Kif1aa* genotyping primers used were as follows: *kif1aa*_FWD 5’-AACACCAAGCTGACCAGTGC-3’ and *kif1aa*_REV 5’-TGCGGTCCTAGGCTTACAAT-3’. *Kif1aa* mutants were compared to either heterozygous and wild-type siblings (sibling controls) or to wild-type siblings. ## Code/software The data from the .csv files in the startle folder was extracted using Python code ‘zantiks processing-0.3s_eachtmpt.ipynb’. This was used in Jupyter Notebook within Anaconda. This code detects whether each animal travels more than 4 pixels or ~1.9 mm within 2 frames after stimulus onset. Then the number of times (out of 5 trials) the animal startles is determined. The table Summary data outlines the date, plate and well for each animal. In addition, this table lists the proportion of times (out of 5 trials) that the animal startled in response to the each vibrational stimulus (HI, MED, LO and control (no stimulus)). This is the data plotted in Figure 11A.