DOI: 10.5061/dryad.crjdfn3gg
title: Microtubule networks in zebrafish hair cells facilitate presynapse transport and fusion during development

description: Sensory cells in the retina and inner ear rely on specialized ribbon
 synapses for neurotransmission. Disruption of these synapses is linked to
 visual and auditory dysfunction, but it is unclear how these unique
 synapses are formed. Ribbon synapses are defined by a presynaptic density
 called a ribbon. Using live-imaging approaches in zebrafish, we find that
 early in hair-cell development, many small ribbon precursors are present
 throughout the cell. Later in development, fewer and larger ribbons
 remain, and localize at the presynaptic active zone (AZ). Using tracking
 analyses, we show that ribbon precursors exhibit directed motion along an
 organized microtubule network towards the presynaptic AZ. In addition, we
 show that ribbon precursors can fuse together on microtubules to form
 larger ribbons. Using pharmacology, we find that microtubule disruption
 interferes with ribbon motion, fusion, and normal synapse formation.
 Overall, this work demonstrates a dynamic series of events that underlies
 formation of a critical synapse required for sensory function.
description: <strong>Zebrafish animals</strong>
 Zebrafish (<em>Danio rerio</em>) were bred and cared
 for at the National Institutes of Health (NIH) under animal study protocol
 #1362-13. Zebrafish larvae raised at 28°C in E3 embryo medium (5 mM NaCl,
 0.17 mM KCl, 0.33 mM CaCl<sub>2</sub>, and 0.33 mM
 MgSO<sub>4</sub>, buffered in HEPES, pH 7.2). All experiments
 were performed on larvae aged 2-3 days post fertilization (dpf). Larvae
 were chosen at random at an age where sex determination is not possible.
 The previously described mutant and transgenic lines were used in this
 study:
 <em>Tg(myo6b:ctbp2a-TagRFP)<sup>idc11Tg</sup></em>
 referred to as <em>myo6b:riba-TagRFP;
 Tg(myo6b:YFP-Hsa.TUBA)<sup>idc16Tg </sup></em>referred
 to as <em>myo6b:YFP-tubulin </em>(Ohta et al., 2020; Wong et
 al., 2019).
 <em>Tg(myo6b:ctbp2a-TagRFP)<sup>idc11Tg</sup></em>
 reliably labels mature ribbons, similar to a pan-CTBP immunolabel at 5 dpf
 (Figure 1-S2B). This transgenic line does not alter the number of hair
 cells or complete synapses per hair cell (Figure 1-S2A-D). In addition,
 <em>myo6b:ctbp2a-TagRFP </em>does not alter the size of
 ribbons (Figure 1-S2E). <strong>Zebrafish
 transgenic and CRISPR-Cas9 mutant generation</strong>
 To create <em>myo6b:EB3-GFP</em> transgenic fish,
 plasmid construction was based on the tol2/gateway zebrafish kit (Kwan et
 al., 2007). The p5E <em>pmyo6b</em> entry clone<sup>
 </sup>(Trapani et al., 2009) was used to drive expression in hair
 cells. A pME-<em>EB3-GFP </em><em>clone was kindly
 provided by Catherine Drerup at University of Wisconsin, Madison.
 </em>pDestTol2pACryGFP was a gift from Joachim Berger &
 Peter Currie (Addgene plasmid # 64022). These clones were used along with
 the following tol2 kit gateway clone, p3E-<em>polyA</em>
 (#302) to create the expression construct:
 <em>myo6b:EB3-GFP</em>. To generate the stable transgenic fish
 line <em>myo6b:EB3-GFP<sup>idc23Tg</sup></em>,
 plasmid DNA and tol2 transposase mRNA were injected into zebrafish embryos
 as previously described (Kwan et al., 2007). The
 <em>myo6b:EB3-GFP<sup>idc23Tg</sup></em>
 transgenic line was selected for single copy and low expression of
 EB3-GFP. A<em> kif1aa</em> germline mutant
 (<em>kif1aa<sup>idc24</sup>)</em> was generated
 in-house using CRISPR-Cas9 technology as previously described (Varshney et
 al., 2016). Exon 6, containing part of the Kinesin motor domain was
 targeted. Guides RNAs (gRNAs) targeted to <em>kif1aa</em> are
 as follows: 5’-<strong>ACGGATGTTCTCGCACACGT(AGG)-3’,
 5’-</strong>GTGCGAGAACATCCGTTGCT(AGG)-3’,
 5’-TGGACTCCGGGAATAAGGCT(AGG)-3’, 5’-AGAATACCTAGCCTTATTCC(CGG)-3’. Founder
 fish were identified using fragment analysis of fluorescent PCR (fPCR)
 products. A founder fish containing a complex INDEL that destroys a BslI
 restriction site in exon 6 was selected (Figure 5-S1B). This INDEL
 disrupts the protein at amino acid 166 (Figure 5-S1A). Subsequent
 genotyping was accomplished using standard PCR and Bsl1 restriction enzyme
 digestion. <em>Kif1aa</em> genotyping primers used were:
 <em>kif1aa</em>_FWD 5’-AACACCAAGCTGACCAGTGC-3’ and
 <em>kif1aa</em>_REV 5’-TGCGGTCCTAGGCTTACAAT-3’.
 We created <em>kif1aa</em> F0 crispants for our live
 imaging analyses. Here we injected the following
 <em>kif1aa</em> gRNAs: 5’-GTGCGAGAACATCCGTTGCT(AGG)-3’ and
 5’-AGAATACCTAGCCTTATTCC(CGG)-3’, along with Cas9 protein, as previously
 described (Hoshijima et al., 2019). We then grew
 <em>kif1aa</em> injected F0 crispants for 2 days and then used
 them for our live imaging analyses. Because <em>kif1aa</em>
 mutants have no phenotype to distinguish them from sibling controls at the
 ages imaged, the low throughput of our live imaging approaches made using
 germline mutants prohibitive. Studies have shown that F0 crispants are a
 fast and effective way to knockdown gene function in any genetic
 background (Hoshijima et al., 2019; Sheets et al., 2021). After live
 imaging, we genotyped all <em>kif1aa</em> F0 crispants (Figure
 6-S1) to ensure that the gRNAs cut the target robustly using fragment
 analysis of fluorescent PCR products and the following primers:
 <em>kif1aa</em>_FWD_fPCR
 5’-TGTAAAACGACGGCCAGT-AAATAGAGATTCACTTTTAATC-3’ and
 <em>kif1aa</em>_REV_fPCR 5’- GTGTCTT-CCTAGGCTTACAATGCTTTTGG-3’
 (Carrington et al., 2015). fPCR fragments were run on a genetic analyzer
 (Applied Biosystems, 3500XL) using LIZ500 (Applied Biosystems, 4322682) as
 a dye standard. Any<em> kif1aa</em> F0 crispants without
 robust genomic cutting were not included in analyses.
 <strong>Zebrafish pharmacology</strong>
 To destabilize or stabilize microtubules, larval zebrafish at 2
 dpf were incubated in either nocodazole (Sigma-Aldrich, SML1665) or
 Paclitaxel (taxol) (Sigma-Aldrich, 5082270001). Both drugs were maintained
 in DMSO. For experiments these drugs were diluted in media for a final
 concentration of 0.1 % DMSO, 250-500 nM nocodazole and 25 µM taxol. For
 controls, larvae were incubated in media containing 0.1 % DMSO. For
 long-term incubation (16 hr), wild-type larvae were incubated in E3 media
 containing 250 nM nocodazole or 25 µM taxol at 54 hpf for 16 hrs
 (overnight). After this long-term treatment, larvae were fixed and
 prepared for immunohistochemistry (see below). For live, short-term
 incubations (for 3-4 hr incubations or ribbon tracking), transgenic larvae
 (<em>myo6b:riba-tagRFP;
 Tg(myo6b:YFP-alpha-tubulin</em>)<sup> </sup>at 48-54 hpf
 were embedded in 1 % low melt agarose prepared in E3 media containing 0.03
 % tricaine (Sigma-Aldrich, A5040, ethyl 3-aminobenzoate methanesulfonate
 salt). 500 nM nocodazole, 25 µM taxol or DMSO were added to the agarose
 and to the E3 media used to hydrate the sample. For short-term treatments,
 hair cells were imaged after 30 min of embedding.
 <strong>Immunohistochemistry of zebrafish
 samples</strong> Immunohistochemistry to label
 acetylated-tubulin, tyrosinated-tubulin, Ribeyeb or pan-CTBP (ribbons and
 precursors), pan-Maguk (postsynaptic densities) and Myosin7a (cell bodies)
 was performed on whole zebrafish larvae similar to previous work. The
 following primary antibodies were used: rabbit anti-Myosin7a (Proteus
 25-6790; 1:1000); mouse anti-pan-Maguk (IgG1) (Millipore MABN7; 1:500);
 mouse anti-Ribeyeb (IgG2a) ((Sheets et al., 2011); 1:10,000); mouse
 anti-CTPB (IgG2a) (Santa Cruz sc-55502; 1:1000); mouse
 anti-acetylated-tubulin (IgG2b) (Sigma-Aldrich T7451; 1:5,000); mouse
 anti-tyrosinated-tubulin (IgG2a) (Sigma-Aldrich MAB1864-I; 1:1,000);
 chicken anti-GFP (to stain YFP-tubulin) (Aves labs GFP-1010; 1:1,000). The
 following secondary antibodies were used at 1:1,000: (ThermoFisher
 Scientific, A-11008; A-21143, A-21131, A-21240; A-11-039, A-21242,
 A-21241, A-11039). Larvae were fixed with 4 % paraformaldehyde in PBS for
 4 hr at 4°C. All wash, block and antibody solutions were prepared in 0.1 %
 Tween in PBS (PBST). After fixation, larvae were washed 5 × 5 min in PBST.
 Prior to block, larvae were permeabilized with acetone. For this
 permeabilization larvae were first washed for 5 min with
 H<sub>2</sub>O. The H<sub>2</sub>O was removed and
 replaced with ice-cold acetone and placed at −20°C for 3 min, followed by
 a 5 min H<sub>2</sub>O wash. The larvae were then washed for 5
 × 5 min in PBST. Larvae were then blocked overnight at 4°C 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 4°C.
 The next day, the larvae were washed for 5 × 5 min in PBST to remove the
 primary antibodies. Secondary antibodies in antibody solution were added
 and larvae were incubated for 3 hrs at room temperature. After 5 × 5 min
 washes min in PBST to remove the secondary antibodies, larvae were rinsed
 in H<sub>2</sub>O and mounted in Prolong gold (ThermoFisher
 Scientific P36930). <strong>Confocal imaging and
 analysis of fixed zebrafish samples</strong>
 After immunostaining, fixed zebrafish samples were imaged on an
 inverted Zeiss LSM 780 (Zen 2.3 SP1) or an upright Zeiss LSM 980 (Zen 3.4)
 laser-scanning confocal microscope with Airyscan using a 63x 1.4 NA oil
 objective lens. Z-stacks encompassing the entire neuromast were acquired
 every 0.17 (LSM 980) or 0.18 (LSM 780) µm with an 0.04 µm x-y pixel size
 and Airyscan autoprocessed in 3D.          Synaptic
 images from fixed samples were further processed using FIJI.
 Acetylated-a-tubulin or Myosin7 label was used to manually count hair
 cells. Complete synapses comprised of both a Ribeyeb/CTBP and Maguk puncta
 were also counted manually. To quantify the area of each ribbon and
 precursor, images were processed in a FIJI
 ‘IJMacro_AIRYSCAN_simple3dSeg_ribbons only.ijm’ as previously described
 (Wong et al., 2019). Here each Airyscan z-stack was max-projected.
 Background was subtracted from each projection using rolling-ball
 subtraction. A threshold was applied to each image, followed by
 segmentation to delineate individual Ribeyeb/CTBP puncta. The watershed
 function was used to separate adjacent puncta. A list of 2D objects of
 individual ROIs (minimum size filter of 0.002 μm<sup>2</sup>)
 was created to measure the 2D areas of each Ribeyeb/CTBP puncta. Areas for
 all Ribeyeb/CTBP puncta within each neuromast were then exported as a csv
 spreadsheet. For comparisons, all fixed images analyzed in FIJI were
 imaged and processed using the same parameters.
          To quantify the mean intensity of acetylated-a-tubulin
 after overnight nocodazole or taxol treatments, 20 slices centered on the
 hair cells were max-projected in FIJI. An ROI was draw around the hair
 cells, and this ROI was used to measure the mean intensity of the
 acetylated-a-tubulin label in each neuromast.
 <strong>Confocal imaging and <em>in vivo</em>
 analysis of ribbon numbers in developing zebrafish hair
 cells</strong> For counting ribbon numbers in
 developing and mature hair cells (Figure 1), double transgenic
 <em>myo6b:riba-TagRFP</em> and
 <em>myo6b:YFP-tubulin</em> larvae at 2 and 3 dpf were imaged.
 Transgenic larvae were pinned to a Sylgard-filled petri dish in E3 media
 containing 0.03 % tricaine and imaged 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, at,
 16x averaging, and 0.05 µm/pixel. Z-stacks of whole neuromasts including
 the kinocilium were acquired in a top-down configuration using 488 and 561
 nm lasers. The 488 nm laser along with a transmitted PMT (T-PMT) detector
 was used to capture the kinocilial heights. For
 quantification of ribbon numbers at different developmental stages (Figure
 1), a custom-written Fiji macro “Live ribbon counter” was used to
 batch-process the z-stacks. The red channel (Riba-TagRFP) of each z-stack
 was thresholded (threshold value = 97). Watershed was applied to the
 thresholded stack to separate ribbons near each other. The resulting mask
 from the thresholding and watershedding was applied to the original red
 channel. The number of ribbons was then counted using ‘3D Objects Counter’
 plugin (Threshold = 1, min size = 0, max size = 183500). The counted
 objects were merged with the green channel (YFP-tubulin). Each z-stack was
 visually inspected to determine the localization of the ribbons. Ribbons
 below the nucleus were classified as ‘basal’ and the rest as ‘apical’. The
 number of apical and basal ribbons were counted in each hair
 cell. To classify the developmental stage of each hair
 cell (Figure 1), the height of the kinocilium was used. The number of
 z-slices between the kinocilium tip and base was determine and multiplied
 by the z-slice interval (0.425 µm) to get the kinocilium height. Hair
 cells with heights &lt; 1.5 µm were classified as ‘Early’, hair cells
 with heights 1.5-10 µm were classified as ‘Intermediate’ and with heights
 10-18 µm were classified as ‘Late’. Hair cells with heights &gt; 18 µm
 were considered ‘Mature’. <strong>Confocal
 imaging and <em>in vivo</em> tracking EB3-GFP dynamics in
 zebrafish</strong> Transgenic
 <em>myo6b:EB3-GFP</em> larvae at 2-3 dpf were mounted in 1 %
 LMP agarose containing 0.03 % tricaine in a glass-bottom dish. Larvae were
 imaged on an inverted Zeiss LSM 780 (Zen 2.3 SP1) confocal microscope
 using a 63x 1.4 NA oil objective lens. For timelapses, confocal z-stacks
 of partial cell volumes (3.5 µm, 7 z slices at 0.5 µm z interval) with an
 0.07 µm x-y pixel size were taken every 7 s for 15-30 min.
 The EB3-GFP timelapses were registered in FIJI using the plugin
 “Correct 3D drift” (Parslow et al., 2014), max-projected, and then tracked
 in 2D in Imaris. For spot detection, we used an estimated xy diameter of
 0.534 µm with background subtraction. The detected spots were filtered by
 ‘Quality’ using the automatic threshold. The timelapses were visually
 checked to make sure the spot detection was accurate. For the tracking
 step, the ‘Autoregressive motion’ algorithm was used, with a maximum
 linking distance of 1 µm and a maximum gap size of 3 frames. To ensure
 accurate track detection, short tracks were removed by filtering for the
 number of spots in a track (&gt; 5) and track displacement length
 (&gt; automatic threshold). To calculate the track
 angles relative to the cell base, we used cells that lie horizontally so
 we only need to consider the angles in the xy place. In Imaris, the tracks
 in each hair cell were selected and exported separately. Using the start
 and end position co-ordinates of the exported tracks, we calculated track
 angles in MATLAB using custom written code called, “EB3 track angle”. The
 angle of each hair cell was measured in Imaris. The final track angle
 distribution plotted was obtained by measuring the difference between each
 track angle and the angle of the hair cell.          
 To create movies of EB3-GFP tracks in Movie S1, the FIJI plugin “Correct
 3D drift” was applied. Z-stacks were then max-projected tracks were
 detected using the FIJI plugin TrackMate (Parslow et al., 2014; Tinevez et
 al., 2017). For Movie S1, the LoG detector in TrackMate was used with an
 estimated object diameter of 0.6 µm, a quality threshold of 8, using a
 median filter and sub-pixel localization. The Linear Assignment Problem
 (LAP) tracker was selected using frame to frame linking max distance of 1
 µm, a track segment gap closing max distance of 1 µm and max frame gap of
 2 µm. Tracks were colored by Track index.  For viewing tracks over time,
 tracks were displayed as “Show tracks backwards in time” with a fade range
 of 5 time-points. To create color-coded temporal map of EB3-GFP tracks
 over a short time window (21 s, Figure 2C-D), the FIJI Hyperstack plugin
 Temporal-Color code was used with the 16 colors LUT.
 <strong>Confocal imaging and <em>in vivo</em>
 analysis of ribbon numbers after short-term pharmacological treatments in
 zebrafish</strong> For counting ribbons after 3-4
 hr drug treatment, transgenic zebrafish expressing
 <em>myo6b:riba-TagRFP</em> and
 <em>myo6b:YFP-tubulin</em> at 2 dpf were examined. Transgenic
 larvae were mounted in 1 % low melt agarose in E3 media containing 0.03 %
 tricaine and one of the following: 500 nM nocodazole, 25 µM taxol or 0.1 %
 DMSO (Control). An inverted Zeiss LSM 780 (Zen 2.3 SP1) confocal
 microscope with Airyscan, along with a 63x NA 1.4 oil objective lens.
 Z-stacks encompassing the entire neuromast were acquired every or 0.18 µm
 with an 0.04 µm x-y pixel size and Airyscan autoprocessed in 3D.
 To quantification of ribbon numbers before and after 3-4 hr
 nocodazole and taxol treatment or in <em>kif1aa</em> F0
 crispants (Figure 6, Figure 6-S1), the custom-written Fiji macro “Live
 ribbon counter” described above was used to batch-processed the z-stacks.
 The red channel (Riba-TagRFP) of each z-stack was thresholded (threshold
 value = 28) and segmented (watershed). The resulting mask was applied to
 the original red channel. The number of ribbons was then counted using ‘3D
 Objects Counter’ (threshold = 1, min size = 0, max size = 183500). The
 counted objects were merged with the green channel (YFP-tubulin). Each
 z-stack was visually inspected to make sure the objects counted were
 within hair cells. The number of ribbons per neuromast was determined and
 the difference in numbers pre- and post-drug treatment was
 plotted. <strong>Confocal imaging and
 <em>in vivo</em> tracking of ribbons </strong>
 To visualize ribbon precursor movement, timelapses of double
 transgenic <em>myo6b:riba-TagRFP</em> and
 <em>myo6b:YFP-tubulin</em> larvae at 2 dpf were imaged. For
 pharmacological treatments, transgenic larvae were mounted in 1 % low melt
 agarose in E3 media containing 0.03 % tricaine and one of the following:
 500 nM nocodazole, 25 µM taxol or 0.1 % DMSO (Control) in a glass-bottom
 dish. Double transgenic <em>kif1aa</em> crispants and
 uninjected controls were mounted in 1 % low melt agarose in E3 media
 containing 0.03 % tricaine in a glass-bottom dish. Larvae were imaged on
 an inverted Zeiss LSM 780 or an upright LSM 980 confocal microscope with
 Airyscan using a 63x 1.4 NA oil objective lens. Airyscan z-stacks of
 partial cell volumes (~3 µm, 15-20 z-slices using 0.18 µm z-interval and
 an 0.04 µm x-y pixel size) were taken on the LSM 780 every 50-100 s for
 30-70 min. Faster LSM 980 Airscan z-stacks of partial cell volumes (~2-3.5
 µm, 12-20 z-slices using 0.17 µm z interval and an 0.04 µm x-y pixel size)
 were taken every 3-20 s for 5-40 minutes. Airyscan timelapses were
 autoprocessed in 3D. In addition, we acquired a subset of LSM 780 Airscan
 z-stacks every 5-8 min for 30-100 min to capture fusion events more for
 clearly for Movies S9-11. The longer timelapses
 acquired on the Zeiss LSM 780 were registered using the FIJI plugin
 “Correct 3D drift”. Drift corrected timelapses were then tracked in 3D in
 Imaris using spot detection with estimated xy diameters of 0.427 µm (with
 background subtraction). The spots were filtered based on ‘Quality’, with
 thresholds between 3-8, chosen after visual inspection of the detected
 spots. For tracking, the ‘Autoregressive motion’ algorithm was used with a
 maximum linking distance of 1.13 µm and a maximum gap size of 1 frame.
 Tracks with number of spots &lt; 5 were not included. Using the Track
 displacement length filter in Imaris, the number of tracks with Track
 displacement length &gt; 1 µm were counted and divided by the total
 number of tracks to get the fractions plotted in Figure 7. For the mean
 squared displacement (MSD) analysis, the xyzt co-ordinates were exported
 in ‘csv’ format for all tracks in a timelapse. The MSD analysis was done
 using the prewritten MATLAB class MSDanalyzer (Tarantino et al., 2014). MSDanalyzer calculates the mean squared displacement for each track, curve-fits the MSD vs time, and provides the value of the exponent ( . The first 25 % of the MSD vs time graph was used for curve-fitting. Tracks with the number of spots &lt; 10 were removed to ensure accuracy of the MSD analysis. Fusion events between ribbons and precursors were scored manually in these timelapses.
description: # Microtubule networks in zebrafish hair cells facilitate presynapse
 transport and fusion during development Dataset DOI:
 [10.5061/dryad.crjdfn3gg](10.5061/dryad.crjdfn3gg) ## Description of the
 data and file structure Paper associated with dataset: Elife (2024)
 [https://doi.org/10.7554/eLife.98119.1](https://doi.org/10.7554/eLife.98119.1) This dataset includes raw data collected at the NIH/NIDCD: Figure 1_Figure 1-S2 Figure 2 Figure 3 Figure 4\_ Figure 4-S1\_ Figure 4-S2 Figure 5 Figure 6 Figure 7_Figure7-S1 Figure 8_Figure8-S1 Figure 1-S1 Figure 2-S1 Figure 2-S2 Figure 3-S1 Figure 6-S1 Figure 6-S2 Movies S1-S11 This dataset includes live imaging experiments that monitor microtubules or presynapse/ribbon movements in zebrafish posterior lateral-line hair cells (D1, M1I,L1-L5) in: wild type, *kif1aa* germline mutants, *kif1aa* crispant mutant animals and wild type animals treated with nocodazole or taxol (Figure 1,2,3,6,7,8 Figure 1-S2,2-S2,3-S1,6-S2,7-S1,8-S1 Movies S1-11).  In addition, this dataset includes immunostaining experiments that examine microtubules, presynapses/ribbons or complete synapses in zebrafish posterior lateral-line hair cells (D1,L1-L5) in: wild type, *kif1aa* germline mutants, and wild type animals treated with nocodazole or taxol (Figure 4,5 Figure 1-S1,2-S1,4-S1,4-S2). Lastly this dataset includes fluorescent PCR ABI traces used to genotype *kif1aa* crispant mutant animals (Figure 6-S1). Folders included in this submission have the following names: RAW_DATA_Figure 1_Figure 1-S2 RAW_DATA_Figure 2 RAW_DATA_Figure 3 RAW_DATA_Figure 4\_ Figure 4-S1\_ Figure 4-S2 RAW_DATA_Figure 5 RAW_DATA_Figure 6 RAW_DATA_Figure 7_Figure 7-S1 RAW_DATA_Figure 8_Figure 8-S1 RAW_DATA_Figure 1-S1 RAW_DATA_Figure 2-S1 RAW_DATA_Figure 2-S2 RAW_DATA_Figure 3-S1 RAW_DATA_Figure 6-S1 RAW_DATA_Figure 6-S2 RAW_DATA_Movies S1-S11 In addition, the following macros and scripts were used to process the data in these folders: calculateMSD.m check_tracklength.m FragAnalysis.R IJMacro_AIRYSCAN_simple3dSeg_ribbons only.ijm Live ribbon counter.ijm modified_loglogfit.m Trackdisplacement_angles.m tracksCSVtoCell.m Usage notes: The following is a list of the different data types by file suffix included in this repository along with brief descriptions for how to work with them **.xlsx**: Data in Excel format that can be viewed and manipulated with programs like LibreOffice Calc or imported into Google Sheets. **.czi**: Data from Carl Zeiss microscopes can be opened using FIJI. **.nd2**: Data from Nikon microscopes can be opened using FIJI. **.avi**: Movie files can be opened using FIJI or VLC media player. **.ijm**: FIJI programing macro files can be opened and used in FIJI. **.m:** An .m file is a script[ ](https://techterms.com/definition/script)written in Matlab, a programming language used for statistical analysis and graphing purposes. It contains code that can be executed within the Matlab software environment. It can be viewed using text editor. **.R:** An R file is a script written in R, a programming language used for statistical analysis and graphing purposes. It contains code that can be executed within the R software environment. It can be viewed using text editor. **.fsa**: fragment analysis data files, that can be opened with Peak Scanner which is available from the Thermo Fisher website. In all Excel sheets “n/a” denotes empty cells. Outlined below is information pertaining to each of these Data folders. **RAW_DATA_Figure 1_Figure 1-S2** Acquisition of images to count ribbon numbers and determine cell stage Summary of folder contents: This folder contains 3 Zeiss z-stack .czi files, 8 Nikon z-stack .nd2 files and 1 excel file. Channel 1= *YFP-tubulin*, Channel 2 = *ctbpa-tagRFP* for czi files Channel 1= *YFP-tubulin*, Channel 2 = DIC, Channel 3 = *ctbpa-tagRFP* for nd2 files The 3 Zeiss z-stack .czi files were max-projected and cropped in FIJI to create example images in Figure 1D and F-I: The 8 z-stack .nd2 images were used to quantify ribbon numbers at different stages. The 8 nd2 files were analyzed for kinocilial heights and ribbon counts in FIJI. For each hair cell, the height of the kinocilium was measured using the Transmitted light channel by counting the number of slices between the tip and base of the kinocilium and multiplying it with the z slice interval (0.425 µm). Hair cells with heights &lt; 2.5 µm were classified as ‘Early’, hair cells with heights &gt; 2.5 and &lt; 10 µm were classified as ‘Intermediate’ and with heights &gt; 10 µm and &lt; 18 µm were classified as ‘Late’. Hair cells with heights &gt; 18 µm were considered ‘Mature’. The total number of ribbons in each cell was obtained using the FIJI macro: Live ribbon counter.ijm. To determine the ‘apical’ or ‘basal’ localization of the ribbons in the hair cell, the counted ribbons were visually inspected and those below the nucleus were classified as ‘basal’, the rest as ‘apical’.  The excel 073020 ribbon analysis by cell_FINAL.xlsx file contains:  Sheet 1, “Quantification by file” summarizes data by filename and each hair cell in each image file. The kinocilium height for each hair cell is in Column B. Total number of ribbons identified for each hair cell obtained using the “Live ribbon counter.ijm” is listed in Column C. The total number of apical ribbons is in Column D, and basal ribbons Column E. Sheet 2, “Quantification by stage” lists the same data as in Sheet 1, except it is sorted by kinocilial height. The data in Sheet 2 was plotted and shown in Figure 1E and Figure 1-S2A-C.   **RAWDATA_Figure 2** Acquisition of timelapses to quantify microtubule growth in hair cells Summary of folder contents: This folder contains 8 Zeiss czi timelapses, and 1 excel file. Channel 1= *EB3-GFP* for the czi files The 8 .czi files were used to quantify and visualize microtubule growth and direction: All EB3-GFP timelapses were registered in FIJI using the plugin “Correct 3D drift”.  The file “042822_F1_L2_EB3-GFP_day2.czi” was processed in FIJI to create the images in Figure 2A-F. Figure 2A is a single time point, max projected in FIJI. In C-D images from single cells are shown overtime. To convert grayscale comets to color overtime in Figure C-D the Hyperstack Temporal color-code option was selected in FIJI and the 16 colors LUT was used. To create the vectors of EB3 tracks in Figure 2B,E and F the EB3-GFP comets were tracked in Imaris as described below. For the remaining .czi files EB3-GFP comets were tracked in Imaris using the following parameters: *Spot detection* Estimated xy diameter of 0.534 µm with background subtraction ‘Quality’ filter using the automatic threshold *Tracking* ‘Autoregressive motion’ algorithm Maximum linking distance of 1.00 µm Maximum gap size of 3 frames. Number of spots in a track &gt; 5 Track displacement length &gt; automatic threshold The tracks in each hair cell in the .czi files were selected and exported separately as csv files and the angle of each hair cell was recorded. Matlab was used to calculate track angles from the position co-ordinates of the tracks. ‘tracksCSVtoCell.m’ reads the exported csv files from Imaris and stores them as a matrix. ‘Trackdisplacement_angles.m’ calculates the angle from start to end of each track. The excel file ‘Track Angles Raw EB3.xlsx’ contains EB3-GFP track angles calculated and displayed in Figure 2G. In Sheet 1 “Tracks and cell angle” each file name is listed. Below each filename are all the cells analyzed for that file name. The “cell angle” and “track angle” are listed for each cell and all EB3-GFP comets tracked for that cell. The difference between the track angle and cell angle is calculated “track to cell” and recorded in this sheet. Values greater that 180 were subtracted from 360 to get a final angle. All “track to cell” angles are combined in Sheet 2 “All track angles summary”. The data in Sheet 2 was plotted in a histogram binned every 20 degrees in Graphpad prism and is shown in Figure 2G.  **RAW_DATA_Figure 3** Acquisition of timelapses to quantify ribbon and precursor movement in lateral line hair cells. Summary of folder contents: This folder contains in total 11 Zeiss czi timelapses, and 2 excel files. Channel 1= *YFP-tubulin*, Channel 2 = *ctbpa-tagRFP* All timelapses were registered in FIJI using the plugin “Correct 3D drift”. After registration, for analyses .czi timelapses were tracked in Imaris using spot detection with estimated xy diameters of 0.427 µm (with background subtraction). The spots were filtered based on ‘Quality’, with thresholds between 3-8, chosen after visual inspection of the detected spots. For tracking, the ‘Autoregressive motion’ algorithm was used with a maximum linking distance of 1.13 µm and a maximum gap size of 3 frames. Tracks with a displacement distance &gt; 1um were filtered in Imaris. For the mean squared displacement (MSD) analysis, the xyzt co-ordinates were exported in ‘csv’ format for all tracks in a timelapse. The MSD analysis was done using the prewritten MATLAB class [MSDanalyzer](https://www.mathworks.com/matlabcentral/fileexchange/40692-mean-square-displacement-analysis-of-particles-trajectories) (Tarantino et al., 2014) “calculateMSD.m, modified_loglogfit.m”. [MSDanalyzer](https://www.mathworks.com/matlabcentral/fileexchange/40692-mean-square-displacement-analysis-of-particles-trajectories) calculates the mean squared displacement for each track, curve-fits the MSD vs time, and provides the value of the exponent (![](file:////Users/kindtks/Library/Group%20Containers/UBF8T346G9.Office/TemporaryItems/msohtmlclip/clip_image002.png). The first 25 % of the MSD vs time graph was used for curve-fitting. Tracks with the number of spots &lt; 10 were removed using the Matlab script “check_tracklength” to ensure accuracy of the MSD analysis. The percent of these tracks with alpha &gt;1 and alpha ≤ 1, these numbers are in the excel file “ctrl_track_directions”, columns K-M and summarized in columns M-O. This data is shown in Figure 3I. Imaris was used to determine the direction (towards the cell apex, base or undetermined direction) and location of tracks (below and above the nucleus) with displacement distances &gt; 1um. For this analysis the trajectory of the tracks was determined by examining the vector of the tracks manually in Imaris. The percent of these track types is shown in Figure 3C. The data used to generate these percentages is listed in the excel file “ctrl_track_directions”, in columns B-I and summarized in columns M-O. The summary percentages were used to create the bar graph in Figure 3K-M. The .czi file F1_L2_yfp-tublaserpoint3_riba-trfplaserpoint2_20sinterval_day2_070822_Airyscan Processing-01.czi was processed in FIJI to show the movement of ribbon precursors along microtubules and is displayed in Figure 3D-E. MSD analysis was also used to analyze the following 2 .czi files: F1_L4_ctrl_yfp-tub_riba-trfp_day2_nointerval_Airyscan Processing.czi F2_L1_ctrl_yfp-tub_riba-trfp_day2_nointerval_Airyscan Processing.czi The excel file, “alpha values 2NM ex” contains 2 sheets, “MSD alpha less than 1” and “MSD alpha greater than 1”. The 15 tracks are listed in the first sheet and 5 tracks are listed in the second sheet. The alpha value is listed for each track, row 4, followed below by the MSD value for each time point for each track. This data was used to create the example plots in Figure 3H.  Tracks detected in Imaris for the following .czi file are shown in Figure 3A-B. F1_L4_ctrl_yfp-tub_riba-trfp_day2_nointerval_Airyscan Processing.czi The following .czi file was z-projected in FIJI and used as an example in Figure 3D-E: F1_L2_yfp-tublaserpoint3_riba-trfplaserpoint2_20sinterval_day2_070822_Airyscan Processing-01.czi **RAW_DATA_Figure 4_Figure 4-S1,S2** Acquisition of images to count and quantify hair cells, synapse and ribbons in our before and after overnight nocodazole or taxol treatment. Summary of folder contents: This folder contains 55 Zeiss z-stack czi files, and 3 excel file. Wild type animals were incubated with 250nM nocodazole or 25uM taxol in E3 or 0.1 % DMSO (control) at day 2 for 16 hrs and fixed at day 3. Larvae were immunolabeled with acetylated tubulin, ribeyeb and pan-Maguk to label hair cell microtubules, presynapses and postsynapses. Lateral line neuromasts L1-L5 and D1 (earNM3) were imaged. 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-pan-MAGUK (IgG1) (Millipore MABN7; 1:500); mouse anti-Ribeyeb (IgG2a) (Sheets et al 2011; 1:10,000) and mouse anti-acetylated-tubulin (IgG2b) (Sigma-Aldrich T7451; 1:5,000). The following secondary antibodies were used at 1:1000: A-21131, A-21143and A-21240, ThermoFisher Scientific). Channel 1= ribeyeb, Channel 2 = Acetylated tubulin and Channel 3 = pan-Maguk for the czi files The following .czi files were used to quantify hair cell, synapse and ribbon number and size in the nocodazole experiments: Airyscan images were processed to extract ribbon (presynapse) areas in FIJI using the following macros: IJMacro_AIRYSCAN_simple3dSeg_ribbons only.ijm with a minimum area of 0.001. Hair cell and complete synapse counts were scored manually.   The excel file “taxol overnight analysis” contains the quantifications. In the Sheet “Quantification per file” column A lists the files names for DMSO control and taxol treated  fish. Column B listed the number of hair cells per image, column C the number of complete synapses per hair cell, and column D, the number of ribbons and precursors per hair cells (ribbons &lt; 0.1um) per image. This data is plotted in Figure 4-S2E-G. The sheet “Histogram areas” contains the area of all ribbons for all images in DMSO control and taxol treated hair cells. These areas were used to plot the distribution of areas in Figure 4-S2H-I. The excel file “nocodazole overnight analysis” contains the quantifications. In the Sheet “Quantification per file” column A lists the files names for control and nocodazole treated  fish. Column B listed the number of hair cells per image, column C the number of complete synapses per hair cell, and column D, the number of ribbons and precursors per hair cells (ribbons &lt; 0.1um) per image. This data is plotted in Figure 4E-G. The sheet “Histogram areas” contains the area of all ribbons for all images in DMSO control and nocodazole treated hair cells. These areas were used to plot the distribution of areas in Figure 4H-I. The excel file “noco_DMSO_on quant” contains the mean intensity measurements of acetylated tubulin in hair cell after nocodazole or taxol compared to control. For this analysis, files were opened in FIJI and 20 planes containing the hair cells were max-projected. An ROI was drawn around the hair cells and the mean intensity in the acetylated tubulin channel was detected. The sheet “noco” lists the filename analyzed in column A, the planes max-projected in column B and the mean intensity measured in column C. The sheet “taxol” lists the filename analyzed in column A, the planes max-projected in column B and the mean intensity measured in column C. This data is plotted in Figure 4-S1A-B.   The following images: 021621_250nM_noco_d3_cs1_F5_L2-Airyscan Processing-05.czi 021421_DMSO_d3_F3_L3-Airyscan Processing-12.czi Were max-projected in FIJI and used as examples in Figure 4A-C. The following images: 100121_25uMtaxol_cs1_r1_F6_earNM3_ribb_acetub_magik-Airyscan Processing-09.czi 100121_control_cs1_r2_F11_earNM3_ribb_acetub_magik-Airyscan Processing-11.czi Were max-projected in FIJI and used as examples in Figure 4-S2A-C. **RAW_DATA_Figure 5** Acquisition of images to count and quantify hair cells, synapse and ribbons in in kif1aa germline mutants at day3 Summary of folder contents: This folder contains 2 subfolders that contain 26 Zeiss z-stack czi files, and 1 excel file. Sibling and *kif1aa* mutant larvae were fixed at day 3. Larvae were immunolabeled with Myosin7a, ribeyeb and pan-Maguk to label hair cells, presynapses and postsynapses. Lateral line neuromasts L1-L5 were imaged. Information on immunostaining: Sibling and *kif1aa* mutant 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-Myosin7a (Proteus 25-6790; 1:1000); mouse anti-Ribeyeb (IgG2a) (Sheets et al 2011; 1:10,000) and mouse anti-Maguk (IgG1) (Millipore MABN7; 1:500).  The following secondary antibodies were used at 1:1000: A-11008, A-21133 and A-21235, ThermoFisher Scientific. Channel 1= Myosin7a, Channel 2 = Ribeyeb and Channel 3 = pan-Maguk for the czi files The following .czi files were used to quantify hair cell, synapse and ribbon number and size in the kif1aa mutants compared to siblings. After imaging fish were genotyped to detect the complex INDEL that destroys a BslI restriction site in exon 6. Genotyping was accomplished using standard PCR and Bsl1 restriction enzyme digestion. *Kif1aa* genotyping primers used were: *kif1aa*_FWD 5’-AACACCAAGCTGACCAGTGC-3’ and *kif1aa*_REV 5’-TGCGGTCCTAGGCTTACAAT-3’. Airyscan images were processed to extract ribbon (presynapse) areas in FIJI using the following macros: IJMacro_AIRYSCAN_simple3dSeg_ribbons only.ijm with a minimum area of 0.001. Hair cell and complete synapse counts were scored manually. The excel file “kif1aa synapses analysis” contains the quantifications. In the Sheet “Quantification per file” column A lists the files names for sibling control and kif1aa germline mutant fish. Column B listed the number of hair cells per image, column C the number of complete synapses per hair cell, and column D, the number of ribbons and precursors per hair cells (ribbons &lt; 0.1um) per image. This data is potted in Figure 5E-G. The sheet “Histogram areas” contains the area of all ribbons for all images in sibling control and kif1aa germline mutant hair cells. These areas were used to plot the distribution of areas in Figure 5H-I. The following images: Slide2_cs2_r1_F5_L5_050621-Airyscan Processing-10.czi Slide2_cs2_r1_F3_L2_050621-Airyscan Processing-05.czi Were max-projected in FIJI and used as examples in Figure 5A-C. **RAW_DATA_Figure 6** Acquisition of z-stacks to quantify ribbon and precursor counts before and after a 4 hr treatment with nocodazole, or taxol in lateral-line hair cells at 2 dpf. Summary of folder contents: The main folder contains 2 main subfolders “Noco raw data” and “Taxol raw data” and 1 excel files. In addition, each of these subfolders contains 2 folders, a dmso control folder and treatment group (noco to taxol) folder. Each of these folders contains images capture at time = 0 and time = 4 hrs. In total there are 91 Zeiss z-stack .czi files in these folders. Information on drug treatment: Zebrafish animals expressing *myo6b:ctbpa-tagRFP* and *myo6b:YFP-tubulin* at day 2 were embedded in low melt agarose containing 500nM nocodazole or 25uM taxol or 0.1 % DMSO (control) for and imaged immediately. The same samples were reimaged 4 hrs later.  Channel 1= *YFP-tubulin*, Channel 2 = *ctbpa-tagRFP*  for the czi files The total number of ribbons in each neuromast at each time point was obtained using the FIJI macro: Live ribbon counter.ijm. The total number of ribbons was divided by the number of hair cells to yield number of ribbons per hair cells. The number of ribbons at T = 0 was subtracted from the number of ribbons at T = 4 hrs to obtain the change in ribbons during this time course. Ribbon and precursor counts for these .czi files are summarized the excel file “4 hr taxol noco data summary”. In sheet 1, “total RibA-tagRFP puncta” the file names for each neuromast are listed for T = 0 and T = 4hrs for each treatment group. Followed by the file names, the total number of ribbons detected using the Live ribbon counter are listed at T = 0 and T = 4hrs. Finally, the “change” in ribbon counts is listed for each neuromast. This data was used to create the plot in Figure 6G. The following images: 500nmolNoc_F1_L2_ribA-tRFP_yfp-tub_d2_Airyscan Processing.czi 4hr_500nmolNoc_F1_L2_ribA-tRFP_yfp-tub_d2_Airyscan Processing.czi F1_L3_yfp-tub_ribA-tRFP_25uMtaxol_day2_Out.czi t4_F1_L3_yfp-tub_ribA-tRFP_25uMtaxol_day2_Out.czi F3_L5_yfp-tub_ribA-tRFP_ctrl_day2_Out.czi t4_F3_L5_yfp-tub_ribA-tRFP_ctrl_day2_Out.czi Were max-projected in FIJI and used as examples in Figure 6A-F. **RAW_DATA_Figure 7_Figure 7-S1** Acquisition of timelapses to quantify ribbon precursor movement in control, taxol treated, nocodazole treated or KIF1aa deficient lateral-line hair cells. Summary of folder contents: This main folder contains 3 subfolders “kif1aa tracking”, “noc tracking” and “taxol tracking”, and 2 excel files. In addition, each of these subfolders contains 2 folders, a control folder and a mutant or treatment group folder. In total there are 49 Zeiss z-stack .czi files in these folders. Transgenic fish expressing *myo6b:ctbpa-tagRFP* and *myo6b:YFP-tubulin* in wildtype or kif1aa crispants, nocodazole or taxol treated animals were imaged at day 2. Neuromasts L1-L4 of the posterior lateral line were examined. Information on creating *kif1aa* crispants: Zebrafish transgenic lines expressing *myo6b:ctbpa-tagRFP* and *myo6b:YFP-tubulin* newly fertilized embryos were injected with crispr guide RNAs (gRNAs) to lesion the *kif1aa* gene. We injected the following *kif1aa* gRNAs: 5’-GTGCGAGAACATCCGTTGCT(AGG)-3’ and 5’-AGAATACCTAGCCTTATTCC(CGG)-3’, along with Cas9 protein. Information on drug treatment: Zebrafish animals expressing *myo6b:ctbpa-tagRFP* and *myo6b:YFP-tubulin* at day 2 were mounted in low melt agarose containing with 500nM nocodazole or 25uM taxol in E3 or 0.1 % DMSO (control) for 30 min prior to imaging. *Kif1aa* crispants and controls were mounted in low melt agarose in E3. Channel 1= *YFP-tubulin*, Channel 2 = *ctbpa-tagRFP* for the czi files Timelapses were registered in FIJI using the plugin “Correct 3D drift”. After registration ribbons were tracked in Imaris using spot detection with estimated xy diameters of 0.427 µm (with background subtraction). The spots were filtered based on ‘Quality’, with thresholds between 3-8, chosen after visual inspection of the detected spots. For tracking, the ‘Autoregressive motion’ algorithm was used with a maximum linking distance of 1.13 µm and a maximum gap size of 3 frames. Using the Track displacement length filter in Imaris, the number of tracks with Track displacement length &gt; 1 µm were counted and divided by the total number of tracks to get the fractions of plots with displacement length &gt; 1 µm. Fusion events between ribbons and precursors were scored manually in these timelapses. For the mean squared displacement (MSD) analysis, the xyzt co-ordinates were exported in ‘csv’ format for all tracks in a timelapse. The MSD analysis was done using the prewritten MATLAB class [MSDanalyzer](https://www.mathworks.com/matlabcentral/fileexchange/40692-mean-square-displacement-analysis-of-particles-trajectories) (Tarantino et al., 2014) “calculateMSD.m, modified_loglogfit.m”. [MSDanalyzer](https://www.mathworks.com/matlabcentral/fileexchange/40692-mean-square-displacement-analysis-of-particles-trajectories) calculates the mean squared displacement for each track, curve-fits the MSD vs time, and provides the value of the exponent (![](file:////Users/kindtks/Library/Group%20Containers/UBF8T346G9.Office/TemporaryItems/msohtmlclip/clip_image002.png). The first 25 % of the MSD vs time graph was used for curve-fitting. Tracks with the number of spots &lt; 10 were removed used the Matlab script “check_tracklength” to ensure accuracy of the MSD analysis. This data extracted in contained in the excel file “noco taxol_kif1aa_tracking_MSDs and displacement”. Sheet 1, “MSD and displacement” lists the filename (column A), time interval (column B), timepoints acquired (column C), fraction of tracks with alpha &gt;1 (column D), fraction of tracks with displacement &gt; 1um (column E). The data in column D and E is plotted in Figure 7C-H. The second excel file “taxol noc alpha histrogram analysis” contains all the MSD values for all tracks for nocodazole, taxol and the linked controls. In sheet 1, “All MSDs” columns A-C list all MSDs detected for this dataset. Columns D-F list all MSD with values greater than 1. This data is plotted in Figure 7-S1A-B. \**RAW_DATA_Figure 8_Figure 8-S1 ** Acquisition of timelapses to quantify ribbon precursor fusion and fusion events in control, taxol treated, nocodazole treated or KIF1aa deficient lateral-line hair cells. Summary of folder contents: This main folder contains 4 subfolders  “data for Figure examples”, “kif1aa tracking”, “noc tracking” and “taxol tracking”, and 2 excel files. In addition, each of these subfolders contains 2 folders, a control folder and a mutant or treatment group folder. In total there are 56 Zeiss z-stack .czi files in these folders.  Transgenic fish expressing *myo6b:ctbpa-tagRFP* and *myo6b:YFP-tubulin* in wildtype or kif1aa crispants, nocodazole or taxol treated animals were imaged at day 2. Neuromasts L1-L4 of the posterior lateral line were examined. Information on drug treatment: Zebrafish animals expressing *myo6b:ctbpa-tagRFP* and *myo6b:YFP-tubulin* at day 2 were mounted in low melt agarose containing with 500nM nocodazole or 25uM taxol in E3 or 0.1 % DMSO (control) for 30 min prior to imaging. *Kif1aa* crispants and controls were mounted in low melt agarose in E3.  Channel 1= *YFP-tubulin*, Channel 2 = *ctbpa-tagRFP* for the czi files Timelapses were registered in FIJI using the plugin “Correct 3D drift”. After registration fusion events were counted manually. The number of fusions counted is plotted in the excel file “Fusion counts”. Here column A contains the file name, and column B contains the number of fusion events scored for each timelapse. The following files in data for Figure examples: F1_L3_Airyscan Processing.czi F1_L2try2_Airyscan Processing.czi Were registered in FIJI using the plugin “Correct 3D drift”. After registration files were max projected and cropped to show ribbon fusion events in Figure 8A-B (F1_L3_Airyscan Processing.czi) and Figure 8-S1A-B (F1_L2try2_Airyscan Processing.czi). The file “F1_L2try2_Airyscan Processing.czi” was further processed in FIJI. Ribbons undergoing fusion were thresholded. The area of the 2 ribbons (within this threshold) before and after ribbon fusion for 2 events in this timelapse were plotted in Figure 8-S1. The areas for these events is in the excel file “area changes with fusion”. In the sheet “Areas”, column B contains the area calculated in pixels, column C contains the areas in um^2. **RAW_DATA_Figure 1-S1** Acquisition of images to count and quantify ribbons in our *myo6b:ctbpb-mcherry* transgenic line. Summary of folder contents: This folder contains 24 Zeiss z-stack czi files, and 1 excel file. Wild type or transgenic lines expressing *myo6b:ctbpa-tagRFP* were fixed at day 5. Larvae were immunolabeled with pan-CTBP to label presynapses and pan-Maguk to label postsynapses. 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-pan-MAGUK (IgG1) (Millipore MABN7; 1:500); and mouse pan-CTBP (IgG2a) (Santa Cruz sc-55502; 1:1,000). The following secondary antibodies were used at 1:1000: A-21240 and a A-21131, ThermoFisher Scientific). Channel 1= pan-CTBP, Channel 2 = *ctbpa-tagRFP* or nothing in wild type, Channel 3 = pan-Maguk for the czi files Airyscan images were processed to extract ribbon (presynapse) areas in FIJI using the following macros: IJMacro_AIRYSCAN_simple3dSeg_ribbons only.ijm with a minimum area of 0.001. Hair cell and complete synapse counts were scored manually. The excel file “control_myo6bribaTagRFP comparison” contains the quantifications. In the Sheet “ribaTagFRP analysis” column A lists the files names for control and MYO6B:ribATagRFP (*myo6b:ctbpa-tagRFP) *transgenic fish. Column B listed the number of hair cells per image, column C the number of complete synapses per hair cell, and column D, the average ribbon area (ribbons &gt; 0.1um) per image. This data is potted in Figure 1-S1C-E. The following images: F8_L3_ctrl_mgk488_ctbp647_day5-Airyscan Processing-27.czi F4_L3_tg_riba-tRFP_mgk488_ctbp647_day5-Airyscan Processing-15.czi Were max-projected in FIJI and used as examples in Figure 1-S1A-B. **RAW_DATA_Figure 2-S1** Acquisition of images to examine microtubule modifications in hair cells. Summary of folder contents: This folder contains 2 Zeiss z-stack czi files. Transgenic fish expressing *myo6b:YFP-tubulin* were fixed at day 5. Larvae were immunolabeled with acetylaed tubulin, tyrosinated tubulin and GFP antibodies. Neuromast D2 of the posterior lateral line was imaged to obtain a side view. 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-Aldrich T7451; 1:5,000) and chicken anti-GFP (Aves labs GFP-1010; 1:1,000) or mouse anti-tyrosinated tubulin (IgG2a) (Sigma-Aldrich MAB1864-I; 1:1,000) and chicken anti-GFP. (Aves labs GFP-1010; 1:1,000). The following secondary antibodies were used at 1:1000: ** **A-11039 and A-21236 (ThermoFisher Scientific). 092122_YFPtub-GFP488_Acetub_647_r1_F4_D2_d5-Airyscan Processing-12.czi Channel 1= GFP (YFP-tubulin), Channel 2 = acetylated tubulin 092122_YFPtub-GFP488_trytub_647_r1_F4_D2_d5-Airyscan Processing-19.czi Channel 1= GFP (YFP-tubulin), Channel 2 = tyrosinated tubulin Both files were max-projected in FIJI and used as examples in Figure 2-S1A-F.  **RAW_DATA_Figure 2-S2** Acquisition of images to examine EB3-GFP localization at kinocilial tips in inner ear (medial crista) and lateral line hair cells. Summary of folder contents: This folder contains 2 Zeiss z-stack czi files. Channel 1= EB3-GFP, Channel 2 = transmitted light for the czi files The following 2 images were acquired: 030923_EB3GFP_F2_D1_zstack.czi 030923_EB3GFP_F2_MC_zstack.czi The 2 Zeiss z-stack .czi files were partially max-projected and cropped in FIJI to create example images in Figure 2-S2A-C.  **RAW_DATA_Figure 3-S1** Acquisition of timelapses to visualize ribbon and precursor movement in lateral line hair cells. Summary of folder contents: This folder contains 3 Zeiss czi timelapses, a folder “filament switching” containing 10 additional Zeiss czi timelapses and 1 excel file. Channel 1= *YFP-tubulin*, Channel 2 = *ctbpa-tagRFP f*or the czi files All timelapses were registered in FIJI using the plugin “Correct 3D drift”. The first 3 timelapses were partially max-projected and cropped to show individual frames in Figure 3-S1A-C. To find and track of ribbon movement in these timelapses the FIJI Plugin Trackmate2 was used. The LoG detector in TrackMate was used with an estimated object diameter of 0.6 µm, a quality threshold of 8, using a median filter and sub-pixel localization. The Linear Assignment Problem (LAP) tracker was selected using frame to frame linking max distance of 1 µm, a track segment gap closing max distance of 1 µm and max frame gap of 2 µm. Tracks were colored in yellow.  These 3 examples were also used to create Movies S5,S6 and S7. In addition, the 10 .czi files in the folder “filament switching” were used to quantify the number of times a ribbon or precursor moved from one filament to another. This information was scored manually, and the counts are in the excel file “filament switching counts”. In this file column A contains the file name, column B the number of switching events per neuromast, column C the number of hair cells in the timelapse, and column contains the number of switching events per hair cell. The data in column B is plotted in Figure 3-S1D. **RAW_DATA_FIGURE 6-S1** Acquisition fluorescent PCR data to genotype *kif1aa* crispants after injection with gRNAs. This folder contains 1 excel file and 2 subfolders, that contain 15 .fsa, 4 .svg files and 4 .csv files.  We genotyped all *kif1aa* F0 crispants to ensure that the gRNAs cut the genomic target robustly using fragment analysis of fluorescent PCR products and the following primers: *kif1aa*_FWD_fPCR 5’-TGTAAAACGACGGCCAGT-AAATAGAGATTCACTTTTAATC-3’ and *kif1aa*_REV_fPCR 5’- GTGTCTT-CCTAGGCTTACAATGCTTTTGG-3’ (Carrington et al., 2015). fPCR fragments were run on a genetic analyzer (Applied Biosystems, 3500XL) using LIZ500 (Applied Biosystems, 4322682) as a dye standard. Any *kif1aa* F0 crispants without robust genomic cutting were not included in analyses. The raw data from the fPCR is the 15 .fsa files in subfolder “raw AB files” was read in the Applied Biosystems Genemapper or Peak Scanner software (Thermo Fisher) to detect the size of the fPCR peak at 357 bp. Peak heights for the 15 .fsa files are listed in the excel file “ABI *kif1aa* crispant genotypes”. Column A lists the files name, column B the genotype, and columns C and D the fragment of interest and its height is listed, which is an intensity measure. This data is plotted in Figure 6-S1E-F. In addition to a smaller peak at 357bp in *kif1aa* crispants, an excess of peaks were present in the crispants, indicative of successful crispr-cas9 cutting. The following .fsa files were further processed in the R program “FragAnalysis.R” to extract the raw fluorescent traces to present as examples in Figure 6-S1A-D: 11152022_Kif_SH_01_A10.fsa 11152022_Kif_SH_03_C10.fsa 11152022_Kif_SH_12_D11.fsa 11152022_Kif_SH_17_A12.fsa The extracted data is found in the folder “R extracted data” and includes a .csv (column A is fragment size detected, column B is the fluorescent intensity at each fragment size, and column C is the fluorescent peaks of the LIZ500 ladder), and an .svg file for each .fsa file analyzed. This extracted data is replotted in Figure 6-S1A-D. This analysis generated the files in subfolder “R extracted data”: **RAW_DATA_FIGURE 6-S2** Acquisition of images to count ribbon numbers in lateral line hair cells over a 4 hour time period in sibling and kif1aa crispants. Summary of folder contents: This folder contains 2 subfolders “control” and kif1aa” and 1 excel file. Each subfolders also contains 2 subfolders with 2 timepoints of Zeiss z-stack .czi files “T=0” and “T=4”. In total there are 48 Zeiss z-stack .czi files. Transgenic fish expressing *myo6b:ctbpa-tagRFP* and *myo6b:YFP-tubulin* in a wildtype or kif1aa crispant were imaged at  day 2. Neuromasts L1-L4 of the posterior lateral line were imaged Information on creating *kif1aa* crispants: Zebrafish transgenic lines expressing *myo6b:ctbpa-tagRFP* and *myo6b:YFP-tubulin* newly fertilized embryos were injected with crispr guide RNAs (gRNAs) to lesion the *kif1aa* gene. We injected the following *kif1aa* gRNAs: 5’-GTGCGAGAACATCCGTTGCT(AGG)-3’ and 5’-AGAATACCTAGCCTTATTCC(CGG)-3’, along with Cas9 protein. Channel 1= *YFP-tubulin*, Channel 2 = *ctbpa-tagRFP f*or the .czi files To determine the total number of ribbons in each neuromast at each timepoint, the FIJI macro: Live ribbon counter.ijm was used. The total number of ribbons was divided by the number of hair cells to yield number of ribbons per hair cells. The number of ribbons at T = 0 was subtracted from the number of ribbons at T = 4 to obtain the change in ribbons during this time course. The excel file “4 hr kif1aa data summary” contains the quantifications. In the Sheet “total RibA-tagRFP puncta” column A and C lists the files names for control and kif1aa crispant fish at T = 0 and T = 4. Columns B  and D lists the number of ribbons per hair cells for each image at T = 0 and T = 4. Column E contains the change in ribbon counts from T = 0 to T = 4. This data is plotted in Figure 6-S2E. The following 3 Zeiss z-stack .czi files were max-projected and cropped in FIJI to create example images in Figure 6-S2A-D: F1_L2_uninjctrl_riba-tRFP_yfp-tub_day2-Airyscan Processing-02.czi t4_F1_L2_uninjctrl_riba-tRFP_yfp-tub_day2-Airyscan Processing-09.czi t0_F5L3_kif1aainj_ribA-tRFP_yfp-tub_day2_Out.czi t4_F5L3_kif1aainj_ribA-tRFP_yfp-tub_day2_Out.czi **RAW_DATA_Movies S1-S11** Acquisition of timelapses to visualize microtubule dynamics and ribbon and precursor movement in lateral line hair cells. Summary of folder contents: This folder contains 12 Zeiss czi timelapses as follows Movie_S1_F1_L2_EB3-GFP_day2.tif Channel 1= *EB3-GFP* The timelapse was registered in FIJI using the plugin “Correct 3D drift”. The timelapse was then max-projected and growing microtubules were tracked in FIJI using the Plugin Trackmate2. The LoG detector in TrackMate was used with an estimated object diameter of 0.6 µm, a quality threshold of 8, using a median filter and sub-pixel localization. The Linear Assignment Problem (LAP) tracker was selected using frame to frame linking max distance of 1 µm, a track segment gap closing max distance of 1 µm and max frame gap of 2 µm. For viewing tracks over time, tracks were displayed as “Show tracks backwards in time” with a fade range of 5 time-points. After tracking the files was converted into an .avi file with jpg compression at 5 fps. Movie S2_F1_L4_uninjctrl_yfp-tub_riba-trfp_day2_nointerval_Out.czi Movie S3_F2_L3_fast_Airyscan Processing.czi Channel 1= *YFP-tubulin*, Channel 2 = *ctbpa-tagRFP* The timelapses were registered in FIJI using the plugin “Correct 3D drift”. After registration, ribbons were tracked in Imaris using spot detection with estimated xy diameters of 0.427 µm (with background subtraction). The spots were filtered based on ‘Quality’, with thresholds between 3-8, chosen after visual inspection of the detected spots. For tracking, the ‘Autoregressive motion’ algorithm was used with a maximum linking distance of 1.13 µm and a maximum gap size of 3 frames. Movies containing tracks were exported from Imaris into movies at 5 fps. Movie S4_F1_L2_yfp-tublaserpoint3_riba-trfplaserpoint2_20sinterval_day2_070822_Airyscan Processing-01.czi Movie S5_F3_L5_yfp-tub_riba-trfp_day2_3sinterval-Airyscan Processing-11.czi Movie S6_ctrl_F1_L4_yfp-tub_ribA-rfp_d2_Airyscan Processing.czi Movie S7_ctrl_DMSO_F1_L1_yfp-tub_ribA-tRFP_day2_Airyscan Processing.czi Movie S8_ctrl_F1_L2_yfp-tub_ribA-tRFP_d2_Airyscan Processing.czi Movie S8_F1_L5_taxol_yfp-tubulin_ribA-tRFP_day2_nointerval_Airyscan Processing.czi Movie S8_noc500nmol_F1_L5_yfp-tub_ribA-rfp_d2_Airyscan Processing.czi Movie S9_F1_L3_Airyscan Processing.czi Movie S10_S11_F1_L2try2_Airyscan Processing.czi Channel 1= *YFP-tubulin*, Channel 2 = *ctbpa-tagRFP* The timelapses were registered in FIJI using the plugin “Correct 3D drift”. Then timelapses were max-projected. Images were cropped and exported as an .avi file with jpeg compressed at 2 or 5 fps. ## Access information Other publicly accessible locations of the data: none, although will be provided upon request from Katie Kindt [katie.kindt@nih.gov](mailto:katie.kindt@nih.gov) Data was derived from the following sources: n/a