Figure 4

Figure 4 Kif1aa mutants enrich less LysoTracker, Vglut3, CSP and Rab3a at the presynapse

A–C, example live image of LysoTracker Red (green) to label synaptic vesicles in neuromasts at 5 dpf in a kif1aa mutant (B) or sibling control (A). The dashed box in each image indicates the hair cells magnified and outlined with a continuous line in C. The dashed box in each image indicates example ROIs of the apical and basal regions used for intensity analysis in D. D, quantification of LysoTracker shows that in kif1aa mutants there is significantly less enrichment at the cell base compared with sibling controls (control: 7.38 ± 1.16; kif1aa: 0.52 ± 0.16, n = 10 kif1aa and control neuromasts, unpaired t test, P < 0.0001). E–G, example immunostain of Vglut3 to label synaptic vesicles in neuromasts at 5 dpf in a kif1aa mutant (F) or sibling control (E). The dashed box in each image indicates the hair cells magnified and outlined with a continuous line in G. H, quantification reveals that Vglut3 is significantly less enriched at the cell base in kif1aa mutants compared with sibling controls (control: 2.24 ± 0.65; kif1aa: 0.45 ± 0.07, n = 12 control and 13 kif1aa neuromasts, unpaired t test, P < 0.0001). I, examples of neuromast hair cells immunolabeled with CSP. Cyan dashed lines indicate CSP label from neighbouring cells. J, quantification of CSP label reveals that in kif1aa mutants, there is significantly less CSP enriched at the cell base (control: 1.86 ± 0.34, kif1aa: 0.58 ± 0.07, n = 10 control and kif1aa neuromasts, unpaired t test, P < 0.0001). K, examples of neuromast hair cells immunolabeled with Rab3a. Residual basal puncta in kif1aa mutants are efferent terminals contacting hair cells that also have high levels of Rab3a. L, quantification reveals that the Rab3a label is significantly less enriched at the cell base (control: 1.57 ± 0.30, kif1aa: 0.63 ± 0.15, n = 9 control and kif1aa neuromasts, unpaired t test, P < 0.0001). The solid lines in the magnified images outline a single hair cell, with the base of the cell at the bottom of the image, and dashed boxes indicate example ROIs of the apical and basal regions used for quantification. n indicates nucleus. Scale bars in A and E = 5 µm and 2 µm in C, G, I, K.