The apico-basally polarized IHC microtubule cytoskeleton is highly acetylated

A Representative confocal yz-projection of an immunohistochemical staining of the MT cytoskeleton in acutely dissected organs of Corti of early postnatal mice (P7). Indicated is the IHC outline (white dashed line), labeled for α-tubulin (yellow), ribbons (RIBEYE, magenta) and IHC context (Calretinin, blue). A1-A3 different axial sections of IHC and MT labeling from A, displaying the MT cytoskeleton localization within the IHCs and in the surrounding tissue. B Representative confocal images and sectioning (B1-3) as in A, but for immunolabeling of acetylated tubulin (green). Please note that in A1-3 and B1-3 the intensity levels of the tubulin channels have been adjusted for optimal visibility. C Immunohistochemical labeling of MT –end binding protein CAMSAP2 (green), and acetylated MT strands (magenta) within IHCs (Calretinin, blue), in acutely dissected organs of Corti (P12). C’ Acetylated tubulin strands reach from the cellular apex into the basolateral synaptic area. C’’ CAMSAP2 labeling is specifically localized in the apical IHC just below the cuticular plate. D Schematic depiction of the mechanical cleaning technique used to remove OHCs, inner pillar cells and phalangeal cells to facilitate unobstructed access to the row of IHCs. Hair cells are labelled for Myo7a (blue), spiral ganglion neurons for β3-tubulin/Tuj1 (green). E-E’ Immunohistochemical labeling of mechanically-cleaned organotypic cultures of the organ of Corti, stained for IHCs (Calretinin, blue), acetylated tubulin (green) and ribbon precursors (RIBEYE, magenta). E’’ Higher magnification single confocal sections of ribbon precursors colocalizing with acetylated MT strands. Scale bars: A-B” & D, 5 μm; C, 50 μm; D’, 2.5 μm; D”, 0.5 μm.

Visualizing MT-based ribbon precursor transport in living IHCs.

A Representative confocal live-cell image of the IHC row of an organotypic culture labeled with SPY555-tubulin, in which the outer hair cells, inner pillar cells and phalangeal cells have been mechanically removed. A’ Higher magnification live-cell image of an exposed IHC, labeled with SPY555-tubulin with surrounding tissue cleaned, expressing RIBEYE-GFP (green). B Three-dimensional reconstruction of live-cell timelapse imaging of the basolateral compartment of a RIBEYE-GFP transduced IHC additionally labelled with SPY555. Single particle tracking of ribbon precursors within the basolateral IHC reveals highly dynamic displacements. Trajectories are color-coded for time. Total imaging time: 40 min. C The majority of traced ribbon precursors were classified as mobile (displacing >1 µm in 30 min). Half of the mobile population could be detected to displace along MTs. D Although static ribbon precursors showed low displacement over time, precursors did undergo moderate spatial fluctuation, leading to a low average track velocity. While both mobile populations showed a considerably higher average velocity than the static precursors, remarkably, the track velocity of precursor displacement independent from MTs was significantly higher than of MT-associated precursors. E Combined plot of the mean squared displacement (MSD) of all MT-associated ribbon precursor trajectories, indicative of multiple types of motion. F Distribution of the exponent α, extracted from the MT-associated precursor tracks, where α=1 equals a diffusive or Brownian motion, α<1 indicates subdiffusion for confined motion, and α >1 directed transport. G, H, I Example trajectories of ribbon precursors in association with the MT cytoskeleton. Three main types of motion could be observed: (G) stop-and-go displacement, including rapid long-distance traversing jumps, as well as intermittent periods of near static behavior, (H) slow continuous, near linear progressive motion in a targeted fashion along the MT strand and (I) confined motion in place but attached to the MT network. Of the three main MT-associated motion types, a three-dimensional representation is plotted (G’, H’, I’), as well as the MSD of the respective trajectories (G’’, H’’, I’’) – please note that individual scales have been adapted for optimal visibility of the respective trajectory. During precursor displacement, we detected significant velocity fluctuations of which representative sample traces are shown per motion subtype (G’’’, H’’’, I’’’). Illustrated examples indicated by consistent coloring and color-coded for time. Values represented as violin plots, with medians and the 25% and 75% interquartile range indicated with solid and dashed lines respectively. Statistical significance: Kruskal-Wallis. ****p<0.0001. N=5, n=8. Scale bars: A-B, 10 μm; A’, B’-C, 2 μm; G-I 1 μm.

Ribbon precursor volume is dynamically modified by bi-directional structural plasticity

A Upper panel: Schematic representation of detected plasticity events using the lineage tracing algorythm (IMARIS). Ribbon precursors could be observed to undergo fusions and fissions. Lower panel: representative example of a lineage tracing graph for all precursors of one IHC, color-coded for time. Total live-cell imaging time: 40 min. As indicated in the upper panel, each horizontal line represents one ribbon precursor at that specific time point. Vertical connections reflect plasticty events. B,C Exemplary confocal live-cell imaging frames, displaying different steps in the plastic process of precursors fusion (B), and fission of one precursor into two (C). B’,C’ Representations of the plasticity event examples in (B) and (C) in simplified style of the lineage tracing graphic. Precursors labeled by viral transduction of RIBEYE-GFP; additonal labeling of the MT cytoskeleton by application of SPY555-tubulin. Scale bars: 1 μm.

Live-cell IHC ribbon precursor dynamics upon pharmacological disruption of the MT cytoskeleton.

A Representative live-cell imaging stills of organotypically-cultured IHCs of Ai32-VC-KI mice, with (ChR2-coupled) YFP expression decorating the IHC membrane, and virally-expressed RIBEYE-tdTomato. Ribbon precursor temporal trajectories are color-coded for time. A’ Graphical representation of the lineage tracing-based ribbon precursor motion over time, illustrating precursor fusion and fission in the cytoplasm. Total imaging time: 40 min. B The velocity of individual precursor particles was slightly reduced upon acute treatment with the MT-destabilizing drug nocodazole (1 μM, 3 h). C The displacement of ribbon precursors over the course of their trajectories was significantly lower upon nocodazole treatment. Displacement length corrected for the duration of the trajectory, calculated as displacement in 1 minute. D,E Interestingly, the frequency of plasticity events within the precursor trajectories was significantly reduced, as precursors were observed to undergo significantly fewer (D) fusion as well as (E) fission events. F The nocodazole-induced reduction in plasticity event frequency resulted in precursors spending an increased percentage of time in individually stable, non-interactive trajectories. On the other hand, the presence of highly dynamic trajectories was reduced. G-G’ Ribbon precursor volume was increased upon acute nocodazole treatment. H The number of ribbon precursors per IHC was not significantly affected by nocodazole treatment. Values represented as violin plots, with medians and the 25% and 75% interquartile range indicated with solid and dashed lines respectively. Statistical significance: Mann-Whitney U. **p<0.01, ****p>0.0001. N=6, n=18. Scale bar: 5 μm.

Analysis of three-dimensional ribbon precursor displacement and directionality of motion.

A Mean square displacement (MSD) of ribbon precursors traced in control conditions (DMSO) and after incubation with nocodazole. N(exp.)=6, n(IHC)=18, n(particles)= 604(DMSO), 462(nocodazole). Nocodazole-induced MT destabilization reduced the MSD. B Assessment of the (an)isometry/asymmetry of precursor motion. C Distribution of the mean track velocity for precursors in vehicle- and nocodazole-treated IHCs. Indicated are the used cutoffs to selectively analyze trajectories with a low (blue) and high velocity (orange) displacement. D MSD analysis of trajectories with a low mean velocity reveals a loss of directed motion upon nocodazole treatment (left panel). Trajectorial asymmetry analysis of slow transport tracks shows a clear directionality for precursors in the vehicle treated condition that is absent in nocodazole-treated IHCs (right panel). n(particles)=40(DMSO), 41(nocodazole). E The MSD of high velocity trajectories shows a moderate reduction in directed transport resulting from nocodazole treatment (left panel), but do not show preferential directionality, as apparent from the lack of trajectorial asymmetry (right panel). n(particles)=295(DMSO), 224(nocodazole)

Kif1a is required for hearing and adequate IHC ribbon synapse volume acquisition.

A Auditory brainstem responses (ABR) of P21-P25 mice carrying the Kif1algdg/lgdg mutation, compared to Wt and heterozygous littermates (Kif1a+/lgdg). Homozygous Kif1algdg/lgdg mutants displayed a moderate ∼10-20 dB increase in ABR thresholds for all tested frequencies, while the heterozygous mice showed intact hearing. n(Wt) = 10; n(Kif1a+/lgdg) =13; n(Kif1algdg/lgdg) = 8. Shown are means ± SD. B Representative confocal maximum projections of acutely-dissected organs of Corti of P21-P25 Wt littermates and Kif1algdg/lgdg mice, immunohistochemically labeled for RIBEYE (magenta), PSD95 (green) and IHC context (Calretinin, blue). C The number of ribbon synapses is comparable between mature Kif1algdg/lgdg mice and Wt littermates. D Ribbon volume of Kif1algdg/lgdg mice is reduced compared to Wt littermates.

Impaired synaptic maturation in developing IHCs of Kif1algdg mutants.

A Representative confocal maximum projections of acutely-dissected organs of Corti from P5 Kif1algdg/lgdg mice, immunohistochemically labeled for RIBEYE (magenta), PSD95(green) and IHC context (Myosin VIIa, blue). B The number of ribbon precursors that localize to the synapse is reduced in Kif1algdg/lgdg mice, whereas the number of cytosolic ribbons remains unaltered. C Ribbon volume in Kif1algdg/lgdg mice is reduced for synaptic as well as cytosolic ribbon precursors. D Representative confocal maximum projections of acutely-dissected organs of Corti from P3 Kif1algdg/lgdgmice, immunohistochemically labeled analogous to (A). E The number of synaptically-engaged and cytosolic ribbon precursors remains unaltered, although a trend towards reduction can be observed in the latter population. F Ribbon volumes in Kif1algdg/lgdgmice show a trend towards reduction for the synaptic population, while the cytosolic ribbon precursor fraction displays reduced volumes. Values represented either as individual datapoints with mean ± SEM, or as violin plots, with medians and the 25% and 75% interquartile range indicated with solid and dashed lines respectively. Statistical significance: Mann-Whitney U. * p<0.05, **p<0.01, ****p>0.0001. P5, N=15, n=30; P3, N=11, n=21. Scale bars: 5 μm.

Experimental paradigm and effects of short-term RIBEYE-GFP overexpression on ribbon count and volumes.

A Wild-type mouse pups were injected with an AAV encoding RIBEYE-GFP at postnatal day P4-6. One day after transduction, organ of Corti explant cultures were prepared and – after additional one to two days in vitro (DIV) – mechanically-cleaned and incubated with the MT dye SPY555-tubulin. B Representative maximum projection of a confocal z-stack showing a transduced IHC, which expresses RIBEYE-GFP (green). Please note the colocalization with the ribbon marker CtBP2 (magenta). C-D Both, ribbon counts (C) and volumes (D) were indistinguishable between RIBEYE-GFP transduced and neighboring non-transduced IHCs, suggesting appropriate integration of the fluorescent construct into endogenous ribbons while not displaying any obvious overexpression artifacts. No statistical significances detected (Mann-Whitney U test). RIBEYE-GFP transduced: N=9, n=9; Control non-transduced: N=12, n=14. Scale bar: 5 μm.

Dilution of nocodazole effects in faster-displacing ribbon precursor populations

A Reproduction of the same dataset as in Figure 5C: Shown is the distribution of the mean track velocity for precursors in vehicle- and nocodazole-treated IHCs. Indicated are the used cutoffs to selectively analyze trajectories with a low (blue) and high velocity (orange) displacement. Color-coded bars indicate the different velocity ranges displayed in B-E. B-E MSD analysis (left panels) and asymmetry assessment (right panels) of trajectories with a mean velocity within the range 0.0056 – 0.0069 μm/s (B), 0.0069 – 0.0088 μm/s (C), 0.0056 – 0.0088 μm/s (D), and below 0.0088 μm/s (E). The inclusion of a moderate-to-fast displacing population of ribbon precursor trajectories dilutes the reducing effect of nocodazole on ribbon precursor displacement and directionality present in the low velocity trajectories (below 0.0056 μm/s, as seen in Figure 5D). B-D Trajectories with a velocity between 0.0056 and 0.0088 μm/s do not appear to be subjected to directed transport in control, nor nocodazole-treated conditions. E Addition of the 0.0056 – 0.0088 μm/s velocity range to the low velocity cutoff range (<0.0056 μm/s) largely eliminates the distinction in 3D displacement and directed transport of ribbon precursors between nocodazole-treated and control IHCs.