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Auditory hair cell centrioles undergo confined Brownian motion throughout the developmental migration of the kinocilium.

Auditory hair cell centrioles undergo confined Brownian motion throughout developmental migration of the kinociliumLepelletier-et-al_additional_info_figure2

Figure Legend: 

(A) Experimental setup. Cochlear explants are let to adhere on a custom matrigel-coated podium (Top). The podium is turned upside down and placed onto two rubber pieces fixed at the bottom of a culture dish (Middle). After relaxation in medium at 37°C (> 5 hours) the podium’s edges are  fixed against the rubber pieces with an adjustable cover, allowing the explant’s surface to be positioned for imaging by video-microscopy (Bottom). (B) Images of the preparation at embryonic day E14.5 + 1 in vitro day, taken from two long time-lapse sequences (3-4 hours, 15-20 min per frame). Pairs formed by the mother (basal body) and daughter centrioles in inner hair cells (IHC) do not change in position nor in orientation relative to the IHC apical surfaces (arrows). Note the slow drift of the tissue reflecting the ongoing extension of the cochlear epithelium at this stage. Scale bars: 2 μm. (C) Images of IHCs with their centriole trajectories (superimposed and in closeups) obtained from short time-lapse sequences (10-15 min, 2 s between frames) at two developmental stages before and after kinocilium migration. Plotted in the lower graphs are the mean-square displacement (MSD) curves computed for each centriole after correction by subtraction of the drift due to cochlear elongation (thick continuous curves), and without correction (thin dashed curves). Note the significantly reduced drift at the later stage (when the elongation of the cochlear epithelium is near complete), reflected by a slower quadratic growth of the MSD. After the drift subtraction the centriole trajectories at both stages display a saturation of their MSD values after a fraction of second, indicating confinement of the centrioles in a region ~ 0.3 µm in diameter at the IHC apical surface. Scale bars: 0.5 µm in the images, 100 nm in the trajectory closeups.

 

 

Journal Reference

Lepelletier L1, de Monvel JB, Buisson J, Desdouets C, Petit C.

Biophys J. 2013 Jul 2;105(1):48-58.

Unité de Génétique et Physiologie de l’Audition, Institut Pasteur, Paris, France.

Abstract
Planar polarization of the forming hair bundle, the mechanosensory antenna of auditory hair cells, depends on the poorly characterized center-to-edge displacement of a primary cilium, the kinocilium, at their apical surface. Taking advantage of the gradient of hair cell differentiation along the cochlea, we reconstituted a map of the kinocilia displacements in the mouse embryonic cochlea. We then developed a cochlear organotypic culture and video-microscopy approach to monitor the movements of the kinocilium basal body (mother centriole) and its daughter centriole, which we analyzed using particle tracking and modeling. We found that both hair cell centrioles undergo confined Brownian movements around their equilibrium positions, under the apparent constraint of a radial restoring force of ∼0.1 pN. This magnitude depended little on centriole position, suggesting nonlinear interactions with constraining, presumably cytoskeletal elements. The only dynamic change observed during the period of kinocilium migration was a doubling of the centrioles’ confinement area taking place early in the process. It emerges from these static and dynamic observations that kinocilia migrate gradually in parallel with the organization of hair cells into rows during cochlear neuroepithelium extension. Analysis of the confined motion of hair cell centrioles under normal and pathological conditions should help determine which structures contribute to the restoring force exerting on them.

Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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