Par3 regulates Rac1 signaling and microtubule organization during planar polarization of auditory hair cells

In the inner ear sensory epithelia, hair bundles atop sensory hair cells are mechanosensory apparati with planar polarized structure and orientation. This is established during development by the concerted action of tissue-level planar cell polarity (PCP) signaling and a hair cell-intrinsic, microtubule-mediated machinery. However, how various polarity signals are integrated during hair bundle morphogenesis is poorly understood. Here, we show that the conserved cell polarity protein Par3 plays a key role in planar polarization of hair cells. Par3 deletion in the inner ear resulted in defects in cochlear length, hair bundle orientation and kinocilium positioning. During PCP establishment, Par3 promotes localized Rac-Pak signaling through an interaction with Tiam1. Par3 regulates microtubule dynamics and organization, which is crucial for basal body positioning. Moreover, there is reciprocal regulation of Par3 and the core PCP molecule Vangl2. Thus, we conclude that Par3 is an effector and integrator of cell-intrinsic and tissue-level PCP signaling. One sentence summary Par3 regulates planar polarity of auditory hair cells


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The sense of hearing and balance is critically dependent on the sensory hair cells of the inner ear. Hair cells 35 are specialized epithelial cells that are polarized along both the apical-basal axis and within the plane of the 36 epithelial cell sheet, referred to as planar cell polarity (PCP). Importantly, the hair bundle, the 37 mechanotransduction organelle atop auditory hair cells, adopts a planar-polarized structure with rows of 38 stereocilia arranged in a staircase-like V shape, and thus is directionally sensitive to mechanical stimuli

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Interestingly, in the maculae, the transcription factor Emx2 has been shown to reverse hair bundle polarity in its . In this study, we found that Par3 regulates PCP but 77 not apical-basal polarity in the OC. Par3 is asymmetrically localized in the OC during PCP establishment.

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Deletion of Par3 disrupted microtubule organization and basal body positioning, leading to hair bundle polarity 79 and orientation defects. Surprisingly, Par3 has distinct localizations from its canonical partner Par6/aPKC and 80 is not required for asymmetric localization of LGN/Gi; instead, we present evidence that Par3 regulates 81 microtubule cortical capture in hair cells through a Tiam1-Rac-Pak axis.

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Par3 is asymmetrically localized in the developing OC

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To investigate the involvement of Par3 in hair cell PCP, we first analyzed Par3 protein localization in the OC at 87 early stages of hair bundle morphogenesis. At embryonic day (E) 16.5 Par3 is localized to apical junctions of 88 hair cells and supporting cells and significantly enriched along the lateral borders of hair cells ( Figure 1A, B).

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By postnatal day (P) 0, when hair cell PCP has been established, Par3 could still be detected around cell 90 junctions, however its distribution no longer showed planar asymmetry ( Figure 1E

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We next analyzed cochlear morphogenesis of Par3 cKO mutants, which were alive at birth but died at P1. The

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Therefore, we conclude that Par3 is not required for PCP establishment in the utricular macula.

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LGN was localized asymmetrically in the hair cell, both in the microvilli-free zone (bare zone) of the apical 57 surface, and on the lateral hair cell cortex ( Figure 6A

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To determine the mechanisms by which Par3 promotes Rac-Pak signaling in the cochlea, we next focused on 74 Tiam1 as a candidate. Tiam1 is a guanine nucleotide exchange factor (GEF) specific for Rac and is highly     . Surprisingly, we found that Par3 is dispensable for asymmetric cortical localization of

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LGN and Gi, in contrast to its classic role in mitotic spindle orientation.

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In addition to acting downstream of the core PCP pathway, Par3 also regulates the asymmetric localization of 75 the core PCP protein Vangl2 in the cochleae, likely by an indirect mechanism. Of note, Par3 has also been 12 shown to regulate subcellular localization of Vangl1/2 during neural tube closure and in cultured epithelia cells 77 (Kharfallah et al., 2017). Thus, Par3 regulates intercellular PCP signaling in diverse developmental processes.

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In the cochlea, Dishevelled-associating protein with a high frequency of leucine residues (Daple) was recently 79 identified as interacting with Gαi, Dishevelled as well as Par3, and therefore may act in conjunction with Par3

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Therefore, for analysis of protein localizations, care was taken to ensure that an equivalent mid-basal region of 34 the cochlea was compared between experimental groups. Experimental data sets were tested for significance 35 using a Student's t-test, and data are presented as mean ± standard deviation of the mean.

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Quantifications were calculated with LI-COR Image Studio™ Software. Data were presented as mean ± 57 standard error of the mean, and P-value calculated using a Student's t-test.                                      were labeled by phalloidin staining (red). All images were taken from the mid-basal region of the cochlea. 60 Arrowheads indicate the pillar cell row and brackets indicate outer hair cell rows. Scale bars: 6 μm.