The Actin Nucleator Cobl Is Critical for Centriolar Positioning, Postnatal Planar Cell Polarity Reﬁnement, and Function of the Cochlea

Proper cochlear hair cell array development and sensory apparatus positioning are achieved by planar cell polarity signaling. Effectors executing proper tissue development and maturation pro-grams are largely unknown. We show that the actin nucleator Cobl is an important effector in postnatal reﬁnement and maintenance of planar cell polarity. these polarity defects coincided with reduced beneath the sensory apparatus and with premature kinocilium retraction. These defects were accompanied by organizational defects of the pericentriolar scaffold that coincided with basal body and centriolar mispositionings. Importantly, the pericentriolar defects observed in Cobl KO mice were demonstrated to be actin polymerization dependent and and the central tip stereocilia of the bundle (the V-shaped bundles usually show indented tips, i.e., do not exhibit a pointed V shape) – e.g., a doubling of the distance may only lead to about 30% in the measurements of the neighboring side of the triangle formed between central ‘‘tip’’ stereocilium, kinocilium and the ﬁrst side stereocilium when tips are broad – these measurements were most useful, as the distance between ﬁrst side stereocilium and kinocilium could always reliably be addressed, whereas sight on the base of the central tip stereocilium is often blocked due to the tip indention of the stereocilia bundle. Analyzed were n = 60 OHCs/row from 4 animals/cochleae each genotype.


INTRODUCTION
Establishment and changes of cell morphology and polarity depend on forces created by actin and microtubule cytoskeletal structures. Early embryonic planar cell polarity (PCP) plays a central role in tissue patterning (Montcouquiol et al., 2003;Curtin et al., 2003). PCP signaling, for example, brings about a preliminary alignment of outer hair cells (OHCs) in the inner ear and of their stereocilia bundles serving as mechanosensors. Auditory perception in mice starts about 2 weeks after birth (Song et al., 2006). Sound-evoked basilar membrane vibrations cause stereociliar deflections and open mechanotransduction channels (Fettiplace and Hackney, 2006).
While microtubule-based ciliary structures are indicative of PCP and also the orientation of OHC stereociliar bundles in the cochlea precisely reflects PCP in the tissue (Kelley et al., 2009;Ezan and Montcouquiol, 2013), little is known about PCP effectors and about postnatal processes subsequent to classical embryonic PCP signaling.
Stereocilia of OHCs are organized as V-shaped bundles inserted into the cortical actin network, the cuticular plate (Tilney et al., 1980;Kitajiri et al., 2010;Szarama et al., 2012). They show tight correlation with the microtubule-based kinocilium intimately associated with the tip of the stereocilia bundle. The kinocilium originates from a mother centriole-derived basal body and may play some guiding role in PCP and stereocilia positioning, as it migrates to the distal (i.e., abneural) hair cell side during embryonic development. Recent work in simplified systems suggested some, albeit not yet fully understood F-actin cross talk with centrosomes and centrioles Carvajal-Gonzalez et al., 2016;Obino et al., 2016).
Here, we demonstrate at the whole-animal level that Cobl (Cordon-Bleu) (Ahuja et al., 2007)-a WH2 domain-based actin nucleator (Qualmann and Kessels, 2009)-is critical for cross talk of microtubule-based and F-actin-based structures in OHCs of the cochlea. Cobl knockout (KO) hereby specifically impaired F-actin structures and functions in the cuticular plate beneath stereocilia and the kinocilium. Cobl KO disrupted the organization of the pericentriolar material (PCM) and its intimate spatial alignments with the kinocilium and the stereocilia bundle. Interestingly, the associated stereociliar positioning defects mostly manifested postnatally, that is, were not related to classical embryonic PCP signaling. Cobl KO phenotypes thereby unveiled postnatal refinements and also revealed that PCP has to be actively maintained during adulthood. Physiological studies uncovered that Cobl-mediated functions in the cochlea are important for the OHC amplifier function in hearing.

Cobl KO Mice Show a Lack of a Specific Cortical Subset of Actin Filaments
Cobl promotes F-actin formation and is critical for dendrite formation and branching in neurons (Ahuja et al., 2007;Hou et al., 2015Hou et al., , 2018. In situ hybridizations of zebrafish larvae suggested an additional role in the auditory system. Strong Cobl signals also occurred in the developing ear ( Figure 1A). In mice, cochlear Cobl expression was detected, too ( Figure 1B). Cobl was prominent in OHCs and enriched in their cuticular plates (Figures 1C and 1D).
To analyze its functional importance, we generated Cobl KO mice. The murine Cobl gene comprises 15 exons (GenBank: NM_172496.3). Database analyses predict that besides the actin nucleator Cobl also a splice variant lacking exons 10-15, that is, all WH2 domains, may exist (CoblDE10-15). Antibodies against the N-terminal part of Cobl (anti-Cobl ARA ;Schwintzer et al., 2011), which would detect both Cobl and the putative truncated variant not representing an actin nucleator, did not yield any immunosignals in samples from up to ten pooled cochleae due to the minimal tissue amounts (not shown). Therefore, these analyses could not exclude that a CoblDE10-15 protein may exist. RT-PCRs could also not rule out that indeed some actin nucleation-incompetent DE10-15 variant may exist, as some weak mRNA trace was obtained with E9 and I9 primers (Figures S1A-S1C).
To eradicate specifically all actin nucleating products of the Cobl gene but not the putative truncated form, which lacks all actin-binding WH2 domains and thus would have functions unrelated to actin nucleation, we targeted exon 11 (Cre/lox). Using mice ubiquitously expressing Cre (Schwenk et al., 1995), we removed exon 11 together with the selection cassette yielding a mouse line with an ubiquitous and constitutive .
RT-PCRs demonstrated that exon 11 deletion affected Cobl mRNA stability in cochlea and brain (Figures 1I and 1J). Western blotting as well as immunofluorescence analyses of cochleae and brain material with antibodies against a C-terminal epitope ( Figure S1A; Haag et al., 2012) confirmed that the actin nucleator Cobl was successfully knocked out ( Figures 1K-1N).
Homozygous Cobl KO mice displayed normal viability during >1 year of monitoring. Body weights of both sexes were unaltered. Litter sizes were significantly reduced when homozygous mice were bred. These impairments, however, did not seem to reflect a critical role of Cobl during embryonic development, as suggested by work in zebrafish (Ravanelli and Klingensmith, 2011;Sch€ uler et al., 2013), because genotypes did not differ from Mendelian distribution (Figures S1D-S1G).
Cobl KO embryos did not display any obvious defects in embryogenesis, in body laterality establishment (data not shown), or in neural tube closure, and did also not lead to exencephaly, which would have been an obvious readout for defects in cerebrospinal fluid movements through the ventricular system (Figures S1H and S1I). Thus, although Cobl is expressed in the floor plate and in the notochord of the neural tube at embryonic day 9.5 (E9.5) (Figures S1J-S1M), as demonstrated before at the mRNA level (Gasca et al., 1995;Carroll et al., 2003), Cobl KO mice did not show any phenotypes reflecting impairments of motile cilia or defects in early development based on the parameters examined.
To address a putative Cobl role in the cochlea, we first examined the F-actin-rich cuticular plates of OHCs ( Figures 1O-1U). Their width increases during cochlear maturation (Szarama et al., 2012). This was not affected upon Cobl . However, specifically areas beneath the sensory apparatus showed a significantly reduced F-actin content in Cobl KO OHCs ( Figures 1R-1U). The overall reduction across the entire line scan was about 10% ( Figure 1R). Interestingly, distribution analyses showed that the F-actin loss occurred specifically in an about 1-mm-thick apical layer underneath OHC stereocilia. In this substereociliar layer, the F-actin loss reached $20% ( Figure 1S).

Altered Organization of PCM in Cobl KO Cochleae
The cell cortex embeds both stereocilia and kinocilium base structures. Three-dimensional (3D) surface models computed from postnatal day 8 (P8) cochleae immunostained for pericentrin, a marker for the PCM surrounding the centrioles at the kinocilium base (Doxsey et al., 1994), showed a dramatically increased volume of the pericentriolar scaffold. In all three OHC rows, it more than doubled (Figures 2A-2E). The sum intensity of the anti-pericentrin labeling was also increased ( Figure 2F). This may reflect an increased pericentrin recruitment in Cobl KO mice, an improved immunodetection upon a loss of PCM compression, or both. Since the density of the anti-pericentrin labeling in the enlarged PCM was decreased ( Figure 2G), PCM organization clearly was impaired. Similar defects-albeit not yet as pronounced-were seen in newborn mice (P2) (Figures S2A-S2G).
In contrast to the defects in PCM organization, the centrioles encased by the PCM (marked by immunolabeling of g-tubulin; Jones et al., 2008) did not show any significant defects ( Figures  S2H and S2I). Cobl KO thus specifically disrupted the organization of the pericentrin-marked PCM around centrioles.

Proper PCM Organization in OHCs Depends on Actin Filament Formation and Ca 2+ /CaM Signaling
To address whether the PCM organization defects observed upon KO of the actin nucleator Cobl reflect a critical role of F-actin formation, we applied 1 mM latrunculin A to freshly dissected cochlear whole mounts of P8 WT animals. Strikingly, this resulted in an increase in PCM volume and sum anti-pericentrin signal ( Figures 2H-2M) as well as in a decrease of anti-pericentrin immunolabeling density ( Figure 2N). Thus, suppression of F-actin formation by latrunculin A mirrored the defects observed in Cobl KO mice very well ( Cobl's actin functions in neurons depend on Ca 2+ /CaM signaling (Hou et al., 2015). Strikingly, CaM inhibitors CGS9343B and W7 both also led to a dramatic loss of PCM organization reminiscent of the Cobl KO phenotypes. The PCM volume more than doubled and the sum anti-pericentrin signal increased strongly ( Figures 2O-2T).
Together, these experiments demonstrate that Cobl and proper F-actin formation play key roles in organizing the PCM surrounding the centrioles, that is, the kinociliar base, and that this cell biological process is dependent on Ca 2+ /CaM signaling.

The Defects in PCM Organization Coincide with Impaired Centriole Positioning
Proper positioning of centrioles is considered key in the development of organized cellular arrays. Three consequences of PCM disorganization for the OHC sensory array seemed plausible: First, since the PCM harbors the two centrioles, their positioning to each other could be affected. Second, impairments of the PCM at the base of the kinocilium may affect kinocilium organization and stability. Third, consequences for the stereocilia bundle that is linked to the kinocilium during development seemed possible-especially, if the tight alignment of the mother centriole (basal body) and/or of the PCM with the tip position of the stereocilia bundle would be disrupted.
First, Cobl KO indeed coincided with impaired centriole positioning. 3D analyses of immunolabelings of g-tubulin showed that the intercentriolar distance was increased in P2 Cobl KO OHCs (Figures 3A-3C and S3A).
Cobl KO Impairs PCM-Kinocilium Alignment and Causes Premature Kinocilium Retraction prior to Hearing Onset Second, we addressed whether the PCM disorganization would coincide with impairments in kinocilium linkage and subsequently with defects in kinocilium stability. Strikingly, 3D recon-structions of OHCs of P2 cochlea stained for pericentrin and for acetylated tubulin (axoneme marker;Figures S3B and S3C) showed that basal body docking to the axoneme of the kinocilium was affected upon Cobl KO. Specifically in the more mature OHC1 and OHC2 cells, the PCM-to-kinocilium base distance was increased by about 25% (Figures 3D-3I).
The fate of kinocilia was examined next. Cobl KO animals showed a premature kinocilium retraction. While at P2 and P6 invariably every OHC still had a kinocilium and at P15 the kinocilium had disappeared from almost all WT and Cobl KO OHCs, we observed significantly fewer kinocilia-bearing OHC3s in Cobl KO cochleae during the critical time window of cochlear maturation prior to hearing onset (P8) (Figures 3J-3T). At P9, premature kinocilium retraction was even more severe. Now, all three OHCs rows of the apico-medial turn of Cobl KO cochleae showed significantly fewer remaining kinocilia ( Figures 3P, 3Q, and 3T).
Cobl KO Leads to Disrupted Spatial Correlation of the Kinocilium with the Stereocilia Bundle We then addressed the third possibility and explored whether the Cobl-and actin filament formation-dependent defects in PCM organization may disrupt the normally tight kinocilium/stereocilia bundle alignment. As the kinocilium showed premature regression at P8 and P9 in Cobl KO mice and both PCM as well as centriole positioning defects were already observable in newborn Cobl KO mice (Figures 2 and 3), we addressed this question in newborn mice. Strikingly, scanning electron microscopy (EM) revealed that the tight spatial correlation of the kinociliar base and the stereocilia bundle was disrupted upon Cobl KO (Figures 4A and 4B). Analyses of images with the same rotational positioning of the cochlear array (Figures 4C and 4D) showed that the distance of the kinociliar base to the stereocilia bundle was significantly increased. In Cobl KO OHC3s, the distance increased by almost 30% (to >420 nm) when compared to WT-a significant disruption of the normally very intimate assembly of the kinocilium with the tip of the stereocilia bundle ( Figure 4E).
3D analyses of surface-rendered anti-pericentrin immunostained PCMs and of phalloidin-highlighted stereocilia bundles demonstrated that also the PCM and the base of the stereocilia bundle tip were misaligned in all three OHC rows ( Figures  4F-4J).
PCM to one side of the stereocilia bundle ( Figure 4K), as described for defects in classical embryonic PCP signaling (Gegg et al., 2014). Instead, the disruption of the alignment of the PCM (i.e., the kinocilia base) with the stereocilia bundle occurred along the symmetry axis.
The fact that the proportion of OHCs that showed a disorganized PCM as well as structural defects in kinocilia docking to the stereocilia bundle tip was about 80% suggests that these Cobl KO phenotypes do not occur separately from each other but may be correlated ( Figure 4L).

Cobl KO Leads to Rotational Defects in OHC Arrays of Adult Cobl KO Cochleae that Reflect Impairments of Postnatal PCP Refinements and Maintenance
Considering that proper ciliary functions and a tight coupling of kinocilia to stereocilia are thought to be linked to PCP signaling and/or establishment in some way (Frolenkov et al., 2004;Kelley et al., 2009;Ezan and Montcouquiol, 2013), the Cobl KO phenotypes identified thus far could have consequences for stereocilia bundle positioning. Scanning EM analyses indeed showed that the sensory arrays of OHCs were altered upon Cobl KO ( Figure 5). Whereas neither individual stereocilia in the bundles nor the bundles themselves showed any obvious defects (erect, normal three-row staircase pattern, normal height gradation, all three rows with normal extent and undisturbed packing and no distortions in the row linearity or any gaps; Figure S4), we observed a broader range of orientation angles of OHC stereocilia bundles in cochlea of young adult Cobl KO mice ( Figures 5A and 5B). Quantitation of orientation angles of stereocilia bundles according to literature procedures (absolute deviations from 0 ) (e.g., Dabdoub et al., 2003;Copley et al., 2013; showed a less regular alignment in Cobl KO cochleae when compared to WT. The orientation deviations reached 13 (OHC3) and were 40%-70% above WT and highly statistically significant in all three OHC rows ( Figure 5D). Interestingly, Cobl KO hereby led to a superimposition of two effects (Figures 5E-5G and S5). First, the ranges of orientations of OHC stereocilia were increased, as shown by measuring the total range of angle distributions. In absolute numbers, the ranges of deviations from the corresponding means were 18-20 , that is, 37%-69% larger than in WT ( Figures 5H and S5).
Second, in contrast to the often erratically directed deviations found upon disrupting embryonic PCP signaling (e.g., Copley et al., 2013;, the orientation deficits in Cobl KO mice showed directionality of the deviations. Cobl KO OHCs showed a general rotational distortion of stereocilia bundles toward the apical turn of the cochlea (+). This rotation was in clear contrast to WT, which rather displayed rotations toward the basal turn (À) (Figures 5I-5K).
Kinocilia and/or basal body disruptions in, for example, Kif3a, Bbs8, and Ift20 KO coincided with impairments in cochlear development and were linked to grossly misoriented stereocilia bundles indicative of disrupted embryonic PCP signaling (Sipe and Lu, 2011;May-Simera et al., 2015). It was therefore of utmost importance to clarify whether the stereociliar orientation defects seen in young adult Cobl KO mice represent defects in classical embryonic PCP. Interestingly, this was not the case. Scanning EM demonstrated that OHC stereocilia bundles in newborn (P2) Cobl KO pups were correctly located, orientated, and aligned after the completion of embryonic development (Figures S6A-S6I).
Only row 3 OHCs showed a widened range of distributions, which may reflect the fact that OHC maturation follows a gradient from OHC1 to OHC3 (Frolenkov et al., 2004) ( Figures  S6F-S6I). However, in average, the OHCs of all three rows were orientated around 0 in both Cobl KO and WT newborns ( Figures S6J-S6L). This average orientation is in line with a report describing a minor, postnatal minus-directed rotation of specifically OHC3 stereocilia bundles between P0 and P4 with OHC3s being orientated close to 0 at P4 (Copley et al., 2013).
Consistent with undisturbed classical embryonic PCP signaling and successful distinction between distal and proximal sides, also the PCP markers Par6b and aPKC (Ezan and Montcouquiol, 2013) were properly localized to the proximal side in both WT and Cobl KO OHCs (Figures S6M and S6N).
Taken together, classical embryonic PCP signaling seems not to be affected by Cobl KO. This provided an excellent opportunity to unveil postnatal processes important for stereocilia bundle orientation. Our examinations clearly unveiled two PCP refinement processes that operate postnatally, general narrowing of angle deviations and directed rotation. The postnatal narrowing of the orientation ranges from 33.2 in P2 to only 25.9 in adult WT OHC1s was completely impaired in Cobl KO mice (P2, 40.0 ; adult OHC1s, 43.8 ; Figure S6I versus Figure 5H).
Furthermore, orientations already successfully obtained by classical embryonic PCP signaling were not maintained in Cobl KO cochleae after birth. The orientation range of 42.4 for newborn Cobl KO OHC2s subsequently widened to 62.3 in adult OHC2s. OHC2 in adult Cobl KO cochleae thereby reached levels of misalignment almost as severe as those observed for OHC3s (67.7 ) ( Figure S6I versus Figure 5H).

Cobl-Dependent Postnatal PCP Refinements Are Executed during Cochlear Maturation Preceding Hearing Onset
Since PCP has to be actively maintained lifelong instead of being a robust tissue achievement provided by embryonic PCP signaling, we asked whether phenotypes seen in adult mice exclusively reflect maintenance defects during hearing or reflect impairments of postnatal refinements. We therefore conducted scanning EM analyses of P8 cochleae. At this stage, the auditory canal is still closed (Figures 6A and 6B) and the middle ear still filled with fluid. Interestingly, although not yet exposed to the physical stresses of sound, the ranges of stereocilia bundle deviations were already increased in the OHCs of Cobl KO pups ( Figure 6C). Also, stereocilia bundle orientations in all three P8 OHC rows already showed a directed shift toward positive directions as in adult cochleae. The absolute differences were less pronounced (about 2 each) than at later times. However, already at P8, they were statistically highly significant in all rows ( Figures 6D-6I).
Cobl therefore plays an important role in postnatal PCP refinements that are part of a cochlear maturation program initiated prior to hearing onset.
Cobl KO Mice Show Hearing Impairments Reflecting Defects in the Cochlear Amplifier Finally, we addressed whether the defects in the orientations of OHC stereocilia bundles may be associated with any defects in integrity and functionality of the cochlea. The OHC arrays of cochleae from WT and Cobl KO mice showed that Cobl KO cochleae were not marked by array distortions by insertions of extra OHCs. Also distortions by lacking OHCs (free positions in array, gaps) were not frequent (2%-4%) and mostly comparable to irregularities also occurring in WT cochlear arrays. Only in OHC row 3, an increased frequency of gaps (1.5% in WT versus 4% in KO) became statistically significant in Cobl KO mice (Figures S7A-S7D). Since these findings were correlated with OHC3 also showing the severest early misrotations ( Figures S6F-S6I), the slightly increased gap frequency in row 3 may suggest that misorientations of the sensory apparatus are mechanically detrimental for hair cell integrity. The observed gaps indeed reflected a lack of the entire OHC at the respective array position ( Figures  S7E and S7F), as it, for example, also occurs during age-related hearing loss (Bowl and Dawson, 2015). A trend toward a loss of hair cells was also observed for IHCs, whereas insertions again were at WT levels ( Figures S7G and S7H).
At P2, however, no hair cell gaps were observed ( Figure S7I). The gaps observed in adult Cobl KO cochleae are thus unrelated to classical embryonic PCP signaling defects but represent lowfrequency deletions from previously properly patterned arrays.
Whereas Cobl KO did not seem to have effects on the integrity of hair cells that go much beyond the slow hair cell loss that also occurs with age, distortions in the carefully aligned cochlear array are more likely to have direct physiological consequences for hearing. To address this, we first excluded that Cobl KO mice may suffer from general hearing loss. The general waveform of evoked responses in auditory brainstem response (ABR) measurements was unaffected ( Figure 7A). Also the latencies and the amplitudes of the individual ABR waves were not significantly different between Cobl KO and WT littermates for click stimulation ranging from 10 to 80 dB (not shown). ABR thresholds to tone burst stimulation were only mildly elevated, especially for low-frequency stimuli, such as 4 kHz ( Figure 7B). Thus, general sound detection was not strongly impaired but at low frequencies showed some defects.
We next analyzed cochlear amplification. In the healthy cochlea, electromotility of OHCs leads to enhanced basilar membrane vibrations, which are back-propagated through the middle ear and can be measured as otoacoustic emissions. Over the entire frequency range, the intensities of distortion product otoacoustic emissions (DPOAEs) from adult (12-14 weeks) Cobl KO mice consistently were significantly lower than those of WT mice ( Figure 7C). Cobl may contribute to shaping the tonotopic spread of cochlear amplification, for which distorsion product otoacoustic emissions are a sensitive measure. Taken together, the Cobl KO-mediated defects in cochlear arrays are accompanied by defective cochlear amplification. Together, our data unveil that Cobl is critical for in part previously unnoticed, postnatal PCP refinement processes and for correlated structural organizations beneath stereocilia, which are important for proper functioning of the cochlear amplifier during hearing ( Figure 7D).

DISCUSSION
The organ of Corti is a highly organized array of sensory cells generated by different developmental cell polarity programs. Here, we report that KO of the actin nucleator Cobl leads to defects in postnatal PCP refinement and to impaired OHC amplifier function in hearing.
Analyses of Cobl KO mice unveiled the existence of a specialized, Cobl-dependent F-actin subset in OHCs. F-actin formation in OHCs was required for proper PCM organization. The specific Cobl function identified is in line with Cobl being an evolutionary young, vertebrate-specific actin nucleator. In addition, Cobl functions may be backed up by its distant relative Cobl-like (Izadi et al., 2018). In contrast to the specialized functions of Cobl beneath the sensory structures of OHCs, the general F-actinrich cell cortex represents a very basic cell-biological structure and can be expected to depend on actin nucleators, which are evolutionarily much older and less specialized.
The identified Cobl-dependent substereociliar F-actin subset seemed to be distinct from the F-actin bundles in OHC stereocilia, which protrude into the cell cortex as rootlets conferring stereocilia stability and proper morphology (Tilney et al., 1980;Kitajiri et al., 2010;Vranceanu et al., 2012;Szarama et al., 2012). Stereociliar length is sensitive to changes in F-actin bundling and dynamics. Loss of F-actin-bundling proteins localized to the stereocilia core usually causes stereocilia degeneration. Interference with actin cytoskeletal components localized to the tips of stereocilia, such as the myosin XVa/whirlin/eps8 complex, reduces stereociliar length and diminishes the height differences between stereocilia rows (Manor et al., 2011;Ebrahim et al., 2016). Interferences with proteins at the stereociliar base, such as CLIC5 and ERM proteins, lead to stereocilia enlargement and fusion (Salles et al., 2014). Finally, deletion of band g-actin severely shortened stereocilia and led to losses of individual stereocilia, respectively (Perrin et al., 2010;McGrath et al., 2017). At the physiological level, such defects are associated with fast progressive hearing loss or deafness at birth. Our analyses showed that Cobl KO did not lead to any of such more classical phenotypes. Instead, Cobl KO phenotypes affected the cuticular plate actin cytoskeleton and structures embedded in it (see Figure 7D for summary).
The identified, very specific role of Cobl in OHCs is in line with Cobl's relatively high abundance in inner ear OHCs and especially in the cuticular plate. The decrease of Cobl-dependent, substereociliar F-actin in the cuticular plate was accompanied with several hair cell phenotypes. Cobl KO led to an impaired organization of the PCM of OHCs. These impairments were observed both prior and during the critical postnatal period of structural and functional maturation of the cochlea prior to onset of hearing (P8). Importantly, they were F-actin formation dependent as demonstrated by latrunculin A-mediated inhibition of F-actin formation in WT cochleae. Interestingly, proper PCM organization furthermore turned out to be dependent on Ca 2+ /calmodulin signaling. That both different inhibitors are phenocopying the PCM organization defects in Cobl KO is consistent with Cobl being (1) an actin nucleator (Ahuja et al., 2007) and (2) Ca 2+ /calmodulin controlled (Hou et al., 2015). These defects were accompanied by a loss of the spatial correlation of the centrioles, which are encased by the disorganized PCM. This Cobl KO phenotype may be in line with a suggested role of F-actin in centriole positioning (Tang and Marshall, 2012) and maybe also with recent reports describing some cross talk of centrosomes with the actin cytoskeleton-at least in less adherent cells, such as Jurkat, RPE1 cells and lymphocytes Obino et al., 2016). We demonstrate the importance of the actin nucleator Cobl for the spatial organization of the PCM encasing the centriole-derived basal body of OHC kinocilia in the cochlea.
Mechanistically, it seems likely that Cobl-dependent, rather dynamic F-actin structures beneath the sensory apparatus help to encase and thereby spatially constrain the PCM. A rather static F-actin scaffold directly in the PCM to properly build the PCM or prevent the PCM from fragmentation seems unlikely because no F-actin can be observed in the PCM volume. It will therefore be interesting to evaluate whether Cobl has interaction partners in the PCM and/or at the basal body that may help to promote Cobl-mediated F-actin formation around the PCM. The PCM relations to cortical F-actin that we observed may furthermore relate to recent reports describing some sort of structural connections emanating from microtubular structures in the cuticular plate (Antonellis et al., 2014), some cross talk of the actin cytoskeleton with cilia during their embryonic migration , RNAi against the PCM component PCM1 affecting the cloud-like F-actin mesh around related microtubule-based structures (centrosomes) in Jurkat cells, and the ability of isolated centrosomes to grow actin asters in vitro .
While the physiological relevance of the latter findings for centrosomes await further examinations, our data clearly show that Cobl-mediated disruption of cortical F-actin and of pericentriolar organization is linked to cell-biological processes that underlie the proper spatial arrangement of the sensory structures of OHCs. First, the distance of the disorganized PCM to the axoneme of the kinocilium was increased. Second, the distance of the PCM to the stereocilia bundle tip was increased. Accordingly, these defects coincided with an impaired spatial correlation of the normally intimately linked kinocilium base and the stereocilia bundle in Cobl KO mice. It seems possible that the defects in basal body docking of the kinocilium with the stereocilia lead to the observed premature loss of kinocilia in Cobl KO mice during the critical time period prior to hearing onset ( Figure 7D).
Cobl KO-mediated defects in kinocilium maintenance were observed at P8 and P9. This is in line with the observation that the Cobl KO defects in stereocilia bundle orientation also did not occur during classical embryonic PCP signaling, which is critical for PCP establishment and rough alignment of sensory cells prior to birth, but almost exclusively manifested postnatally. This provided an excellent possibility to specifically unveil thus far largely unknown postnatal planar polarity processes occurring subsequent to embryonic PCP establishment. In mutant mice lacking the functions of classical core PCP components, such as Frizzled or Celsr1, postnatal refinements are difficult to reveal because they are masked by the severe embryonal defects (Wang et al., 2006;Curtin et al., 2003). Comparisons of WT and Cobl KO cochleae from P2 and adult mice at ultra-high resolution clearly demonstrated that stereocilia bundle positioning angles are refined after birth to reach an even more aligned status in mature cochleae. An existence of postnatal refinement processes has already been suggested in earlier studies (Dabdoub et al., 2003;Copley et al., 2013). Our work shows that postnatal refinement narrowing the spread of orientation angles is completely disrupted upon Cobl KO.
Interestingly, all Cobl KO defects manifested before hearing onset, that is, between P2 and P8. During this time frame, hair cells are just probing transmission by self-activation (Johnson et al., 2013), but the hearing channel is still closed, the middle ear is still filled with fluid, and outside sounds cannot reach the inner ear. Thus, all forms of postnatal refinement we identified studying Cobl KO mice thus seem to be cochlea-autonomous processes independent of the physical pressures of sound.
Our analyses of Cobl KO mice furthermore revealed that the angle deviation status successfully reached by the embryonic PCP program has to be actively maintained after birth. The fact that both maintenance of embryonic PCP achievements and further postnatal refinements (angle fine-tuning and rotational refinement) are Cobl dependent strongly suggests that these processes are mechanistically related ( Figure 7D).
The existence of postnatal planar polarity refinement and maintenance processes independent from classical embryonic PCP unraveled by Cobl KO is further supported by a previous observation of unknown, Vangl2-independent patterning mechanisms operating postnatally in cochleae from mice lacking the PCP core component Vangl2 (Montcouquiol et al., 2003;Copley et al., 2013). Parallel, somewhat related, but independent functions of Vangl2 and Cobl may finally also provide an explanation for Vangl2's genetic interaction with the Coblc101 gene trap mutation (Carroll et al., 2003).
The finding that misorientation of stereocilia bundles in Cobl KO worsened after hearing onset suggests that Cobl-mediated processes actively counteract the mechanical stress occurring during mechanotransduction and thereby prevent rotational shifts of the tip of the V-shaped bundle toward the apical turn of the cochlea. Thus, the achievements of embryonic PCP signaling have to be both refined and actively maintained after birth.
Two findings underscore the physiological importance of the postnatal processes executed by Cobl in the inner ear. First, OHC3, which showed the strongest defect in rotational refinement and premature kinocilium retraction during the critical time window of hearing onset, also showed the highest frequency in hair cell deletions in adult Cobl KO mice. Cobl-dependent postnatal refinements thus seem important for withstanding the mechanical stress during mechanotransduction after the onset of hearing and thereby ensure the integrity of the organ of Corti. It is therefore likely that defects in refinement and maintenance processes, such as those dependent on Cobl, are one of the cellular causes underlying the insidious loss of hair cells in age-related hearing loss.
Second, our analyses of otoacoustic emissions of Cobl KO mice showed that distortions of stereocilia bundle orientations caused by impaired, Cobl-dependent planar polarity refinement processes, which were linked to impaired PCM organization, were accompanied with disrupted alignment of the kinocilium with the stereociliar bundle, and with premature kinocilia retraction, and coincided with defects in the cochlear amplifier.
Thus, the ability to execute Cobl-dependent processes, which precisely fine-tune stereocilia bundle orientations postnatally to ensure a correct patterning of the inner ear sensory epithelium, is essential for cochlear amplification during hearing.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

Mice
Tissue material for RT-PCRs and Western blot analyses as well as brain sections and cochleae for immunohistochemistry were taken from WT (C57BL/6J) and Cobl KO mice of different developmental stages (see figure legends and Method Details). Ubiquitously Cre recombinase expressing mice (Schwenk et al., 1995) were used for Cobl exon11 deletion.
Cobl KO mouse generation and backcrossings were done using C57BL/6J mice.

Generation of Cobl KO Mice
The targeting vector was generated using a clone isolated from a 129/SvJ mouse genomic l library (Agilent). An approximately 10 kb EcoRV fragment of this clone including exons 11-12 of the Cobl gene (GenBank: NC_000077.5, Mus musculus, chromosome 11) was cloned into the SmaI site of the pKO-DTA plasmid (Lexicon Genetics) with a phosphoglycerate kinase promoter-driven diphtheria toxin A cassette as a negative selection marker. A phosphoglycerate kinase promoter-driven neomycin resistance cassette (positive selection marker) flanked by frt sites and a loxP site was inserted into the AflII site of intron 10. A second loxP site together with an additional BamHI site was inserted into the SwaI site of intron 11. R1 mouse embryonic stem (ES) cells were electroporated with the NotI-linearized targeting vector. Genomic DNA of 288 Neomycin-resistant ES cell clones was screened by Southern blot with a P 32labeled DNA probe (379 bp, nt 213457-213835, GenBank: NC_000077.5) after BamHI restriction (WT fragment: 9.3 kbp, nt 205809-215151; transgene fragment: 6.2 kbp, 208983-215151). One correctly targeted ES cell clone (B8) was injected into C57BL/6 blastocysts to generate chimeras. Cobl KO mice were obtained via mating with mice ubiquitously expressing Cre recombinase (Schwenk et al., 1995) to remove exon11 together with the selection cassette. Germline transmission successfully gave rise to a mouse line lacking the actin nucleator Cobl. For genotyping, DNA of mouse tail biopsies was extracted with 10 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.4 mg/ml proteinase K. After inactivation (10 min, 95 C), a high-speed supernatant was analyzed by PCR (fwd-primer F1 acacagccctggcatcat, rev-primers R1 (atacgggcaatcacgttttc) and R2 (tgctccacactgaggtgttc)). Primer combination F1/R1 amplified a 388 bp WT allele and primer combination F1/R2 a 235 bp KO allele.
The generation of Cobl KO mice and initial characterization was performed in strict compliance with the EU guidelines for animal experiments and was approved by the local government (permission number: 02-011/10; Th€ uringer Landesamt, Bad Langensalza; Germany).
Experiments were done with both male and female mice 3-16 weeks (body weight) and with 12-14 weeks old male and female mice (hearing physiology), respectively.
To measure OHC bundle orientation, the two ends of each individual V-shaped cilia bundle were connected and the deviation of this line from the 0 axis defined by the cell borders of row 1 OHCs and the Inner Pillar cells (0 axis, see scheme in Figure 5C) was determined.
Averaged stereocilia bundle orientations (bundle deviations from 0 C) were calculated by using the modulus of the orientation angles, i.e., irrespective of direction. This literature procedure was complemented by additional analyses, in which the direction of deviation was considered, too.
The distributions of stereocilia bundle orientations in adult WT and Cobl KO cochleae were plotted as percentage of hair cell bundles in a given deviation angle class with steps of 5 (considering the direction of deviation from 0 C).
Quantitative determinations of the ranges of stereocilia orientation angles were calculated considering 98% of the angles closest to mean.
The spatial correlation of the kinocilium position in relation to the stereocilia bundle of was addressed for P2 OHCs by measuring the distances between the bases of the kinocilium and the first side stereocilium of the V-shaped structure in top views of cochlear arrays. Although this type of measurement may grossly under-evaluate increases in the distance between kinocilium and the central tip stereocilia of the bundle (the V-shaped bundles usually show indented tips, i.e., do not exhibit a pointed V shape) -e.g., a doubling of the distance may only lead to about 30% in the measurements of the neighboring side of the triangle formed between central ''tip'' stereocilium, kinocilium and the first side stereocilium when tips are broad -these measurements were most useful, as the distance between first side stereocilium and kinocilium could always reliably be addressed, whereas sight on the base of the central tip stereocilium is often blocked due to the tip indention of the stereocilia bundle. Analyzed were n = 60 OHCs/row from 4 animals/cochleae each genotype.

Recordings of Auditory Brainstem Response and DPOAE
The physiological examinations of hearing thresholds and cochlear amplification were essentially done as described before (Jing et al., 2013). In brief, 12-14 weeks old animals (male and female) were anesthetized intraperitoneally with a combination of ketamine (125 mg/kg) and xylazine (2.5 mg/kg). The heart rate was monitored constantly and the core temperature was maintained constant at 37 C using a rectal temperature-controlled heating blanket (Hugo Sachs Elektronik; Harvard Apparatus).
For stimulus generation, presentation, and data acquisition a TDT System II (Tucker-Davis Technologies) run by BioSig32 software (TDT) was used. Sound pressure levels are provided in dB SPL RMS (tonal stimuli) or dB SPL peak equivalent (PE, clicks) and were calibrated using a 1/4 00 microphone (Br€ uel & Kjaer). Tone bursts (12 kHz, 10 ms plateau, 1 ms cos2 rise/fall) or clicks of 0.03 ms were presented at 20 Hz or 100 Hz in the free field ipsilaterally using a JBL 2402 speaker (JBL & Co.).
The difference potential between vertex and mastoid subdermal needles was amplified (50,000 times), filtered (low pass: 4 kHz, high pass: 400 Hz) and sampled at a rate of 50 kHz for 20 ms, 2 3 2000 times to obtain two mean auditory brainstem responses (ABRs) for each sound intensity.
Hearing thresholds were determined with 10 dB precision as the lowest stimulus intensity that evoked a reproducible response waveform in both traces by visual inspection.
For DPOAEs, MF1 speakers (Tucker-Davis) were used to generate two primary tones (frequency ratio f2/f1: 1.2, intensity f2 = intensity f1 + 10 dB). Primary tones were coupled into the ear canal by a custom-made probe containing an MKE-2 microphone (Sennheiser) and adjusted to the desired sound intensities at the position of the ear drum as mimicked in a mouse ear coupler.
The microphone signal was amplified and digitalized (DMX 6 Fire; Terratec) and analyzed by fast Fourier transformation (MATLAB; MathWorks).

QUANTIFICATION AND STATISTICAL ANALYSIS
No statistical methods were used to predetermine sample size. All quantitative data shown represent mean and SEM. Statistical analyses were done using GraphPad Prism software using the tests specified in the figure legends. Comparisons of two conditions were tested by either D'Agostino normality test/Mann Whitney U or unpaired t test (normal data distribution). Multiple comparisons were tested by two-way-ANOVA with post-test (Tukey's, Sidak's, Bonferroni's). Connected datasets were analyzed using 1way ANOVA with post-test (Dunnett's, Dunn's).