The Rho GTPase Cell Division Cycle 42 Regulates Stereocilia Development in Cochlear Hair Cells
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ABSTRACT:
SUBMITTER: Du H
PROVIDER: S-EPMC8570139 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Plant RHO GTPases (RAC/ROPs) mediate multiple extracellular signals ranging from hormone to stress and regulate diverse cellular processes important for polarized cell growth, differentiation, development, reproduction, and responses to the environment. They shuttle between the GDP-bound inactive state and the GTP-bound activated state and their activation is predominantly mediated by a family of guanine nucleotide exchange factors (GEFs) referred to as ROPGEFs. Using the Arabidopsis ROPGEF1 as bait, we identified members of a receptor-like kinase (RLK) family as potential upstream regulators for RAC/ROP signaling. NADPH oxidase-derived reactive oxygen species (ROS) are emerging as important regulators for growth and development and play a crucial role in mediating RAC/ROP-regulated root hair development, a polarized cell growth process. We therefore screened T-DNA insertion mutants in these RLKs for root hair defects and found that mutations in one of them, At3g51550 encoding the FERONIA (FER) receptor-like kinase, induced severe root hair defects. We show that the fer phenotypes correlated with reduced levels of active RAC/ROPs and NADPH oxidase-dependent, auxin-regulated ROS accumulation in roots and root hairs and that up-regulating RAC/ROP signaling in fer countered the mutant phenotypes. Taken together, these observations strongly support FER as an upstream regulator for the RAC/ROP-signaled pathway that controls ROS-mediated root hair development. Moreover, FER was pulled down by ROP2 GTPase in a guanine nucleotide-regulated manner implying a dynamic signaling complex involving FER, a ROPGEF, and a RAC/ROP.
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Project description:Cochlear hair cells transduce mechanical stimuli into electrical activity. The site of hair cell transduction is the hair bundle, an array of stereocilia with different height arranged in a staircase. Tip links connect the apex of each stereocilium to the side of its taller neighbor. The hair bundle and tip links of hair cells are susceptible to acoustic trauma and ototoxic drugs. It has been shown that hair cells in lower vertebrates and in the mammalian vestibular system may survive bundle loss and undergo self-repair of the stereocilia. Our goals were to determine whether cochlear hair cells could survive the trauma and whether the tip link and/or the hair bundle could be regenerated. We simulated the acoustic trauma-induced tip link damage or stereociliary loss by disrupting tip links or ablating the hair bundles in the cultured organ of Corti from neonatal gerbils. Hair-cell fate and stereociliary morphology and function were examined using confocal and scanning electron microscopies and electrophysiology. Most bundleless hair cells survived and developed for approximately 2 weeks. However, no spontaneous hair-bundle regeneration was observed. When tip links were ruptured, repair of tip links and restoration of mechanotransduction were observed in <24 h. Our study suggests that the dynamic nature of the hair cell's transduction apparatus is retained despite the fact that regeneration of the hair bundle is lost in mammalian cochlear hair cells.
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Project description:Outer hair cell (OHC) stereocilia bundle deflection opens mechanoelectrical transduction channels at the tips of the stereocilia from the middle and short rows, while bundle cohesion is maintained owing to the presence of horizontal top connectors. Here, we used a quantitative noncontact atomic force microscopy method to investigate stereocilia bundle stiffness and damping, when stimulated at acoustic frequencies and nanometer distances from the bundle. Stereocilia bundle mechanics were determined in stereocilin-deficient mice lacking top connectors and with detached tectorial membrane (Strc -/-/Tecta -/- double knockout) and heterozygous littermate controls (Strc +/-/Tecta -/-). A substantial decrease in bundle stiffness and damping by ~60 and ~74% on postnatal days P13 to P15 was observed when top connectors were absent. Additionally, we followed bundle mechanics during OHC top connectors development between P9 and P15 and quantified the observed increase in OHC bundle stiffness and damping in Strc +/-/Tecta -/- mice while no significant change was detected in Strc -/-/Tecta -/- animals.
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Project description:Inner ear sensory hair cells convert mechanical stimuli into electrical signals. This conversion happens in the exquisitely mechanosensitive hair bundle that protrudes from the cell's apical surface. In mammals, cochlear hair bundles are composed of 50-100 actin-filled stereocilia, which are organized in three rows in a staircase manner. Stereocilia actin filaments are uniformly oriented with their barbed ends toward stereocilia tips. During development, the actin core of each stereocilium undergoes elongation due to addition of actin monomers to the barbed ends of the filaments. Here we show that in the mouse cochlea the barbed end capping protein twinfilin 2 is present at the tips of middle and short rows of stereocilia from postnatal day 5 (P5) onward, which correlates with a time period when these rows stop growing. The tall stereocilia rows, which do not display twinfilin 2 at their tips, continue to elongate between P5 and P15. When we expressed twinfilin 2 in LLC/PK1-CL4 (CL4) cells, we observed a reduction of espin-induced microvilli length, pointing to a potent function of twinfilin 2 in suppressing the elongation of actin filaments. Overexpression of twinfilin 2 in cochlear inner hair cells resulted in a significant reduction of stereocilia length. Our results suggest that twinfilin 2 plays a role in the regulation of stereocilia elongation by restricting excessive elongation of the shorter row stereocilia thereby maintaining the mature staircase architecture of cochlear hair bundles.
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Project description:Hair cells of the organ of Corti (OC) of the cochlea exhibit distinct planar polarity, both at the tissue and cellular level. Planar polarity at tissue level is manifested as uniform orientation of the hair cell stereociliary bundles. Hair cell intrinsic polarity is defined as structural hair bundle asymmetry; positioning of the kinocilium/basal body complex at the vertex of the V-shaped bundle. Consistent with strong apical polarity, the hair cell apex displays prominent actin and microtubule cytoskeletons. The Rho GTPase Cdc42 regulates cytoskeletal dynamics and polarization of various cell types, and, thus, serves as a candidate regulator of hair cell polarity. We have here induced Cdc42 inactivation in the late-embryonic OC. We show the role of Cdc42 in the establishment of planar polarity of hair cells and in cellular patterning. Abnormal planar polarity was displayed as disturbances in hair bundle orientation and morphology and in kinocilium/basal body positioning. These defects were accompanied by a disorganized cell-surface microtubule network. Atypical protein kinase C (aPKC), a putative Cdc42 effector, colocalized with Cdc42 at the hair cell apex, and aPKC expression was altered upon Cdc42 depletion. Our data suggest that Cdc42 together with aPKC is part of the machinery establishing hair cell planar polarity and that Cdc42 acts on polarity through the cell-surface microtubule network. The data also suggest that defects in apical polarization are influenced by disturbed cellular patterning in the OC. In addition, our data demonstrates that Cdc42 is required for stereociliogenesis in the immature cochlea.
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Project description:ELMOD3, an ARL2 GTPase-activating protein, is implicated in causing hearing impairment in humans. However, the specific role of ELMOD3 in auditory function is still far from being elucidated. In the present study, we used the CRISPR/Cas9 technology to establish an Elmod3 knockout mice line in the C57BL/6 background (hereinafter referred to as Elmod3-/- mice) and investigated the role of Elmod3 in the cochlea and auditory function. Elmod3-/- mice started to exhibit hearing loss from 2 months of age, and the deafness progressed with aging, while the vestibular function of Elmod3-/- mice was normal. We also observed that Elmod3-/- mice showed thinning and receding hair cells in the organ of Corti and much lower expression of F-actin cytoskeleton in the cochlea compared with wild-type mice. The deafness associated with the mutation may be caused by cochlear hair cells dysfunction, which manifests with shortening and fusion of inner hair cells stereocilia and progressive degeneration of outer hair cells stereocilia. Our finding associates Elmod3 deficiencies with stereocilia dysmorphologies and reveals that they might play roles in the actin cytoskeleton dynamics in cochlear hair cells, and thus relate to hearing impairment.
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Project description:The mechanism behind the spatiotemporal control of cancer cell dynamics and its possible association with cell proliferation has not been well established. By exploiting the intravital imaging technique, we found that cancer cell motility and invasive properties were closely associated with the cell cycle. In vivo inoculation of human colon cancer cells bearing fluorescence ubiquitination-based cell cycle indicator (Fucci) demonstrated an unexpected phenomenon: S/G2/M cells were more motile and invasive than G1 cells. Microarray analyses showed that Arhgap11a, an uncharacterized Rho GTPase-activating protein (RhoGAP), was expressed in a cell-cycle-dependent fashion. Expression of ARHGAP11A in cancer cells suppressed RhoA-dependent mechanisms, such as stress fiber formation and focal adhesion, which made the cells more prone to migrate. We also demonstrated that RhoA suppression by ARHGAP11A induced augmentation of relative Rac1 activity, leading to an increase in the invasive properties. RNAi-based inhibition of Arhgap11a reduced the invasion and in vivo expansion of cancers. Additionally, analysis of human specimens showed the significant up-regulation of Arhgap11a in colon cancers, which was correlated with clinical invasion status. The present study suggests that ARHGAP11A, a cell cycle-dependent RhoGAP, is a critical regulator of cancer cell mobility and is thus a promising therapeutic target in invasive cancers.
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Project description:Key pointsMechanoelectrical transduction at auditory hair cells requires highly specialized stereociliary bundles that project from their apical surface, forming a characteristic graded 'staircase' structure. The morphogenesis and maintenance of these stereociliary bundles is a tightly regulated process requiring the involvement of several actin-binding proteins, many of which are still unidentified. We identify a new stereociliary protein, the I-BAR protein BAIAP2L2, which localizes to the tips of the shorter transducing stereocilia in both inner and outer hair cells (IHCs and OHCs). We find that Baiap2l2 deficient mice lose their second and third rows of stereocilia, their mechanoelectrical transducer current, and develop progressive hearing loss, becoming deaf by 8 months of age. We demonstrate that BAIAP2L2 localization to stereocilia tips is dependent on the motor protein MYO15A and its cargo EPS8. We propose that BAIAP2L2 is a new key protein required for the maintenance of the transducing stereocilia in mature cochlear hair cells.AbstractThe transduction of sound waves into electrical signals depends upon mechanosensitive stereociliary bundles that project from the apical surface of hair cells within the cochlea. The height and width of these actin-based stereocilia is tightly regulated throughout life to establish and maintain their characteristic staircase-like structure, which is essential for normal mechanoelectrical transduction. Here, we show that BAIAP2L2, a member of the I-BAR protein family, is a newly identified hair bundle protein that is localized to the tips of the shorter rows of transducing stereocilia in mouse cochlear hair cells. BAIAP2L2 was detected by immunohistochemistry from postnatal day 2.5 (P2.5) throughout adulthood. In Baiap2l2 deficient mice, outer hair cells (OHCs), but not inner hair cells (IHCs), began to lose their third row of stereocilia and showed a reduction in the size of the mechanoelectrical transducer current from just after P9. Over the following post-hearing weeks, the ordered staircase structure of the bundle progressively deteriorates, such that, by 8 months of age, both OHCs and IHCs of Baiap2l2 deficient mice have lost most of the second and third rows of stereocilia and become deaf. We also found that BAIAP2L2 interacts with other key stereociliary proteins involved in normal hair bundle morphogenesis, such as CDC42, RAC1, EPS8 and ESPNL. Furthermore, we show that BAIAP2L2 localization to the stereocilia tips depends on the motor protein MYO15A and its cargo EPS8. We propose that BAIAP2L2 is key to maintenance of the normal actin structure of the transducing stereocilia in mature mouse cochlear hair cells.
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Project description:Acoustical excitation of the organ of Corti induces radial fluid flow in the subtectorial space (STS) that excites the hair bundles (HBs) of the sensory inner hair cell of the mammalian cochlea. The inner hair cell HBs are bathed in endolymphatic fluid filling a thin gap in the STS between the tectorial membrane and the reticular lamina. According to the fluctuation dissipation theorem, the fluid viscosity gives rise to mechanical fluctuations that are transduced into current noise. Conversely, the stochastic fluctuations of the mechanically gated channels of the HBs also induce dissipation. We develop an analytic model of the STS complex in a cross section of the gerbil organ of Corti. We predict that the dominant noise at the apex is due to the channel stochasticity whereas viscous effects dominate at the base. The net root mean square fluctuation of the HB motion is estimated to be at least 1.18 nm at the base and 2.72 nm at the apex. By varying the HB height for a fixed STS gap, we find that taller HBs are better sensors with lower thresholds. An integrated active HB model is shown to reduce the hydrodynamic resistance through a cycle-by-cycle power addition through adaptation, reducing the thresholds of hearing, hinting at one potential role for HB activity in mammalian hearing. We determine that a Couette flow approximation in the STS underestimates the dissipation and that modeling the entire STS complex is necessary to correctly predict the low-frequency dissipation in the cochlea. Finally, the difference in the noise budget at the base and the apex of the cochlea indicate that a sensing modality other than the shear motion of the TM that may be used to achieve low-noise acoustic sensing at the apex.
| S-EPMC5984990 | biostudies-literature
Project description:Assembly of the hair bundle, the sensory organelle of the inner ear, depends on differential growth of actin-based stereocilia. Separate rows of stereocilia, labeled 1 through 3 from tallest to shortest, lengthen or shorten during discrete time intervals during development. We used lattice structured illumination microscopy and surface rendering to measure dimensions of stereocilia from mouse apical inner hair cells during early postnatal development; these measurements revealed a sharp transition at postnatal day 8 between stage III (row 1 and 2 widening; row 2 shortening) and stage IV (final row 1 lengthening and widening). Tip proteins that determine row 1 lengthening did not accumulate simultaneously during stages III and IV; while the actin-bundling protein EPS8 peaked at the end of stage III, GNAI3 peaked several days later-in early stage IV-and GPSM2 peaked near the end of stage IV. To establish the contributions of key macromolecular assemblies to bundle structure, we examined mouse mutants that eliminated tip links (Cdh23v2J or Pcdh15av3J), transduction channels (TmieKO), or the row 1 tip complex (Myo15ash2). Cdh23v2J/v2J and Pcdh15av3J/av3J bundles had adjacent stereocilia in the same row that were not matched in length, revealing that a major role of these cadherins is to synchronize lengths of side-by-side stereocilia. Use of the tip-link mutants also allowed us to distinguish the role of transduction from effects of transduction proteins themselves. While levels of GNAI3 and GPSM2, which stimulate stereocilia elongation, were greatly attenuated at the tips of TmieKO/KO row 1 stereocilia, they accumulated normally in Cdh23v2J/v2J and Pcdh15av3J/av3J stereocilia. These results reinforced the suggestion that the transduction proteins themselves facilitate localization of proteins in the row 1 complex. By contrast, EPS8 concentrates at tips of all TmieKO/KO, Cdh23v2J/v2J, and Pcdh15av3J/av3J stereocilia, correlating with the less polarized distribution of stereocilia lengths in these bundles. These latter results indicated that in wild-type hair cells, the transduction complex prevents accumulation of EPS8 at the tips of shorter stereocilia, causing them to shrink (rows 2 and 3) or disappear (row 4 and microvilli). Reduced rhodamine-actin labeling at row 2 stereocilia tips of tip-link and transduction mutants suggests that transduction's role is to destabilize actin filaments there. These results suggest that regulation of stereocilia length occurs through EPS8 and that CDH23 and PCDH15 regulate stereocilia lengthening beyond their role in gating mechanotransduction channels.
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