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Rho GTPases Mediate the Regulation of Cochlear Outer Hair Cell Motility by Acetylcholine*

Open AccessPublished:September 08, 2000DOI:https://doi.org/10.1074/jbc.M004917200
      Outer hair cells are the mechanical effectors of the cochlear amplifier, an active process that improves the sensitivity and frequency discrimination of the mammalian ear. In vivo, the gain of the cochlear amplifier is regulated by the efferent neurotransmitter acetylcholine through the modulation of outer hair cell motility. Little is known, however, regarding the molecular mechanisms activated by acetylcholine. In this study, intracellular signaling pathways involving the small GTPases RhoA, Rac1, and Cdc42 have been identified as regulators of outer hair cell motility. Changes in cell length (slow motility) and in the amplitude of electrically induced movement (fast motility) were measured in isolated outer hair cells patch clamped in whole-cell mode, internally perfused through the patch pipette with different inhibitors and activators of these small GTPases while being externally stimulated with acetylcholine. We found that acetylcholine induces outer hair cell shortening and a simultaneous increase in the amplitude of fast motility through Rac1 and Cdc42 activation. In contrast, a RhoA- and Rac1-mediated signaling pathway induces outer hair cell elongation and decreases fast motility amplitude. These two opposing processes provide the basis for a regulatory mechanism of outer hair cell motility.
      OHC
      outer hair cell
      ACh
      acetylcholine
      GTPγS
      guanosine 5′-O-(3-thiotriphosphate)
      GDPβS
      guanosine 5′-O-2-thiodiphosphate)
      dn
      dominant negative
      Inside the mammalian inner ear, the mechanical stimulus provided by sound is amplified up to 100 times by a mechanism known as the “cochlear amplifier.” As a consequence of this active process, the sensitivity and the frequency discrimination of the hearing system are greatly increased (
      • Dallos P.
      ). Damage of this mechanism, for instance by acoustic trauma, aminoglycoside antibiotics, or simply aging, is a common cause of sensory neural hearing loss afflicting millions of people around the world.
      At the core of the cochlear amplifier are the outer hair cells (OHCs).1 OHCs are specific to the mammalian cochlea, probably reflecting an adaptation to the frequency and dynamic range demands of mammalian hearing (
      • Lim D.J.
      • Kalinec F.
      ). They are cylindrical with lengths ranging between 10 and 100 μm and a rather constant diameter of ∼8 μm. Cochlear OHCs can reversibly change their length by two different mechanisms: slow and fast OHC motility (
      • Holley M.C.
      ). Slow OHC motility occurs in seconds and involves cytoskeletal reorganization (
      • Dulon D.
      • Schacht J.
      ). In contrast, fast motility works in the microsecond range and is voltage-driven, with hyperpolarization causing elongation and depolarization shortening of the OHCs (
      • Brownell W.E.
      • Bader C.R.
      • Bertrand D.
      • de Ribaupierre Y.
      ,
      • Ashmore J.F.
      ,
      • Kachar B.
      • Brownell W.E.
      • Altschuler R.
      • Fex J.
      ). We and others have recently demonstrated that OHC fast motility is mediated by the concerted direct action of a large number of independent molecular motors embedded in the OHC lateral plasma membrane (
      • Holley M.C.
      • Ashmore J.F.
      ,
      • Dallos P.
      • Evans B.N.
      • Hallworth R.
      ,
      • Kalinec F.
      • Holley M.C.
      • Iwasa K.
      • Lim D.J.
      • Kachar B.
      ,
      • Huang G.
      • Santos-Sacchi J.
      ) and funneled along the cell longitudinal axis by the prominent actin-spectrin cortical cytoskeleton (
      • Holley M.C.
      • Kalinec F.
      • Kachar B.
      ).
      Compelling evidence suggests that the gain of the cochlear amplifier is regulated in vivo through the modulation of OHC motility by acetylcholine (ACh) released from terminals of the medial efferent system (for review, see Refs.
      • Wiederhold M.L.
      and
      • Guinan J.J.
      ). Little is known, however, regarding the molecular mechanisms activated by ACh in OHCs. Several lines of evidence have led us to consider the involvement of members of the Rho (Ras homologous) family of small GTPases in this process. For instance, early studies have established that RhoA, Rac1, and Cdc42 play a crucial role in cytoskeletal reorganization and mediate different types of motility in nonauditory cell populations (for review see Refs.
      • Van Aelst L.
      • D'Souza-Schorey C.
      and
      • Hall A.
      ). In addition, recent evidence has indicated that ACh activates Rho-mediated signaling pathways in neuroblastoma cells (
      • Kozma R.
      • Sarner S.
      • Ahmed S.
      • Lim L.
      ). More importantly, targets of Rho GTPases have been associated to sensorineural hearing loss. For example, a mutation in the Dia1 protein (a profilin ligand and target of Rho (
      • Watanabe N.
      • Iwamura T.
      • Shinoda T.
      • Fujita T.
      ,
      • Watanabe N.
      • Kato T.
      • Fujita A.
      • Ishizaki T.
      • Narumiya S.
      )) is the cause of the autosomal dominant nonsyndromic deafness DFNA1 (
      • Lynch E.D.
      • Lee M.K.
      • Morrow J.E.
      • Welcsh P.L.
      • Leon P.E.
      • King M.-C.
      ), and mutations in another potential target of Rho, myosin VIIa, are responsible for human Usher syndrome type 1B (
      • Gibson F.
      • Walsh J.
      • Mburu P.
      • Varela A.
      • Brown K.A.
      • Antonio M.
      • Beisel K.W.
      • Steel K.P.
      • Brown S.D.M.
      ,
      • Weil D.
      • Blanchard S.
      • Kaplan J.
      • Guilford P.
      • Gibson F.
      • Walsh J.
      • Mburu P.
      • Varela A.
      • Levilliers J.
      • Weston M.D.
      • et al.
      ,
      • Weil D.
      • Levy G.
      • Sahly I.
      • Levi-Acobas F.
      • Blanchard S.
      • El-Amraoui A.
      • Crozet F.
      • Philippe H.
      • Abitbol M.
      • Petit C.
      ). The existence of a direct link between these pathways, OHC motility, and the physiology of the hearing system, however, remains unexplored. Thus, this is the first study to demonstrate that Rho proteins participate in the signaling cascade that ultimately regulates OHC motility in response to ACh. This finding is crucial for our understanding of a basic mechanism for both normal human hearing and deafness.

      DISCUSSION

      By using specific activators and inhibitors of RhoA, Rac1, and Cdc42, we have revealed the first evidence that modulation of OHC motility by ACh is mediated by Rho GTPases. Our results suggest that Rac1 is a crucial regulator of ACh-induced OHC motility. In cooperation with Cdc42, Rac1 mediates OHC shortening and a simultaneous increase in the amplitude of OHC fast motility. In contrast, in cooperation with RhoA, Rac1 mediates OHC elongation and a decrease in the amplitude of fast motility. Furthermore, these results indicate the existence of a Rac1-controlled feedback mechanism responsible for the fine tuning of OHC fast motility and able to rapidly revert the changes induced by ACh. These processes are essential for maintaining the homeostasis of the cochlear amplifier and thereby for normal hearing.

      Localization of RhoA, Rac1, and Cdc42 in OHCs

      In OHCs, RhoA, Rac1, and Cdc42 co-localize with cytoskeletal structures. Immunolabeling was stronger at the cuticular plate, infracuticular network, and along the lateral wall of OHCs (Fig. 1). The cuticular plate is a dense meshwork of actin and spectrin in the OHC apex, thought to be a stiff nonflexible plate in which the stereocilia are anchored (
      • Drenckhahn D.
      • Engel K.
      • Höfer D.
      • Merte C.
      • Tilney L.
      • Tilney M.
      ). The infracuticular network, in turn, is an expansion of the cuticular plate into the cytoplasm found only in OHCs from the apical end of the guinea pig cochlea (such as those illustrated in Fig.1) (
      • Raphael Y.
      • Athey B.D.
      • Wang Y.
      • Lee M.K.
      • Altschuler R.A.
      ). The OHC lateral wall, on the other hand, is a unique structure composed of three distinct layers: the plasma membrane, the cortical cytoskeleton, and the lateral cisternae (
      • Holley M.C.
      ). The lateral cisternae are multiple, highly ordered layers (as many as twelve in guinea pig) lining up the lateral cytoplasmic surface of OHCs from the apical tight junction to the infranuclear region (
      • Gulley R.L.
      • Reese T.S.
      ,
      • Saito K.
      ). Whereas in OHCs the Golgi apparatus is small and confined to a restricted region in the apical, subcuticular area of the cell, specific labeling suggests that lateral cisternae membranes share characteristics of Golgi and smooth endoplasmic reticulum (
      • Pollice P.A.
      • Brownell W.E.
      ,
      • Forge A.
      • Zajic G.
      • Li L.
      • Nevill G.
      • Schacht J.
      ). This is particularly relevant, because localization analyses in other laboratories have shown association of Cdc42 with Golgi membranes (
      • Erickson J.W.
      • Zhang C.
      • Kahn R.A.
      • Evans T.
      • Cerione R.A.
      ,
      • McCallum S.J.
      • Erickson J.W.
      • Cerione R.A.
      ).
      The cortical cytoskeleton, located in the narrow space (∼30 nm wide) between the plasma membrane and the outermost cisternal membrane, is a two-dimensional structure responsible for the shape and most of the mechanical properties of the OHCs (
      • Holley M.C.
      ,
      • Holley M.C.
      • Kalinec F.
      • Kachar B.
      ). It is composed essentially by roughly circumferential actin filaments up to 1 μm long, cross-linked by shorter (∼50 nm) spectrin tetramers (
      • Holley M.C.
      • Kalinec F.
      • Kachar B.
      ). The actin filaments are connected to the plasma membrane through thousands of 25-nm-long, rod-like structures (pillars) placed about 40 nm from each other (
      • Holley M.C.
      • Kalinec F.
      • Kachar B.
      ,
      • Flock A.
      • Flock B.
      • Ulfendahl M.
      ,
      • Raphael Y.
      • Wroblewski R.
      ). Even though no changes in the distribution of Rho GTPases or actin were detected in the lateral wall of OHC after stimulation with ACh, Rho-mediated changes in the cortical cytoskeleton remain one of the most attractive candidate mechanisms for the regulation of OHC motility. For instance, subtle biochemical changes in the OHC cortical cytoskeleton may be undetectable by the techniques used in the present work. Future detailed biochemical analyses of this phenomena will likely provide critical insights into the identity of the molecular targets of the Rho GTPases.

      ACh, Rho GTPases, and OHC Motility

      OHC slow motility is an actin-mediated process. Thus, the putative involvement of RhoA, Rac1, and Cdc42 in its regulation should hardly be a surprise. OHC fast motility, on the other hand, is independent of ATP, Ca2+, and, presumptively, of any second messenger-mediated process (
      • Holley M.C.
      ,
      • Kachar B.
      • Brownell W.E.
      • Altschuler R.
      • Fex J.
      ,
      • Holley M.C.
      • Ashmore J.F.
      ). The motor function is very robust, and neither drugs like cytochalasins, colchicine, and nocodazole nor complete disruption of cytoplasmic structures by internal perfusion of the cells with high concentrations of trypsin, can inhibit it (
      • Holley M.C.
      ,
      • Holley M.C.
      • Ashmore J.F.
      ,
      • Kalinec F.
      • Holley M.C.
      • Iwasa K.
      • Lim D.J.
      • Kachar B.
      ,
      • Huang G.
      • Santos-Sacchi J.
      ). However, regulation does not imply inhibition, and OHC fast motility could be regulated without inhibition of the motor function. The mechanical load on the membrane-embedded motor proteins, for instance, could be modulated through changes in number and strength of the thousands of periodically distributed “pillars” that connect the cortical cytoskeleton to the plasma membrane in OHCs. Interestingly, the membrane cytoskeleton linkage through members of the 4.1/Ezrin/Radixin/Moesin protein family seems to be regulated by Rho GTPases (
      • Hall A.
      ,
      • Tsukita S.
      • Yonemura S.
      • Tsukita S.
      ,
      • Girault J.-A.
      • Labesse G.
      • Mornon J.-P.
      • Callebaut I.
      ), and 4.1/Ezrin/Radixin/Moesin proteins have been associated with the pillars in guinea pig OHCs (
      • Kalinec F.
      • Jaeger R.
      • Kachar B.
      ).
      In nonauditory cell types, Rho GTPases have been associated with a variety of motile processes such as filopodia, lamellipodia, and stress fiber formation (
      • Van Aelst L.
      • D'Souza-Schorey C.
      ,
      • Hall A.
      ). Mature OHCs, however, are terminally differentiated, highly specialized cells that do not migrate, divide, or form these structures. Therefore, it is likely that in OHCs, Rho proteins have adapted to regulate functions that are unique to these cells. The recent report that mutations in known targets of Rho GTPases result in deafness further substantiates this idea. In this regard, our results demonstrate that Rho GTPases are involved in the regulation of OHC motility by ACh, a crucial mechanism for acoustic signal amplification and frequency discrimination in the mammalian inner ear. The role of RhoA, Rac1, and Cdc42 in this process, however, may be much more complex than the work models depicted in Figs. 2 F and3 F suggest. For instance, dominant-negative mutants of Rho GTPases inhibit the catalytic domain of Rho-GEFs rather than Rho themselves (
      • Feig L.A.
      ). Because it has been demonstrated that some of these GEFs may activate more than one Rho family member (
      • Zheng Y.
      • Hart M.J.
      • Cerione R.A.
      ,
      • Cerione R.A.
      • Zheng Y.
      ), dominant negative mutants might be interfering with the activation of other components of the family in addition to its normal counterpart, generating a more complex scenario for GTPase interplay. Future studies focused on identifying and characterizing the Rho targets in OHCs will undoubtedly help to refine these models as well as to provide critical insights into the basic mechanisms of both normal human hearing and deafness.

      Acknowledgments

      We thank T. Cook, B. Gebelein, and A. Andalibi for critically reading the manuscript.

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