**6.1 Hair bundle cohesion**

308 Hearing Loss

+: existence of mislocalization, -: normal localization, +/-, contradictory results

*Sans-/-* 

*Ush2a-/-* 

*Gpr98-/-* 

*Whrn-/-* 

+

(Michalski et

+

(Michalski et

al., 2007)

(Michalski et

(Michalski

et al., 2007;

Yang et al.,

+

al., 2007;

Yang et al.,

2010)

2010)

al., 2007)

+

+

(Lefevre et al.,

2008)

+

(Yang et al.,

(Yang et

al., 2010)

+

2010)

+

(Michalski

et al., 2007)

Table 3. Interdependence of USH proteins in hair cells

*Myo7a-/-* + (Boeda et al., 2002; Lefevre et al., 2008) - (Boeda et al., 2002; Senften et al., 2006) + (Senften et al., 2006) - (Caberlotto et al., 2011) + (Michalski et al., 2007) + (Michalski et al., 2007) + (Michalski et al., 2007) *Ush1c-/-* +/- (Lefevre et al., 2008; Yan et al., 2011) - (Lefevre et al., 2008) +/- (Lefevre et al., 2008; Yan et al., 2011) +/- (Caberlotto et al., 2011; Yan et al., 2011) *Cdh23-/-* + (Bahloul et al., 2010) + (Lefevre et al., 2008; Bahloul et al., 2010) - (Senften et al., 2006) + (Caberlotto et al., 2011) *Pcdh15-/-* + (Senften et al., 2006) + (Lefevre et al., 2008) - (Senften et al., 2006) + (Caberlotto et al., 2011)

**MYO7A USH1C CDH23 PCDH15 SANS USH2A GPR98 WHRN** 

During development, at the apex of hair cells, microvilli grow into stereocilia by recruiting more actin filaments. These stereocilia are bundled with transient lateral links and are connected with the kinocilium through kinociliary links. Following the establishment of the planar cell polarity, the kinocilium moves from the center to the periphery of the cell, and the stereocilia elongate differentially. The staircase-shape hair bundle is eventually formed. At the same time, the transient lateral links are gradually substituted by two distinct sets of interstereociliary links. They are the horizontal top connectors and the ankle links, close to the tip and base of the hair bundle, respectively (Figure 4). The tip links emerge, which are fibrous connections between the tip of medium and low stereocilia and the side of the neighboring taller stereocilia (Figure 4). Finally, the stereocilia grow both in length and in width and reach their mature size. In rodent mature cochlear hair cells, the ankle links and the kinociliary links disappear with the regression of the kinocilium (Frolenkov et al., 2004; Goodyear et al., 2005; Nayak et al., 2007).

CDH23 (Siemens et al., 2004; Lagziel et al., 2005; Michel et al., 2005; Rzadzinska et al., 2005; Lefevre et al., 2008) and PCDH15 (Goodyear et al., 2010; Webb et al., 2011; Lefevre et al., 2008) are localized at the transient lateral links and kinociliary links during early development of hair cells. In their mutant mice, hair bundles are usually splayed into several clumps; kinocilium is mispositioned and disconnected with the hair bundle (Lefevre et al., 2008), indicating that CDH23 and PCDH15, as components of the interstereociliary links, are important for hair bundle cohesion and that loss of the connection between the stereocilia and kinocilium causes the misorientation of the hair bundle. Interestingly, the mutant mouse models of all five USH1 genes share such similar phenotypes. This could be explained by the idea that the five USH1 proteins coordinate in this function. The PST domain of harmonin b binds to and bundles actin filaments (Boeda et al., 2002). MYO7A is a high duty ratio motor, which binds to actin filament strongly. Therefore, these two actinbinding proteins may anchor their interacting partners, CDH23 and PCDH15, to the actin bundle in the stereocilia of hair cells (Table 2). In *Ush1g-/-* mice, cohesion of stereocilia is disrupted. In *Ush1gfl/flMyo7a-cre+/-* mice, whose expression of SANS is disturbed only after birth, the stereocilia stay cohesive (Caberlotto et al., 2011). Therefore, SANS plays a role in stereocilia cohesion during the prenatal period. It may be involved in the organization of other USH1 proteins through directly interacting with them (Table 2).

All three USH2 proteins, USH2A, GPR98, and whirlin, are positioned at the ankle links of hair cells. Among these proteins, USH2A and GPR98 probably interact with each other or with some unidentified cell adhesion proteins to form the ankle links. Whirlin interacts with USH2A and GPR98 through the PDZ domain-mediated binding to anchor them at the base of the stereocilia. In the absence of GPR98, the ankle links are missing. Thus far, the

Usher Syndrome: Genes, Proteins, Models, Molecular Mechanisms, and Therapies 311

(Caberlotto et al., 2011). In *Ush1gfl/fl Myo7a-cre+/-* mice, whose hair bundle morphology is intact, only the amplitude of transduction is affected. This finding indicates that SANS is implicated in mechanotransduction and plays a different role from harmonin or MYO7A. *Gpr98* knockout and *Gpr98del7TM* mice also show defects in mechanotransduction, though there are some discrepancies between them (McGee et al., 2006; Michalski et al., 2007). In general, the sensitivity to the stimulation direction is changed in both outer and inner hair cells. The amplitude and sensitivity of the transduction current decrease in the outer hair cells, but are normal in the inner hair cells and the utricular hair cells. It is suggested that the misorganization of hair bundles in *Gpr98* mutant mice accounts of the abnormal sensitivity direction. Alternatively, GPR98 could be indirectly related with the cellular process of

In photoreceptors, the outer segment is a large specialized cilium filled with many flat membrane disks, where phototransduction occurs (Figure 4). This cellular compartment undergoes continuous and rapid renewal (Young, 1967; LaVail, 1976; Young, 1976; Besharse and Hollyfield, 1979), which requires a large amount of proteins and membrane lipids to be synthesized in the inner segment and to be quickly transported to the base of the outer segment through the connecting cilium (Figure 4). The removal of the old outer segment is achieved through phagocytosis by RPE cells. In addition, in both photoreceptors and RPE cells, several proteins, involved in phototransduction and retinoid cycle, translocate between two different cellular compartments in response to light (Artemyev, 2008; Slepak

Among USH proteins, MYO7A is an actin-based motor. In the retina, it is expressed in both RPE cells and photoreceptors. In RPE cells, MYO7A is essential for the transport of phagosomes to their degradation apparatus (Gibbs et al., 2003), tethering melanosomes during their movement (Gibbs et al., 2004), and the translocation of RPE65 responding to light exposure (Lopes et al., 2011). In photoreceptors, MYO7A is present along the connecting cilium. Loss of MYO7A was found to delay the transport of opsin from the inner to the outer segment (Liu et al., 1999) and the transducin translocation from the outer to the inner segment after light exposure (Peng et al., 2011). In hair cells, without MYO7A, all USH2 proteins are mislocalized from the ankle links (Table 3), suggesting that MYO7A may transport the USH2 proteins. These lines of evidence establish the notion that MYO7A may function in protein and organelle transport in various cells in the retina and the inner ear.

USH2 proteins are positioned at the PMC in mammalian photoreceptors, which is an analogous structure to the periciliary ridge complex (PRC) in frogs (Peters et al., 1983). The PRC is a morphologically-specialized structure with a symmetrical array of 9 ridges and 9 grooves. It has been proposed, based on immunocytochemistry and freeze-fracture electron microscopy, as the membrane fusion site for post-Golgi vesicles carrying opsin and docosahexaenoyl (DHA)-phospholipids before these cargos are transported from the inner to the outer segment (Peters et al., 1983; Papermaster et al., 1986; Rodriguez de Turco et al., 1997; Papermaster, 2002). Additionally, Rab8, rac1, Sec8, moesin, syntaxin 3 and SNAP-25 have been localized around the PRC in frog photoreceptors (Deretic et al., 2004; Mazelova et al., 2009). These proteins are proposed, though not verified using mouse genetics, to

mechanotransduction.

**6.3 Protein and organelle transport** 

and Hurley, 2008; Lopes et al., 2011).

dependence of the ankle links on USH2A and whirlin has not been examined. In the wildtype mouse, the stereocilia of outer hair cells are organized into a V-shaped staircase-like hair bundle. However, in all three *Ush2* mutant mice, the outer hair cells show various disorganized stereocilia and abnormal U-shape hair bundles (Mburu et al., 2003; McGee et al., 2006; Liu et al., 2007; Michalski et al., 2007; Yang et al., 2010). Accordingly, as components of the ankle links, the three USH2 proteins probably contribute to hair bundle cohesion as well.
