Учебники / Genetic Hearing Loss Willems 2004

may occur via DIP and IRSp53, which bind both diaphanous and the Cdc42 e ector WASP (60,65,66). Thus, diaphanous provides a point of intersection between the Rho, Cdc42, and Rac pathways.

V.POSSIBLE MOLECULAR MECHANISMS FOR DFNA1 HEARING LOSS

A.Consequences of Loss of the Diaphanous C-Terminus

Complete loss of diaphanous is likely to be devastating to the cytoskeleton and hence lethal. The DFNA1 mutation clearly reflects a more subtle pertubation. Watanabe et al. proposed that the DFNA1 truncation leads to defective diaphanous autoregulation (14). That is, by precluding or reducing the normal intramolecular interaction between diaphanous N- and C-termini, the mutant DFNA1 protein may be open and active even in the absence of activation by Rho (Fig. 4). As a result, the mutant protein may be constitutively active or may interfere with wild-type protein in a dominant negative fashion. However, studies of autoregulation of Diap3 are not fully consistent with this model. The normal diaphanous autoregulatory domain of Diap3 ends at residue 1177 corresponding to DIAPH1 residue 1204. This is the site corresponding to the DFNA1 truncation, suggesting that diaphanous autoregulation may be intact in DFNA1 (31). However, autoregulation of Diap1 may require a larger region. Although the diaphanous autoregulatory domain is highly conserved between Diap3 and Diap1, the region C-terminal to DFNA1 site is not conserved.

In human HeLa cells, overexpression of diaphanous that is full length except for absence of the C-terminal 63 amino acids (i.e., aa 1–1192 present) induces protruding membranes reminiscent of filopodia or microspikes (14). The same e ect is produced by simultaneously inhibiting Rac and ROCK activities (61). That is, these protrusions may be caused by activated diaphanous or by diaphanous uncoupled from ROCK and Rac pathways. Expression of diaphanous constructs missing only the last 39 amino acids (i.e., aa 1–1216 present) do not produce protruding membranes (14). It is not yet known what e ect would be seen when 52 amino acids are deleted from the C-terminus, as in Family M.

B.Why Is DFNA1 Nonsyndromic?

Diaphanous induces actin filaments, organizes actin structures and microtubules, and coordinates cell signaling. These functions are central roles within a cell. Why then does Family M exhibit only hearing loss? This may reflect functional redundancy among the DIAPH paralogs in nonauditory

(indicative of cytokinesis defects), and abnormally formed mechanosensory bristles with disorganized actin bundles (68).

Figure 5 indicates possible hair cell structures that might be perturbed by altered diaphanous activity. These include actin structures with parallel fibers such as hair bundles and the zona adherens, parallel microtubules, or regions of contact between the cuticular plate and actin filaments or microtubules. The function of diaphanous may also be important for maintenance of the zona adherens of the hair cell and for proper localization of E-cadherin and a-catenin, akin to its role in other cell types (39). Furthermore, focal contact formation in response to tension has been observed as a diaphanousdependent event in fibroblasts. A link between integrins and Rho family GTPases at focal contacts is thought to be a crucial feature in localized control of focal contacts (69). These observations suggest that diaphanous might be important for transduction of signals in response to movement or extracellular matrix signals.

Diaphanous might also have a role in maintenance of hair cells. Development of hair bundles in chick initially involves Rac-dependent, then Cdc42dependent events (70). Acoustic trauma to chick cochlea leads to an increase in Cdc42, suggesting that induction of a RhoGTPase may be involved in hair cell regeneration of hair bundle repair (71). Perturbation of diaphanous may directly or indirectly inhibit repair of hair bundles, perhaps via interference with Rac or Cdc42 pathways. For example, mutant diaphanous could titrate DIP, which also binds the Cdc42 e ector WASP.

In conclusion, DFNA1 is associated with sensorineural hearing loss in the first decade that begins as a low-frequency loss and progresses to profound loss at all frequencies. The protein encoded by the diaphanous gene is a formin family protein that serves as an e ector of the small GTPase Rho. Diaphanous regulates formation of actin-based structures (stress fibers, focal contacts, and contractile rings), spatially organizes actin filaments and stable microtubules, and coordinates cytoskeletal changes with cellular signaling. E ects of mutation in DFNA1 in hearing loss are likely to reflect a subtle perturbation of these functions in the cochlea.

ACKNOWLEDGMENTS

We thank the members of Family M, whose generous participation has been essential for work on DFNA1. We thank Karen Avraham, John Kemner, Piri Welsch, and Terry Young for comments during the preparation of this chapter. We thank Jim Thomas for providing the Bonsai software used for creating neighbor-joining trees and multiple alignments of proteins. Research

in the laboratory of M.C.K. is supported by the National Institutes of Health (NIDCD Grant RO1 DC01076).

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