Учебники / Genetic Hearing Loss Willems 2004

land, Canada (DFNA38) (8). Hearing loss in all these families proved to be due to mutations in the Wolfram syndrome gene WFS1 (8,9). Additional families with hearing loss due to WFS1 have been observed in Japan, Belgium, Germany, and the United Kingdom (10,11). Homozygosity or compound heterozygosity for loss-of-function mutations in WFS1 led to Wolfram syndrome, a severe recessive disorder characterized by diabetes mellitus and optic atrophy but only occasionally including mild-to-moderate high frequency hearing loss (12). In contrast to mutations causing Wolfram syndrome, WFS1 mutations leading to LFHL are dominantly inherited missense substitutions a ecting the C-terminus of the wolframin protein. Multiple di erent WFS1 mutations leading to low-frequency hearing loss have been described, but only one DFNA1 mutation is known. It is possible that only mutations in a restricted region of diaphanous can lead to a hearing loss phenotype.

B.DFNA1 Mutation

DFNA1 was the first autosomal dominant nonsyndromic hearing loss locus mapped in the human genome. Linkage analysis localized hearing loss in Family M to chromosome 5q31 with a lod score of 13.55 at D5S2119 and D5S2010 (13). Positional cloning subsequently revealed a mutation in the human homolog of the Drosophila melanogaster diaphanous gene cosegregating with deafness in Family M (3). The human diaphanous gene comprises 27 exons spanning 102 kb of genomic DNA. The wild-type human diaphanous gene encodes a transcript of 4.7 kb and a protein of 1283 amino acids.

The DFNA1 mutation in Family M is 3634(+1)G !T, a nucleotide substitution in the first base of the last intron, immediately 3V of codon 1211

(3). The DFNA1 mutation abrogates the 5V splice site of intron 26 so that splicing occurs at a cryptic site between bp +4 and +5 of the intron. Four additional base pairs are introduced into the mutant transcript, leading to premature truncation of the mutant protein at codon 1233. Mutant diaphanous has 21 aberrant amino acids and lacks 52 amino acids (codon 1212– 1263 inclusive) relative to the normal diaphanous protein product. Both normal and mutant alleles are transcribed in lymphoblast cells from a ected family members (3). Wild-type diaphanous has no alternative splicing in this region. All carriers of the mutation older than age 20 are a ected, suggesting that penetrance of the allele is complete. No gender bias is observed. To date, the Family M mutation has not been observed in several hundred hearing controls or in any other family with hearing loss (3).

Expression of the DFNA1 transcript occurs in all tissues tested including cochlea, brain, heart, lung, placenta, kidney, pancreas, liver, and skeletal

muscle (the last of which showed the highest level of expression) (3). RT-PCR of the DFNA1 transcript from cochlear RNA demonstrates that this mRNA is transcribed in the inner ear (3). Expression of diaphanous protein has also been observed in multiple cell lines including fibroblasts (14,15), lymphoblasts (3), kidney cells (16–18), colon epithelial and carcinoma lines (16), fibrosarcomas (18), and several neural carcinoma lines (19).

III.DIAPHANOUS PROTEINS

DFNA1 is a member of the formin gene family. Formins have roles in cytoskeletal organization and cell polarity (20). Proteins encoded by formin genes appear in animals (nematodes, flies, mammals), plants (mustard, tobacco), fungi (yeast, Aspergillus), and protozoa (Entamoeba histolytica). The first formin gene identified was the mouse limb deformity (ld) gene (21,22). More than 10 members of the extended formin gene family are now known in humans.

Mammalian formins and their Drosophila diaphanous homolog share conserved formin homology (FH) domains flanked by coiled-coil domains (CCD) (23) (Fig. 2). Formin homology domain 1 (FH1) is a polyproline rich region of f200 amino acids with multiple repeats of 5–12 consecutive prolines (24). The FH1 region binds both profilin and the SH3 domains of a variety of proteins (25). Formin homology domain 2 (FH2) is highly conserved but its molecular role is less characterized. Sequences of FH1 and FH2 and spacing between them are conserved in formins. The N-terminus contains a third, more loosely conserved domain (FH3) originally defined in the yeast diaphanous homolog Bni1p as essential for protein localization (26). The FH3 domain acts as a binding site and overlaps with a portion of the Rho-binding domain.

Figure 3 illustrates the evolutionary relationships among selected formin family protein sequences. Formin subfamilies include the diapha- nous-related genes (DIAPH and Diap), the dishevelled-activator-of- morphogenesis group (DAAM), formin-like homologs (FMNL), formin- homolog-overexpressed-in-spleen (FHOS), and the classic formins. The homologs of Drosophila diaphanous include human and mouse diaphanous homologs, C elegans cyk-1, S. cerevisiae Bni1p, S. pombe cdc12, and E. histolytica diaphanous.

Three diaphanous homologs exist in humans and mouse. The human and mouse genome informatics groups have adopted DIAPH1–3 and Diap1– 3 as gene names for human and mouse homologs, respectively (Table 1). Each human DIAPH gene is more similar to its mouse counterpart than are the

Figure 2 Diaphanous is a member of the formin family defined by conserved formin homology (FH) domains. Protein domains of Diap1 include FH3 (aa 157–457), polyproline-rich FH1 (aa 570–761), and FH2 (aa 945–1010). Also present are two coiled-coil domains, CCDI and CCDII (aa 457–570 and 1011–1192, respectively), and a putative nuclear localization signal (NLS aa 1196–1202). Rho GTPase binds diaphanous at the N-terminus (aa 63–260). Profilin binds the FH1 domain of diaphanous via polyproline stretches. The SH3 domains of Src, IRSp53, and DIP bind diaphanous via the SH3 binding sites in FH1. The C-terminal portion of the FH3 domain and the first coiled-coil domain (aa 431–603) target diaphanous to the mitotic spindle. The FH2 domain binds stable microtubules in nondividing cells. The diaphanous N-terminus (aa 63–413) and the C-terminal intramolecular interaction domain (CIID, aa 1116–1255) interact. The CIID includes a conserved 23-amino-acid region (aa 1177–1199 in Diap1) that overlaps the diaphanous autoregulatory domain (DAD) defined in Diap3.

DIAPH genes to each other (Fig. 3). DFNA1 is caused by a mutation in human DIAPH1 on chromosome 5q31. Human and mouse diaphanous1 proteins are 90% identical and 93% similar at the amino acid level. Functional domains are conserved (Fig. 2). The mouse diaphanous1 coding region (1255 amino acids) is nearly identical in size to the human (1263 amino acids) (18). Human DIAPH2 was initially identified during cloning of DFNA1 and is located at Xq22 (3,27). Disruption of DIAPH2 is associated with premature ovarian failure in a patient carrying translocation t(X;12)(q21;p2.3) (28,29). Identification of the breakpoint in this and a second patient with premature ovarian failure showed that both breakpoints occur in the last intron of DIAPH2 (28,30). These individuals exhibit mild amenorrhea or premature menopause with associated elevated gonadotropin levels. Neither reports any hearing abnormalities. The association of disruption of DIAPH2 with ovarian failure is consistent with the observation that mutations in Drosophila diaphanous lead to defects in oogenesis. As yet little is known about human DIAPH3 on chromosome 13. Expression of DIAPH3 is observed

Figure 3 Relationships between selected members of the formin family are illustrated with an unrooted neighbor-joining tree constructed from comparison of protein sequences using Bonsai software (72). Asterisks indicate the mammalian diaphanous homologs. Two-letter prefixes indicate species: Hs for Homo sapiens, Mm for Mus musculus, Dm for Drosophila melanogaster, Sc for Saccharomyces cerevisiae, and Sc for Schizosaccharomyces pombe.

Table 1 Diaphanous Homologs in Human and Mouse

predominantly among hematopoetic lineages and carcinoma cell lines, indicating that DIAPH3 has a narrower range of expression than DIAPH1. No mutations associated with disease have yet been found in DIAPH3. Thorough examination of overlap and distinctions between the diaphanous paralogous proteins in vivo remains to be done.

The presence of three diaphanous homologs in humans and mice (Fig. 3) may allow functional redundancy. Diaphanous 1 and 3 can substitute for each other for some functions, such as formation of stress fibers in fibroblasts (16,31). However, there are indications that the diaphanous paralogs have diverged. Not all cell types contain both DIAPH1 and DIAPH3 protein. Also, Diap3 may be activated in response to broader types of stimuli than Diap1 (18). The mutation associated with premature ovarian failure in DIAPH2 is a C-terminal truncation, similar to the Family M mutation in DIAPH1, and is likely to be a subtle hypomorphic or dominant negative mutation rather than a null mutation. It remains to be seen what phenotype occurs with a null mutation in any mouse or human diaphanous homolog.

IV. DIAPHANOUS FUNCTION

A.Role of Diaphanous in Regulation of Actin Structures

Diaphanous is a component of a Rho GTPase pathway (Fig. 4) (reviewed in Ref. (32–35)). Rho GTPase pathways are ubiquitous and coordinate external stimuli with internal cellular responses, ultimately regulating organization of the cytoskeleton. Of the 17 members of the Rho family of small GTPase molecules in mammals, the best studied are Rho, Cdc42, and Rac1 (Fig. 4). These related proteins promote di erent downstream responses by activating various combinations of e ectors. Rho is involved in induction of stress fibers and focal contacts, Cdc42 in induction of filopodia, and Rac in induction of membrane ru ng or lamellipodia. Both Rho and Cdc42 are required for cytokinesis. Recent work indicates there may be considerable cross-talk and use of multiple pathways in formation of these cytoskeletal structures. Diaphanous is a target of Rho and performs most, if not all, of its functions via this pathway. When activated by Rho, diaphanous functions in formation and spatial organization of actin-based structures (e.g., stress fibers, focal contacts, contractile rings), organization of microtubules, and transduction of cellular signals.

Rho mediates formation of cytoskeletal structures by binding the diaphanous N-terminus and activating the protein. In the absence of Rho, diaphanous N- and C-termini bind to each other (Fig. 2) and preclude diaphanous activity (Fig. 4) (14,31,36). Activated Rho disrupts this intramolecular

Figure 4 Extracellular stimuli activate Rho GTPase pathways to induce cytoskeletal reorganization and coordinated cellular events. Rho activates diaphanous. Cdc42 and Rac GTPases Induce di erent actin structures through activation of other e ector molecules.

interaction, apparently leaving diaphanous in an open, active conformation (14). Diaphanous interacts via its FH1 domain with profilin, an actin-binding protein (Fig. 2), thereby regulating actin polymerization (Fig. 4) (18,36). Most of the structures induced by diaphanous are composed of actin-myosin filaments. The Diap1 Rho-binding domain (RBD) (Fig. 2) is specifically bound by active Rho but not by inactive Rho or by either form of Rac1 or Cdc42 (Fig. 4) (18). In contrast, both Rho and Cdc42 are reported to bind Diap3 (31). Unlike most Rho targets, diaphanous is not a kinase and contains a distinct Rho binding site (32,35,37). However, Rho often simultaneously activates diaphanous and ROCK, a Rho-associated protein kinase (Fig. 4). Diaphanous and ROCK can act cooperatively, as seen with the induction of stress

fibers (14,16,38), or can act antagonistically, as seen with formation of adherens junctions (39).

When activated by Rho, diaphanous regulates formation of stress fi- bers and focal contacts at the end of stress fibers. Stress fibers are linear, contractile structures of bundled actin and myosin II filaments that influence cell shape and strength. Focal contacts, specialized sites of contact between the plasma membrane and the extracellular matrix, function in adhesion and cell signaling to coordinate cellular response to the extracellular matrix (40). The role of diaphanous in formation of these structures was demonstrated by creating a mutant diaphanous protein lacking the N-terminus. This mutant protein was constitutively active and, when overexpressed, led to formation of parallel stress fibers and focal contacts (14,17). In response to external force, activated diaphanous enables focal contacts to form from focal complexes, smaller integrin-based actin complexes containing distinct proteins (40,41). Riveline et al. noted that focal complexes act as ‘‘individual mechanosensors,’’ transducing stimulation by force in a local area into focal contacts (41) if diaphanous is present to mediate their production.

In addition to inducing focal contacts, activated diaphanous regulates formation of other anchoring junctions important for attachment of actin filaments to the plasma membrane: adherens junctions, anchoring sites mediating cell-cell contacts, and fibrillar adhesions, fibronectin-induced sites associated with wound healing and phagocytosis. These anchoring junctions are abundant in tissues subject to mechanical stress and are important for the transmission of force. Diaphanous and actin filaments enable colocalization of E-cadherin and alpha-catenin at adherens junctions (39). Inhibition of diaphanous leads to dynamic instability of the cell periphery, seen as extension and retraction of membrane projections (39). This suggests that diaphanous stabilizes the cell periphery via its e ect on cadherins at adherens junctions.

Rho signals both diaphanous and a second e ector, Rho-associated kinase or ROCK. ROCK promotes formation of myosin filaments by phosphorylating myosin light chain and myosin light chain phosphatase (Fig. 4) (19). Disassembly and reassembly of stress fibers and focal contacts respectively require inactivation and reactivation of both ROCK and diaphanous (38). Thus, formation of these actinomyosin structures results from cooperative interaction of diaphanous and ROCK. ROCK and diaphanous act antagonistically to determine the spatial organization of these same structures. Overexpression of active diaphanous favors formation of parallel stress fibers and small focal contacts at the cell periphery, whereas overexpression of active ROCK favors formation of radial or stellate stress fibers and large basally located focal contacts. Overexpression of both diaphanous and ROCK yields intermediate stress fiber conformations and moderately sized, evenly distrib-

uted focal contacts (14,16,38). Antagonism between diaphanous and ROCK is also seen in the formation of adherens junctions, where diaphanous acts to cluster integrins and stabilize the cell periphery while ROCK destablizes the cell periphery (39).

B.Role of Diaphanous in Cytokinesis

Diaphanous is required for normal cytokinesis. Perturbing interactions of mammalian diaphanous Rho-binding domain disrupts cytokinesis by preventing contractile ring formation and results in binucleated cells (16). Mutations of diaphanous orthologs in D. melanogaster, C. elegans, S. cerevisiae,

S. pombe, and A. nidulans are associated with defects in cytokinesis (23,42– 47). Furthermore, diaphanous itself localizes to the midbody and cleavage furrow of dividing fibroblasts (16).

The most comprehensive information about the role of diaphanous in vivo comes from Drosophila diaphanous mutants. Null alleles of Drosophila diaphanous confer a recessive lethal phenotype in pupae characterized by absence of cleavage furrows and presence of hyperploid cells (23). Weaker alleles allow flies to progress further in development, but these mutants also show defects in cells undergoing rapid division including imaginal discs, ovarian follicle cells, ommatidia of the eye, and neuroblasts.

Absence of diaphanous in flies disrupts both mitosis and meiosis. The mildest Drosophila diaphanous allele, dia1, leads to recessive male sterility. Spermatid production rapidly ceases following an initial burst of division (23,48). Initial rounds of spermatogenesis generate multinucleate spermatids, indicating a failure of cytokinesis following meiosis. Female flies carrying dia1 and a diaphanous deletion exhibit oogenesis defects as the result of failure of cytokinesis in follicle cells. These gametogenesis defects in flies are reminiscent of the association of DIAPH2 with premature ovarian failure.

Diaphanous is critical to normal cell division in Drosophila embryos (47). It coordinates actin-membrane interactions during cytokinesis. Diaphanous protein is abundant at the leading edge of invaginating cell membranes and appears on the basal surface of each new cell. Diaphanous is essential for proper reorganization of actin during cell division (47). Fly embryos lacking maternal diaphanous have disorganized actin arrays and mislocalize myosin II (47).

Diaphanous functions as a major e ector of Rho during cytokinesis. Mutations in Drosophila diaphanous act as dominant suppressors of constitutively active mutations of Rho 1 or pebble, a Rho guanine nucleotide exchange factor. Diaphanous mutations dominantly enhance loss of function of pebble (49). The ability of a diaphanous null allele to act dominantly in Rho1 or pebble mutant flies suggests that a precise molecular balance exists.

If the mutation in human DFNA1 results in an unstable protein, decreased levels of diaphanous may cause a similar dominant impact on function of the human Rho pathway.

C.Role of Diaphanous in Regulating Microtubules

Diaphanous is an organizer of microtubules as well as of actin. Diaphanous localizes to spindle microtubules in prophase through telophase and associates with all three types of spindle microtubules: kinetochore, nonkinetochore, and astral microtubules (15). These interactions are independent of Rho activity or the presence of actin structures. A region including part of the diaphanous FH3 and CCDI domains is responsible for microtubule association. However, the FH3-CCDI region of diaphanous does not bind polymerizing microtubules in vitro, indicating that the association of diaphanous with microtubules is indirect (15).

Diaphanous a ects organization of spindle microtubules. During Drosophila spermatogenesis, diaphanous mutants do not form actin rings and also show defects in the central spindle including fewer microtubules, decreased microtubule interdigitation, and delayed aster positioning. During mitotic divisions, fly embryos lacking maternal diaphanous have disorganized microtubule baskets around nuclei and exhibit abnormal centrosome movements in addition to severe actin defects (47). Signaling from the central spindle is generally required for orienting the contractile ring (50,51). Diaphanous has been proposed to mediate cross-talk between microtubules and actin filaments (47). Such coordinate regulation of microtubules and actin via diaphanous is observed in yeast. In S. cerevisiae, Bni1p, the homolog of diaphanous, a ects positioning of the central spindle in the bud neck as well as establishment of the contractile ring (52).

In nondividing cells, diaphanous a ects the formation and maintenance of microtubules and hence influences cell shape. Overexpression of activated Diap1 leads to significant elongation of cells (53). Within these elongated cells, actin bundles are aligned in parallel, coordinately oriented with microtubules, and terminate in focal contacts at the cell periphery that contain diaphanous (53). Overexpression of dominant negative mouse Diap1 protein reduces parallel microtubule formation, eliminates formation of focal contacts, and leads to disorganized stress fibers.

Microtubules induced by diaphanous are stable rather than dynamic (54). Stable microtubules have detyrosinated ends and persist for hours in contrast to dynamic, tyrosinated microtubules, which turn over in 5–10 min (55). Mouse diaphanous interacts directly with stable microtubules (54). The action of mouse Diap1 on microtubules is mediated by the FH2 domain (53). The conservation of the FH1 and FH2 regions among formin homologs

may allow coordinate regulation of actin (via FH1) and microtubule processes (via FH2) (53). Similarly, Bni1p, the diaphanous homolog in S. cerevislae, is involved in capture and transient capping of microtubules in the cell cortex, thereby positioning the mitotic spindle and moving the yeast nucleus to the budding daughter cell (44,56). Thus diaphanous-induced actin (39) and microtubule structures (54) aid stabilization of the cell periphery.

D.Role of Diaphanous in Transducing Cellular Signals

The role of diaphanous in Rho-mediated transduction of external stimuli involves cellular signaling in addition to cytoskeletal alterations. Accordingly, diaphanous binds several proteins via its SH3 binding sites, which are themselves regulatory molecules: diaphanous interacting protein (DIP) (57), insulin receptor substrate p53 or IRSp53 (58), and Src (16) (Fig. 2). Interaction of IRSp53 and DIP homologs with diaphanous homologs have been conserved from yeast to mammals (45,59). DIP and diaphanous colocalize at the cell periphery and in membrane ru es in human HeLa cells. Mutant DIP suppresses diaphanous induction of stress fibers (57), suggesting that the interaction of DIP with diaphanous is important for stress fiber formation. In yeast, mutations in DIP or IRSp53 homologs lead to cells with cytokinesis defects akin to mutations in the diaphanous homologs, Bni1p and Bnr1p (60). The phenotypes of these yeast mutants suggest that diaphanousDIP and diaphanous-IRSp53 interactions are critical both to cell division and to formation of actin structures.

Src interaction with diaphanous is required for some diaphanousmediated events including cytokinesis and response to serum (16,61). In response to serum, diaphanous activates serum response factor (SRF) via Src (16). SRF is a transcription factor regulating expression of vinculin, h- actin, and sm-a-actin (62,63). Diaphanous-dependent Src activity leads to increased expression of components required for diaphanous-induced structures. Diaphanous is su cient for SRF activation in fibroblasts (16,39,61) but requires the ROCK target LIMK, in neural cells (61). Src activity is also needed for stress fiber organization (16). Inhibition of Src eliminates cooperative stress fiber formation by diaphanous and ROCK and produces a stellate actin filament pattern reminiscent of activated ROCK alone. Thus the spatial organization of actin by diaphanous requires Src activity (16).

Cross-talk between Rho GTPase pathways can occur via diaphanous. Interaction of Rho and Rac pathways occurs via diaphanous and Src interaction. When ROCK is suppressed, diaphanous activates Rac and induces membrane ru es via Src (64). When both Rac and ROCK are suppressed, expression of diaphanous leads to filopodia-like protrusions (64) akin to Cdc42-induced filopodia. Cross-talk between the Rho and Cdc42 pathways