Учебники / Genetic Hearing Loss Willems 2004

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(10,11). Mutations in myosin VI are found in the deaf and circling mouse mutant, Snell’s waltzer (12). Before delving into the main subject of this chapter, myosin VI in human and mouse hearing loss, we will describe myosins in general.

II.MYOSIN: A MOLECULAR MOTOR

The unconventional myosins are divided into several classes based on analysis of their motor and tail domains. To date at least 18 classes of conventional myosins have been defined and they are numbered in the order in which they were discovered (for complete list, see Myosin Homepage, http://www.mrclmb.cam.ac.uk/myosin/myosin.html).

The myosins are comprised of a heavy chain with a conserved f80-kDa catalytic domain (the head or motor domain), and most are followed by an a- helical light-chain-binding region (the neck region). Most myosins contain a C-terminal tail and, in some cases, an N-terminal extension as well. The amino acid sequence of the head domain is conserved within each classspecific myosin. All myosins contain an actin-binding domain and an ATPbinding domain in their head region, allowing them to move along actin filaments. The tail domains diverge from one another between myosin classes and are believed to confer the function of each di erent myosin. The myosin VIIA and myosin XVA tail share MyTH4 (myosin tail homology 4) domains and FERM membrane-binding domains (13,14), whereas the myosin VI tail is unique among the myosins. Both myosin XVA and myosin IIIA contain N- terminal extensions; the latter is a kinase domain that has been shown to play an important role in phototransduction in Drosophila (15).

The neck domain in almost all myosins consists of a variable number (1– 6) of light-chain-binding motifs, called IQ motifs. These motifs are usually formed from six tandem repeats of a basic and hydrophobic f23 amino acid sequence (reviewed in Ref. 16). Calmodulin is used most frequently as a light chain, as well as other members of the EF hand superfamily. Many calmodulins have been shown to bind to di erent myosins in this region, suggesting that certain calmodulins were formed to bind di erent myosins throughout evolution. The actual presence of calmodulin light chains in many unconventional myosins suggests that their activity may be regulated by calcium. Finally, many myosins share a coiled-coil a-helix motif in the tail region that is predicted to form dimers.

Myosins have a class-specific tail domain, which is the main feature that distinguishes between them. It is hypothesized that this variation in structure is what dictates the varied cellular localization and function of each class of myosin (17). One myosin I, myoB, for example, is thought to control the

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activity of the actin-rich cortex of the cell and has a Src-homology 3 (SH3) domain at its C-terminus, suggesting that it might be regulated through tyrosine-kinase-associated receptors (18). Evidence from yeast and slime demonstrates that the SH3 domain recruits the Arp complex and may play a role in regulating actin polymerization (19). Myosin Va, associated with neurological and melanosome defects in dilute mice and Griscelli disease in humans, possesses a tail that specifies its localization to various organelles in yeast and targets it to the melanosome in vertebrate melanocytes (reviewed in Ref. 20).

III.MYOSIN VI

Myosin VI was first discovered in Drosophila (21) but has since been identified in pig (22), bullfrog (23), mice (12), humans (24), C. elegans (25), striped bass (26), sea urchin (27), and zebrafish (K.B. Avraham and T. Nicolson, unpublished observations). Myosin VI, like other unconventional myosins, has a class-conserved head, neck, and tail region (Fig. 1). Myosin VI has a number of defined features in the head region, including a f25-amino-acid insertion at the position of a surface loop (16) and a conserved threonine residue (residue 405 in humans and 406 in mice) (24). Studies suggest that myosin VI may be activated by a heavy-chain kinase at this conserved threonine residue (28). In the neck domain, myosin VI has a 53-amino-acid segment adjacent to its single IQ motif that di ers from any known N-terminal junction of the neck domain of other myosins. The tail domain has a coiled-coil domain, like a number of other myosins, followed by a globular domain that is unique and highly conserved between the di erent organisms’ myosin VI.

The presence of a unique domain in the neck region of myosin VI led to the suspicion that myosin VI moves in the opposite direction along actin relative to other myosins, toward the ‘‘minus’’ end of actin filaments (29). By use of in vitro motility assays, myosin VI was shown to move toward the pointed (minus) end of actin. Cryoelectron microscopy and image analysis demonstrated that the lever arm, the putative calmodulin light-chain-binding

Figure 1 The myosin VI protein is composed of a head or motor domain, a neck region containing one IQ motif, and a tail composed of a coiled-coil region and a globular domain.

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domain, is indeed in the opposite orientation in myosin VI relative to other myosins. The rotation of the lever arm may determine direction of movement, and thus enable this molecule to move ‘‘backward’’ along actin. Other reports have suggested that the motor core domain of myosin VI determines its directionality (30); this is further supported by the fact that myosin IXb was recently found to also move toward the pointed end of actin, although it does not possess an insert in its head domain (31). Regardless of what portion of the protein determines its directionality, this feature may have significance in polarized cells, where the pointed ends of actin are in the center of the cell, and in hair cells, where the pointed ends of actin face the rootlets of the stereocilia. Myosin VI is expressed in both types of cells (see Sec. VI.A).

IV. MYOSIN VI MUTATIONS IN ANIMAL MODELS

The most informative information regarding myosin VI function has been obtained from either blocking myosin VI function or studying spontaneous or induced mutations.

A.Myosin VI in Drosophila and C. elegans

The Drosophila myosin VI, also named 95F, was found to be required for the normal formation and organization of the syncytial blastoderm. It was shown that when 95F function was inhibited by injecting inhibitory-95F antibodies into syncytial blastoderm Drosophila embryos, profound defects in syncytial blastoderm organization occurred, including colliding mitotic spindles that resulted in misassortment of chromosomes and fused multipolar nuclei (32). 95F is located in cytoplasmic domains around the nuclei during interphase and is found concentrated in the region where actin-based invaginations will form during metaphase (33). It is thought that 95F myosin may be indirectly involved in furrow formation during mitosis by delivery of required furrowforming components. Myosin VI is associated with motile particles and this movement is inhibited by the same antibodies in both Drosophila oocytes and embryos; this was the first discovery that led to speculation that myosin VI might play a role in vesicle tra cking.

Myosin VI also appears to have a conserved role in sperm development. C. elegans myosin VI deletion mutants do not properly segregate cell components, leading to aberrant spermatogenesis (34). Mitochondria, endoplasmic/ reticulum/Golgi-derived fibrous-body membranous organelle complexes, actin filaments, and microtubules are not properly sorted to developing spermatids. These data suggest that in addition to playing a role in the segregation of organelles, myosin VI may also participate in the organization of both

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microtubules and actin. Two Drosophila myosin VI mutants are known to have defects in spermatid individualization, a process where each spermatid is enclosed in its own membrane (35). Myosin VI is therefore required for transport and reorganization of membranes in the individualization complex within Drosophila developing spermatids.

Recent experiments in Drosophila demonstrate that myosin VI is required for E-cadherin-mediated border cell migration (36). Myosin VI is expressed in the outer migratory border cells of Drosophila, and depletion of this protein is associated with a reduction of E-cadherin, which is required for migration of these cells. This finding demonstrates yet more evidence of a function for myosin VI in cell motility.

B.Myosin VI in the Mouse

Mutations in the unconventional myosin VI gene, Myo6, are associated with deafness and vestibular dysfunction in the Snell’s waltzer mouse (12). The Snell’s waltzer mouse mutant was discovered in the late 1960s in Dr. George Snell’s colony at the Jackson Laboratory in Bar Harbor, Maine. Crosses set up to identify the chromosomal location of the defective locus determined that Snell’s waltzer was located on chromosome 9, within the dilute-short-ear region (37). Twenty years later, a positional cloning approach was undertaken to identify the causative gene for deafness in this mouse. Fortunately, one of the Snell’s waltzer alleles, sesv, had an inversion on chromosome 9 that led to access of the sv region (Fig. 2). Yeast artificial chromosome (YAC) clones were obtained from this region and used in an exon-trapping approach to identify an exon that shared 87% homology to the pig myosin VI (22). Myosin VI was an excellent candidate for hearing loss since mutations in the unconventional myosin VIIa had been found a short time earlier in the deaf shaker 1 mouse (10). Soon thereafter, a 130-bp deletion was found in the myosin VI coding region in the second Snell’s waltzer allele, sv (12). This deletion leads to a frameshift in the neck portion of the protein that results in a stop codon (Fig. 1). Consequently, no myosin VI protein has been detected in any tissues examined in the sv allele. The inversion in the sesv allele leads to a reduction in myosin VI expression. The ‘‘break’’ does not occur within the sv gene; rather, it occurs between 30 and 220 kb upstream of the first coding exon. Presumably, promoter regulatory sequences associated with controlling myosin VI expression levels are lost owing to the inversion. Despite the almost ubiquitous expression pattern of myosin VI, the only phenotypes of the Snell’s waltzer mice are deafness and circling.

The major advantage of the mouse myosin VI mutant is the ability to have a mouse model for hearing loss caused by myosin VI mutations. Mice are proving to be an invaluable tool for researchers to study the morphology

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Figure 2 Myosin VI mutations lead to deafness and circling in two Snell’s waltzer alleles, sv and sesv. The sv mouse mutant arose spontaneously and contains an intragenic deletion in the Myo6 gene. The sesv mouse arose by radiation; the Myo6 gene is inverted, presumably leading to the loss of regulatory sequences. The gray box depicts the myosin VI gene, the arrow under the gray boxes shows transcriptional orientation of the gene, and the black rectangle in the sv allele gene (Myo6sv) demonstrates Myo6 deletion.

and physiology of the inner ear owing to the similarity between the mouse and human genomes and the physiology and morphology of the auditory system (reviewed in Ref. 38). With use of a variety of techniques, such as the generation of transgenic mice, scanning electron microscopy (SEM), patch clamping on individual hair cells, and culturing of the organ of Corti, much can be learned about the function of a protein in the inner ear.

V.HUMAN MYOSIN VI

A.The Human Myosin VI Gene and Protein

Since myosin VIIA was involved in both mouse and human deafness, it seemed plausible that the same would be the case for myosin VI. The human myosin VI (MYO6) human cDNA was isolated and characterized (24). Human MYO6 has a 3789-bp open reading frame coding for the human myosin VI protein that shares 90.2% identity with the mouse myosin VI protein. With fluorescent in situ hybridization (FISH), human myosin VI was mapped to chromosome 6q13 (24). At this time, no deafness loci had been identified in this region, suggesting that myosin VI mutations may be rare in the human deaf population or, alternatively, may be found in a population not yet examined. As this gene was still an attractive candidate for human deafness, the map position of human MYO6 was refined by radiation hybrid mapping and its genomic structure was characterized (39).

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Human myosin VI is composed of 32 coding exons, spanning a genomic region of approximately 70 kb. Exon 30, which contains a putative CKII phosphorylation site, was found to be alternatively spliced and expressed in fetal and adult human brain and testis. Another alternatively spliced exon was discovered in the tail region of myosin VI following the coiled-coil domain (40). This exon is highly conserved when compared to chicken and rat and was found to be expressed in tissues such as kidney, liver, and small intestine, all of which have a high proportion of polarized cells with microvilli at their apical surface. The myosin VI isoform containing this exon is localized to clathrincoated vesicles, suggesting a role for myosin VI in clathrin-mediated endocytosis.

B.Human Myosin VI–Associated Hearing Loss

The successful use of a mouse model in predicting human disease was shown when a mutation in the MYO6 gene was found to be associated with hearing loss in a large Italian family (4). A ected members of this family su er from progressive autosomal dominant sensorineural hearing loss, with a variable age of onset mainly during childhood (8–10 years old) (Fig. 3). Audiograms showed di erent degrees of hearing impairment, ranging from moderate to profound sensorineural hearing loss. A genome scan revealed linkage to an 11-cM region on human chromosome 6. This locus was named DFNA22, since it was the twenty-second autosomal dominant locus to be identified (for a list of all loci, see the Hereditary Hearing Loss Homepage, http:// dnalab-www.uia.ac.be/dnalab/hhh/). The human MYO6 gene maps within this region and was an excellent candidate based on its chromosomal location,

Figure 3 A myosin VI mutation leads to progressive hearing loss in an Italian family (9). (A) Pedigree of family, demonstrating dominant inheritance of hearing loss. (B) Representative pure-tone thresholds of a ected members of the Italian family with a myosin VI mutation. Only the left ear is shown. (Adapted with permission from Ref. 4.)

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cochlear expression, and function. The entire MYO6 coding sequence and all exon-intron boundaries were scanned for mutations both by sequencing and by single-strand conformation polymorphism (SSCP) analysis (39). A G!A transition was detected in exon 12 at position 1325 of the cDNA sequence (relative to the ATG, designated +1), which replaces a cysteine (TGT) with a tyrosine (TAT) at residue 442 of the protein (C442Y) (Fig. 4). All a ected members of this family carried the C442Y mutation. The C442Y mutation was not identified in 200 normal chromosomes derived from Italian individuals with no family history of hearing loss.

The C442Y mutation a ects a cysteine residue in the motor domain that is conserved across other myosin VI species, including human, mouse, chicken, pig, striped bass, and sea urchin. The nematode and fruitfly myosin VI each have a similar hydrophilic amino acid, serine, at this position (Fig. 4). In addition, a pairwise alignment of 143 myosins revealed no changes to tyrosine. Owing to the high homology between the motor domains of myosin VI and myosin II, for which a 3D structure was known (41), a three-dimen- sional model of myosin VI was made to predict what kind of damage the tyrosine could inflict (Fig. 5A). According to the model constructed, the cysteine is partly buried in the protein core, and resides at the beginning of the a-helix HO (Fig. 5B). The a-helix HG, which ends with an arginine, lies antiparallel to the cysteine. Replacing C442 with a tyrosine, which is a much bulkier amino acid, may cause a collision between this tyrosine and arginine (Fig. 5C). This collision may lead to either binding through cation-pie

Figure 4 Alignment of a portion of human myosin VI C442 with myosin VI from various species. Note conservation of cysteine (arrow) in Homo sapiens (human; Accession no. U90236) through Strongylocentrotus (sea urchin; Accession no. AF248485) and a similar hydrophilic residue in Drosophila (fruitfly; Accession no. NM_079754) and C. elegans (worm; Accession no. NM_067528). Other accession numbers are as follows: Sus scrofa (pig; Accession no. Z35331), Mus musculus (mouse: U49739); Gallus gallus (chicken; Accession no. AJ278608), Rana catesbeiana (bullfrog, Accession no. U14370), Morone saxatilis (striped sea bass, Accession no. AF017304). (Adapted with permission from Ref. 4.)

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Figure 5 Three-dimensional model of human myosin VI. (A) Schematic representation of a model of the myosin VI motor domain. (B) Wild-type structure showing C442 amino acid. The a-helix HO and the a-helix HG is at approximately amino acid residue 178–184 and 412–442, respectively (see Myosin Homepage Multiple Alignment, http://www.mrc-lmb.cam.ac.uk/myosin/trees/txalign.html). Arrow depicts space between C442 and A181. (C) Mutant structure showing altered amino acid to tyrosine, with arrow depicting potential collision between T442 and A181. The model was built using the automatic homology modeling facility, Swiss Model (http:// www.expasy.ch/swissmod/SWISS-MODEL.html), and the three-dimensional structures of several myosin motor domains. The mutated model was created using the ‘‘Modeler’’ module in Insight (Accelyrs).

interactions (42) or repulsion, both of which will disrupt the protein structure. It is important to stress that this is a model and these theories are speculative based on this model.

VI. MYOSIN VI AND THE INNER EAR

Myosin VI clearly plays a vital role in the inner ear, since mutations in both humans and mice lead to severe to profound hearing loss. Expression has been studied on both an RNA and protein level in a range of organisms.

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A.Myosin VI Expression

Human MYO6 is present in heart, brain, placenta, lung liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, and colon, demonstrating its ubiquitous expression (24). In the striped bass, two isoforms of myosin VI (FMVIA and FMVIB) are expressed in the photoreceptors, horizontal cells, and Mu¨ller cells in fish, suggesting that myosin VI may play a role in retinal motility events (26). Fish myosin VI seems to have a more restricted expression pattern than that of mouse or rat myosin VI. Despite the fact that myosin VI is found in retina of fish and primates (rhesus monkey) (26), no retinal phenotype has been found in animal models or humans with myosin VI mutations. Several expression studies suggest that myosin VI is a general membrane transport motor and has an essential role in membrane tra cking (reviewed in Ref. 43). Myosin VI is expressed in the intermicrovillar (IMV) coated-pit region of the rat kidney brush border, suggesting a role in the function of the endocytic process of the proximal tubule (44). In fibroblasts, myosin VI is localized to the Golgi complex and the leading, ru ing edge of these cells (28).

In the inner ear, myosin VI is specifically expressed in the sensory hair cells. Using antibodies directed against a portion of the pig myosin VI tail, immunochemical studies in the mouse and guinea pig cochlea localized myosin VI to the outer and inner hair cells of the cochlea (24,45). Myosin

Figure 6 Localization of myosin VI in frog and guinea pig hair cells. (A) Antibodies against myosin VI label the cytoplasm of the hair cells. MVI, myosin VI labeling; OHC, outer hair cells; IHC, inner hair cells. (B) No expression of myosin VI is detected in guinea pig stereocilia (arrows). (C) Myosin VI is expressed in the tapered ends of frog stereocilia (arrows). (Adapted with permission from Ref. 45.)

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VI is present in the cell body, and in particular, in the cuticular plate, the actinrich structure underlying the stereocilia (Fig. 6A). No labelling was observed in the stereocilia (Fig. 6B). Extensive analysis has been performed on the expression of myosin VI in the adult American bullfrog (Rana catesbeiana) hair cells (45). Myosin VI was observed in hair cells but not in supporting cells or peripheral cells of the saccule and the cochlea (Fig. 6C). In the saccule and cochlear hair cell, myosin VI is enriched in the cuticular plate and pericuticular necklace and appears throughout the cell. In the bullfrog, myosin VI is present in the tapered ends of the stereocilia, unlike in mammals.

B.The Consequences of Myosin VI Mutations on the Hair Cell

While we cannot determine the fate of the hair cells in humans with myosin VI–associated deafness, we can make predictions by studying the mutants with unconventional myosin mutations. Scanning and transmission electron microscopy on sv mice cochleas revealed severe stereocilia disorganization, with stereocilia fusing shortly after birth (46) (Fig. 7). The fusion begins at the base of the stereocilia and by 7 days after birth, only a few giant and/or fused stereocilia are found on the top of the cells. By 20 days after birth, all hair cells in the cochlear duct possess abnormal stereocilia. Fewer hair cells are seen, demonstrating degeneration of hair cells. Histological sections of the cochlear duct from sv/sv mice indicate a complete loss of hair cells by 6–8 weeks of age (12).

Based on the study on the sv mutants, it was suggested that myosin VI is involved in anchoring the apical hair cell membrane to the underlying actinrich cuticular plate. Without myosin VI, the plasma membrane is unstable and leads to a ‘‘zipping up’’ of the membrane, observed as stereocilia fusion (46). How does this role coincide with the observation that myosin VI is an actin minus end-directed motor? There may be two roles for myosin VI in

Figure 7 Morphology of stereocilia in hair cells of sv/sv mice. (A) In wild-type cells, there are three rows of outer hair cells and one row of inner hair cells. (B) Disorganization of stereocilia is apparent in 20-day-old mice. (C) Fusion at base of stereocilia can be seen at 7 days after birth. (Adapted with permission from Ref. 46.)