[International expert consensus on gene therapy for hereditary hearing loss: based on clinical trials].
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Journal Article↗
Biomedical subjects
Y Zhao
Publications and source records attributed to Y Zhao.
Starvation and diabetes increase the amount of pyruvate dehydrogenase kinase isoenzyme 4 in rat heart.
This study investigated whether conditions known to alter the activity and phosphorylation state of the pyruvate dehydrogenase complex have specific effects on the levels of isoenzymes of pyruvate dehydrogenase kinase (PDK) in rat heart. Immunoblot analysis revealed a remarkable increase in the amount of PDK4 in the hearts of rats that had been starved or rendered diabetic with streptozotocin. Re-feeding of starved rats and insulin treatment of diabetic rats very effectively reversed the increase in PDK4 protein and restored PDK enzyme activity to levels of chow-fed control rats. Starvation and diabetes also markedly increased the abundance of PDK4 mRNA, and re-feeding and insulin treatment reduced levels of the message to that of controls. In contrast with the findings for PDK4, little or no changes in the amounts of PDK1 and PDK2 protein and the abundance of their messages occurred in response to starvation and diabetes. The observed shift in the relative abundance of PDK isoenzymes probably explains previous studies of the effects of starvation and diabetes on heart PDK activity. The results indicate that control of the amount of PDK4 is important in long-term regulation of the activity of the pyruvate dehydrogenase complex in rat heart.
Animals↗
Identification and characterization of sporulation-dependent promoters upstream of the enterotoxin gene (cpe) of Clostridium perfringens.
Three promoter sites (P1, P2, and P3) responsible for the sporulation-associated synthesis of Clostridium perfringens enterotoxin, a common cause of food poisoning in humans and animals, were identified. Nested and internal deletions of the cpe promoter region were made to narrow down the location of promoter elements. To measure the effects of the deletions on the expression of cpe, translational fusions containing the promoter deletions were made with the gusA gene of Escherichia coli, which codes for beta-glucuronidase; E. coli-C. perfringens shuttle vectors carrying the fusions were introduced into C. perfringens by electroporation. In addition, in vitro transcription assays were performed with the cpe promoter region as the DNA template for extracts made from sporulating cells. DNA sequences upstream of P1 were similar to consensus SigK-dependent promoters, while P2 and P3 were similar to consensus SigE-dependent promoters. SigE and SigK are sporulation-associated sigma factors known to be active in the mother cell compartment of sporulating cells of Bacillus subtilis, the same compartment in which enterotoxin is synthesized in C. perfringens.
Base Sequence↗
Localization of the heme binding region in soluble guanylate cyclase.
Soluble guanylate cyclase (sGC) is a heterodimeric hemoprotein composed of alpha1 and beta1 subunits. sGC is activated by nitric oxide (NO) and therefore plays a central role in NO signal transduction. Activation of sGC by NO is believed to be mediated by the interaction between NO and the heme of sGC. Spectroscopic and kinetic studies have shown that the heme of sGC is in a unique environment. Characterization of the heme environment is critical to the understanding of the mechanism of NO activation. To approach this goal, the beta1 N-terminal fragment consisting of residues 1-385 [beta1(1-385)] of sGC was expressed in E. coli. beta1(1-385) was then purified to homogeneity in two steps by DEAE ion exchange and gel filtration chromatography. Purified beta1(1-385) was found to contain a stoichiometric amount of heme. The UV-visible spectrum of beta1(1-385) is almost identical to that of the native heterodimeric sGC purified from bovine lung. beta1(1-385) binds both NO and CO, leading to a shift in the Soret maximum from 431 nm to 398 and 423 nm, respectively. These spectral shifts are identical to those observed with heterodimeric sGC purified from bovine lung. These results suggest that the heme in the beta1(1-385) is similar to that in the heterodimeric sGC. Therefore, for the first time, the heme binding region of sGC has been unambiguously localized to the N-terminal region of the beta1 subunit. Our data also suggest that the N-terminal region of the beta1 subunit of sGC is itself sufficient for heme binding.
Animals↗
Identification of a second aryl phosphate-binding site in protein-tyrosine phosphatase 1B: a paradigm for inhibitor design.
The structure of the catalytically inactive mutant (C215S) of the human protein-tyrosine phosphatase 1B (PTP1B) has been solved to high resolution in two complexes. In the first, crystals were grown in the presence of bis-(para-phosphophenyl) methane (BPPM), a synthetic high-affinity low-molecular weight nonpeptidic substrate (Km = 16 microM), and the structure was refined to an R-factor of 18. 2% at 1.9 A resolution. In the second, crystals were grown in a saturating concentration of phosphotyrosine (pTyr), and the structure was refined to an R-factor of 18.1% at 1.85 A. Difference Fourier maps showed that BPPM binds PTP1B in two mutually exclusive modes, one in which it occupies the canonical pTyr-binding site (the active site), and another in which a phosphophenyl moiety interacts with a set of residues not previously observed to bind aryl phosphates. The identification of a second pTyr molecule at the same site in the PTP1B/C215S-pTyr complex confirms that these residues constitute a low-affinity noncatalytic aryl phosphate-binding site. Identification of a second aryl phosphate binding site adjacent to the active site provides a paradigm for the design of tight-binding, highly specific PTP1B inhibitors that can span both the active site and the adjacent noncatalytic site. This design can be achieved by tethering together two small ligands that are individually targeted to the active site and the proximal noncatalytic site.
Base Sequence↗
Molecular cloning, characterization and localization of PfPK4, an eIF-2alpha kinase-related enzyme from the malarial parasite Plasmodium falciparum.
PfPK4, a protein kinase gene from the human malarial parasite Plasmodium falciparum, has been cloned utilizing oligonucleotide probing. The gene encodes a protein of a predicted length of 1123 amino acids, and within this amino acid sequence all the conserved regions characteristic of protein kinases can be identified. The catalytic kinase domain possesses highest identities (34-37%) with eukaryotic initiation factor-2alpha (eIF-2alpha) kinases, especially haem-regulated inhibitory (HRI) protein kinases. There are two kinase inserts in PfPK4, located at positions common to eIF-2alpha kinases. The first insert separates kinase subdomains IV and VI by 559 amino acids, and the second subdomains VII and VIII by 41 amino acids. Both inserts are larger than their homologues in eIF-2alpha kinases. The sequence of PfPK4 has one putative haemin-binding site. The recombinant protein, expressed in Escherichia coli, phosphorylates a synthetic peptide representing a substrate of eIF-2alpha kinases. Autophosphorylation and substrate phosphorylation are inhibited by haemin. Thus PfPK4 appears to be the first protozoan protein kinase related to eIF-2alpha kinases and might be the first non-mammalian HRI kinase. Western blots indicated that the protein is expressed as major forms of 80 and 90 kDa. Whereas the 80 kDa form is present throughout the intraerythrocytic development and in merozoites, the two 90 kDa forms are only found in mature parasites. One of the latter is also present in the membrane fraction of erythrocytes harbouring segmenters. Confocal microscopy detected the protein distributed throughout the trophozoite, whereas it was found in discrete foci (punctate distribution) in segmenters. PfPK4 co-localizes with P. falciparum 83 kDa antigen/apical membrane antigen-1 at the apical complex in segmenters and merozoites, but does not co-localize with rhoptry-associated protein-1.
Amino Acid Sequence↗
Xefiltin, a Xenopus laevis neuronal intermediate filament protein, is expressed in actively growing optic axons during development and regeneration.
Neurofilaments are an important structural component of the axonal cytoskeleton and are made of neuronal intermediate filament (nIF) proteins. During axonal development, neurofilaments undergo progressive changes in molecular composition. In mammals, for example, highly phosphorylated forms of the middle- and high-molecular-weight neurofilament proteins (NF-M and NF-H, respectively) are characteristic of mature axons, whereas nIF proteins such as alpha-internexin are typical of young axons. Such changes have been proposed to help growing axons accommodate varying demands for plasticity and stability by modulating the structure of the axonal cytoskeleton. Xefiltin is a recently discovered nIF protein of the frog Xenopus laevis, whose nervous system has a large capacity for regeneration and plasticity. By amino acid identity, xefiltin is closely related to two other nIF proteins, alpha-internexin and gefiltin. alpha-Internexin is found principally in embryonic axons of the mammalian brain, and gefiltin is expressed primarily in goldfish retinal ganglion cells and has been associated with the ability of the goldfish optic nerve to regenerate. Like gefiltin in goldfish, xefiltin in Xenopus is the most abundantly expressed nIF protein of mature retinal ganglion cells. In the present study, we used immunocytochemistry to study the distribution of xefiltin during optic nerve development and regeneration. During development, xefiltin was found in optic axons at stage 35/36, before they reach the tectum at stage 37/38. Similarly, after an orbital crush injury, xefiltin first reemerged in optic axons after the front of regeneration reached the optic chiasm, but before it reached the tectum. Thus, during both development and regeneration, xefiltin was present within actively growing optic axons. In addition, aberrantly projecting retinoretinal axons expressed less xefiltin than those entering the optic tract, suggesting that xefiltin expression is influenced by interactions between regenerating axons and cells encountered along the visual pathway. These results support the idea that changes in xefiltin expression, along with those of other nIF proteins, modulate the structure and stability of actively growing optic axons and that this stability is under the control of the pathway which growing axons follow.
Animals↗
A strategy for rapid, high-confidence protein identification.
A procedure is described for rapid, high-confidence identification of proteins using matrix-assisted laser desorption/ionization tandem ion trap mass spectrometry in conjunction with a genome database searching strategy. The procedure involves excision of copper-stained bands or spots from electrophoretic gels, in-gel trypsin digestion of the proteins, single-stage mass spectrometric analysis of the resultant mixture of tryptic peptides, followed by tandem ion trap mass spectrometric analysis of selected individual peptides, and database searching of the relevant genomic database using the program PepFrag. The scheme provides sensitive, real-time protein identification as well as facile identification of modifications. A single operator can unambiguously identify 5-10 proteins/day from an organism whose genome is known at a level of > 0.5 pmol of protein loaded on a gel. The utility of the technique was demonstrated by the identification and characterization of a band from a human HTLV-I preparation and 11 different proteins from a yeast RNA polymerase II C-terminal repeat domain-affinity preparation. The technology has great potential for postgenome biological science, where it promises to facilitate the dissection and anatomy of macromolecular assemblages, the definition of disease state markers, and the investigation of protein targets in biological processes such as the cell cycle and signal transduction.
Amino Acid Sequence↗
Definition of the substrate specificity of the 'sensing' xylanase of Streptomyces cyaneus using xylooligosaccharide and cellooligosaccharide glycosides of 3,4-dinitrophenol.
The title compounds, (Xylp beta (1-->4))nXylp beta-3,4-DNP (n = 0-4) have been made by selective anomeric deprotection of peracetylated xylose oligosaccharides with hydrazine, followed by formation of the trichloroacetimidate, uncatalysed reaction with 3,4-dinitrophenol, and Zemplén deacetylation. The values of k(cat)/K(m) for 3,4-dinitrophenol release from these substrates by xylanase III of Streptomyces cyaneus, expressed in Escherichia coli, increase with increasing n up to n = 2 and then slightly decrease. Since it is known from previous work that in its normal host, the enzyme is produced constitutively at low levels and excreted, these results suggest that the biological function of the enzyme may be to produce small molecule inducers, predominantly xylotriose, from the non-reducing end of the xylan. Activity on cellooligosaccharide glycosides (Glcp beta (1-->4))nGlcp beta-3,4-DNP (n = 0-3) was detected, at a rate about two-and-a-half orders of magnitude less than that observed on the corresponding xylooligosaccharides, indicating that the enzyme is a true xylanase.
Dinitrophenols↗
Routes of Cl- transport across the trophectoderm of the mouse blastocyst.
The blastocyst stage of embryo development is characterized by a fluid-filled cavity called the blastocoel. Blastocoel formation requires vectorial Na+ and Cl- transport and the accompanying osmotic accumulation of fluid. We found under conditions of low external Cl- that inhibitors of Cl- transport mechanisms inhibited blastocoel expansion, indicating a possible transcellular route for Cl- uptake across the outer epithelial layer (the trophectoderm). Using the Cl--sensitive fluorophore, N-(6-methoxyquinolyl)acetoethyl ester, we found that Cl- efflux from the blastocoel can occur via pathways with properties that resemble both HCO-3/Cl- exchange and Cl- channels, as well as by another yet uncharacterized pathway. In contrast, Cl- re-uptake into Cl--depleted blastocoels (the physiologically relevant direction for Cl- transport during blastocoel expansion) occurred only via the channel-like mechanism. Patch-clamp recordings detected a component of current carried by apical Cl- channels. Intracellular pH measurements during external Cl- removal detected HCO-3/Cl- exchange activity in collapsed blastocysts but little in intact blastocysts, suggesting predominantly basolateral HCO-3/Cl- exchange activity. This was corroborated by the immunolocalization of the AE2 isoform of HCO-3/Cl- exchanger to the basolateral surface of the trophectoderm. Thus, it appears that Cl- transport into the blastocoel may occur via apical Cl- channels, while efflux also involves a basolateral HCO-3/Cl- exchanger.
Animals↗
Positive and negative selection of functional mouse CD4 cells by porcine MHC in pig thymus grafts.
Specific tolerance to discordant xenogeneic donors can be achieved by grafting of fetal pig thymic and liver tissue (FP THY/LIV) to T cell and NK cell-depleted, thymectomized (ATX) mice. Mouse CD4+ T cells develop in FP THY/LIV grafts, and demonstrate remarkably normal immune function, including host-restricted responses to keyhole limpet hemocyanin. We have therefore studied the role of host MHC class II in the development of mouse T cells in FP THY/LIV grafts by comparing their development in ATX MHC class II-deficient (IIKO) and wild-type (H-2b) mice. Mouse CD4+ T cells repopulated T/NK cell-depleted, ATX IIKO mice after grafting with FP THY/LIV, indicating that pig MHC can positively select mouse CD4 cells. Expression of TCR, MHC class I, Qa-2, heat-stable Ag, and CD45RB among double-positive and CD4 single-positive (SP) graft thymocytes in wild-type recipients was similar to that in normal mouse thymi, whereas CD4 SP thymocytes in grafts of IIKO mice showed increased Qa-2 and decreased heat-stable Ag expression, suggesting an increased level of maturity. Double-positive cells in grafts of IIKO mice also expressed higher than normal levels of Qa-2. Deletion within the grafts of Vbeta3+, Vbeta5.1/5.2+, and Vbeta11+ but not Vbeta6+, Vbeta7+, or Vbeta8.1/8.2+ mouse CD4 SP thymocytes in ATX IIKO mice demonstrated that swine leukocyte Ag participates in negative selection of the T cell repertoire. Therefore, porcine MHC mediates positive and negative selection of mouse thymocytes, but host class II MHC molecules also regulate thymocyte maturation in xenogeneic thymic grafts.
Animals↗
Sequence requirement for replication initiation at the rat aldolase B locus implicated in its functional correlation with transcriptional regulation.
Transcription promoter of the aldolase B gene was previously shown to be centered on an initiation region of DNA replication in rat hepatoma cells in vivo. Here, we defined an essential region required for replication in a plasmid form upon transfection. Deletion analyses around the origin region revealed that the proximal 200 bp promoter was necessary, but not sufficient for replication as flanking sequence restored replication activity. Therefore, the 200 bp region seemed to cooperate with the flanking sequence to play an important role in replication. Electrophoretic mobility shift assays using nuclear extracts from synchronously growing hepatoma cells showed that some protein factors bound to this region in a cell cycle-regulated manner. Since transcription of the aldolase B gene is repressed in the hepatoma cells, the cell cycle-regulated protein-binding is considered to be involved in regulation of replication initiation.
Animals↗
Resistance to monoclonal antibody-induced CD8+ T-cell depletion in thymectomized MHC class II-deficient mice.
BACKGROUND: CD8+ T cells are present at higher than normal levels in MHC class II-deficient (IIKO) mice. METHODS: In this study, we have examined the sensitivity of CD8+ T cells to depletion induced by a single injection or multiple injections of an anti-CD8 monoclonal antibody (mAb) (2.43) in IIKO mice in vivo. RESULTS: Thymectomized (ATX) IIKO mice showed the presence of a greater percentage of memory CD8+ T cells (CD44high, CD45RBlow, and MEL-14(-)) in peripheral blood lymphocytes (PBL) by 1 month after ATX compared with age-matched euthymic mice. Although CD8+ cells were not detectable in the periphery at 5 and 14 days after 2.43 injection, CD8+ T cell receptor alpha/beta+ cells expressing the memory phenotype had recovered markedly by 21 days after mAb injection in these ATX IIKO mice. The expression of CD8 beta-chain and Thy-1 as well as the absence of CD4 and of T cell receptor gamma/delta among most recovering CD8+ cells, and their varied Vbeta usage, suggested that these cells were derived from the thymus rather than from extra-thymic T-cell differentiation or from oligoclonal peripheral expansion. In addition, low numbers of CD8+ cells that were coated with mAb (2.43) were detected in the lymph nodes of ATX IIKO mice 7 days after mAb injection. Most of these nondepleted lymph node CD8+ cells expressed the memory phenotype and low levels of CD8beta. Furthermore, the levels of recovering CD8+ cells in PBL of ATX IIKO mice by 21 days after mAb treatment were markedly higher than those in PBL of simultaneously mAb-treated ATX wild-type (B10) mice. CONCLUSION: Together, these studies indicate that memory CD8+ T cells are relatively resistant to mAb-induced depletion in vivo.
Animals↗
Estrogen effects on insulin-like growth factor-I (IGF-I)-induced cell proliferation and IGF-I expression in native and allograft vessels.
BACKGROUND: Estrogen protects against cardiovascular disease in both patients and animal models and regulates insulin-like growth factor-I (IGF-I), an important cell-cycle progression factor. METHODS AND RESULTS: Smooth muscle cells and tissues were harvested from male recipient rabbits that 6 weeks earlier had received a cardiac allograft transplant consisting of a donor heart and ascending aorta. Segments of the ascending aorta from the native and allograft hearts from 9 placebo-treated and 8 estradiol-treated recipients were compared by using IGF-I-stimulated [3H]thymidine incorporation. The responses of the native vessel segments were similar (175.3+/-32% and 166.9+/-41%, respectively; P>.05) whether or not the recipients had been treated for 6 weeks with estradiol. In the grafts, however, estradiol markedly inhibited vascular cell thymidine incorporation (328.04+/-56% compared with 67.3+/-11%; P<.02). Smooth muscle cells were derived from the native aorta of the placebo-treated rabbits to study the effect of estradiol in vitro. IGF-I increased cell counts in a concentration-dependent manner. In serum-starved cells estradiol further decreased cell proliferation; this effect was blocked by the specific estrogen receptor antagonist ZK-119.010. Immunohistochemistry staining for IGF-I protein in the coronary arteries and ascending aorta of the cardiac allograft from the placebo-treated recipients revealed extensive IGF-I expression in the myointima. In contrast, IGF-I protein was not expressed in the coronary arteries and ascending aorta of the cardiac allograft from the estradiol-treated recipients. The IGF-I protein was extensively expressed only in the placebo-treated graft vessels. Myointimal thickening of the coronary arteries was significantly reduced by estradiol treatment (17.9+/-1.5% versus 44.3+/-3.7%; P<.02). CONCLUSIONS: In vivo estradiol treatment abolishes both IGF-I mitogenic effects and IGF-I protein expression in the vascular wall, which may be causally related to the inhibitory effect of estradiol on transplant arteriosclerosis.
Animals↗
The cytoplasmic domain of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha subunit is essential for both GM-CSF-mediated growth and differentiation.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates differentiation, survival, and proliferation of colony-forming unit-granulocyte-macrophage progenitor cells. The biologic actions of GM-CSF are mediated by binding to a specific receptor consisting of two chains designated as alpha and beta subunits. We have demonstrated that the murine FDC-P1-derived cell line WT-19 transfected with the human GM-CSF receptor alpha and beta subunits (GM-CSFRalpha and beta) can be induced to differentiate by the addition of human GM-CSF (hGM-CSF). By expressing a series of GM-CSFRalpha mutants in WT19 cells, we have determined the amino acid domains of the GM-CSFRalpha cytoplasmic domain that regulate cell differentiation, proliferation, and survival. We found that the membrane proximal proline-rich domain and adjacent 16 residues are essential for both hGM-CSF-dependent cell proliferation and differentiation. In contrast, the C-terminal region of the GM-CSFRalpha cytoplasmic domain was not necessary for cell differentiation mediated by hGM-CSF, but the removal of this region severely impaired the ability of hGM-CSF to support cell survival. While the activation of JAK2, Shc, Erk, and STAT5 proteins correlated with hGM-CSF-mediated cell growth, cellular differentiation occurred in the absence of activation of these signal transduction pathways.
Adaptor Proteins, Signal Transducing↗
Examination of the transition state of the low-molecular mass small tyrosine phosphatase 1. Comparisons with other protein phosphatases.
The reactions of p-nitrophenyl phosphate (pNPP) with the low-molecular mass tyrosine phosphatase Stp1 and with the mutants D128N, D128A, D128E, and S18A have been studied by measurement of heavy-atom isotope effects in the substrate. The isotope effects were measured at the nonbridging oxygen atoms [18(V/K)nonbridge], at the bridging oxygen atom (the site of bond cleavage) [18(V/K)bridge], and at the nitrogen atom in the nitrophenol leaving group [15(V/K)]. The results with native Stp1 were 1.0160 +/- 0.0005 for 18(V/K)bridge, 1.0007 +/- 0.0001 for 15(V/K), and 1.0018 +/- 0.0003 for 18(V/K)nonbridge. The values for 18(V/K)nonbridge and 15(V/K) differ from those previously measured with other protein-tyrosine phosphatases and from those of the aqueous hydrolysis reaction of pNPP. The values indicate that in the transition state of the native Stp1 reaction the leaving group bears a partial negative charge, and there is nucleophilic interaction between the Cys nucleophile, and the phosphoryl group, causing some decrease in the nonbridge P-O bond order. The transition state remains highly dissociative with respect to the degree of bond cleavage to the leaving group. Mutation of the general acid from aspartic acid to glutamic acid slows catalysis but causes no change in the isotope effects and thus does not alter the degree of proton transfer to the leaving group in the transition state. Mutations of this residue to asparagine or alanine give values for 18(V/K)bridge of about 1.029, for 15(V/K) of about 1.003, and for 18(V/K)nonbridge of 1.0010 (D128A) to 1.0024 (D128N). These data indicate a dissociative transition state with the leaving group departing as the nitrophenolate anion and indicate more nucleophilic participation than in the aqueous hydrolysis of the pNPP dianion, just as in the native enzyme. The isotope effects with the S18A mutant, in which a hydrogen bonding stabilization of the anionic Cys nucleophile has been removed, were within experimental error of those with the native enzyme, indicating that this alteration has no effect on the transition state for phosphoryl transfer from pNPP.
Hydrogen-Ion Concentration↗
Stress proteins on the yeast cell surface determine resistance to osmotin, a plant antifungal protein.
Strains of the yeast Saccharomyces cerevisiae differ in their sensitivities to tobacco osmotin, an antifungal protein of the PR-5 family. However, cells sensitive to tobacco osmotin showed resistance to osmotin-like proteins purified from the plant Atriplex nummularia, indicating a strict specificity between the antifungal protein and its target cell. A member of a gene family encoding stress proteins induced by heat and nitrogen limitation, collectively called Pir proteins, was isolated among the genes that conveyed resistance to tobacco osmotin to a susceptible strain. We show that overexpression of Pir proteins increased resistance to osmotin, whereas simultaneous deletion of all PIR genes in a tolerant strain resulted in sensitivity. Pir proteins have been immunolocalized to the cell wall. The enzymatic digestion of the cell wall of sensitive and resistant cells rendered spheroplasts equally susceptible to the cytotoxic action of tobacco osmotin but not to other osmotin-like proteins, indicating that the cell membrane interacts specifically with osmotin and facilitates its action. Our results demonstrate that fungal cell wall proteins are determinants of resistance to antifungal PR-5 proteins.
Cloning, Molecular↗
Unconventional myosins in inner-ear sensory epithelia.
To understand how cells differentially use the dozens of myosin isozymes present in each genome, we examined the distribution of four unconventional myosin isozymes in the inner ear, a tissue that is particularly reliant on actin-rich structures and unconventional myosin isozymes. Of the four isozymes, each from a different class, three are expressed in the hair cells of amphibia and mammals. In stereocilia, constructed of cross-linked F-actin filaments, myosin-Ibeta is found mostly near stereociliary tips, myosin-VI is largely absent, and myosin-VIIa colocalizes with crosslinks that connect adjacent stereocilia. In the cuticular plate, a meshwork of actin filaments, myosin-Ibeta is excluded, myosin-VI is concentrated, and modest amounts of myosin-VIIa are present. These three myosin isozymes are excluded from other actin-rich domains, including the circumferential actin belt and the cortical actin network. A member of a fourth class, myosin-V, is not expressed in hair cells but is present at high levels in afferent nerve cells that innervate hair cells. Substantial amounts of myosins-Ibeta, -VI, and -VIIa are located in a pericuticular necklace that is largely free of F-actin, squeezed between (but not associated with) actin of the cuticular plate and the circumferential belt. Our localization results suggest specific functions for three hair-cell myosin isozymes. As suggested previously, myosin-Ibeta probably plays a role in adaptation; concentration of myosin-VI in cuticular plates and association with stereociliary rootlets suggest that this isozyme participates in rigidly anchoring stereocilia; and finally, colocalization with cross-links between adjacent stereocilia indicates that myosin-VIIa is required for the structural integrity of hair bundles.
Animals↗