RESUMO
Transporting epithelial cells build apical microvilli to increase membrane surface area and enhance absorptive capacity. The intestinal brush border provides an elaborate example with tightly packed microvilli that function in nutrient absorption and host defense. Although the brush border is essential for physiological homeostasis, its assembly is poorly understood. We found that brush border assembly is driven by the formation of Ca(2+)-dependent adhesion links between adjacent microvilli. Intermicrovillar links are composed of protocadherin-24 and mucin-like protocadherin, which target to microvillar tips and interact to form a trans-heterophilic complex. The cytoplasmic domains of microvillar protocadherins interact with the scaffolding protein, harmonin, and myosin-7b, which promote localization to microvillar tips. Finally, a mouse model of Usher syndrome lacking harmonin exhibits microvillar protocadherin mislocalization and severe defects in brush border morphology. These data reveal an adhesion-based mechanism for brush border assembly and illuminate the basis of intestinal pathology in patients with Usher syndrome. PAPERFLICK:
Assuntos
Caderinas/metabolismo , Enterócitos/metabolismo , Microvilosidades/metabolismo , Animais , Células COS , Células CACO-2 , Proteínas Relacionadas a Caderinas , Cálcio/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Chlorocebus aethiops , Proteínas do Citoesqueleto , Modelos Animais de Doenças , Enterócitos/citologia , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Microvilosidades/ultraestrutura , Miosinas/metabolismo , Síndromes de Usher/patologiaRESUMO
Membrane fusion and fission are vital for eukaryotic life. For three decades, it has been proposed that fusion is mediated by fusion between the proximal leaflets of two bilayers (hemi-fusion) to produce a hemi-fused structure, followed by fusion between the distal leaflets, whereas fission is via hemi-fission, which also produces a hemi-fused structure, followed by full fission. This hypothesis remained unsupported owing to the lack of observation of hemi-fusion or hemi-fission in live cells. A competing fusion hypothesis involving protein-lined pore formation has also been proposed. Here we report the observation of a hemi-fused Ω-shaped structure in live neuroendocrine chromaffin cells and pancreatic ß-cells, visualized using confocal and super-resolution stimulated emission depletion microscopy. This structure is generated from fusion pore opening or closure (fission) at the plasma membrane. Unexpectedly, the transition to full fusion or fission is determined by competition between fusion and calcium/dynamin-dependent fission mechanisms, and is notably slow (seconds to tens of seconds) in a substantial fraction of the events. These results provide key missing evidence in support of the hemi-fusion and hemi-fission hypothesis in live cells, and reveal the hemi-fused intermediate as a key structure controlling fusion and fission, as fusion and fission mechanisms compete to determine the transition to fusion or fission.
Assuntos
Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Fusão de Membrana/fisiologia , Modelos Biológicos , Animais , Ligação Competitiva , Cálcio/metabolismo , Bovinos , Membrana Celular/química , Membrana Celular/metabolismo , Sobrevivência Celular , Células Cultivadas , Células Cromafins/citologia , Dinaminas/metabolismo , Células Secretoras de Insulina/citologia , Microscopia Confocal , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
The polycistronic miR-183/96/182 cluster is preferentially and abundantly expressed in terminally differentiating sensory epithelia. To clarify its roles in the terminal differentiation of sensory receptors in vivo, we deleted the entire gene cluster in mouse germline through homologous recombination. The miR-183/96/182 null mice display impairment of the visual, auditory, vestibular, and olfactory systems, attributable to profound defects in sensory receptor terminal differentiation. Maturation of sensory receptor precursors is delayed, and they never attain a fully differentiated state. In the retina, delay in up-regulation of key photoreceptor genes underlies delayed outer segment elongation and possibly mispositioning of cone nuclei in the retina. Incomplete maturation of photoreceptors is followed shortly afterward by early-onset degeneration. Cell biologic and transcriptome analyses implicate dysregulation of ciliogenesis, nuclear translocation, and an epigenetic mechanism that may control timing of terminal differentiation in developing photoreceptors. In both the organ of Corti and the vestibular organ, impaired terminal differentiation manifests as immature stereocilia and kinocilia on the apical surface of hair cells. Our study thus establishes a dedicated role of the miR-183/96/182 cluster in driving the terminal differentiation of multiple sensory receptor cells.
Assuntos
Células Ciliadas Auditivas/citologia , Células Ciliadas Vestibulares/citologia , MicroRNAs/genética , Mucosa Olfatória/citologia , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Animais , Regulação da Expressão Gênica no Desenvolvimento/genética , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Vestibulares/metabolismo , Transtornos da Audição/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Família Multigênica , Transtornos do Olfato/genética , Mucosa Olfatória/metabolismo , Equilíbrio Postural/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Transtornos de Sensação/genética , Transtornos da Visão/genéticaRESUMO
Class III myosins (MYO3A and MYO3B) are proposed to function as transporters as well as length and ultrastructure regulators within stable actin-based protrusions such as stereocilia and calycal processes. MYO3A differs from MYO3B in that it contains an extended tail domain with an additional actin-binding motif. We examined how the properties of the motor and tail domains of human class III myosins impact their ability to enhance the formation and elongation of actin protrusions. Direct examination of the motor and enzymatic properties of human MYO3A and MYO3B revealed that MYO3A is a 2-fold faster motor with enhanced ATPase activity and actin affinity. A chimera in which the MYO3A tail was fused to the MYO3B motor demonstrated that motor activity correlates with formation and elongation of actin protrusions. We demonstrate that removal of individual exons (30-34) in the MYO3A tail does not prevent filopodia tip localization but abolishes the ability to enhance actin protrusion formation and elongation in COS7 cells. Interestingly, our results demonstrate that MYO3A slows filopodia dynamics and enhances filopodia lifetime in COS7 cells. We also demonstrate that MYO3A is more efficient than MYO3B at increasing formation and elongation of stable microvilli on the surface of cultured epithelial cells. We propose that the unique features of MYO3A, enhanced motor activity, and an extended tail with tail actin-binding motif, allow it to play an important role in stable actin protrusion length and ultrastructure maintenance.
Assuntos
Actinas/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo III/metabolismo , Pseudópodes/metabolismo , Actinas/genética , Animais , Células COS , Chlorocebus aethiops , Humanos , Cadeias Pesadas de Miosina/genética , Miosina Tipo III/genética , Pseudópodes/genéticaRESUMO
Approximately one-third of known deafness genes encode proteins located in the hair bundle, the sensory hair cell's mechanoreceptive organelle. In previous studies, we used mass spectrometry to characterize the hair bundle's proteome, resulting in the discovery of novel bundle proteins. One such protein is Xin-actin binding repeat containing 2 (XIRP2), an actin-cross-linking protein previously reported to be specifically expressed in striated muscle. Because mutations in other actin-cross-linkers result in hearing loss, we investigated the role of XIRP2 in hearing function. In the inner ear, XIRP2 is specifically expressed in hair cells, colocalizing with actin-rich structures in bundles, the underlying cuticular plate, and the circumferential actin belt. Analysis using peptide mass spectrometry revealed that the bundle harbors a previously uncharacterized XIRP2 splice variant, suggesting XIRP2's role in the hair cell differs significantly from that reported in myocytes. To determine the role of XIRP2 in hearing, we applied clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated genome-editing technology to induce targeted mutations into the mouse Xirp2 gene, resulting in the elimination of XIRP2 protein expression in the inner ear. Functional analysis of hearing in the resulting Xirp2-null mice revealed high-frequency hearing loss, and ultrastructural scanning electron microscopy analyses of hair cells demonstrated stereocilia degeneration in these mice. We thus conclude that XIRP2 is required for long-term maintenance of hair cell stereocilia, and that its dysfunction causes hearing loss in the mouse.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Ciliadas Auditivas/metabolismo , Audição , Proteínas com Domínio LIM/metabolismo , Proteínas Nucleares/metabolismo , Estereocílios/metabolismo , Animais , Células Cultivadas , Embrião de Galinha , Proteínas do Citoesqueleto , Proteínas de Ligação a DNA/genética , Células Ciliadas Auditivas/fisiologia , Perda Auditiva/genética , Proteínas com Domínio LIM/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Proteínas Nucleares/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Ratos , Estereocílios/ultraestruturaRESUMO
All inner ear organs possess extracellular matrix appendices over the sensory epithelia that are crucial for their proper function. The tectorial membrane (TM) is a gelatinous acellular membrane located above the hearing sensory epithelium and is composed mostly of type II collagen, and α and ß tectorins. TM molecules self-assemble in the endolymph fluid environment, interacting medially with the spiral limbus and distally with the outer hair cell stereocilia. Here, we used immunogold labeling in freeze-substituted mouse cochleae to assess the fine localization of both tectorins in distinct TM regions. We observed that the TM adheres to the spiral limbus through a dense thin matrix enriched in α- and ß-tectorin, both likely bound to the membranes of interdental cells. Freeze-etching images revealed that type II collagen fibrils were crosslinked by short thin filaments (4±1.5nm, width), resembling another collagen type protein, or chains of globular elements (15±3.2nm, diameter). Gold-particles for both tectorins also localized adjacent to the type II collagen fibrils, suggesting that these globules might be composed essentially of α- and ß-tectorins. Finally, the presence of gold-particles at the TM lower side suggests that the outer hair cell stereocilia membrane has a molecular partner to tectorins, probably stereocilin, allowing the physical connection between the TM and the organ of Corti.
Assuntos
Colágeno Tipo II/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas de Membrana/metabolismo , Órgão Espiral/metabolismo , Membrana Tectorial/metabolismo , Animais , Colágeno Tipo II/genética , Colágeno Tipo II/ultraestrutura , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/ultraestrutura , Técnica de Congelamento e Réplica , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Proteínas Ligadas por GPI/ultraestrutura , Expressão Gênica , Cobaias , Imuno-Histoquímica , Proteínas de Membrana/genética , Proteínas de Membrana/ultraestrutura , Camundongos , Microscopia Eletrônica de Transmissão , Miosinas/deficiência , Miosinas/genética , Órgão Espiral/ultraestrutura , Ligação Proteica , Ratos , Membrana Tectorial/ultraestruturaRESUMO
During development of the chick cochlea, actin crosslinkers and barbed-end cappers presumably influence growth and remodeling of the actin paracrystal of hair cell stereocilia. We used mass spectrometry to identify and quantify major actin-associated proteins of the cochlear sensory epithelium from E14 to E21, when stereocilia widen and lengthen. Tight actin crosslinkers (i.e. fascins, plastins, and espin) are expressed dynamically during cochlear epithelium development between E7 and E21, with FSCN2 replacing FSCN1 and plastins remaining low in abundance. Capping protein, a barbed-end actin capper, is located at stereocilia tips; it is abundant during growth phase II, when stereocilia have ceased elongating and are increasing in diameter. Capping protein levels then decline during growth phase III, when stereocilia reinitiate barbed-end elongation. Although actin crosslinkers are readily detected by electron microscopy in developing chick cochlea stereocilia, quantitative mass spectrometry of stereocilia isolated from E21 chick cochlea indicated that tight crosslinkers are present there in stoichiometric ratios relative to actin that are much lower than their ratios for vestibular stereocilia. These results demonstrate the value of quantitation of global protein expression in chick cochlea during stereocilia development.
Assuntos
Proteínas de Capeamento de Actina/metabolismo , Actinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Estereocílios/metabolismo , Proteínas de Capeamento de Actina/genética , Animais , Embrião de Galinha/metabolismo , Cóclea/embriologia , Cóclea/metabolismo , Desenvolvimento Embrionário/fisiologia , Epitélio/embriologia , Epitélio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas/metabolismo , Espectrometria de Massas/métodos , Proteínas dos Microfilamentos/genética , Ligação Proteica , Estereocílios/fisiologiaRESUMO
Mechanotransduction in the mammalian auditory system depends on mechanosensitive channels in the hair bundles that project from the apical surface of the sensory hair cells. Individual stereocilia within each bundle contain a core of tightly packed actin filaments, whose length is dynamically regulated during development and in the adult. We show that the actin-binding protein epidermal growth factor receptor pathway substrate 8 (Eps8)L2, a member of the Eps8-like protein family, is a newly identified hair bundle protein that is localized at the tips of stereocilia of both cochlear and vestibular hair cells. It has a spatiotemporal expression pattern that complements that of Eps8. In the cochlea, whereas Eps8 is essential for the initial elongation of stereocilia, Eps8L2 is required for their maintenance in adult hair cells. In the absence of both proteins, the ordered staircase structure of the hair bundle in the cochlea decays. In contrast to the early profound hearing loss associated with an absence of Eps8, Eps8L2 null-mutant mice exhibit a late-onset, progressive hearing loss that is directly linked to a gradual deterioration in hair bundle morphology. We conclude that Eps8L2 is required for the long-term maintenance of the staircase structure and mechanosensory function of auditory hair bundles. It complements the developmental role of Eps8 and is a candidate gene for progressive age-related hearing loss.
Assuntos
Células Ciliadas Auditivas/patologia , Perda Auditiva/genética , Proteínas dos Microfilamentos/deficiência , Análise de Variância , Animais , Audiometria de Resposta Evocada , Células Ciliadas Auditivas/fisiologia , Células Ciliadas Auditivas/ultraestrutura , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Microscopia Eletrônica , Técnicas de Patch-ClampRESUMO
Age-related hearing loss and noise-induced hearing loss are major causes of human morbidity. Here we used genetics and functional studies to show that a shared cause of these disorders may be loss of function of the ATP-gated P2X(2) receptor (ligand-gated ion channel, purinergic receptor 2) that is expressed in sensory and supporting cells of the cochlea. Genomic analysis of dominantly inherited, progressive sensorineural hearing loss DFNA41 in a six-generation kindred revealed a rare heterozygous allele, P2RX2 c.178G > T (p.V60L), at chr12:133,196,029, which cosegregated with fully penetrant hearing loss in the index family, and also appeared in a second family with the same phenotype. The mutation was absent from more than 7,000 controls. P2RX2 p.V60L abolishes two hallmark features of P2X(2) receptors: ATP-evoked inward current response and ATP-stimulated macropore permeability, measured as loss of ATP-activated FM1-43 fluorescence labeling. Coexpression of mutant and WT P2X(2) receptor subunits significantly reduced ATP-activated membrane permeability. P2RX2-null mice developed severe progressive hearing loss, and their early exposure to continuous moderate noise led to high-frequency hearing loss as young adults. Similarly, among family members heterozygous for P2RX2 p.V60L, noise exposure exacerbated high-frequency hearing loss in young adulthood. Our results suggest that P2X(2) function is required for life-long normal hearing and for protection from exposure to noise.
Assuntos
Perda Auditiva Provocada por Ruído/genética , Perda Auditiva Neurossensorial/genética , Mutação de Sentido Incorreto , Receptores Purinérgicos P2X2/genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Modelos Animais de Doenças , Potenciais Evocados Auditivos , Feminino , Genes Dominantes , Perda Auditiva Provocada por Ruído/etiologia , Perda Auditiva Provocada por Ruído/fisiopatologia , Perda Auditiva Neurossensorial/etiologia , Perda Auditiva Neurossensorial/fisiopatologia , Heterozigoto , Humanos , Ativação do Canal Iônico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Linhagem , Penetrância , Receptores Purinérgicos P2X2/deficiência , Receptores Purinérgicos P2X2/fisiologia , Homologia de Sequência de Aminoácidos , Adulto JovemRESUMO
Sensory processing in the auditory system requires that synapses, neurons, and circuits encode information with particularly high temporal and spectral precision. In the amphibian papillia, sound frequencies up to 1 kHz are encoded along a tonotopic array of hair cells and transmitted to afferent fibers via fast, repetitive synaptic transmission, thereby promoting phase locking between the presynaptic and postsynaptic cells. Here, we have combined serial section electron microscopy, paired electrophysiological recordings, and Monte Carlo diffusion simulations to examine novel mechanisms that facilitate fast synaptic transmission in the inner ear of frogs (Rana catesbeiana and Rana pipiens). Three-dimensional anatomical reconstructions reveal specialized spine-like contacts between individual afferent fibers and hair cells that are surrounded by large, open regions of extracellular space. Morphologically realistic diffusion simulations suggest that these local enlargements in extracellular space speed transmitter clearance and reduce spillover between neighboring synapses, thereby minimizing postsynaptic receptor desensitization and improving sensitivity during prolonged signal transmission. Additionally, evoked EPSCs in afferent fibers are unaffected by glutamate transporter blockade, suggesting that transmitter diffusion and dilution, and not uptake, play a primary role in speeding neurotransmission and ensuring fidelity at these synapses.
Assuntos
Células Ciliadas Auditivas/citologia , Neurotransmissores/metabolismo , Transdução de Sinais/fisiologia , Transmissão Sináptica/fisiologia , Algoritmos , Animais , Ácido Aspártico/farmacologia , Benzotiadiazinas/farmacologia , Cálcio/metabolismo , Simulação por Computador , Relação Dose-Resposta a Droga , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Ácido Glutâmico/metabolismo , Células Ciliadas Auditivas/ultraestrutura , Masculino , Microscopia Eletrônica , Modelos Neurológicos , Técnicas de Patch-Clamp , Rana catesbeiana , Transdução de Sinais/efeitos dos fármacosRESUMO
Class III myosins are unique members of the myosin superfamily in that they contain both a motor and kinase domain. We have found that motor activity is decreased by autophosphorylation, although little is known about the regulation of the kinase domain. We demonstrate by mass spectrometry that Thr-178 and Thr-184 in the kinase domain activation loop and two threonines in the loop 2 region of the motor domain are autophosphorylated (Thr-908 and Thr-919). The kinase activity of MYO3A 2IQ with the phosphomimic (T184E) or phosphoblock (T184A) mutations demonstrates that kinase activity is reduced 30-fold as a result of the T184A mutation, although the Thr-178 site only had a minor impact on kinase activity. Interestingly, the actin-activated ATPase activity of MYO3A 2IQ is slightly reduced as a result of the T178A and T184A mutations suggesting coupling between motor and kinase domains. Full-length GFP-tagged T184A and T184E MYO3A constructs transfected into COS7 cells do not disrupt the ability of MYO3A to localize to filopodia structures. In addition, we demonstrate that T184E MYO3A reduces filopodia elongation in the presence of espin-1, whereas T184A enhances filopodia elongation in a similar fashion to kinase-dead MYO3A. Our results suggest that as MYO3A accumulates at the tips of actin protrusions, autophosphorylation of Thr-184 enhances kinase activity resulting in phosphorylation of the MYO3A motor and reducing motor activity. The differential regulation of the kinase and motor activities allows for MYO3A to precisely self-regulate its concentration in the actin bundle-based structures of cells.
Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo III/metabolismo , Pseudópodes/enzimologia , Actinas/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , Substituição de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Humanos , Mutação de Sentido Incorreto , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/genética , Miosina Tipo III/química , Miosina Tipo III/genética , Fosforilação , Estrutura Terciária de Proteína , Pseudópodes/genética , Células Sf9 , SpodopteraRESUMO
Protocadherin 15 (PCDH15) is expressed in hair cells of the inner ear and in photoreceptors of the retina. Mutations in PCDH15 cause Usher Syndrome (deaf-blindness) and recessive deafness. In developing hair cells, PCDH15 localizes to extracellular linkages that connect the stereocilia and kinocilium into a bundle and regulate its morphogenesis. In mature hair cells, PCDH15 is a component of tip links, which gate mechanotransduction channels. PCDH15 is expressed in several isoforms differing in their cytoplasmic domains, suggesting that alternative splicing regulates PCDH15 function in hair cells. To test this model, we generated three mouse lines, each of which lacks one out of three prominent PCDH15 isoforms (CD1, CD2 and CD3). Surprisingly, mice lacking PCDH15-CD1 and PCDH15-CD3 form normal hair bundles and tip links and maintain hearing function. Tip links are also present in mice lacking PCDH15-CD2. However, PCDH15-CD2-deficient mice are deaf, lack kinociliary links and have abnormally polarized hair bundles. Planar cell polarity (PCP) proteins are distributed normally in the sensory epithelia of the mutants, suggesting that PCDH15-CD2 acts downstream of PCP components to control polarity. Despite the absence of kinociliary links, vestibular function is surprisingly intact in the PCDH15-CD2 mutants. Our findings reveal an essential role for PCDH15-CD2 in the formation of kinociliary links and hair bundle polarization, and show that several PCDH15 isoforms can function redundantly at tip links.
Assuntos
Processamento Alternativo/fisiologia , Caderinas/metabolismo , Células Ciliadas Auditivas/metabolismo , Precursores de Proteínas/metabolismo , Processamento Alternativo/genética , Animais , Proteínas Relacionadas a Caderinas , Caderinas/genética , Cóclea/citologia , Cóclea/metabolismo , Cóclea/ultraestrutura , Células Ciliadas Auditivas/ultraestrutura , Imuno-Histoquímica , Hibridização In Situ , Mecanotransdução Celular/genética , Mecanotransdução Celular/fisiologia , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Ligação Proteica , Precursores de Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We have generated 3 mouse lines, each with a different mutation in the nonmuscle myosin II-A gene, Myh9 (R702C, D1424N, and E1841K). Each line develops MYH9-related disease similar to that found in human patients. R702C mutant human cDNA fused with green fluorescent protein was introduced into the first coding exon of Myh9, and D1424N and E1841K mutations were introduced directly into the corresponding exons. Homozygous R702C mice die at embryonic day 10.5-11.5, whereas homozygous D1424N and E1841K mice are viable. All heterozygous and homozygous mutant mice show macrothrombocytopenia with prolonged bleeding times, a defect in clot retraction, and increased extramedullary megakaryocytes. Studies of cultured megakaryocytes and live-cell imaging of megakaryocytes in the BM show that heterozygous R702C megakaryocytes form fewer and shorter proplatelets with less branching and larger buds. The results indicate that disrupted proplatelet formation contributes to the macrothrombocytopenia in mice and most probably in humans. We also observed premature cataract formation, kidney abnormalities, including albuminuria, focal segmental glomerulosclerosis and progressive kidney disease, and mild hearing loss. Our results show that heterozygous mice with mutations in the myosin motor or filament-forming domain manifest similar hematologic, eye, and kidney phenotypes to humans with MYH9-related disease.
Assuntos
Catarata/etiologia , Modelos Animais de Doenças , Perda Auditiva/etiologia , Nefropatias/etiologia , Megacariócitos/patologia , Mutação/genética , Miosina não Muscular Tipo IIA/fisiologia , Trombocitopenia/etiologia , Animais , Catarata/metabolismo , Catarata/patologia , Feminino , Imunofluorescência , Genes Letais , Perda Auditiva/metabolismo , Perda Auditiva/patologia , Heterozigoto , Homozigoto , Humanos , Immunoblotting , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Megacariócitos/metabolismo , Camundongos , Camundongos Transgênicos , Cadeias Pesadas de Miosina , Contagem de Plaquetas , Trombocitopenia/metabolismo , Trombocitopenia/patologiaRESUMO
In the most accepted model for hair cell mechanotransduction, a cluster of myosin motors located at the stereocilia upper tip-link density (UTLD) keeps the tip-link under tension at rest. Both myosin VIIa (MYO7A) and myosin 1c have been implicated in mechanotransduction based on functional studies. However, localization studies are conflicting, leaving open the question of which myosin localizes at the UTLD and generates the tip-link resting tension. Using immunofluorescence, we now show that MYO7A and sans, a MYO7A-interacting protein, cluster at the UTLD. Analysis of the immunofluorescence intensity indicates that eight or more MYO7A molecules are present at each UTLD, consistent with a direct role for MYO7A in maintaining tip-link tension. MYO7A and sans localization at the UTLD is confirmed by transfection of hair cells with GFP-tagged constructs for these proteins. Cotransfection studies in a heterologous system show that MYO7A, sans, and the UTLD protein harmonin-b form a tripartite complex and that each protein is capable of interacting with one another independently. We propose that MYO7A, sans, and harmonin-b form the core components of the UTLD molecular complex. In this complex, MYO7A is likely the motor element that pulls on CDH23 to exert tension on the tip-link.
Assuntos
Cílios/metabolismo , Mecanotransdução Celular/fisiologia , Miosinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Chlorocebus aethiops , Cílios/ultraestrutura , Proteínas do Citoesqueleto , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cobaias , Células Ciliadas Vestibulares/metabolismo , Células Ciliadas Vestibulares/ultraestrutura , Humanos , Camundongos , Camundongos Mutantes , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Dados de Sequência Molecular , Complexos Multiproteicos , Miosina VIIa , Miosinas/química , Miosinas/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Órgão Espiral/metabolismo , Órgão Espiral/ultraestrutura , Domínios e Motivos de Interação entre Proteínas , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Síndromes de Usher/fisiopatologiaRESUMO
Superresolution imaging techniques based on the precise localization of single molecules, such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), achieve high resolution by fitting images of single fluorescent molecules with a theoretical Gaussian to localize them with a precision on the order of tens of nanometers. PALM/STORM rely on photoactivated proteins or photoswitching dyes, respectively, which makes them technically challenging. We present a simple and practical way of producing point localization-based superresolution images that does not require photoactivatable or photoswitching probes. Called bleaching/blinking assisted localization microscopy (BaLM), the technique relies on the intrinsic bleaching and blinking behaviors characteristic of all commonly used fluorescent probes. To detect single fluorophores, we simply acquire a stream of fluorescence images. Fluorophore bleach or blink-off events are detected by subtracting from each image of the series the subsequent image. Similarly, blink-on events are detected by subtracting from each frame the previous one. After image subtractions, fluorescence emission signals from single fluorophores are identified and the localizations are determined by fitting the fluorescence intensity distribution with a theoretical Gaussian. We also show that BaLM works with a spectrum of fluorescent molecules in the same sample. Thus, BaLM extends single molecule-based superresolution localization to samples labeled with multiple conventional fluorescent probes.
Assuntos
Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Software , Animais , Células COS , Chlorocebus aethiops , Corantes Fluorescentes , Proteínas de Fluorescência Verde/metabolismo , FotodegradaçãoRESUMO
The hair bundle of auditory and vestibular hair cells converts mechanical stimuli into electrical signals through mechanoelectrical transduction (MET). The MET apparatus is built around a tip link that connects neighboring stereocilia that are aligned in the direction of mechanosensitivity of the hair bundle. Upon stimulation, the MET channel complex responds to changes in tip-link tension and allows a cation influx into the cell. Ca 2+ influx in stereocilia has been used as a signature of MET activity. Using genetically encoded Ca 2+ sensors (GCaMP3, GCaMP6s) and high-performance fluorescence confocal microscopy, we detect spontaneous Ca 2+ transients in individual stereocilia in developing and fully formed hair bundles. We demonstrate that this activity is abolished by MET channel blockers and thus likely originates from putative MET channels. We observe Ca 2+ transients in the stereocilia of mice in tissue explants as well as in vivo in zebrafish hair cells, indicating this activity is functionally conserved. Within stereocilia, the origin of Ca 2+ transients is not limited to the canonical MET site at the stereocilia tip but is also present along the stereocilia length. Remarkably, we also observe these Ca 2+ transients in the microvilli-like structures on the hair cell surface in the early stages of bundle development, prior to the onset of MET. Ca 2+ transients are also present in the tallest rows of stereocilia in auditory hair cells, structures not traditionally thought to contain MET channels. We hypothesize that this newly described activity may reflect stochastic and spontaneous MET channel opening. Localization of these transients to other regions of the stereocilia indicates the presence of a pool of channels or channel precursors. Our work provides insights into MET channel assembly, maturation, function, and turnover.
RESUMO
Usher syndrome is the leading cause of genetic deaf-blindness. Monoallelic mutations in PDZD7 increase the severity of Usher type II syndrome caused by mutations in USH2A and GPR98, which respectively encode usherin and GPR98. PDZ domain-containing 7 protein (PDZD7) is a paralog of the scaffolding proteins harmonin and whirlin, which are implicated in Usher type 1 and type 2 syndromes. While usherin and GPR98 have been reported to form hair cell stereocilia ankle-links, harmonin localizes to the stereocilia upper tip-link density and whirlin localizes to both tip and ankle-link regions. Here, we used mass spectrometry to show that PDZD7 is expressed in chick stereocilia at a comparable molecular abundance to GPR98. We also show by immunofluorescence and by overexpression of tagged proteins in rat and mouse hair cells that PDZD7 localizes to the ankle-link region, overlapping with usherin, whirlin, and GPR98. Finally, we show in LLC-PK1 cells that cytosolic domains of usherin and GPR98 can bind to both whirlin and PDZD7. These observations are consistent with PDZD7 being a modifier and candidate gene for USH2, and suggest that PDZD7 is a second scaffolding component of the ankle-link complex.
Assuntos
Proteínas de Transporte/química , Redes Reguladoras de Genes/fisiologia , Domínios PDZ/fisiologia , Estereocílios/química , Síndromes de Usher , Sequência de Aminoácidos , Animais , Células COS , Proteínas de Transporte/genética , Embrião de Galinha , Chlorocebus aethiops , Feminino , Humanos , Células LLC-PK1 , Masculino , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Ratos , Estereocílios/genética , Estereocílios/metabolismo , Suínos , Síndromes de Usher/genética , Síndromes de Usher/metabolismoRESUMO
We examined the structure and biomineralization of prismatic magnetosomes in the magnetotactic marine vibrio Magnetovibrio blakemorei strain MV-1 and a non-magnetotactic mutant derived from it, using a combination of cryo-electron tomography and freeze-fracture. The vesicles enveloping the Magnetovibrio magnetosomes were elongated and detached from the cell membrane. Magnetosome crystal formation appeared to be initiated at a nucleation site on the membrane inner surface. Interestingly, while scattered filaments were observed in the surrounding cytoplasm, their association with the magnetosome chains could not be unequivocally established. Our data suggest fundamental differences between prismatic and octahedral magnetosomes in their mechanisms of nucleation and crystal growth as well as in their structural relationships with the cytoplasm and plasma membrane.
Assuntos
Cristalização/métodos , Magnetossomos/fisiologia , Magnetossomos/ultraestrutura , Rhodospirillaceae/ultraestrutura , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Técnica de Fratura por CongelamentoRESUMO
BACKGROUND: Myelinated axons are organized into distinct subcellular and molecular regions. Without proper organization, electrical nerve conduction is delayed, resulting in detrimental physiological outcomes. One such region is the paranode where axo-glial septate junctions act as a molecular fence to separate the sodium (Na+) channel-enriched node from the potassium (K+) channel-enriched juxtaparanode. A significant lack of knowledge remains as to cytoskeletal proteins which stabilize paranodal domains and underlying cytoskeleton. Whirlin (Whrn) is a PDZ domain-containing cytoskeletal scaffold whose absence in humans results in Usher Syndromes or variable deafness-blindness syndromes. Mutant Whirlin (Whrn) mouse model studies have linked such behavioral deficits to improper localization of critical transmembrane protein complexes in the ear and eye. Until now, no reports exist about the function of Whrn in myelinated axons. RESULTS: RT-PCR and immunoblot analyses revealed expression of Whrn mRNA and Whrn full-length protein, respectively, in several stages of central and peripheral nervous system development. Comparing wild-type mice to Whrn knockout (Whrn-/-) mice, we observed no significant differences in the expression of standard axonal domain markers by immunoblot analysis but observed and quantified a novel paranodal compaction phenotype in 4 to 8 week-old Whrn-/- nerves. The paranodal compaction phenotype and associated cytoskeletal disruption was observed in Whrn-/- mutant sciatic nerves and spinal cord fibers from early (2 week-old) to late (1 year-old) stages of development. Light and electron microscopic analyses of Whrn knockout mice reveal bead-like swellings in cerebellar Purkinje axons containing mitochondria and vesicles by both. These data suggest that Whrn plays a role in proper cytoskeletal organization in myelinated axons. CONCLUSIONS: Domain organization in myelinated axons remains a complex developmental process. Here we demonstrate that loss of Whrn disrupts proper axonal domain organization. Whrn likely contributes to the stabilization of paranodal myelin loops and axonal cytoskeleton through yet unconfirmed cytoskeletal proteins. Paranodal abnormalities are consistently observed throughout development (2 wk-1 yr) and similar between central and peripheral nervous systems. In conclusion, our observations suggest that Whrn is not required for the organization of axonal domains, but once organized, Whrn acts as a cytoskeletal linker to ensure proper paranodal compaction and stabilization of the axonal cytoskeleton in myelinated axons.
Assuntos
Citoesqueleto/ultraestrutura , Proteínas de Membrana/metabolismo , Fibras Nervosas Mielinizadas/ultraestrutura , Animais , Axônios/metabolismo , Axônios/ultraestrutura , Citoesqueleto/metabolismo , Eletrofisiologia , Immunoblotting , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Fibras Nervosas Mielinizadas/metabolismo , Nós Neurofibrosos/metabolismo , Nós Neurofibrosos/ultraestrutura , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Hair cells of the inner ear are mechanosensors that transduce mechanical forces arising from sound waves and head movement into electrochemical signals to provide our sense of hearing and balance. Each hair cell contains at the apical surface a bundle of stereocilia. Mechanoelectrical transduction takes place close to the tips of stereocilia in proximity to extracellular tip-link filaments that connect the stereocilia and are thought to gate the mechanoelectrical transduction channel. Recent reports on the composition, properties and function of tip links are conflicting. Here we demonstrate that two cadherins that are linked to inherited forms of deafness in humans interact to form tip links. Immunohistochemical studies using rodent hair cells show that cadherin 23 (CDH23) and protocadherin 15 (PCDH15) localize to the upper and lower part of tip links, respectively. The amino termini of the two cadherins co-localize on tip-link filaments. Biochemical experiments show that CDH23 homodimers interact in trans with PCDH15 homodimers to form a filament with structural similarity to tip links. Ions that affect tip-link integrity and a mutation in PCDH15 that causes a recessive form of deafness disrupt interactions between CDH23 and PCDH15. Our studies define the molecular composition of tip links and provide a conceptual base for exploring the mechanisms of sensory impairment associated with mutations in CDH23 and PCDH15.