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1.
J Biol Chem ; 294(29): 11333-11341, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31175157

RESUMO

Vertebrate myosin-5a is an ATP-utilizing processive motor associated with the actin network and responsible for the transport and localization of several vesicle cargoes. To transport cargo efficiently and prevent futile ATP hydrolysis, myosin-5a motor function must be tightly regulated. The globular tail domain (GTD) of myosin-5a not only functions as the inhibitory domain but also serves as the binding site for a number of cargo adaptor proteins, including melanophilin (Mlph) and Rab-interacting lysosomal protein-like 2 (RILPL2). In this study, using various biochemical approaches, including ATPase, single-molecule motility, GST pulldown assays, and analytical ultracentrifugation, we demonstrate that the binding of both Mlph and RILPL2 to the GTD of myosin-5a is required for the activation of myosin-5a motor function under physiological ionic conditions. We also found that this activation is regulated by the small GTPase Rab36, a binding partner of RILPL2. In summary, our results indicate that RILPL2 is required for Mlph-mediated activation of Myo5a motor activity under physiological conditions and that Rab36 promotes this activation. We propose that Rab36 stimulates RILPL2 to interact with the myosin-5a GTD; this interaction then induces exposure of the Mlph-binding site in the GTD, enabling Mlph to interact with the GTD and activate myosin-5a motor activity.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas Motores Moleculares/fisiologia , Miosina Tipo V/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Camundongos , Proteínas Motores Moleculares/metabolismo , Miosina Tipo V/metabolismo , Concentração Osmolar , Ligação Proteica
2.
Dev Dyn ; 248(4): 284-295, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30801852

RESUMO

BACKGROUND: During heart morphogenesis, the cardiac chambers undergo ballooning: a process involving regionalized elongation of cardiomyocytes. Cardiomyocyte shape changes require reorganization of the actin cytoskeleton; however, the genetic regulation of this process is not well understood. RESULTS: From a forward genetic screen, we identified the zebrafish uq 23ks mutant which manifests chamber ballooning defects. Whole-genome sequencing-mapping identified a truncating mutation in the gene, myo5b. myo5b encodes an atypical myosin required for endosome recycling and, consistent with this, increased vesicles were observed in myo5b mutant cardiomyocytes. Expression of RFP-Rab11a (a recycling endosome marker) confirmed increased recycling endosomes in cardiomyocytes of myo5b mutants. To investigate potential cargo of MyoVb-associated vesicles, we examined the adherens junction protein, N-cadherin. N-cadherin appeared mispatterned at cell junctions, and an increase in the number of intracellular particles was also apparent. Co-localization with RFP-Rab11a confirmed increased N-cadherin-positive recycling endosomes, demonstrating N-cadherin trafficking is perturbed in myo5b mutants. Finally, phalloidin staining showed disorganized F-actin in myo5b cardiomyocytes, suggesting the cytoskeleton fails to remodel, obstructing chamber ballooning. CONCLUSIONS: MyoVb is required for cardiomyocyte endosomal recycling and appropriate N-cadherin localization during the onset of chamber ballooning. Cardiomyocytes lacking MyoVb are unable to reorganize their actin cytoskeleton, resulting in failed chamber ballooning. Developmental Dynamics 248:284-295, 2019. © 2019 Wiley Periodicals, Inc.


Assuntos
Caderinas/metabolismo , Citoesqueleto/ultraestrutura , Coração/crescimento & desenvolvimento , Miócitos Cardíacos/metabolismo , Miosina Tipo V/fisiologia , Animais , Forma Celular , Citoesqueleto/metabolismo , Endossomos/metabolismo , Humanos , Miocárdio/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/ultraestrutura , Miosina Tipo V/genética , Miosinas/genética , Miosinas/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia
3.
Nat Commun ; 9(1): 2844, 2018 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-30030431

RESUMO

Myosin-5B is one of three members of the myosin-5 family of actin-based molecular motors. Despite its fundamental role in recycling endosome trafficking and in collective actin network dynamics, the molecular mechanisms underlying its motility are inherently unknown. Here we combine single-molecule imaging and high-speed laser tweezers to dissect the mechanoenzymatic properties of myosin-5B. We show that a single myosin-5B moves processively in 36-nm steps, stalls at ~2 pN resistive forces, and reverses its directionality at forces >2 pN. Interestingly, myosin-5B mechanosensitivity differs from that of myosin-5A, while it is strikingly similar to kinesin-1. In particular, myosin-5B run length is markedly and asymmetrically sensitive to force, a property that might be central to motor ensemble coordination. Furthermore, we show that Ca2+ does not affect the enzymatic activity of the motor unit, but abolishes myosin-5B processivity through calmodulin dissociation, providing important insights into the regulation of postsynaptic cargoes trafficking in neuronal cells.


Assuntos
Cálcio/química , Cadeias Pesadas de Miosina/química , Miosina Tipo V/química , Miosinas/química , Animais , Biotinilação , DNA/química , Homeostase , Cinesina/química , Cinética , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Miosinas/fisiologia , Neurônios/metabolismo , Pontos Quânticos , Ratos , Estresse Mecânico , Potenciais Sinápticos
4.
Circ Res ; 114(6): 982-92, 2014 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-24508725

RESUMO

RATIONALE: Kv1.5 (KCNA5) mediates the ultra-rapid delayed rectifier current that controls atrial action potential duration. Given its atrial-specific expression and alterations in human atrial fibrillation, Kv1.5 has emerged as a promising target for the treatment of atrial fibrillation. A necessary step in the development of novel agents that selectively modulate trafficking pathways is the identification of the cellular machinery controlling Kv1.5 surface density, of which little is yet known. OBJECTIVE: To investigate the role of the unconventional myosin-V (MYO5A and MYO5B) motors in determining the cell surface density of Kv1.5. METHODS AND RESULTS: Western blot analysis showed MYO5A and MYO5B expression in the heart, whereas disruption of endogenous motors selectively reduced IKur current in adult rat cardiomyocytes. Dominant negative constructs and short hairpin RNA silencing demonstrated a role for MYO5A and MYO5B in the surface trafficking of Kv1.5 and connexin-43 but not potassium voltage-gated channel, subfamily H (eag-related), member 2 (KCNH2). Live-cell imaging of Kv1.5-GFP and retrospective labeling of phalloidin demonstrated motility of Kv1.5 vesicles on actin tracts. MYO5A participated in anterograde trafficking, whereas MYO5B regulated postendocytic recycling. Overexpression of mutant motors revealed a selective role for Rab11 in coupling MYO5B to Kv1.5 recycling. CONCLUSIONS: MYO5A and MYO5B control functionally distinct steps in the surface trafficking of Kv1.5. These isoform-specific trafficking pathways determine Kv1.5-encoded IKur in myocytes to regulate repolarizing current and, consequently, cardiac excitability. Therapeutic strategies that manipulate Kv1.5 selective trafficking pathways may prove useful in the treatment of arrhythmias.


Assuntos
Membrana Celular/metabolismo , Canal de Potássio Kv1.5/metabolismo , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Miosinas/fisiologia , Transporte Proteico/fisiologia , Citoesqueleto de Actina/fisiologia , Animais , Arritmias Cardíacas/fisiopatologia , Linhagem Celular , Conexina 43/análise , Canal de Potássio ERG1 , Endocitose , Canais de Potássio Éter-A-Go-Go/análise , Junções Comunicantes , Genes Reporter , Sistema de Condução Cardíaco/fisiopatologia , Transporte de Íons , Canal de Potássio Kv1.5/genética , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Cardiovasculares , Cadeias Pesadas de Miosina/deficiência , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/deficiência , Miosina Tipo V/genética , Miosinas/deficiência , Miosinas/genética , Potássio/metabolismo , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/metabolismo , Proteínas rab de Ligação ao GTP/fisiologia
5.
J Cell Sci ; 127(Pt 5): 1007-17, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24413175

RESUMO

Microvilli at the apical surface of enterocytes allow the efficient absorption of nutrients in the intestine. Ezrin activation by its phosphorylation at T567 is important for microvilli development, but how such ezrin phosphorylation is controlled is not well understood. We demonstrate that a subset of kinases that phosphorylate ezrin closely co-distributes with apical recycling endosome marker Rab11a in the subapical domain. Expression of dominant-negative Rab11a mutant or depletion of the Rab11a-binding motor protein myosin Vb prevents the subapical enrichment of Rab11a and these kinases and inhibits ezrin phosphorylation and microvilli development, without affecting the polarized distribution of ezrin itself. We observe a similar loss of the subapical enrichment of Rab11a and the kinases and reduced phosphorylation of ezrin in microvillus inclusion disease, which is associated with MYO5B mutations, intestinal microvilli atrophy and malabsorption. Thus, part of the machinery for ezrin activation depends on recycling endosomes controlled by myosin Vb and Rab11a which, we propose, might act as subapical signaling platforms that enterocytes use to regulate development of microvilli and maintain human intestinal function.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Enterócitos/metabolismo , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Processamento de Proteína Pós-Traducional , Proteínas rab de Ligação ao GTP/fisiologia , Linhagem Celular Tumoral , Polaridade Celular , Códon sem Sentido , Endossomos/metabolismo , Células HEK293 , Humanos , Isoenzimas/metabolismo , Síndromes de Malabsorção/genética , Microvilosidades/genética , Microvilosidades/metabolismo , Microvilosidades/patologia , Mucolipidoses/genética , Fosforilação , Proteína Quinase C/metabolismo , Transporte Proteico , Proteínas Serina-Treonina Quinases/metabolismo
6.
J Cell Biol ; 203(6): 971-84, 2013 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-24368805

RESUMO

The assembly and composition of ribonucleic acid (RNA)-transporting particles for asymmetric messenger RNA (mRNA) localization is not well understood. During mitosis of budding yeast, the Swi5p-dependent HO expression (SHE) complex transports a set of mRNAs into the daughter cell. We recombinantly reconstituted the core SHE complex and assessed its properties. The cytoplasmic precomplex contains only one motor and is unable to support continuous transport. However, a defined interaction with a second, RNA-bound precomplex after its nuclear export dimerizes the motor and activates processive RNA transport. The run length observed in vitro is compatible with long-distance transport in vivo. Surprisingly, SHE complexes that either contain or lack RNA cargo show similar motility properties, demonstrating that the RNA-binding protein and not its cargo activates motility. We further show that SHE complexes have a defined size but multimerize into variable particles upon binding of RNAs with multiple localization elements. Based on these findings, we provide an estimate of number, size, and composition of such multimeric SHE particles in the cell.


Assuntos
Transporte de RNA/fisiologia , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/fisiologia , Dimerização , Cadeias Pesadas de Miosina/metabolismo , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/metabolismo , Miosina Tipo V/fisiologia , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição
7.
Biochem Soc Trans ; 40(6): 1416-20, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-23176491

RESUMO

Mammals express three class V myosins. Myosin Va is widely expressed, but enriched in the brain, testes and melanocytes, myosin Vb is expressed ubiquitously, and myosin Vc is believed to be epithelium-specific. Myosin Va is the best characterized of the three and plays a key role in the transport of cargo to the plasma membrane. Its cargo includes cell-surface receptors, pigment and organelles such as the endoplasmic reticulum. It is also emerging that RNA and RNA-BPs (RNA-binding proteins) make up another class of myosin Va cargo. It has long been established that the yeast class V myosin, Myo4p, transports mRNAs along actin cables into the growing bud, and now several groups have reported a similar role for class V myosins in higher eukaryotes. Myosin Va has also been implicated in the assembly and maintenance of P-bodies (processing bodies), cytoplasmic foci that are involved in mRNA storage and degradation. The present review examines the evidence that myosin Va plays a role in the transport and turnover of mRNA.


Assuntos
Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Transporte de RNA , RNA Mensageiro/metabolismo , Animais , Espinhas Dendríticas/metabolismo , Humanos , Modelos Biológicos , Biossíntese de Proteínas , Estrutura Quaternária de Proteína
8.
Curr Biol ; 22(15): R606-8, 2012 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-22877783

RESUMO

Cytoskeletal trafficking systems are becoming more complex at every turn. A new study reports that a yeast myosin V walks on only a select few actin filaments - those that are decorated with tropomyosin.


Assuntos
Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Tropomiosina/fisiologia
9.
J Cell Biol ; 198(4): 545-60, 2012 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-22908308

RESUMO

Rab proteins are important regulators of insulin-stimulated GLUT4 translocation to the plasma membrane (PM), but the precise steps in GLUT4 trafficking modulated by particular Rab proteins remain unclear. Here, we systematically investigate the involvement of Rab proteins in GLUT4 trafficking, focusing on Rab proteins directly mediating GLUT4 storage vesicle (GSV) delivery to the PM. Using dual-color total internal reflection fluorescence (TIRF) microscopy and an insulin-responsive aminopeptidase (IRAP)-pHluorin fusion assay, we demonstrated that Rab10 directly facilitated GSV translocation to and docking at the PM. Rab14 mediated GLUT4 delivery to the PM via endosomal compartments containing transferrin receptor (TfR), whereas Rab4A, Rab4B, and Rab8A recycled GLUT4 through the endosomal system. Myosin-Va associated with GSVs by interacting with Rab10, positioning peripherally recruited GSVs for ultimate fusion. Thus, multiple Rab proteins regulate the trafficking of GLUT4, with Rab10 coordinating with myosin-Va to mediate the final steps of insulin-stimulated GSV translocation to the PM.


Assuntos
Adipócitos/metabolismo , Vesículas Citoplasmáticas/fisiologia , Transportador de Glucose Tipo 4/metabolismo , Insulina/fisiologia , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Proteínas rab de Ligação ao GTP/fisiologia , Células 3T3-L1 , Animais , Membrana Celular/metabolismo , Camundongos , Transporte Proteico/fisiologia
10.
Biophys J ; 103(1): 48-58, 2012 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-22828331

RESUMO

Intracellular trafficking of organelles often involves cytoskeletal track switching. Organelles such as melanosomes are transported by multiple motors including kinesin-2, dynein, and myosin-V, which drive switching between microtubules and actin filaments during dispersion and aggregation. Here, we used optical trapping to determine the unitary and ensemble forces of kinesin-2, and to reconstitute cargo switching at cytoskeletal intersections in a minimal system with kinesin-2 and myosin-V motors bound to beads. Single kinesin-2 motors exerted forces up to ∼5 pN, similar to kinesin-1. However, kinesin-2 motors were more likely to detach at submaximal forces, and the duration of force maintenance was short as compared to kinesin-1. In multimotor assays, force increased with kinesin-2 density but was not affected by the presence of myosin-V. In crossed filament assays, switching frequencies of motor-bound beads were dependent on the starting track. At equal average forces, beads tended to switch from microtubules onto overlying actin filaments consistent with the relatively faster detachment of kinesin-2 at near-maximal forces. Thus, in addition to relative force, switching probability at filament intersections is determined by the dynamics of motor-filament interaction, such as the quick detachment of kinesin-2 under load. This may enable fine-tuning of filament switching in the cell.


Assuntos
Citoesqueleto de Actina/fisiologia , Cinesina/fisiologia , Microtúbulos/fisiologia , Proteínas de Xenopus/fisiologia , Citoesqueleto de Actina/química , Animais , Cinesina/química , Microscopia de Fluorescência , Simulação de Dinâmica Molecular , Miosina Tipo V/química , Miosina Tipo V/fisiologia , Conformação Proteica , Coelhos , Xenopus , Proteínas de Xenopus/química
11.
Cell Rep ; 1(5): 483-94, 2012 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-22832273

RESUMO

mRNAs encoding polarity and secretion factors (POLs) target the incipient bud site in yeast for localized translation during division. In pheromone-treated cells we now find that these mRNAs are also localized to the yeast-mating projection (shmoo) tip. However, in contrast to the budding program, neither the She2 nor She3 proteins are involved. Instead, the Scp160 RNA-binding protein binds POL and mating pathway mRNAs and regulates their spatial distribution in a Myo4- and cortical ER-dependent fashion. RNA binding by Scp160 is stimulated by activation of Gpa1, the G protein α subunit regulated by the pheromone receptor, and is required for pheromone gradient sensing, as well as subsequent chemotropic growth and cell-cell mating. These effects are incurred independently of obvious changes in translation; thus, mRNA trafficking is required for chemotropism and completion of the mating program. This is, to our knowledge, the first demonstration of ligand-activated RNA targeting in the development of a simple eukaryote.


Assuntos
Quimiotaxia/fisiologia , Feromônios/fisiologia , RNA Mensageiro/fisiologia , Proteínas de Ligação a RNA/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , Tropismo/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Divisão Celular/fisiologia , Polaridade Celular/fisiologia , Subunidades alfa de Proteínas de Ligação ao GTP/fisiologia , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Proteínas de Ligação a RNA/genética , Proteínas de Saccharomyces cerevisiae/genética
12.
Comput Math Methods Med ; 2012: 781456, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22675402

RESUMO

We describe the development of a hierarchic modelling method applied to simulating the processive movement of the myosin-V molecular motor protein along an actin filament track. In the hierarchic model, three different levels of protein structure resolution are represented: secondary structure, domain, and protein, with the level of detail changing according to the degree of interaction among the molecules. The integrity of the system is maintained using a tree of spatially organised bounding volumes and distance constraints. Although applied to an actin-myosin system, the hierarchic framework is general enough so that it may easily be adapted to a number of other large biomolecular systems containing in the order of 100 proteins. We compared the simulation results with biophysical data, and despite the lack of atomic detail in our model, we find good agreement and can even suggest some refinements to the current model of myosin-V motion.


Assuntos
Modelos Biológicos , Proteínas Motores Moleculares/fisiologia , Miosina Tipo V/fisiologia , Citoesqueleto de Actina/química , Citoesqueleto de Actina/fisiologia , Algoritmos , Animais , Simulação por Computador , Dimerização , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas Motores Moleculares/química , Movimento/fisiologia , Miosina Tipo V/química , Ligação Proteica
13.
Curr Biol ; 22(15): 1410-6, 2012 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-22704989

RESUMO

Myosin V is an actin-based motor protein involved in intracellular cargo transport [1]. Given this physiological role, it was widely assumed that all class V myosins are processive, able to take multiple steps along actin filaments without dissociating. This notion was challenged when several class V myosins were characterized as nonprocessive in vitro, including Myo2p, the essential class V myosin from S. cerevisiae [2-6]. Myo2p moves cargo including secretory vesicles and other organelles for several microns along actin cables in vivo. This demonstrated cargo transporter must therefore either operate in small ensembles or behave processively in the cellular context. Here we show that Myo2p moves processively in vitro as a single motor when it walks on an actin track that more closely resembles the actin cables found in vivo. The key to processivity is tropomyosin: Myo2p is not processive on bare actin but highly processive on actin-tropomyosin. The major yeast tropomyosin isoform, Tpm1p, supports the most robust processivity. Tropomyosin slows the rate of MgADP release, thus increasing the time the motor spends strongly attached to actin. This is the first example of tropomyosin switching a motor from nonprocessive to processive motion on actin.


Assuntos
Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Tropomiosina/fisiologia , Actinas/fisiologia , Isoformas de Proteínas
14.
PLoS One ; 7(2): e31218, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22359575

RESUMO

Because of the industrial and medical importance of members of the fungal genus Aspergillus, there is considerable interest in the functions of cytoskeletal components in growth and secretion in these organisms. We have analyzed the genome of Aspergillus nidulans and found that there are two previously unstudied myosin genes, a myosin II homolog, myoB (product = MyoB) and a myosin V homolog, myoE (product = MyoE). Deletions of either cause significant growth defects. MyoB localizes in strings that coalesce into contractile rings at forming septa. It is critical for septation and normal deposition of chitin but not for hyphal extension. MyoE localizes to the Spitzenkörper and to moving puncta in the cytoplasm. Time-lapse imaging of SynA, a v-SNARE, reveals that in myoE deletion strains vesicles no longer localize to the Spitzenkörper. Tip morphology is slightly abnormal and branching occurs more frequently than in controls. Tip extension is slower than in controls, but because hyphal diameter is greater, growth (increase in volume/time) is only slightly reduced. Concentration of vesicles into the Spitzenkörper before incorporation into the plasma membrane is, thus, not required for hyphal growth but facilitates faster tip extension and a more normal hyphal shape.


Assuntos
Aspergillus nidulans/crescimento & desenvolvimento , Miosina Tipo II/fisiologia , Miosina Tipo V/fisiologia , Aspergillus nidulans/ultraestrutura , Proteínas Fúngicas , Genoma Fúngico , Imagem com Lapso de Tempo
15.
Proc Natl Acad Sci U S A ; 109(5): E218-24, 2012 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-22228305

RESUMO

Myosin Va (myoV) is a processive molecular motor that transports intracellular cargo along actin tracks with each head taking multiple 72-nm hand-over-hand steps. This stepping behavior was observed with a constitutively active, truncated myoV, in which the autoinhibitory interactions between the globular tail and motor domains (i.e., heads) that regulate the full-length molecule no longer exist. Without cargo at near physiologic ionic strength (100 mM KCl), full-length myoV adopts a folded (approximately 15 S), enzymatically-inhibited state that unfolds to an extended (approximately 11 S), active conformation at higher salt (250 mM). Under conditions favoring the folded, inhibited state, we show that Quantum-dot-labeled myoV exhibits two types of interaction with actin in the presence of MgATP. Most motors bind to actin and remain stationary, but surprisingly, approximately 20% are processive. The moving motors transition between a strictly gated and hand-over-hand stepping pattern typical of a constitutively active motor, and a new mode with a highly variable stepping pattern suggestive of altered gating. Each head of this partially inhibited motor takes longer-lived, short forward (35 nm) and backward (28 nm) steps, presumably due to globular tail-head interactions that modify the gating of the individual heads. This unique mechanical state may be an intermediate in the pathway between the inhibited and active states of the motor.


Assuntos
Actinas/fisiologia , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Animais , Camundongos , Concentração Osmolar , Ultracentrifugação
16.
Traffic ; 12(10): 1371-82, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21740491

RESUMO

Von-Willebrand factor (vWF) is a highly multimerized hemostatic glycoprotein that is stored in endothelial Weibel-Palade bodies (WPB) and secreted upon cell stimulation to act in recruiting platelets to sites of vessel injury. Only fully matured multimeric vWF represents an efficient anchor for platelets, and endothelial cells have developed mechanisms to prevent release of immature vWF. Full maturation of vWF occurs within WPB following their translocation from a perinuclear site of emergence at the trans-Golgi network (TGN) to the cell periphery. The WPB-associated small GTPase Rab27a is involved in restricting immature WPB exocytosis and we searched for links between Rab27a and the actin cytoskeleton that could anchor WPB inside endothelial cells until they are fully matured. We here identify myosin Va as such link. Myosin Va forms a tripartite complex with Rab27a and its effector MyRIP and depletion of or dominant-negative interference with myosin Va leads to an increase in the ratio of perinuclear to more peripheral WPB. Concomitantly, myosin Va depletion results in an elevated secretion of less-oligomeric vWF from histamine-stimulated endothelial cells. These results indicate that a Rab27a/MyRIP/myosin Va complex is involved in linking WPB to the peripheral actin cytoskeleton of endothelial cells to allow full maturation and prevent premature secretion of vWF.


Assuntos
Células Endoteliais/metabolismo , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Proteínas de Transporte Vesicular/fisiologia , Proteínas rab de Ligação ao GTP/fisiologia , Fator de von Willebrand/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , Células Endoteliais/fisiologia , Humanos , Imunoprecipitação , Microscopia Confocal , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Plasmídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Proteínas de Transporte Vesicular/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTP
17.
Am J Physiol Gastrointest Liver Physiol ; 301(3): G498-507, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21680773

RESUMO

Nitrergic neurotransmission at the smooth muscle neuromuscular junctions requires nitric oxide (NO) release that is dependent on the transport and docking of neuronal NO synthase (nNOS) α to the membrane of nerve terminals. However, the mechanism of translocation of nNOSα in actin-rich varicosities is unknown. We report here that the processive motor protein myosin Va is necessary for nitrergic neurotransmission. In wild-type mice, nNOSα-stained enteric varicosities colocalized with myosin Va and its tail constituent light chain 8 (LC8). In situ proximity ligation assay showed close association among nNOSα, myosin Va, and LC8. nNOSα was associated with varicosity membrane. Varicosities showed nitric oxide production upon stimulation with KCl. Intracellular microelectrode studies showed nitrergic IJP and smooth muscle hyperpolarizing responses to NO donor diethylenetriamine-NO (DNO). In contrast, enteric varicosities from myosin Va-deficient DBA (for dilute, brown, non-agouti) mice showed near absence of myosin Va but normal nNOSα and LC8. Membrane-bound nNOSα was not detectable, and the varicosities showed reduced NO production. Intracellular recordings in DBA mice showed reduced nitrergic IJPs but normal hyperpolarizing response to DNO. The nitrergic slow IJP was 9.1 ± 0.7 mV in the wild-type controls and 3.4 ± 0.3 mV in the DBA mice (P < 0.0001). Deficiency of myosin Va resulted in loss of nitrergic neuromuscular neurotransmission despite normal presence of nNOSα in the varicosities. These studies reveal the critical importance of myosin Va in nitrergic neurotransmission by facilitating transport of nNOSα to the varicosity membrane.


Assuntos
Sistema Nervoso Entérico/fisiologia , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Junção Neuromuscular/fisiologia , Óxido Nítrico Sintase Tipo I/metabolismo , Transmissão Sináptica/fisiologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos DBA , Microscopia Confocal , Cadeias Leves de Miosina/fisiologia
18.
PLoS One ; 6(2): e17087, 2011 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-21359212

RESUMO

The neurofilament light subunit (NF-L) binds to myosin Va (Myo Va) in neurons but the sites of interaction and functional significance are not clear. We show by deletion analysis that motor domain of Myo Va binds to the NF-L rod domain that forms the NF backbone. Loss of NF-L and Myo Va binding from axons significantly reduces the axonal content of ER, and redistributes ER to the periphery of axon. Our data are consistent with a novel function for NFs as a scaffold in axons for maintaining the content and proper distribution of vesicular organelles, mediated in part by Myo Va. Based on observations that the Myo Va motor domain binds to intermediate filament (IF) proteins of several classes, Myo Va interactions with IFs may serve similar roles in organizing organelle topography in different cell types.


Assuntos
Axônios/metabolismo , Retículo Endoplasmático/metabolismo , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/metabolismo , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/química , Miosina Tipo V/metabolismo , Miosina Tipo V/fisiologia , Proteínas de Neurofilamentos/metabolismo , Animais , Axônios/fisiologia , Filamentos Intermediários/metabolismo , Filamentos Intermediários/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Neurônios/metabolismo , Neurônios/fisiologia , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Distribuição Tecidual
19.
J Neurochem ; 116(2): 177-91, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21077886

RESUMO

In neuroscience, myosin V motor proteins have attracted attention since they are highly expressed in brain, and absence of myosin Va in man leads to a severe neurological disease called Griscelli syndrome. While in some cells myosin V is described to act as a vesicle transport motor, an additional role in exocytosis has emerged recently. In neurons, myosin V has been linked to exocytosis of secretory vesicles and recycling endosomes. Through these functions, it is implied in regulating important brain functions including the release of neuropeptides by exocytosis of large dense-core vesicles and the insertion of neurotransmitter receptors into post-synaptic membranes. This review focuses on the role of myosin V in (i) axonal transport and stimulated exocytosis of large dense-core vesicles to regulate the secretion of neuroactive substances, (ii) tethering of the endoplasmic reticulum at cerebellar synapses to permit long-term depression, (iii) recycling of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors at hippocampal synapses during long-term potentiation, and (iv) recycling of nicotinic acetylcholine receptors at the neuromuscular junction. Myosin V is thus discussed as an important modulator of synaptic plasticity.


Assuntos
Exocitose/fisiologia , Miosina Tipo V/fisiologia , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Animais , Humanos , Cadeias Pesadas de Miosina/deficiência , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/química , Miosina Tipo V/deficiência , Miosina Tipo V/genética , Plasticidade Neuronal/genética , Sinapses/genética , Sinapses/patologia
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