RESUMEN
EphB2-ephrinB signalling, which plays a major role in cell segregation during embryonic development and tissue homeostasis, induces an important reorganization of the cortical actin network. We have previously reported that myosin 1b contributes to reorganization of the cortical actin network upon EphB2 signalling. In this report, we identify Plekhh1 as a new partner of members of the myosin 1 family and EphB2 receptors. Plekhh1 interacts with myosin 1b via its N-terminal domain and with EphB2 via its C-terminal domain. Furthermore, Plekhh1 is tyrosine phosphorylated, and this depends on EphB2 kinase activity. Similar to the effects of manipulating levels of myosin 1b and myosin 1c, manipulation of Plekhh1 expression levels alters the formation of filopodia, the length of focal adhesions and the formation of blebs. Furthermore, binding of the Plekhh1 interacting domain to myosin 1b increases the motor activity of myosin 1b in vitro. Taken together, our data show that Plekhh1 is an effector of EphB2 and suggest that Plekhh1 regulates the cortical actin network via the interaction of its N-terminal domain with myosin 1 upon EphB2-ephrinB signalling.
Asunto(s)
Actinas , Receptor EphB2 , Actinas/genética , Comunicación Celular , Fosforilación , Receptor EphB2/genética , Transducción de SeñalRESUMEN
The cell membrane is an inhomogeneous system composed of phospholipids, sterols, carbohydrates, and proteins that can be directly attached to underlying cytoskeleton. The protein linkers between the membrane and the cytoskeleton are believed to have a profound effect on the mechanical properties of the cell membrane and its ability to reshape. Here, we investigate the role of membrane-cortex linkers on the extrusion of membrane tubes using computer simulations and experiments. In simulations, we find that the force for tube extrusion has a nonlinear dependence on the density of membrane-cortex attachments: at a range of low and intermediate linker densities, the force is not significantly influenced by the presence of the membrane-cortex attachments and resembles that of the bare membrane. For large concentrations of linkers, however, the force substantially increases compared with the bare membrane. In both cases, the linkers provided membrane tubes with increased stability against coalescence. We then pulled tubes from HEK cells using optical tweezers for varying expression levels of the membrane-cortex attachment protein Ezrin. In line with simulations, we observed that overexpression of Ezrin led to an increased extrusion force, while Ezrin depletion had a negligible effect on the force. Our results shed light on the importance of local protein rearrangements for membrane reshaping at nanoscopic scales.
Asunto(s)
Membrana Celular , Citoesqueleto , Células HEK293 , Humanos , Fenómenos Mecánicos , Proteínas de la Membrana , FosfolípidosRESUMEN
In the early secretory pathway, the delivery of anterograde cargoes from the endoplasmic reticulum (ER) exit sites (ERES) to the Golgi apparatus is a multi-step transport process occurring via the ER-Golgi intermediate compartment (IC, also called ERGIC). While the role microtubules in ER-to-Golgi transport has been well established, how the actin cytoskeleton contributes to this process remains poorly understood. Here, we report that Arp2/3 inhibition affects the network of acetylated microtubules around the Golgi and induces the accumulation of unusually long RAB1/GM130-positive carriers around the centrosome. These long carriers are less prone to reach the Golgi apparatus, and arrival of anterograde cargoes to the Golgi is decreased upon Arp2/3 inhibition. Our data suggest that Arp2/3-dependent actin polymerization maintains a stable network of acetylated microtubules, which ensures efficient cargo trafficking at the late stage of ER to Golgi transport.
Asunto(s)
Actinas/metabolismo , Retículo Endoplásmico/metabolismo , Vías Secretoras , Acetilación , Complejo 2-3 Proteico Relacionado con la Actina , Autoantígenos/metabolismo , Centrosoma/metabolismo , Aparato de Golgi/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Proteínas de la Membrana/metabolismo , Microtúbulos/metabolismo , Proteínas de Unión al GTP rab1/metabolismoRESUMEN
Motile and morphological cellular processes require a spatially and temporally coordinated branched actin network that is controlled by the activity of various regulatory proteins, including the Arp2/3 complex, profilin, cofilin and tropomyosin. We have previously reported that myosin 1b regulates the density of the actin network in the growth cone. Here, by performing in vitro F-actin gliding assays and total internal reflection fluorescence (TIRF) microscopy, we show that this molecular motor flattens (reduces the branch angle) in the Arp2/3-dependent actin branches, resulting in them breaking, and reduces the probability of new branches forming. This experiment reveals that myosin 1b can produce force sufficient enough to break up the Arp2/3-mediated actin junction. Together with the former in vivo studies, this work emphasizes the essential role played by myosins in the architecture and dynamics of actin networks in different cellular regions.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Citoesqueleto de Actina , Complejo 2-3 Proteico Relacionado con la Actina , Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Humanos , Miosinas/genética , Miosinas/metabolismo , Unión ProteicaRESUMEN
The regulation of actin dynamics is essential for various cellular processes. Former evidence suggests a correlation between the function of non-conventional myosin motors and actin dynamics. Here we investigate the contribution of myosin 1b to actin dynamics using sliding motility assays. We observe that sliding on myosin 1b immobilized or bound to a fluid bilayer enhances actin depolymerization at the barbed end, while sliding on myosin II, although 5 times faster, has no effect. This work reveals a non-conventional myosin motor as another type of depolymerase and points to its singular interactions with the actin barbed end.
Asunto(s)
Actinas/química , Actinas/metabolismo , Miosina Tipo I/metabolismo , Citoesqueleto de Actina/enzimología , Citoesqueleto de Actina/metabolismo , Actinas/genética , Animales , Humanos , Miosina Tipo I/genética , Miosina Tipo II/química , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Polimerizacion , ConejosRESUMEN
In this study, we aimed to identify the myosin motor proteins that control trafficking at the Golgi complex. In addition to the known Golgi-associated myosins MYO6, MYO18A and MYH9 (myosin IIA), we identified MYO1C as a novel player at the Golgi in a human cell line. We demonstrate that depletion of MYO1C induces Golgi complex fragmentation and decompaction. MYO1C accumulates at dynamic structures around the Golgi complex that colocalize with Golgi-associated actin dots. MYO1C depletion leads to loss of cellular F-actin, and Golgi complex decompaction is also observed after inhibition or loss of the actin-related protein 2/3 complex, Arp2/3 (also known as ARPC). We show that the functional consequence of MYO1C depletion is a delay in the arrival of incoming transport carriers, both from the anterograde and retrograde routes. We propose that MYO1C stabilizes actin at the Golgi complex, facilitating the arrival of incoming transport carriers at the Golgi.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Aparato de Golgi/metabolismo , Miosina Tipo I/metabolismo , Línea Celular , Movimiento Celular , Humanos , Miosina Tipo I/genética , Transporte de ProteínasRESUMEN
One challenge in cell biology is to decipher the biophysical mechanisms governing protein enrichment on curved membranes and the resulting membrane deformation. The ERM protein ezrin is abundant and associated with cellular membranes that are flat, positively or negatively curved. Using in vitro and cell biology approaches, we assess mechanisms of ezrin's enrichment on curved membranes. We evidence that wild-type ezrin (ezrinWT) and its phosphomimetic mutant T567D (ezrinTD) do not deform membranes but self-assemble anti-parallelly, zipping adjacent membranes. EzrinTD's specific conformation reduces intermolecular interactions, allows binding to actin filaments, which reduces membrane tethering, and promotes ezrin binding to positively-curved membranes. While neither ezrinTD nor ezrinWT senses negative curvature alone, we demonstrate that interacting with curvature-sensing I-BAR-domain proteins facilitates ezrin enrichment in negatively-curved membrane protrusions. Overall, our work demonstrates that ezrin can tether membranes, or be targeted to curved membranes, depending on conformations and interactions with actin and curvature-sensing binding partners.
Asunto(s)
Membrana Celular/química , Proteínas del Citoesqueleto/química , Proteínas Mutantes/química , Conformación Proteica , Actinas/química , Actinas/genética , Membrana Celular/genética , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosforilación , Unión Proteica/genética , Dominios Proteicos/genéticaRESUMEN
Single-headed myosin 1 has been identified in neurons, but its function in these cells is still unclear. We demonstrate that depletion of myosin 1b (Myo1b), inhibition of its motor activity, or its binding to phosphoinositides impairs the formation of the axon, whereas overexpression of Myo1b increases the number of axon-like structures. Myo1b is associated with growth cones and actin waves, two major contributors to neuronal symmetry breaking. We show that Myo1b controls the dynamics of the growth cones and the anterograde propagation of the actin waves. By coupling the membrane to the actin cytoskeleton, Myo1b regulates the size of the actin network as well as the stability and size of filopodia in the growth cones. Our data provide the first evidence that a myosin 1 plays a major role in neuronal symmetry breaking and argue for a mechanical control of the actin cytoskeleton both in actin waves and in the growth cones by this myosin.
Asunto(s)
Actinas/metabolismo , Axones/metabolismo , Conos de Crecimiento/metabolismo , Miosina Tipo I/metabolismo , Secuencias de Aminoácidos , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Cinesinas/metabolismo , Ratones , Actividad Motora , Miosina Tipo I/química , Neuritas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Seudópodos/metabolismo , ARN Interferente Pequeño/metabolismoRESUMEN
Hormone secretion relies on secretory granules which store hormones in endocrine cells and release them upon cell stimulation. The molecular events leading to hormone sorting and secretory granule formation at the level of the TGN are still elusive. Our proteomic analysis of purified whole secretory granules or secretory granule membranes uncovered their association with the actomyosin components myosin 1b, actin and the actin nucleation complex Arp2/3. We found that myosin 1b controls the formation of secretory granules and the associated regulated secretion in both neuroendocrine cells and chromogranin A-expressing COS7 cells used as a simplified model of induced secretion. We show that F-actin is also involved in secretory granule biogenesis and that myosin 1b cooperates with Arp2/3 to recruit F-actin to the Golgi region where secretory granules bud. These results provide the first evidence that components of the actomyosin complex promote the biogenesis of secretory granules and thereby regulate hormone sorting and secretion.
Asunto(s)
Actinas/genética , Miosina Tipo I/genética , Vesículas Secretoras/metabolismo , Actinas/metabolismo , Animales , Transporte Biológico , Células COS , Proteínas Portadoras , Chlorocebus aethiops , Aparato de Golgi/metabolismo , Ratones , Miosina Tipo I/metabolismo , Células Neuroendocrinas/metabolismo , Sistemas Neurosecretores/metabolismo , Células PC12 , Unión Proteica , RatasRESUMEN
Eph receptors and their membrane-tethered ligands, the ephrins, have important functions in embryo morphogenesis and in adult tissue homeostasis. Eph/ephrin signaling is essential for cell segregation and cell repulsion. This process is accompanied by morphological changes and actin remodeling that drives cell segregation and tissue patterning. The actin cortex must be mechanically coupled to the plasma membrane to orchestrate the cell morphology changes. Here, we demonstrate that myosin 1b that can mechanically link the membrane to the actin cytoskeleton interacts with EphB2 receptors via its tail and is tyrosine phosphorylated on its tail in an EphB2-dependent manner. Myosin 1b regulates the redistribution of myosin II in actomyosin fibers and the formation of filopodia at the interface of ephrinB1 and EphB2 cells, which are two processes mediated by EphB2 signaling that contribute to cell repulsion. Together, our results provide the first evidence that a myosin 1 functions as an effector of EphB2/ephrinB signaling, controls cell morphology, and thereby cell repulsion.
Asunto(s)
Miosina Tipo I/fisiología , Receptor EphB2/metabolismo , Secuencia de Aminoácidos , Comunicación Celular , Efrina-B1/fisiología , Células HCT116 , Células HEK293 , Humanos , Datos de Secuencia Molecular , Miosina Tipo II/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Seudópodos/fisiología , Transducción de SeñalRESUMEN
Myosin 1b is a single-headed membrane-associated motor that binds to actin filaments with a catch-bond behaviour in response to load. In vivo, myosin 1b is required to form membrane tubules at both endosomes and the trans-Golgi network. To establish the link between these two fundamental properties, here we investigate the capacity of myosin 1b to extract membrane tubes along bundled actin filaments in a minimal reconstituted system. We show that single-headed non-processive myosin 1b can extract membrane tubes at a biologically relevant low density. In contrast to kinesins we do not observe motor accumulation at the tip, suggesting that the underlying mechanism for tube formation is different. In our theoretical model, myosin 1b catch-bond properties facilitate tube extraction under conditions of increasing membrane tension by reducing the density of myo1b required to pull tubes.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Microtúbulos/metabolismo , Miosina Tipo I/metabolismo , Red trans-Golgi/metabolismo , Endosomas/metabolismo , Humanos , Cinesinas/metabolismoRESUMEN
Myosin VI has been implicated in various steps of organelle dynamics. However, the molecular mechanism by which this myosin contributes to membrane traffic is poorly understood. Here, we report that myosin VI is associated with a lysosome-related organelle, the melanosome. Using an actin-based motility assay and video microscopy, we observed that myosin VI does not contribute to melanosome movements. Myosin VI expression regulates instead the organization of actin networks in the cytoplasm. Using a cell-free assay, we showed that myosin VI recruited actin at the surface of isolated melanosomes. Myosin VI is involved in the endocytic-recycling pathway, and this pathway contributes to the transport of a melanogenic enzyme to maturing melanosomes. We showed that depletion of myosin VI accumulated a melanogenic enzyme in enlarged melanosomes and increased their melanin content. We confirmed the requirement of myosin VI to regulate melanosome biogenesis by analysing the morphology of melanosomes in choroid cells from of the Snell's waltzer mice that do not express myosin VI. Together, our results provide new evidence that myosin VI regulates the organization of actin dynamics at the surface of a specialized organelle and unravel a novel function of this myosin in regulating the biogenesis of this organelle.
Asunto(s)
Actinas/metabolismo , Melanosomas/metabolismo , Cadenas Pesadas de Miosina/fisiología , Actinas/química , Animales , Membrana Celular/metabolismo , Coroides/citología , Citoplasma/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Ratones , Ratones Transgénicos , Microscopía Electrónica/métodos , Microscopía por Video/métodos , Microtúbulos/metabolismo , Modelos Biológicos , Cadenas Pesadas de Miosina/química , Miosinas/metabolismo , PigmentaciónRESUMEN
The function of organelles is intimately associated with rapid changes in membrane shape. By exerting force on membranes, the cytoskeleton and its associated motors have an important role in membrane remodelling. Actin and myosin 1 have been implicated in the invagination of the plasma membrane during endocytosis. However, whether myosin 1 and actin contribute to the membrane deformation that gives rise to the formation of post-Golgi carriers is unknown. Here we report that myosin 1b regulates the actin-dependent post-Golgi traffic of cargo, generates force that controls the assembly of F-actin foci and, together with the actin cytoskeleton, promotes the formation of tubules at the TGN. Our results provide evidence that actin and myosin 1 regulate organelle shape and uncover an important function for myosin 1b in the initiation of post-Golgi carrier formation by regulating actin assembly and remodelling TGN membranes.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Membranas Intracelulares/metabolismo , Miosina Tipo I/metabolismo , Forma de los Orgánulos , Red trans-Golgi/metabolismo , Células HeLa , Humanos , Glicoproteínas de Membrana/metabolismo , Microscopía Fluorescente , Microscopía por Video , Miosina Tipo I/genética , Interferencia de ARN , Receptor IGF Tipo 2 , Receptor de Factor de Crecimiento Nervioso/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Toxinas Shiga/metabolismo , Factores de Tiempo , Transfección , Red trans-Golgi/ultraestructuraRESUMEN
Cellular functions are intimately associated with rapid changes in membrane shape. Different mechanisms interfering with the lipid bilayer, such as the insertion of proteins with amphipatic helices or the association of a protein scaffold, trigger membrane bending. By exerting force on membranes, molecular motors can also contribute to membrane remodeling. Previous studies have shown that actin and myosin 1 participate in the invagination of the plasma membrane during endocytosis while kinesins and dynein with microtubules provide the force to elongate membrane buds at recycling endosomes and at the trans-Golgi network (TGN). Using live cell imaging we have recently shown that a myosin 1 (myosin 1b) regulates the actin dependent post-Golgi traffic of cargo and generates force that controls the assembly of F-actin foci and promotes with the actin cytoskeleton the formation of tubules at the TGN. Our data provide evidence that actin and myosin 1 can regulate membrane remodeling of organelles as well as having an unexpected role in the spatial organization of the actin cytoskeleton. Here, we discuss our results together with the role of actin and other myosins that have been implicated in the traffic of cargo.
RESUMEN
Important progress has been made during the past decade in the identification of molecular motors required in the distribution of early and late endosomes and the proper trafficking along the endocytic pathway. There is little direct evidence, however, that these motors drive movement of the endosomes. To evaluate the contributions of kinesin-1, dynein and kinesin-2 to the movement of early and late endosomes along microtubules, we made use of a cytosol-free motility assay using magnetically isolated early and late endosomes as well as biochemical analyses and live-cell imaging. By making use of specific antibodies, we confirmed that kinesin-1 and dynein move early endosomes and we found that kinesin-2 moves both early and late endosomes in the cell-free assay. Unexpectedly, dynein did not move late endosomes in the cell-free assay. We provide evidence from disruption of dynein function and latrunculin A treatment, suggesting that dynein regulates late endosome movement indirectly, possibly through a mechanism involving the actin cytoskeleton. These data provide new insights into the complex regulation of endosomes' motility and suggest that dynein is not the major motor required to move late endosomes toward the minus end of microtubules.
Asunto(s)
Endocitosis/fisiología , Endosomas/metabolismo , Proteínas de Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Motoras Moleculares/metabolismo , Animales , Dineínas/metabolismo , Endosomas/ultraestructura , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Cinesinas/metabolismo , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/metabolismo , Magnetismo , Proteínas de Microtúbulos/genética , Proteínas Motoras Moleculares/genética , Nanopartículas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
The actin network has been implicated in the intracellular transport and positioning of the melanosomes, organelles that are specialized in the biosynthesis and the storage of melanin. It contributes also to molecular mechanisms that underlie the intracellular membrane dynamics and thereby can control the biogenesis of melanosomes. Two mechanisms for actin-based movements have been identified: one is dependent on the motors associated to actin namely the myosins; the other is dependent on actin polymerization. This review will focus on to the role of the actin cytoskeleton and myosins in the transport and in the biogenesis of melanosomes. Myosins involved in membrane traffic are largely seen as transporters of organelles or membrane vesicles containing cargos along the actin networks. Yet increasing evidence suggests that some of the myosins contribute to the dynamics of internal membrane by using other mechanisms. The role of the myosins and the different molecular mechanisms by which they contribute or may contribute to the distribution, the movement and the biogenesis of the melanosomes in epidermal melanocytes and retinal pigmented epithelial (RPE) cells will be discussed.
Asunto(s)
Melanocitos/metabolismo , Miosinas/metabolismo , Actinas/metabolismo , Animales , Citoesqueleto/metabolismo , Dineínas/metabolismo , Humanos , Melanosomas/metabolismo , Modelos Biológicos , Movimiento , Miosina Tipo V/fisiología , Miosina VIIa , Fagosomas/metabolismo , Epitelio Pigmentado Ocular/metabolismoRESUMEN
Members of at least four classes of myosin (I, II, V and VI) have been implicated in the dynamics of a large variety of organelles. Despite their common motor domain structure, some of these myosins, however, are non processive and cannot move organelles along the actin tracks. Here, we demonstrate in the human pigmented MNT-1 cell line that, (1) the overexpression of one of these myosins, myosin 1b, or the addition of cytochalasin D affects the morphology of the sorting multivesicular endosomes; (2) the overexpression of myosin 1b delays the processing of Pmel17 (the product of murine silver locus also named GP100), which occurs in these multivesicular endosomes; (3) myosin 1b associated with endosomes coimmunoprecipitates with Pmel17. All together, these observations suggest that myosin 1b controls the traffic of protein cargo in multivesicular endosomes most probably through its ability to modulate with actin the morphology of these sorting endosomes.
Asunto(s)
Endosomas/metabolismo , Miosina Tipo I/metabolismo , Citoesqueleto de Actina/metabolismo , Citocalasina B/farmacología , Endosomas/efectos de los fármacos , Endosomas/ultraestructura , Humanos , Melanosomas/ultraestructura , Unión Proteica , Transporte de Proteínas , Proteínas Recombinantes de Fusión , Fracciones SubcelularesRESUMEN
Rab GTPases have been implicated in the regulation of specific microtubule- and actin-based motor proteins. We devised an in vitro motility assay reconstituting the movement of melanosomes on actin bundles in the presence of ATP to investigate the role of Rab proteins in the actin-dependent movement of melanosomes. Using this assay, we confirmed that Rab27 is required for the actin-dependent movement of melanosomes, and we showed that a second Rab protein, Rab8, also regulates this movement. Rab8 was partially associated with mature melanosomes. Expression of Rab8Q67L perturbed the cellular distribution and increased the frequency of microtubule-independent movement of melanosomes in vivo. Furthermore, anti-Rab8 antibodies decreased the number of melanosomes moving in vitro on actin bundles, whereas melanosomes isolated from cells expressing Rab8Q67L exhibited 70% more movements than wild-type melanosomes. Together, our observations suggest that Rab8 is involved in regulating the actin-dependent movement of melanosomes.
Asunto(s)
Actinas/metabolismo , Melanosomas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Transporte Biológico , Línea Celular , Humanos , Ratones , Mutación/genética , Proteínas de Unión al GTP rab/genética , Proteínas rab27 de Unión a GTPRESUMEN
The molecular structure of the brush-border of enterocytes has been investigated since the 1980s, but the dynamics of this highly specialized subcellular domain have been difficult to study due to its small size. To perform a detailed analysis of the dynamics of cytoskeleton proteins in this domain, we developed two-photon fluorescence recovery after photobleaching and a theoretical framework for data analysis. With this method, fast dynamics of proteins in the microvilli of the brush border of epithelial intestinal cells can be measured on the millisecond timescale in volumes smaller than 1 microm3. Two major proteins of the cytoskeleton of the microvilli, actin and myosin 1a (Myo1a; formerly named brush border myosin I), are mobile in the brush-border of Caco-2 cells, an enterocyte-like cellular model. However, the mobility of actin is very different from that of Myo1a and they appear to be unrelated (diffusion coefficient of 15 microm2 s(-1) with a mobile fraction of 60% for actin, and 4 microm2 s(-1) with a mobile fraction of 90% for Myo1a). Furthermore, we show for the first time, in vivo, that the dynamics of Myo1a in microvilli reflect its motor activity.