RESUMEN
Tissue folds are structural motifs critical to organ function. In the intestine, bending of a flat epithelium into a periodic pattern of folds gives rise to villi, finger-like protrusions that enable nutrient absorption. However, the molecular and mechanical processes driving villus morphogenesis remain unclear. Here, we identify an active mechanical mechanism that simultaneously patterns and folds the intestinal epithelium to initiate villus formation. At the cellular level, we find that PDGFRA+ subepithelial mesenchymal cells generate myosin II-dependent forces sufficient to produce patterned curvature in neighboring tissue interfaces. This symmetry-breaking process requires altered cell and extracellular matrix interactions that are enabled by matrix metalloproteinase-mediated tissue fluidization. Computational models, together with in vitro and in vivo experiments, revealed that these cellular features manifest at the tissue level as differences in interfacial tensions that promote mesenchymal aggregation and interface bending through a process analogous to the active dewetting of a thin liquid film.
Asunto(s)
Matriz Extracelular , Mucosa Intestinal , Animales , Ratones , Mucosa Intestinal/metabolismo , Mucosa Intestinal/citología , Matriz Extracelular/metabolismo , Miosina Tipo II/metabolismo , Mesodermo/metabolismo , Mesodermo/citología , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Morfogénesis , Metaloproteinasas de la Matriz/metabolismoRESUMEN
Nonmuscle myosin II (NMII) is a multimeric protein complex that generates most mechanical force in eukaryotic cells. NMII function is controlled at three main levels. The first level includes events that trigger conformational changes that extend the complex to enable its assembly into filaments. The second level controls the ATPase activity of the complex and its binding to microfilaments in extended NMII filaments. The third level includes events that modulate the stability and contractility of the filaments. They all work in concert to finely control force generation inside cells. NMII is a common endpoint of mechanochemical signaling pathways that control cellular responses to physical and chemical extracellular cues. Specific phosphorylations modulate NMII activation in a context-dependent manner. A few kinases control these phosphorylations in a spatially, temporally, and lineage-restricted fashion, enabling functional adaptability to the cellular microenvironment. Here, we review mechanisms that control NMII activity in the context of cell migration and division.
Asunto(s)
Citoesqueleto , Miosina Tipo II , Citoesqueleto de Actina/metabolismo , Movimiento Celular/genética , Citoesqueleto/metabolismo , Miosina Tipo II/química , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Transducción de SeñalRESUMEN
The synchronous cleavage divisions of early embryogenesis require coordination of the cell-cycle oscillator, the dynamics of the cytoskeleton, and the cytoplasm. Yet, it remains unclear how spatially restricted biochemical signals are integrated with physical properties of the embryo to generate collective dynamics. Here, we show that synchronization of the cell cycle in Drosophila embryos requires accurate nuclear positioning, which is regulated by the cell-cycle oscillator through cortical contractility and cytoplasmic flows. We demonstrate that biochemical oscillations are initiated by local Cdk1 inactivation and spread through the activity of phosphatase PP1 to generate cortical myosin II gradients. These gradients cause cortical and cytoplasmic flows that control proper nuclear positioning. Perturbations of PP1 activity and optogenetic manipulations of cortical actomyosin disrupt nuclear spreading, resulting in loss of cell-cycle synchrony. We conclude that mitotic synchrony is established by a self-organized mechanism that integrates the cell-cycle oscillator and embryo mechanics.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Ciclo Celular/fisiología , División del Núcleo Celular/fisiología , Proteínas de Drosophila/metabolismo , Actomiosina/metabolismo , Animales , Núcleo Celular/metabolismo , Citocinesis/fisiología , Citoplasma , Citoesqueleto/metabolismo , Drosophila melanogaster/embriología , Embrión no Mamífero/metabolismo , Desarrollo Embrionario/fisiología , Microtúbulos/metabolismo , Mitosis , Miosina Tipo II/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismoRESUMEN
ROCK-Myosin II drives fast rounded-amoeboid migration in cancer cells during metastatic dissemination. Analysis of human melanoma biopsies revealed that amoeboid melanoma cells with high Myosin II activity are predominant in the invasive fronts of primary tumors in proximity to CD206+CD163+ tumor-associated macrophages and vessels. Proteomic analysis shows that ROCK-Myosin II activity in amoeboid cancer cells controls an immunomodulatory secretome, enabling the recruitment of monocytes and their differentiation into tumor-promoting macrophages. Both amoeboid cancer cells and their associated macrophages support an abnormal vasculature, which ultimately facilitates tumor progression. Mechanistically, amoeboid cancer cells perpetuate their behavior via ROCK-Myosin II-driven IL-1α secretion and NF-κB activation. Using an array of tumor models, we show that high Myosin II activity in tumor cells reprograms the innate immune microenvironment to support tumor growth. We describe an unexpected role for Myosin II dynamics in cancer cells controlling myeloid function via secreted factors.
Asunto(s)
Movimiento Celular/fisiología , Miosina Tipo II/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular/inmunología , Proteínas del Citoesqueleto , Femenino , Humanos , Interleucina-1alfa/metabolismo , Masculino , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Persona de Mediana Edad , FN-kappa B/metabolismo , Neoplasias/inmunología , Neoplasias/metabolismo , Fosforilación , Proteómica , Receptor Cross-Talk/fisiología , Transducción de Señal , Microambiente Tumoral/inmunologíaRESUMEN
Transformation from morula to blastocyst is a defining event of preimplantation embryo development. During this transition, the embryo must establish a paracellular permeability barrier to enable expansion of the blastocyst cavity. Here, using live imaging of mouse embryos, we reveal an actin-zippering mechanism driving this embryo sealing. Preceding blastocyst stage, a cortical F-actin ring assembles at the apical pole of the embryo's outer cells. The ring structure forms when cortical actin flows encounter a network of polar microtubules that exclude F-actin. Unlike stereotypical actin rings, the actin rings of the mouse embryo are not contractile, but instead, they expand to the cell-cell junctions. Here, they couple to the junctions by recruiting and stabilizing adherens and tight junction components. Coupling of the actin rings triggers localized myosin II accumulation, and it initiates a tension-dependent zippering mechanism along the junctions that is required to seal the embryo for blastocyst formation.
Asunto(s)
Actinas/química , Blastocisto/metabolismo , Microtúbulos/metabolismo , Miosina Tipo II/química , Animales , Comunicación Celular , Proteínas del Citoesqueleto/química , Embrión de Mamíferos , Desarrollo Embrionario , Femenino , Proteínas Fluorescentes Verdes , Imagenología Tridimensional , Ratones , Ratones Endogámicos C57BL , Mórula , ARN Interferente Pequeño/metabolismo , Uniones EstrechasRESUMEN
Microtubules regulate cell polarity and migration via local activation of focal adhesion turnover, but the mechanism of this process is insufficiently understood. Molecular complexes containing KANK family proteins connect microtubules with talin, the major component of focal adhesions. Here, local optogenetic activation of KANK1-mediated microtubule/talin linkage promoted microtubule targeting to an individual focal adhesion and subsequent withdrawal, resulting in focal adhesion centripetal sliding and rapid disassembly. This sliding is preceded by a local increase of traction force due to accumulation of myosin-II and actin in the proximity of the focal adhesion. Knockdown of the Rho activator GEF-H1 prevented development of traction force and abolished sliding and disassembly of focal adhesions upon KANK1 activation. Other players participating in microtubule-driven, KANK-dependent focal adhesion disassembly include kinases ROCK, PAK, and FAK, as well as microtubules/focal adhesion-associated proteins kinesin-1, APC, and αTAT. Based on these data, we develop a mathematical model for a microtubule-driven focal adhesion disruption involving local GEF-H1/RhoA/ROCK-dependent activation of contractility, which is consistent with experimental data.
Asunto(s)
Adhesiones Focales , Cinesinas , Microtúbulos , Factores de Intercambio de Guanina Nucleótido Rho , Adhesiones Focales/metabolismo , Microtúbulos/metabolismo , Humanos , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética , Cinesinas/metabolismo , Cinesinas/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Miosina Tipo II/metabolismo , Talina/metabolismo , Talina/genética , AnimalesRESUMEN
The planar orientation of cell division (OCD) is important for epithelial morphogenesis and homeostasis. Here, we ask how mechanics and antero-posterior (AP) patterning combine to influence the first divisions after gastrulation in the Drosophila embryonic epithelium. We analyse hundreds of cell divisions and show that stress anisotropy, notably from compressive forces, can reorient division directly in metaphase. Stress anisotropy influences the OCD by imposing metaphase cell elongation, despite mitotic rounding, and overrides interphase cell elongation. In strongly elongated cells, the mitotic spindle adapts its length to, and hence its orientation is constrained by, the cell long axis. Alongside mechanical cues, we find a tissue-wide bias of the mitotic spindle orientation towards AP-patterned planar polarised Myosin-II. This spindle bias is lost in an AP-patterning mutant. Thus, a patterning-induced mitotic spindle orientation bias overrides mechanical cues in mildly elongated cells, whereas in strongly elongated cells the spindle is constrained close to the high stress axis.
Asunto(s)
División Celular , Polaridad Celular , Drosophila melanogaster , Células Epiteliales , Metafase , Huso Acromático , Estrés Mecánico , Animales , Metafase/fisiología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Huso Acromático/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/citología , Polaridad Celular/fisiología , Tipificación del Cuerpo , Miosina Tipo II/metabolismo , Embrión no Mamífero/citología , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Gastrulación/fisiologíaRESUMEN
As tissues grow and change shape during animal development, they physically pull and push on each other, and these mechanical interactions can be important for morphogenesis. During Drosophila gastrulation, mesoderm invagination temporally overlaps with the convergence and extension of the ectodermal germband; the latter is caused primarily by Myosin II-driven polarised cell intercalation. Here, we investigate the impact of mesoderm invagination on ectoderm extension, examining possible mechanical and mechanotransductive effects on Myosin II recruitment and polarised cell intercalation. We find that the germband ectoderm is deformed by the mesoderm pulling in the orthogonal direction to germband extension (GBE), showing mechanical coupling between these tissues. However, we do not find a significant change in Myosin II planar polarisation in response to mesoderm invagination, nor in the rate of junction shrinkage leading to neighbour exchange events. We conclude that the main cellular mechanism of axis extension, polarised cell intercalation, is robust to the mesoderm invagination pull. We find, however, that mesoderm invagination slows down the rate of anterior-posterior cell elongation that contributes to axis extension, counteracting the tension from the endoderm invagination, which pulls along the direction of GBE.
Asunto(s)
Drosophila melanogaster , Ectodermo , Gastrulación , Mesodermo , Miosina Tipo II , Animales , Mesodermo/embriología , Mesodermo/citología , Gastrulación/fisiología , Ectodermo/citología , Ectodermo/embriología , Ectodermo/metabolismo , Miosina Tipo II/metabolismo , Drosophila melanogaster/embriología , Polaridad Celular , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Embrión no Mamífero , Morfogénesis , Tipificación del Cuerpo/fisiología , Drosophila/embriologíaRESUMEN
The development of ectodermal organs begins with the formation of a stratified epithelial placode that progressively invaginates into the underlying mesenchyme as the organ takes its shape. Signaling by secreted molecules is critical for epithelial morphogenesis, but how that information leads to cell rearrangement and tissue shape changes remains an open question. Using the mouse dentition as a model, we first establish that non-muscle myosin II is essential for dental epithelial invagination and show that it functions by promoting cell-cell adhesion and persistent convergent cell movements in the suprabasal layer. Shh signaling controls these processes by inducing myosin II activation via AKT. Pharmacological induction of AKT and myosin II can also rescue defects caused by the inhibition of Shh. Together, our results support a model in which the Shh signal is transmitted through myosin II to power effective cellular rearrangement for proper dental epithelial invagination.
Asunto(s)
Adhesión Celular , Movimiento Celular , Proteínas Hedgehog , Miosina Tipo II , Transducción de Señal , Animales , Ratones , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Adhesión Celular/genética , Miosina Tipo II/metabolismo , Miosina Tipo II/genética , Movimiento Celular/genética , Epitelio/metabolismo , Morfogénesis/genética , Diente/metabolismo , Diente/crecimiento & desarrollo , Células Epiteliales/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Regulación del Desarrollo de la Expresión GénicaRESUMEN
S100A11 is a small Ca2+-activatable protein known to localize along stress fibers (SFs). Analyzing S100A11 localization in HeLa and U2OS cells further revealed S100A11 enrichment at focal adhesions (FAs). Strikingly, S100A11 levels at FAs increased sharply, yet transiently, just before FA disassembly. Elevating intracellular Ca2+ levels with ionomycin stimulated both S100A11 recruitment and subsequent FA disassembly. However, pre-incubation with the non-muscle myosin II (NMII) inhibitor blebbistatin or with an inhibitor of the stretch-activatable Ca2+ channel Piezo1 suppressed S100A11 recruitment, implicating S100A11 in an actomyosin-driven FA recruitment mechanism involving Piezo1-dependent Ca2+ influx. Applying external forces on peripheral FAs likewise recruited S100A11 to FAs even if NMII activity was inhibited, corroborating the mechanosensitive recruitment mechanism of S100A11. However, extracellular Ca2+ and Piezo1 function were indispensable, indicating that NMII contraction forces act upstream of Piezo1-mediated Ca2+ influx, in turn leading to S100A11 activation and FA recruitment. S100A11-knockout cells display enlarged FAs and had delayed FA disassembly during cell membrane retraction, consistent with impaired FA turnover in these cells. Our results thus demonstrate a novel function for S100A11 in promoting actomyosin contractility-driven FA disassembly.
Asunto(s)
Actomiosina , Adhesiones Focales , Humanos , Adhesiones Focales/metabolismo , Actomiosina/metabolismo , Calcio/metabolismo , Proteínas del Citoesqueleto/metabolismo , Miosina Tipo II/metabolismo , Proteínas S100/genética , Proteínas S100/metabolismoRESUMEN
The Rho family of GTPases plays a crucial role in cellular mechanics by regulating actomyosin contractility through the parallel induction of actin and myosin assembly and function. Using exocytosis of large vesicles in the Drosophila larval salivary gland as a model, we followed the spatiotemporal regulation of Rho1, which in turn creates distinct organization patterns of actin and myosin. After vesicle fusion, low levels of activated Rho1 reach the vesicle membrane and drive actin nucleation in an uneven, spread-out pattern. Subsequently, the Rho1 activator RhoGEF2 distributes as an irregular meshwork on the vesicle membrane, activating Rho1 in a corresponding punctate pattern and driving local myosin II recruitment, resulting in vesicle constriction. Vesicle membrane buckling and subsequent crumpling occur at local sites of high myosin II concentrations. These findings indicate that distinct thresholds for activated Rho1 create a biphasic mode of actomyosin assembly, inducing anisotropic membrane crumpling during exocrine secretion.
Asunto(s)
Proteínas de Drosophila , Exocitosis , Miosina Tipo II , Proteínas de Unión al GTP rho , Animales , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Miosina Tipo II/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Proteínas de Unión al GTP rho/genética , Exocitosis/fisiología , Drosophila melanogaster/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Larva/metabolismo , Glándulas Salivales/metabolismo , Glándulas Salivales/citología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Vesículas Secretoras/metabolismoRESUMEN
Myosin II is the motor protein that enables muscle cells to contract and nonmuscle cells to move and change shape1. The molecule has two identical heads attached to an elongated tail, and can exist in two conformations: 10S and 6S, named for their sedimentation coefficients2,3. The 6S conformation has an extended tail and assembles into polymeric filaments, which pull on actin filaments to generate force and motion. In 10S myosin, the tail is folded into three segments and the heads bend back and interact with each other and the tail3-7, creating a compact conformation in which ATPase activity, actin activation and filament assembly are all highly inhibited7,8. This switched-off structure appears to function as a key energy-conserving storage molecule in muscle and nonmuscle cells9-12, which can be activated to form functional filaments as needed13-but the mechanism of its inhibition is not understood. Here we have solved the structure of smooth muscle 10S myosin by cryo-electron microscopy with sufficient resolution to enable improved understanding of the function of the head and tail regions of the molecule and of the key intramolecular contacts that cause inhibition. Our results suggest an atomic model for the off state of myosin II, for its activation and unfolding by phosphorylation, and for understanding the clustering of disease-causing mutations near sites of intramolecular interaction.
Asunto(s)
Microscopía por Crioelectrón , Miosina Tipo II/antagonistas & inhibidores , Miosina Tipo II/ultraestructura , Animales , Sitios de Unión , Modelos Moleculares , Músculo Liso/química , Mutación , Miosina Tipo II/química , Miosina Tipo II/genética , Fosforilación , Unión Proteica , Conformación Proteica , Desplegamiento Proteico , PavosRESUMEN
Myosin-2 is essential for processes as diverse as cell division and muscle contraction. Dephosphorylation of its regulatory light chain promotes an inactive, 'shutdown' state with the filament-forming tail folded onto the two heads1, which prevents filament formation and inactivates the motors2. The mechanism by which this happens is unclear. Here we report a cryo-electron microscopy structure of shutdown smooth muscle myosin with a resolution of 6 Å in the head region. A pseudo-atomic model, obtained by flexible fitting of crystal structures into the density and molecular dynamics simulations, describes interaction interfaces at the atomic level. The N-terminal extension of one regulatory light chain interacts with the tail, and the other with the partner head, revealing how the regulatory light chains stabilize the shutdown state in different ways and how their phosphorylation would allow myosin activation. Additional interactions between the three segments of the coiled coil, the motor domains and the light chains stabilize the shutdown molecule. The structure of the lever in each head is competent to generate force upon activation. This shutdown structure is relevant to all isoforms of myosin-2 and provides a framework for understanding their disease-causing mutations.
Asunto(s)
Microscopía por Crioelectrón , Miosina Tipo II/química , Miosina Tipo II/ultraestructura , Animales , Activación Enzimática , Estabilidad de Enzimas , Modelos Moleculares , Músculo Liso/química , Cadenas Ligeras de Miosina/química , Cadenas Ligeras de Miosina/metabolismo , Cadenas Ligeras de Miosina/ultraestructura , Miosina Tipo II/metabolismo , Fosforilación , Dominios Proteicos , PavosRESUMEN
In most mollusks (conchiferans), the early tissue responsible for shell development, namely, the shell field, shows a common process of invagination during morphogenesis. Moreover, lines of evidence indicated that shell field invagination is not an independent event, but an integrated output reflecting the overall state of shell field morphogenesis. Nevertheless, the underlying mechanisms of this conserved process remain largely unknown. We previously found that actomyosin networks (regularly organized filamentous actin (F-actin) and myosin) may play essential roles in this process by revealing the evident aggregation of F-actin in the invaginated region and demonstrating that nonmuscle myosin II (NM II) is required for invagination in the gastropod Lottia peitaihoensis (= Lottia goshimai). Here, we investigated the roles of the Rho family of small GTPases (RhoA, Rac1, and Cdc42) to explore the upstream regulators of actomyosin networks. Functional assays using small molecule inhibitors suggested that Cdc42 modulates key events of shell field morphogenesis, including invagination and cell rearrangements, while the roles of RhoA and Rac1 may be nonspecific or negligible. Further investigations revealed that the Cdc42 protein was concentrated on the apical side of shell field cells and colocalized with F-actin aggregation. The aggregation of these two molecules could be prevented by treatment with Cdc42 inhibitors. These findings suggest a possible regulatory cascade of shell field morphogenesis in which Cdc42 recruits F-actin (actomyosin networks) on the apical side of shell field cells, which then generates resultant mechanical forces that mediate correct shell field morphogenesis (cell shape changes, invagination and cell rearrangement). Our results emphasize the roles of the cytoskeleton in early shell development and provide new insights into molluscan shell evolution.
Asunto(s)
Actinas , Actomiosina , Exoesqueleto , Gastrópodos , Morfogénesis , Proteína de Unión al GTP cdc42 , Animales , Gastrópodos/embriología , Gastrópodos/metabolismo , Exoesqueleto/metabolismo , Exoesqueleto/crecimiento & desarrollo , Exoesqueleto/embriología , Actinas/metabolismo , Actomiosina/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Miosina Tipo II/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Non-muscle myosin 2 (NM2) is known to play an important role in myofibroblast transdifferentiation, a hallmark of fibrotic disorders. In a recent JBC article, Southern et al. demonstrate that endogenous S100A4, a calcium- and NM2-binding protein acts as a mechanoeffector in this process. Since extracellular S100A4 is also involved in fibrogenesis by triggering the inflammatory response, this small protein appears to contribute to fibrosis via at least two distinct mechanisms.
Asunto(s)
Fibrosis , Proteína de Unión al Calcio S100A4 , Proteínas S100 , Humanos , Proteína de Unión al Calcio S100A4/metabolismo , Proteína de Unión al Calcio S100A4/genética , Fibrosis/metabolismo , Animales , Proteínas S100/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/patología , Transdiferenciación Celular , Ratones , Miosina Tipo II/metabolismoRESUMEN
The regulation of mechanical tension exerted at cell junctions guides cell behavior during tissue formation and homeostasis. Cell junctions along compartment boundaries, which are lineage restrictions separating cells with different fates and functions within tissues, are characterized by increased mechanical tension compared to that of cell junctions in the bulk of the tissue. Mechanical tension depends on the actomyosin cytoskeleton; however, the mechanisms by which mechanical tension is locally increased at cell junctions along compartment boundaries remain elusive. Here, we show that non-muscle Myosin II and F-actin transiently accumulate and mechanical tension is increased at cell junctions along the forming anteroposterior compartment boundary in the Drosophila melanogaster pupal abdominal epidermis. Fluorescence recovery after photobleaching experiments showed that Myosin II accumulation correlated with its increased stabilization at these junctions. Moreover, photoconversion experiments indicated that Myosin II is preferentially recruited within cells to junctions along the compartment boundary. Our results indicate that the preferential recruitment and stabilization of Myosin II contribute to the initial build-up of mechanical tension at compartment boundaries.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Drosophila melanogaster , Estrés Mecánico , Miosina Tipo II , ActomiosinaRESUMEN
Spatial organization within an organ is essential and needs to be maintained during development. This is largely implemented via compartment boundaries that serve as barriers between distinct cell types. Biased accumulation of junctional non-muscle Myosin II along the interface between differently fated groups of cells contributes to boundary integrity and maintains its shape via increased tension. Here, using the Drosophila wing imaginal disc, we tested whether interfacial tension driven by accumulation of Myosin is responsible for the elimination of aberrantly specified cells that would otherwise compromise compartment organization. To this end, we genetically reduced Myosin II levels in three different patterns: in both wild-type and misspecified cells, only in misspecified cells, and specifically at the interface between wild-type and aberrantly specified cells. We found that the recognition and elimination of aberrantly specified cells do not strictly rely on tensile forces driven by interfacial Myosin cables. Moreover, apical constriction of misspecified cells and their separation from wild-type neighbours occurred even when Myosin levels were greatly reduced. Thus, we conclude that the forces that drive elimination of aberrantly specified cells are largely independent of Myosin II accumulation.
Asunto(s)
Miosina Tipo II , Animales , Células Clonales , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Morfogénesis , Miosina Tipo II/metabolismo , Discos Imaginales/metabolismoRESUMEN
Cyclic guanosine 3',5'-monophosphate (cGMP) is a ubiquitous important second messenger involved in various physiological functions. Here, intracellular cGMP (cGMPi) was visualized in chemotactic Dictyostelium cells using the fluorescent probe, D-Green cGull. When wild-type cells were stimulated with a chemoattractant, fluorescence transiently increased, but guanylate cyclase-null cells did not show a change in fluorescence, suggesting that D-Green cGull is a reliable indicator of cGMPi. In the aggregation stage, the responses of cGMPi propagated in a wave-like fashion from the aggregation center. The oscillation of the cGMPi wave was synchronized almost in phase with those of other second messengers, such as the intracellular cAMP and Ca2+. The phases of these waves preceded those of the oscillations of actomyosin and cell velocity, suggesting that these second messengers are upstream of the actomyosin and chemotactic migration. An acute increase in cGMPi concentration released from membrane-permeable caged cGMP induced a transient shuttle of myosin II between the cytosol and cell cortex, suggesting a direct link between cGMP signaling and myosin II dynamics.
Asunto(s)
Dictyostelium , Dictyostelium/fisiología , Quimiotaxis/fisiología , Actomiosina , GMP Cíclico/farmacología , GMP Cíclico/fisiología , Miosina Tipo IIRESUMEN
In common with other actomyosin contractile cellular machineries, actin turnover is required for normal function of the cytokinetic contractile ring. Cofilin is an actin-binding protein contributing to turnover by severing actin filaments, required for cytokinesis by many organisms. In fission yeast cofilin mutants, contractile rings suffer bridging instabilities in which segments of the ring peel away from the plasma membrane, forming straight bridges whose ends remain attached to the membrane. The origin of bridging instability is unclear. Here, we used molecularly explicit simulations of contractile rings to examine the role of cofilin. Simulations reproduced the experimentally observed cycles of bridging and reassembly during constriction, and the occurrence of bridging in ring segments with low density of the myosin II protein Myo2. The lack of cofilin severing produced â¼2-fold longer filaments and, consequently, â¼2-fold higher ring tensions. Simulations identified bridging as originating in the boosted ring tension, which increased centripetal forces that detached actin from Myo2, which was anchoring actin to the membrane. Thus, cofilin serves a critical role in cytokinesis by providing protection from bridging, the principal structural threat to contractile rings.
Asunto(s)
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Citoesqueleto de Actina/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Citocinesis , Proteínas de Microfilamentos/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
Apical constriction, or a reduction in size of the apical domain, underlies many morphogenetic events during development. Actomyosin complexes play an essential role in apical constriction; however, the detailed analysis of molecular mechanisms is still pending. Here, we show that Lim domain only protein 7 (Lmo7), a multidomain adaptor at apical junctions, promotes apical constriction in the Xenopus superficial ectoderm, whereas apical domain size increases in Lmo7-depleted cells. Lmo7 is primarily localized at apical junctions and promotes the formation of the dense circumferential actomyosin belt. Strikingly, Lmo7 binds non-muscle myosin II (NMII) and recruits it to apical junctions and the apical cortex. This NMII recruitment is essential for Lmo7-mediated apical constriction. Lmo7 knockdown decreases NMIIA localization at apical junctions and delays neural tube closure in Xenopus embryos. Our findings suggest that Lmo7 serves as a scaffold that regulates actomyosin contractility and apical domain size.