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1.
Int J Mol Sci ; 22(19)2021 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-34638710

RESUMO

Organization of intracellular content is affected by multiple simultaneous processes, including diffusion in a viscoelastic and structured environment, intracellular mechanical work and vibrations. The combined effects of these processes on intracellular organization are complex and remain poorly understood. Here, we studied the organization and dynamics of a free Ca++ probe as a small and mobile tracer in live T cells. Ca++, highlighted by Fluo-4, is localized in intracellular organelles. Inhibiting intracellular mechanical work by myosin II through blebbistatin treatment increased cellular dis-homogeneity of Ca++-rich features in length scale < 1.1 µm. We detected a similar effect in cells imaged by label-free bright-field (BF) microscopy, in mitochondria-highlighted cells and in ATP-depleted cells. Blebbistatin treatment also reduced the dynamics of the Ca++-rich features and generated prominent negative temporal correlations in their signals. Following Guggenberger et al. and numerical simulations, we suggest that diffusion in the viscoelastic and confined medium of intracellular organelles may promote spatial dis-homogeneity and stability of their content. This may be revealed only after inhibiting intracellular mechanical work and related cell vibrations. Our described mechanisms may allow the cell to control its organization via balancing its viscoelasticity and mechanical activity, with implications to cell physiology in health and disease.


Assuntos
Trifosfato de Adenosina/metabolismo , Miosina Tipo II/metabolismo , Organelas/metabolismo , Compostos de Anilina/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Células Jurkat , Xantenos/metabolismo
2.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360739

RESUMO

Changes in plasma membrane curvature and intracellular ionic strength are two key features of cell volume perturbations. In this hypothesis we present a model of the responsible molecular apparatus which is assembled of two molecular motors [non-muscle myosin II (NMMII) and protrusive actin polymerization], a spring [a complex between the plasma membrane (PM) and the submembrane actin-based cytoskeleton (smACSK) which behaves like a viscoelastic solid] and the associated signaling proteins. We hypothesize that this apparatus senses changes in both the plasma membrane curvature and the ionic strength and in turn activates signaling pathways responsible for regulatory volume increase (RVI) and regulatory volume decrease (RVD). During cell volume changes hydrostatic pressure (HP) changes drive alterations in the cell membrane curvature. HP difference has opposite directions in swelling versus shrinkage, thus allowing distinction between them. By analogy with actomyosin contractility that appears to sense stiffness of the extracellular matrix we propose that NMMII and actin polymerization can actively probe the transmembrane gradient in HP. Furthermore, NMMII and protein-protein interactions in the actin cortex are sensitive to ionic strength. Emerging data on direct binding to and regulating activities of transmembrane mechanosensors by NMMII and actin cortex provide routes for signal transduction from transmembrane mechanosensors to cell volume regulatory mechanisms.


Assuntos
Citoesqueleto de Actina/metabolismo , Membrana Celular/metabolismo , Tamanho Celular , Miosina Tipo II/metabolismo , Transdução de Sinais , Actomiosina/metabolismo , Animais , Humanos , Pressão Hidrostática
3.
Elife ; 102021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34374341

RESUMO

Nonmuscle myosin II (NM II) is an integral part of essential cellular processes, including adhesion and migration. Mammalian cells express up to three isoforms termed NM IIA, B, and C. We used U2OS cells to create CRISPR/Cas9-based knockouts of all three isoforms and analyzed the phenotypes on homogenously coated surfaces, in collagen gels, and on micropatterned substrates. In contrast to homogenously coated surfaces, a structured environment supports a cellular phenotype with invaginated actin arcs even in the absence of NM IIA-induced contractility. A quantitative shape analysis of cells on micropatterns combined with a scale-bridging mathematical model reveals that NM IIA is essential to build up cellular tension during initial stages of force generation, while NM IIB is necessary to elastically stabilize NM IIA-generated tension. A dynamic cell stretch/release experiment in a three-dimensional scaffold confirms these conclusions and in addition reveals a novel role for NM IIC, namely the ability to establish tensional homeostasis.


Assuntos
Elasticidade , Miosina Tipo II/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Miosina não Muscular Tipo IIB/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Homeostase , Humanos , Modelos Teóricos , Miosina Tipo II/classificação , Miosina Tipo II/genética , Miosina não Muscular Tipo IIA/genética , Miosina não Muscular Tipo IIB/genética , Isoformas de Proteínas
4.
Development ; 148(18)2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34409448

RESUMO

Light-sheet or selective plane illumination microscopy (SPIM) is ideally suited for in toto imaging of living specimens at high temporal-spatial resolution. In SPIM, the light scattering that occurs during imaging of opaque specimens brings about limitations in terms of resolution and the imaging field of view. To ameliorate this shortcoming, the illumination beam can be engineered into a highly confined light sheet over a large field of view and multi-view imaging can be performed by applying multiple lenses combined with mechanical rotation of the sample. Here, we present a Multiview tiling SPIM (MT-SPIM) that combines the Multi-view SPIM (M-SPIM) with a confined, multi-tiled light sheet. The MT-SPIM provides high-resolution, robust and rotation-free imaging of living specimens. We applied the MT-SPIM to image nuclei and Myosin II from the cellular to subcellular spatial scale in early Drosophila embryogenesis. We show that the MT-SPIM improves the axial-resolution relative to the conventional M-SPIM by a factor of two. We further demonstrate that this axial resolution enhancement improves the automated segmentation of Myosin II distribution and of nuclear volumes and shapes.


Assuntos
Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Animais , Drosophila/metabolismo , Drosophila/fisiologia , Embrião não Mamífero/metabolismo , Embrião não Mamífero/fisiologia , Desenvolvimento Embrionário/fisiologia , Miosina Tipo II/metabolismo
5.
Cells ; 10(8)2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34440749

RESUMO

The cellular mechanisms of basement membrane (BM) invasion remain poorly understood. We investigated the invasion-promoting mechanisms of actin cytoskeleton reorganization in BM-covered MCF10A breast acini. High-resolution confocal microscopy has characterized actin cell protrusion formation and function in response to tumor-resembling ECM stiffness and soluble EGF stimulation. Traction force microscopy quantified the mechanical BM stresses that invasion-triggered acini exerted on the BM-ECM interface. We demonstrate that acini use non-proteolytic actin microspikes as functional precursors of elongated protrusions to initiate BM penetration and ECM probing. Further, these microspikes mechanically widened the collagen IV pores to anchor within the BM scaffold via force-transmitting focal adhesions. Pre-invasive basal cells located at the BM-ECM interface exhibited predominantly cortical actin networks and actin microspikes. In response to pro-invasive conditions, these microspikes accumulated and converted subsequently into highly contractile stress fibers. The phenotypical switch to stress fiber cells matched spatiotemporally with emerging high BM stresses that were driven by actomyosin II contractility. The activation of proteolytic invadopodia with MT1-MMP occurred at later BM invasion stages and only in cells already disseminating into the ECM. Our study demonstrates that BM pore-widening filopodia bridge mechanical ECM probing function and contractility-driven BM weakening. Finally, these EMT-related cytoskeletal adaptations are critical mechanisms inducing the invasive transition of benign breast acini.


Assuntos
Actinas/metabolismo , Membrana Basal/metabolismo , Miosina Tipo II/metabolismo , Fibras de Estresse/metabolismo , Células Acinares/citologia , Células Acinares/metabolismo , Mama/citologia , Mama/metabolismo , Adesão Celular , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Matriz Extracelular/metabolismo , Feminino , Humanos , Microscopia Confocal , Podossomos/metabolismo , Pseudópodes/metabolismo , Fibras de Estresse/química
6.
Int J Mol Sci ; 22(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206505

RESUMO

Myosins are a remarkable superfamily of actin-based motor proteins that use the energy derived from ATP hydrolysis to translocate actin filaments and to produce force. Myosins are abundant in different types of tissues and involved in a large variety of cellular functions. Several classes of the myosin superfamily are expressed in the nervous system; among them, non-muscle myosin II (NM II) is expressed in both neurons and non-neuronal brain cells, such as astrocytes, oligodendrocytes, endothelial cells, and microglia. In the nervous system, NM II modulates a variety of functions, such as vesicle transport, phagocytosis, cell migration, cell adhesion and morphology, secretion, transcription, and cytokinesis, as well as playing key roles during brain development, inflammation, repair, and myelination functions. In this review, we will provide a brief overview of recent emerging roles of NM II in resting and activated microglia cells, the principal regulators of immune processes in the central nervous system (CNS) in both physiological and pathological conditions. When stimulated, microglial cells react and produce a number of mediators, such as pro-inflammatory cytokines, free radicals, and nitric oxide, that enhance inflammation and contribute to neurodegenerative diseases. Inhibition of NM II could be a new therapeutic target to treat or to prevent CNS diseases.


Assuntos
Microglia/metabolismo , Miosina Tipo II/metabolismo , Animais , Biomarcadores , Movimento Celular/imunologia , Citoesqueleto/metabolismo , Humanos , Microglia/imunologia , Fagocitose/imunologia
7.
Elife ; 102021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34269679

RESUMO

Actin filaments are central to numerous biological processes in all domains of life. Driven by the interplay with molecular motors, actin binding and actin modulating proteins, the actin cytoskeleton exhibits a variety of geometries. This includes structures with a curved geometry such as axon-stabilizing actin rings, actin cages around mitochondria and the cytokinetic actomyosin ring, which are generally assumed to be formed by short linear filaments held together by actin cross-linkers. However, whether individual actin filaments in these structures could be curved and how they may assume a curved geometry remains unknown. Here, we show that 'curly', a region from the IQGAP family of proteins from three different organisms, comprising the actin-binding calponin-homology domain and a C-terminal unstructured domain, stabilizes individual actin filaments in a curved geometry when anchored to lipid membranes. Although F-actin is semi-flexible with a persistence length of ~10 µm, binding of mobile curly within lipid membranes generates actin filament arcs and full rings of high curvature with radii below 1 µm. Higher rates of fully formed actin rings are observed in the presence of the actin-binding coiled-coil protein tropomyosin and when actin is directly polymerized on lipid membranes decorated with curly. Strikingly, curly induced actin filament rings contract upon the addition of muscle myosin II filaments and expression of curly in mammalian cells leads to highly curved actin structures in the cytoskeleton. Taken together, our work identifies a new mechanism to generate highly curved actin filaments, which opens a range of possibilities to control actin filament geometries, that can be used, for example, in designing synthetic cytoskeletal structures.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas dos Microfilamentos/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Animais , Citoesqueleto/metabolismo , Células HEK293 , Humanos , Microtúbulos/metabolismo , Músculos/metabolismo , Miosina Tipo II/metabolismo , Ligação Proteica , Tropomiosina/metabolismo
8.
Sci Rep ; 11(1): 14803, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285275

RESUMO

Many types of adherent cells are known to reorient upon uniaxial cyclic stretching perpendicularly to the direction of stretching to facilitate such important events as wound healing, angiogenesis, and morphogenesis. While this phenomenon has been documented for decades, the underlying mechanism remains poorly understood. Using an on-stage stretching device that allowed programmable stretching with synchronized imaging, we found that the reorientation of NRK epithelial cells took place primarily during the relaxation phase when cells underwent rapid global retraction followed by extension transverse to the direction of stretching. Inhibition of myosin II caused cells to orient along the direction of stretching, whereas disassembly of microtubules enhanced transverse reorientation. Our results indicate distinct roles of stretching and relaxation in cell reorientation and implicate a role of myosin II-dependent contraction via a microtubule-modulated mechanism. The importance of relaxation phase also explains the difference between the responses to cyclic and static stretching.


Assuntos
Rim/citologia , Microtúbulos/metabolismo , Miosina Tipo II/metabolismo , Animais , Adesão Celular , Linhagem Celular , Movimento Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Rim/metabolismo , Fenômenos Mecânicos , Microscopia de Força Atômica , Ratos , Estresse Mecânico
9.
Nat Commun ; 12(1): 3333, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099704

RESUMO

Lysosomes are involved in nutrient sensing via the mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 is tethered to lysosomes by the Ragulator complex, a heteropentamer in which Lamtor1 wraps around Lamtor2-5. Although the Ragulator complex is required for cell migration, the mechanisms by which it participates in cell motility remain unknown. Here, we show that lysosomes move to the uropod in motile cells, providing the platform where Lamtor1 interacts with the myosin phosphatase Rho-interacting protein (MPRIP) independently of mTORC1 and interferes with the interaction between MPRIP and MYPT1, a subunit of myosin light chain phosphatase (MLCP), thereby increasing myosin II-mediated actomyosin contraction. Additionally, formation of the complete Ragulator complex is required for leukocyte migration and pathophysiological immune responses. Together, our findings demonstrate that the lysosomal Ragulator complex plays an essential role in leukocyte migration by activating myosin II through interacting with MPRIP.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Leucócitos/metabolismo , Lisossomos/metabolismo , Miosina Tipo II/metabolismo , Actomiosina/efeitos dos fármacos , Animais , Linhagem Celular , Células Dendríticas , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosfatase de Miosina-de-Cadeia-Leve , Neutrófilos , Transdução de Sinais
10.
Development ; 148(11)2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34124762

RESUMO

During development, gene expression regulates cell mechanics and shape to sculpt tissues. Epithelial folding proceeds through distinct cell shape changes that occur simultaneously in different regions of a tissue. Here, using quantitative imaging in Drosophila melanogaster, we investigate how patterned cell shape changes promote tissue bending during early embryogenesis. We find that the transcription factors Twist and Snail combinatorially regulate a multicellular pattern of lateral F-actin density that differs from the previously described Myosin-2 gradient. This F-actin pattern correlates with whether cells apically constrict, stretch or maintain their shape. We show that the Myosin-2 gradient and F-actin depletion do not depend on force transmission, suggesting that transcriptional activity is required to create these patterns. The Myosin-2 gradient width results from a gradient in RhoA activation that is refined through the balance between RhoGEF2 and the RhoGAP C-GAP. Our experimental results and simulations of a 3D elastic shell model show that tuning gradient width regulates tissue curvature.


Assuntos
Actinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Actomiosina , Animais , Proteínas de Ciclo Celular , Forma Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Ativadoras de GTPase/metabolismo , Morfogênese/fisiologia , Miosina Tipo II/metabolismo , Proteínas rho de Ligação ao GTP/genética
11.
Nat Cell Biol ; 23(7): 733-744, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34155381

RESUMO

Intestinal organoids derived from single cells undergo complex crypt-villus patterning and morphogenesis. However, the nature and coordination of the underlying forces remains poorly characterized. Here, using light-sheet microscopy and large-scale imaging quantification, we demonstrate that crypt formation coincides with a stark reduction in lumen volume. We develop a 3D biophysical model to computationally screen different mechanical scenarios of crypt morphogenesis. Combining this with live-imaging data and multiple mechanical perturbations, we show that actomyosin-driven crypt apical contraction and villus basal tension work synergistically with lumen volume reduction to drive crypt morphogenesis, and demonstrate the existence of a critical point in differential tensions above which crypt morphology becomes robust to volume changes. Finally, we identified a sodium/glucose cotransporter that is specific to differentiated enterocytes that modulates lumen volume reduction through cell swelling in the villus region. Together, our study uncovers the cellular basis of how cell fate modulates osmotic and actomyosin forces to coordinate robust morphogenesis.


Assuntos
Diferenciação Celular , Linhagem da Célula , Mucosa Intestinal/fisiologia , Mecanotransdução Celular , Osmorregulação , Celulas de Paneth/fisiologia , Células-Tronco/fisiologia , Animais , Movimento Celular , Células Cultivadas , Simulação por Computador , Feminino , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Microscopia de Vídeo , Modelos Biológicos , Morfogênese , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Organoides , Pressão Osmótica , Celulas de Paneth/metabolismo , Proteínas de Transporte de Sódio-Glucose/genética , Proteínas de Transporte de Sódio-Glucose/metabolismo , Células-Tronco/metabolismo , Estresse Mecânico , Fatores de Tempo
12.
Cells ; 10(5)2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067877

RESUMO

Wound repair of cell membranes is essential for cell survival. Myosin II contributes to wound pore closure by interacting with actin filaments in larger cells; however, its role in smaller cells is unclear. In this study, we observed wound repair in dividing cells for the first time. The cell membrane in the cleavage furrow, where myosin II localized, was wounded by laserporation. Upon wounding, actin transiently accumulated, and myosin II transiently disappeared from the wound site. Ca2+ influx from the external medium triggered both actin and myosin II dynamics. Inhibition of calmodulin reduced both actin and myosin II dynamics. The wound closure time in myosin II-null cells was the same as that in wild-type cells, suggesting that myosin II is not essential for wound repair. We also found that disassembly of myosin II filaments by phosphorylation did not contribute to their disappearance, indicating a novel mechanism for myosin II delocalization from the cortex. Furthermore, we observed that several furrow-localizing proteins such as GAPA, PakA, myosin heavy chain kinase C, PTEN, and dynamin disappeared upon wounding. Herein, we discuss the possible mechanisms of myosin dynamics during wound repair.


Assuntos
Divisão Celular , Dictyostelium/metabolismo , Miosina Tipo II/metabolismo , Proteínas de Protozoários/metabolismo , Cicatrização , Cálcio/metabolismo , Sinalização do Cálcio , Dictyostelium/genética , Dictyostelium/crescimento & desenvolvimento , Cinética , Microscopia de Fluorescência , Microscopia de Vídeo , Mutação , Miosina Tipo II/genética , Proteínas de Protozoários/genética , Imagem com Lapso de Tempo
13.
Development ; 148(18)2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34097729

RESUMO

Proper organ development often requires nuclei to move to a specific position within the cell. To determine how nuclear positioning affects left-right (LR) development in the Drosophila anterior midgut (AMG), we developed a surface-modeling method to measure and describe nuclear behavior at stages 13-14, captured in three-dimensional time-lapse movies. We describe the distinctive positioning and a novel collective nuclear behavior by which nuclei align LR symmetrically along the anterior-posterior axis in the visceral muscles that overlie the midgut and are responsible for the LR-asymmetric development of this organ. Wnt4 signaling is crucial for the collective behavior and proper positioning of the nuclei, as are myosin II and the LINC complex, without which the nuclei fail to align LR symmetrically. The LR-symmetric positioning of the nuclei is important for the subsequent LR-asymmetric development of the AMG. We propose that the bilaterally symmetrical positioning of these nuclei may be mechanically coupled with subsequent LR-asymmetric morphogenesis.


Assuntos
Padronização Corporal/fisiologia , Núcleo Celular/fisiologia , Sistema Digestório/fisiopatologia , Drosophila/fisiologia , Morfogênese/fisiologia , Animais , Núcleo Celular/metabolismo , Sistema Digestório/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Músculos/metabolismo , Músculos/fisiologia , Miosina Tipo II/metabolismo , Transdução de Sinais/fisiologia
14.
Development ; 148(10)2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33999996

RESUMO

Movement of epithelial cells in a tissue occurs through neighbor exchange and drives tissue shape changes. It requires intercellular junction remodeling, a process typically powered by the contractile actomyosin cytoskeleton. This has been investigated mainly in homogeneous epithelia, where intercalation takes minutes. However, in some tissues, intercalation involves different cell types and can take hours. Whether slow and fast intercalation share the same mechanisms remains to be examined. To address this issue, we used the fly eye, where the cone cells exchange neighbors over ∼10 h to shape the lens. We uncovered three pathways regulating this slow mode of cell intercalation. First, we found a limited requirement for MyosinII. In this case, mathematical modeling predicts an adhesion-dominant intercalation mechanism. Genetic experiments support this prediction, revealing a role for adhesion through the Nephrin proteins Roughest and Hibris. Second, we found that cone cell intercalation is regulated by the Notch pathway. Third, we show that endocytosis is required for membrane removal and Notch activation. Taken together, our work indicates that adhesion, endocytosis and Notch can direct slow cell intercalation during tissue morphogenesis.


Assuntos
Adesão Celular/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Endocitose/fisiologia , Receptores Notch/metabolismo , Retina/embriologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Actomiosina/metabolismo , Junções Aderentes/fisiologia , Animais , Padronização Corporal/fisiologia , Moléculas de Adesão Celular Neuronais/metabolismo , Comunicação Celular , Proteínas de Drosophila/genética , Células Epiteliais/citologia , Proteínas do Olho/metabolismo , Adesões Focais/fisiologia , Proteínas de Membrana/metabolismo , Miosina Tipo II/metabolismo , Receptores Notch/genética , Transdução de Sinais/fisiologia
15.
PLoS One ; 16(5): e0248197, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34038442

RESUMO

Canonical Wnt/ß-catenin (cWnt) signaling is a crucial regulator of development and Dishevelled (Dsh/Dvl) functions as an integral part of this pathway by linking Wnt binding to the Frizzled:LRP5/6 receptor complex with ß-catenin-stimulated gene expression. In many cell types Dsh has been localized to ill-defined cytoplasmic puncta, however in sea urchin eggs and embryos confocal fluorescence microscopy has shown that Dsh is localized to puncta present in a novel and development-essential vegetal cortex domain (VCD). In the present study, we used super-resolution light microscopy and platinum replica transmission electron microscopy (TEM) to provide the first views of the ultrastructural organization of Dsh within the sea urchin VCD. 3D structured illumination microscopy (SIM) imaging of isolated egg cortices demonstrated the graded distribution of Dsh in the VCD, whereas higher resolution stimulated emission depletion (STED) imaging revealed that some individual Dsh puncta consisted of more than one fluorescent source. Platinum replica immuno-TEM localization showed that Dsh puncta on the cytoplasmic face of the plasma membrane consisted of aggregates of pedestal-like structures each individually labeled with the C-terminus specific Dsh antibody. These aggregates were resistant to detergent extraction and treatment with drugs that disrupt actin filaments or inhibit myosin II contraction, and coexisted with the first cleavage actomyosin contractile ring. These results confirm and extend previous studies and reveal, for the first time in any cell type, the nanoscale organization of plasma membrane tethered Dsh. Our current working hypothesis is that these Dsh pedestals represent a prepositioned scaffold organization that is important for the localized activation of the cWnt pathway at the sea urchin vegetal pole. These observations in sea urchins may also be relevant to the submembranous Dsh puncta present in other eggs and embryos.


Assuntos
Proteínas Desgrenhadas/metabolismo , Ouriços-do-Mar/metabolismo , Via de Sinalização Wnt/fisiologia , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/patologia , Animais , Padronização Corporal , Proteínas Desgrenhadas/genética , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Microscopia Eletrônica de Transmissão/métodos , Miosina Tipo II/metabolismo , Óvulo/metabolismo , Óvulo/ultraestrutura , Ouriços-do-Mar/crescimento & desenvolvimento
16.
Am J Physiol Cell Physiol ; 320(6): C1153-C1163, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33881935

RESUMO

Cells adapt to applied cyclic stretch (CS) to circumvent chronic activation of proinflammatory signaling. Currently, the molecular mechanism of the selective disassembly of actin stress fibers (SFs) in the stretch direction, which occurs at the early stage of the cellular response to CS, remains controversial. Here, we suggest that the mechanosensitive behavior of myosin II, a major cross-linker of SFs, primarily contributes to the directional disassembly of the actomyosin complex SFs in bovine vascular smooth muscle cells and human U2OS osteosarcoma cells. First, we identified that CS with a shortening phase that exceeds in speed the inherent contractile rate of individual SFs leads to the disassembly. To understand the biological basis, we investigated the effect of expressing myosin regulatory light-chain mutants and found that SFs with less actomyosin activities disassemble more promptly upon CS. We consequently created a minimal mathematical model that recapitulates the salient features of the direction-selective and threshold-triggered disassembly of SFs to show that disassembly or, more specifically, unbundling of the actomyosin bundle SFs is enhanced with sufficiently fast cell shortening. We further demonstrated that similar disassembly of SFs is inducible in the presence of an active LIM-kinase-1 mutant that deactivates cofilin, suggesting that cofilin is dispensable as opposed to a previously proposed mechanism.


Assuntos
Citoesqueleto de Actina/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Miosina Tipo II/metabolismo , Fibras de Estresse/metabolismo , Actomiosina/metabolismo , Animais , Bovinos , Linhagem Celular Tumoral , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Humanos , Contração Muscular/fisiologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Osteossarcoma/metabolismo , Estresse Mecânico
17.
Nat Commun ; 12(1): 2226, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33850145

RESUMO

At the basis of cell shape and behavior, the organization of actomyosin and its ability to generate forces are widely studied. However, the precise regulation of this contractile network in space and time is unclear. Here, we study the role of the epithelial-specific protein EpCAM, a contractility modulator, in cell shape and motility. We show that EpCAM is required for stress fiber generation and front-rear polarity acquisition at the single cell level. In fact, EpCAM participates in the remodeling of a transient zone of active RhoA at the cortex of spreading epithelial cells. EpCAM and RhoA route together through the Rab35/EHD1 fast recycling pathway. This endosomal pathway spatially organizes GTP-RhoA to fine tune the activity of actomyosin resulting in polarized cell shape and development of intracellular stiffness and traction forces. Impairment of GTP-RhoA endosomal trafficking either by silencing EpCAM or by expressing Rab35/EHD1 mutants prevents proper myosin-II activity, stress fiber formation and ultimately cell polarization. Collectively, this work shows that the coupling between co-trafficking of EpCAM and RhoA, and actomyosin rearrangement is pivotal for cell spreading, and advances our understanding of how biochemical and mechanical properties promote cell plasticity.


Assuntos
Endossomos/metabolismo , Molécula de Adesão da Célula Epitelial/metabolismo , Células Epiteliais/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Actomiosina/metabolismo , Células CACO-2 , Movimento Celular/fisiologia , Polaridade Celular , Forma Celular , Células HeLa , Humanos , Miosina Tipo II/metabolismo , Fibras de Estresse/metabolismo
18.
Dev Cell ; 56(10): 1469-1483.e5, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-33891900

RESUMO

During embryo development, tissues often undergo multiple concomitant changes in shape. It is unclear which signaling pathways and cellular mechanisms are responsible for multiple simultaneous tissue shape transformations. We focus on the process of concomitant tissue folding and extension that is key during gastrulation and neurulation. We use the Drosophila embryo as model system and focus on the process of mesoderm invagination. Here, we show that the prospective mesoderm simultaneously folds and extends. We report that mesoderm cells, under the control of anterior-posterior and dorsal-ventral gene patterning synergy, establish two sets of adherens junctions at different apical-basal positions with specialized functions: while apical junctions drive apical constriction initiating tissue bending, lateral junctions concomitantly drive polarized cell intercalation, resulting in tissue convergence-extension. Thus, epithelial cells devise multiple specialized junctional sets that drive composite morphogenetic processes under the synergistic control of apparently orthogonal signaling sources.


Assuntos
Junções Aderentes/metabolismo , Drosophila melanogaster/embriologia , Mesoderma/embriologia , Morfogênese , Animais , Fenômenos Biomecânicos , Padronização Corporal , Proteínas de Ciclo Celular/metabolismo , Polaridade Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Embrião não Mamífero/metabolismo , Mesoderma/citologia , Miosina Tipo II/metabolismo , Fatores de Transcrição da Família Snail/metabolismo
19.
FASEB J ; 35(5): e21418, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33774873

RESUMO

Constitutively expressed by innate immune cells, the cytokine macrophage migration inhibitory factor (MIF) initiates host immune responses and drives pathogenic responses in infectious, inflammatory, and autoimmune diseases. Dendritic cells (DCs) express high levels of MIF, but the role of MIF in DC function remains poorly characterized. As migration is critical for DC immune surveillance, we investigated whether MIF promoted the migration of DCs. In classical transwell experiments, MIF-/- bone marrow-derived DCs (BMDCs) or MIF+/+ BMDCs treated with ISO-1, an inhibitor of MIF, showed markedly reduced spontaneous migration and chemotaxis. CD74-/- BMDCs that are deficient in the ligand-binding component of the cognate MIF receptor exhibited a migration defect similar to that of MIF-/- BMDCs. Adoptive transfer experiments of LPS-matured MIF+/+ and MIF-/- and of CD74+/+ and CD74-/- BMDCs injected into the hind footpads of homologous or heterologous mice showed that the autocrine and paracrine MIF activity acting via CD74 contributed to the recruitment of DCs to the draining lymph nodes. Mechanistically, MIF activated the Src/PI3K signaling pathway and myosin II complexes, which were required for the migration of BMDCs. Altogether, these data show that the cytokine MIF exerts chemokine-like activity for DC motility and trafficking.


Assuntos
Antígenos de Diferenciação de Linfócitos B/fisiologia , Quimiotaxia , Células Dendríticas/fisiologia , Antígenos de Histocompatibilidade Classe II/fisiologia , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Miosina Tipo II/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Quinases da Família src/metabolismo , Animais , Células Cultivadas , Quimiocinas/metabolismo , Células Dendríticas/citologia , Imunidade , Oxirredutases Intramoleculares/genética , Fatores Inibidores da Migração de Macrófagos/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miosina Tipo II/genética , Fosfatidilinositol 3-Quinases/genética , Transdução de Sinais , Quinases da Família src/genética
20.
Proc Natl Acad Sci U S A ; 118(10)2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33653949

RESUMO

Charcot-Marie-Tooth type 4B1 (CMT4B1) is a severe autosomal recessive demyelinating neuropathy with childhood onset, caused by loss-of-function mutations in the myotubularin-related 2 (MTMR2) gene. MTMR2 is a ubiquitously expressed catalytically active 3-phosphatase, which in vitro dephosphorylates the 3-phosphoinositides PtdIns3P and PtdIns(3,5)P 2, with a preference for PtdIns(3,5)P 2 A hallmark of CMT4B1 neuropathy are redundant loops of myelin in the nerve termed myelin outfoldings, which can be considered the consequence of altered growth of myelinated fibers during postnatal development. How MTMR2 loss and the resulting imbalance of 3'-phosphoinositides cause CMT4B1 is unknown. Here we show that MTMR2 by regulating PtdIns(3,5)P 2 levels coordinates mTORC1-dependent myelin synthesis and RhoA/myosin II-dependent cytoskeletal dynamics to promote myelin membrane expansion and longitudinal myelin growth. Consistent with this, pharmacological inhibition of PtdIns(3,5)P 2 synthesis or mTORC1/RhoA signaling ameliorates CMT4B1 phenotypes. Our data reveal a crucial role for MTMR2-regulated lipid turnover to titrate mTORC1 and RhoA signaling thereby controlling myelin growth.


Assuntos
Doença de Charcot-Marie-Tooth/metabolismo , Bainha de Mielina/metabolismo , Fosfatos de Fosfatidilinositol/biossíntese , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Transdução de Sinais , Animais , Doença de Charcot-Marie-Tooth/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Knockout , Bainha de Mielina/genética , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Fosfatos de Fosfatidilinositol/genética , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
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