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1.
Nat Commun ; 11(1): 4112, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807784

RESUMO

Macropinocytosis is essential for myeloid cells to survey their environment and for growth of RAS-transformed cancer cells. Several growth factors and inflammatory stimuli are known to induce macropinocytosis, but its endogenous inhibitors have remained elusive. Stimulation of Roundabout receptors by Slit ligands inhibits directional migration of many cell types, including immune cells and cancer cells. We report that SLIT2 inhibits macropinocytosis in vitro and in vivo by inducing cytoskeletal changes in macrophages. In mice, SLIT2 attenuates the uptake of muramyl dipeptide, thereby preventing NOD2-dependent activation of NF-κB and consequent secretion of pro-inflammatory chemokine, CXCL1. Conversely, blocking the action of endogenous SLIT2 enhances CXCL1 secretion. SLIT2 also inhibits macropinocytosis in RAS-transformed cancer cells, thereby decreasing their survival in nutrient-deficient conditions which resemble tumor microenvironment. Our results identify SLIT2 as a physiological inhibitor of macropinocytosis and challenge the conventional notion that signals that enhance macropinocytosis negatively regulate cell migration, and vice versa.


Assuntos
Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/metabolismo , Animais , Quimiocina CXCL1/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Peptídeos e Proteínas de Sinalização Intercelular/genética , Macrófagos/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/genética , Fagócitos/metabolismo , Pinocitose/genética , Pinocitose/fisiologia , Receptores Imunológicos/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteína rhoA de Ligação ao GTP/metabolismo
2.
3.
Phys Rev Lett ; 125(7): 078101, 2020 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-32857554

RESUMO

The friction between cytoskeletal filaments is of central importance for the formation of cellular structures such as the mitotic spindle and the cytokinetic ring. This friction is caused by passive cross-linkers, yet the underlying mechanism and the dependence on cross-linker density are poorly understood. Here, we use theory and computer simulations to study the friction between two filaments that are cross-linked by passive proteins, which can hop between discrete binding sites while physically excluding each other. The simulations reveal that filaments move via rare discrete jumps, which are associated with free-energy barrier crossings. We identify the reaction coordinate that governs the relative microtubule movement and derive an exact analytical expression for the free-energy barrier and the friction coefficient. Our analysis not only elucidates the molecular mechanism underlying cross-linker-induced filament friction, but also predicts that the friction coefficient scales superexponentially with the density of cross-linkers.


Assuntos
Citoesqueleto/química , Citoesqueleto/fisiologia , Modelos Biológicos , Modelos Químicos , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/fisiologia , Sítios de Ligação , Citoesqueleto/metabolismo , Fricção , Microtúbulos/química , Microtúbulos/metabolismo , Proteínas Motores Moleculares/metabolismo , Termodinâmica
4.
Phys Rev Lett ; 125(5): 058101, 2020 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-32794890

RESUMO

Diffusion of tracer particles in the cytoplasm of mammalian cells is often anomalous with a marked heterogeneity even within individual particle trajectories. Despite considerable efforts, the mechanisms behind these observations have remained largely elusive. To tackle this problem, we performed extensive single-particle tracking experiments on quantum dots in the cytoplasm of living mammalian cells at varying conditions. Analyses of the trajectories reveal a strong, microtubule-dependent subdiffusion with antipersistent increments and a substantial heterogeneity. Furthermore, particles stochastically switch between different mobility states, most likely due to transient associations with the cytoskeleton-shaken endoplasmic reticulum network. Comparison to simulations highlight that all experimental observations can be fully described by an intermittent fractional Brownian motion, alternating between two states of different mobility.


Assuntos
Citoplasma/metabolismo , Modelos Biológicos , Citoesqueleto de Actina/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Simulação por Computador , Citocalasina D/farmacologia , Citoplasma/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Difusão , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Nocodazol/farmacologia , Pontos Quânticos , Processos Estocásticos , Tiazolidinas/farmacologia
5.
Proc Natl Acad Sci U S A ; 117(29): 17399-17408, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32641513

RESUMO

The cytoskeleton plays a key role in establishing robust cell shape. In animals, it is well established that cell shape can also influence cytoskeletal organization. Cytoskeletal proteins are well conserved between animal and plant kingdoms; nevertheless, because plant cells exhibit major structural differences to animal cells, the question arises whether the plant cytoskeleton also responds to geometrical cues. Recent numerical simulations predicted that a geometry-based rule is sufficient to explain the microtubule (MT) organization observed in cells. Due to their high flexural rigidity and persistence length of the order of a few millimeters, MTs are rigid over cellular dimensions and are thus expected to align along their long axis if constrained in specific geometries. This hypothesis remains to be tested in cellulo Here, we explore the relative contribution of geometry to the final organization of actin and MT cytoskeletons in single plant cells of Arabidopsis thaliana We show that the cytoskeleton aligns with the long axis of the cells. We find that actin organization relies on MTs but not the opposite. We develop a model of self-organizing MTs in three dimensions, which predicts the importance of MT severing, which we confirm experimentally. This work is a first step toward assessing quantitatively how cellular geometry contributes to the control of cytoskeletal organization in living plant cells.


Assuntos
Fenômenos Fisiológicos Celulares , Forma Celular/fisiologia , Citoesqueleto/fisiologia , Células Vegetais/fisiologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinas , Arabidopsis/metabolismo , Citocalasina D/farmacologia , Microtúbulos/metabolismo , Células Vegetais/efeitos dos fármacos , Células Vegetais/ultraestrutura , Protoplastos
6.
Nat Commun ; 11(1): 3457, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651364

RESUMO

Glioblastoma is a deadly cancer, with no effective therapies. Better understanding and identification of selective targets are urgently needed. We found that advillin (AVIL) is overexpressed in all the glioblastomas we tested including glioblastoma stem/initiating cells, but hardly detectable in non-neoplastic astrocytes, neural stem cells or normal brain. Glioma patients with increased AVIL expression have a worse prognosis. Silencing AVIL nearly eradicated glioblastoma cells in culture, and dramatically inhibited in vivo xenografts in mice, but had no effect on normal control cells. Conversely, overexpressing AVIL promoted cell proliferation and migration, enabled fibroblasts to escape contact inhibition, and transformed immortalized astrocytes, supporting AVIL being a bona fide oncogene. We provide evidence that the tumorigenic effect of AVIL is partly mediated by FOXM1, which regulates LIN28B, whose expression also correlates with clinical prognosis. AVIL regulates the cytoskeleton through modulating F-actin, while mutants disrupting F-actin binding are defective in its tumorigenic capabilities.


Assuntos
Glioblastoma/metabolismo , Glioblastoma/patologia , Proteínas dos Microfilamentos/metabolismo , Animais , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Citoesqueleto/metabolismo , Imunofluorescência , Glioblastoma/genética , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas dos Microfilamentos/genética , Microscopia Confocal , Reação em Cadeia da Polimerase em Tempo Real
7.
Nat Commun ; 11(1): 3495, 2020 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-32661310

RESUMO

Cell biologists generally consider that microtubules and actin play complementary roles in long- and short-distance transport in animal cells. On the contrary, using melanosomes of melanocytes as a model, we recently discovered that the motor protein myosin-Va works with dynamic actin tracks to drive long-range organelle dispersion in opposition to microtubules. This suggests that in animals, as in yeast and plants, myosin/actin can drive long-range transport. Here, we show that the SPIRE-type actin nucleators (predominantly SPIRE1) are Rab27a effectors that co-operate with formin-1 to generate actin tracks required for myosin-Va-dependent transport in melanocytes. Thus, in addition to melanophilin/myosin-Va, Rab27a can recruit SPIREs to melanosomes, thereby integrating motor and track assembly activity at the organelle membrane. Based on this, we suggest a model in which organelles and force generators (motors and track assemblers) are linked, forming an organelle-based, cell-wide network that allows their collective activity to rapidly disperse the population of organelles long-distance throughout the cytoplasm.


Assuntos
Actinas/metabolismo , Proteínas rab27 de Ligação ao GTP/metabolismo , Biologia Celular , Citoesqueleto/metabolismo , Células HEK293 , Humanos , Microtúbulos/metabolismo , Organelas , Filogenia , Proteínas rab27 de Ligação ao GTP/genética
8.
Nat Commun ; 11(1): 3068, 2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32555155

RESUMO

Surgical adhesions are bands of scar tissues that abnormally conjoin organ surfaces. Adhesions are a major cause of post-operative and dialysis-related complications, yet their patho-mechanism remains elusive, and prevention agents in clinical trials have thus far failed to achieve efficacy. Here, we uncover the adhesion initiation mechanism by coating beads with human mesothelial cells that normally line organ surfaces, and viewing them under adhesion stimuli. We document expansive membrane protrusions from mesothelia that tether beads with massive accompanying adherence forces. Membrane protrusions precede matrix deposition, and can transmit adhesion stimuli to healthy surfaces. We identify cytoskeletal effectors and calcium signaling as molecular triggers that initiate surgical adhesions. A single, localized dose targeting these early germinal events completely prevented adhesions in a preclinical mouse model, and in human assays. Our findings classifies the adhesion pathology as originating from mesothelial membrane bridges and offer a radically new therapeutic approach to treat adhesions.


Assuntos
Cálcio/química , Epitélio/metabolismo , Aderências Teciduais/metabolismo , Animais , Sinalização do Cálcio , Adesão Celular , Linhagem Celular , Membrana Celular/metabolismo , Biologia Computacional , Citoesqueleto/metabolismo , Citosol/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Imageamento Tridimensional , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Complicações Pós-Operatórias , Análise de Componente Principal , RNA Interferente Pequeno/metabolismo , Análise de Célula Única
9.
Braz J Med Biol Res ; 53(7): e9207, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32520207

RESUMO

The objective of this study was to investigate the relationship between PI3K/mTOR/RhoA signaling regulated cytoskeletal rearrangements and phagocytic capacity of macrophages. RAW264.7 macrophages were divided into four groups; blank control, negative control, PI3K-RNAi, and mTOR-RNAi. The cytoskeletal changes in the macrophages were observed. Furthermore, the phagocytic capacity of macrophages against Escherichia coli is reported as mean fluorescence intensity (MFI) and percent phagocytosis. Transfection yielded 82.1 and 81.5% gene-silencing efficiencies against PI3K and mTOR, respectively. The PI3K-RNAi group had lower mRNA and protein expression levels of PI3K, mTOR, and RhoA than the blank and negative control groups (Р<0.01). The mTOR-RNAi group had lower mRNA and protein levels of mTOR and RhoA than the blank and the negative control groups (Р<0.01). Macrophages in the PI3K-RNAi group exhibited stiff and inflexible morphology with short, disorganized filopodia and reduced number of stress fibers. Macrophages in the mTOR-RNAi group displayed pronounced cellular deformations with long, dense filopodia and an increased number of stress fibers. The PI3K-RNAi group exhibited lower MFI and percent phagocytosis than blank and negative control groups, whereas the mTOR-RNAi group displayed higher MFI and percent phagocytosis than the blank and negative controls (Р<0.01). Before and after transfection, the mRNA and protein levels of PI3K were both positively correlated with mTOR and RhoA (Р<0.05), but the mRNA and protein levels of mTOR were negatively correlated with those of RhoA (Р<0.05). Changes in the phagocytic capacity of macrophages were associated with cytoskeletal rearrangements and were regulated by the PI3K/mTOR/RhoA signaling pathway.


Assuntos
Citoesqueleto/metabolismo , Macrófagos/metabolismo , Fagocitose/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Western Blotting , Inativação Gênica , Vetores Genéticos , Humanos , Camundongos , Células RAW 264.7 , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Transfecção
11.
Chem Biol Interact ; 325: 109109, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32376239

RESUMO

A series of 10 natural and semisynthetic flavonoids (1 to 10) were obtained from Gardenia oudiepe (Rubiaceae), an endemic plant from New Caledonia. Most of them were polymethoxylated flavones (PMFs) of rare occurrence. After a cell viability screening test, PMFs 2 and 3 showed significant cytotoxic activity against A2058 human melanoma cells (IC50 = 3.92 and 8.18 µM, respectively) and were selected for in-depth pharmacological assays. Both compounds inhibited cell migration and induced apoptosis and cell cycle arrest after 72h of treatment. Immunofluorescence assays indicated that these outcomes were possibly related to the induction of cytoskeleton disruption associated to actin and tubulin depolymerization. These data were confirmed by molecular docking studies, which showed a good interaction between PMFs 2 and 3 and tubulin, particularly at the colchicine binding site. As A2058 are considered as chemoresistant to conventional chemotherapy, compounds 2 and 3 (½IC50) were associated to clinically-used antimelanoma drugs (vemurafenib and dacarbazine) and combined therapies efficacy was assessed by the MTT assay. PMFs 2 restored the sensitivity of A2058 cells to dacarbazine treatment (IC50 = 49.38 µM vs. >100 µM). Taken together, these data suggest that PMFs from G. oudiepe could be potential leaders for the design of new antimelanoma drugs.


Assuntos
Apoptose/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Flavonas/farmacologia , Gardenia/química , Melanoma/patologia , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Antineoplásicos/farmacologia , Caspase 3/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citoesqueleto/metabolismo , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Flavonas/química , Flavonas/metabolismo , Humanos , Simulação de Acoplamento Molecular , Conformação Proteica , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo
12.
PLoS Pathog ; 16(5): e1008106, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32463830

RESUMO

Toxoplasma gondii possesses an armada of secreted virulent factors that enable parasite invasion and survival into host cells. These factors are contained in specific secretory organelles, the rhoptries, micronemes and dense granules that release their content upon host cell recognition. Dense granules are secreted in a constitutive manner during parasite replication and play a crucial role in modulating host metabolic and immune responses. While the molecular mechanisms triggering rhoptry and microneme release upon host cell adhesion have been well studied, constitutive secretion remains a poorly explored aspect of T. gondii vesicular trafficking. Here, we investigated the role of the small GTPase Rab11A, a known regulator of exocytosis in eukaryotic cells. Our data revealed an essential role of Rab11A in promoting the cytoskeleton driven transport of dense granules and the release of their content into the vacuolar space. Rab11A also regulates transmembrane protein trafficking and localization during parasite replication, indicating a broader role of Rab11A in cargo exocytosis at the plasma membrane. Moreover, we found that Rab11A also regulates extracellular parasite motility and adhesion to host cells. In line with these findings, MIC2 secretion was altered in Rab11A-defective parasites, which also exhibited severe morphological defects. Strikingly, by live imaging we observed a polarized accumulation of Rab11A-positive vesicles and dense granules at the apical pole of extracellular motile and invading parasites suggesting that apically polarized Rab11A-dependent delivery of cargo regulates early secretory events during parasite entry into host cells.


Assuntos
Vesículas Transportadoras/metabolismo , Vacúolos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Adesão Celular , Linhagem Celular , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Humanos , Proteínas de Membrana/metabolismo , Microtúbulos/metabolismo , Parasitos/metabolismo , Transporte Proteico , Proteínas de Protozoários , Toxoplasma/metabolismo , Toxoplasmose/metabolismo , Proteínas rab de Ligação ao GTP/fisiologia
13.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(3): 193-197, 2020 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-32389165

RESUMO

Objective To investigate the role of Ras homolog gene (Rho) A/Rho-associated coiled-coil containing protein kinase (ROCK) signaling pathway in tumor necrosis factor α (TNF-α) promoting hyper-permeability of vascular endothelial cells infected by Listeria monocytogenes (Lm) . Methods The cultured human umbilical vein endothelial cells (HUVECs) were divided into a control group (uninfected cells), TNF-α treatment group (100 ng/mL TNF-α, for 2 hours), Lm infection group (infected with MOI=10 Lm for 2 hours, then added gentamicin for 0.5 hour), Lm infection and TNF-α treatment group (infected with Lm and then treated with 100 ng/mL TNF-α for 2 hours), and Y-27632 inhibitor group combined with Lm infection and TNF-α treatment (treated with 50 µmol/L ROCK inhibitor Y-27632 for 30 minutes, and then Lm infection and TNF-α treatment as above). The protein levels of RhoA, zonula occluden-1 (ZO-1), occludin and ROCK in HUVECs were detected by Western blot analysis; the permeability of HUVECs was analyzed by the horseradish peroxidase (HRP) leakage; and the distribution of F-actin in HUVECs was detected by fluorescein isothiocyanate (FITC)-labeled phalloidine staining. Results TNF-α reduced the expression of tight junction protein ZO-1 and occludin in Lm-infected HUVECs, promoted its hyper-permeability and cytoskeletal rearrangement, and up-regulated the expression of RhoA and ROCK. ROCK inhibitor Y-27632 obviously inhibited the cytoskeleton rearrangement and hyper-permeability of HUVECs induced by TNF-α. Conclusion TNF-α can enhance hyper-permeability of HUVECs infected by Lm, which may be regulated by RhoA/Rock signaling pathway.


Assuntos
Células Endoteliais da Veia Umbilical Humana/microbiologia , Listeria monocytogenes , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologia , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Permeabilidade
14.
Adv Exp Med Biol ; 1246: 55-70, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32399825

RESUMO

Phagocytosis is a remarkably complex process, requiring simultaneous organisation of the cell membrane, the cytoskeleton, receptors and various signalling molecules. As can often be the case, mathematical modelling is able to penetrate some of this complexity, identifying the key biophysical components and generating understanding that would take far longer with a purely experimental approach. This chapter will review a particularly important class of phagocytosis model, championed in recent years, that primarily focuses on the role of receptors during the engulfment process. These models are pertinent to a host of unsolved questions in the subject, including the rate of cup growth during uptake, the role of both intra- and extracellular noise, and the precise differences between phagocytosis and other forms of endocytosis. In particular, this chapter will focus on the effect of target shape and orientation, including how these influence the rate and final outcome of phagocytic engulfment.


Assuntos
Fagócitos , Fagocitose , Membrana Celular , Citoesqueleto
15.
Int J Nanomedicine ; 15: 2971-2986, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32431496

RESUMO

Background: Due to their extraordinary physical and chemical properties, MoS2 nanosheets (MSNs) are becoming more widely used in nanomedicine. However, their influence on immune systems remains unclear. Materials and Methods: Two few-layered MSNs at sizes of 100-250 nm (S-MSNs) and 400-500 nm (L-MSNs) were used in this study. Bone marrow-derived dendritic cells (DCs) were exposed to both MSNs at different doses (0, 8, 16, 32, 64, 128 µg/mL) for 48 h and subjected to analyses of surface marker expression, cytokine secretion, lymphoid homing and in vivo T cell priming. Results: Different-sized MSNs of all doses did not affect the viability of DCs. The expression of CD40, CD80, CD86 and CCR7 was significantly higher on both S-MSN- and L-MSN-treated DCs at a dose of 128 µg/mL. As the dose of MSN increased, the secretion of IL-12p70 remained unchanged, the secretion of IL-1ß decreased, and the production of TNF-α increased. A significant increase in IL-6 was observed in the 128 µg/mL L-MSN-treated DCs. In particular, MSN treatment dramatically improved the ex vivo movement and in vivo homing ability of both the local resident and blood circulating DCs. Furthermore, the cytoskeleton rearrangement regulated by ROS elevation was responsible for the enhanced homing ability of the MSNs. More robust CD4+ and CD8+ T cell proliferation and activation (characterized by high expression of CD107a, CD69 and ICOS) was observed in mice vaccinated with MSN-treated DCs. Importantly, exposure to MSNs did not interrupt LPS-induced DC activation, homing and T cell priming. Conclusion: Few-layered MSNs ranging from 100 to 500 nm in size could play an immunostimulatory role in enhancing DC maturation, migration and T cell elicitation, making them a good candidate for vaccine adjuvants. Investigation of this study will not only expand the applications of MSNs and other new transition metal dichalcogenides (TMDCs) but also shed light on the in vivo immune-risk evaluation of MSN-based nanomaterials.


Assuntos
Diferenciação Celular , Movimento Celular , Células Dendríticas/citologia , Células Dendríticas/imunologia , Dissulfetos/farmacologia , Molibdênio/farmacologia , Nanopartículas/química , Linfócitos T/citologia , Linfócitos T/imunologia , Animais , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Células Dendríticas/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Nanopartículas/ultraestrutura , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T/efeitos dos fármacos
16.
PLoS One ; 15(4): e0232025, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32353019

RESUMO

The actin cytoskeleton plays a central role in establishing cell polarity and shape during embryonic morphogenesis. Daam1, a member of the Formin family of actin cytoskeleton regulators, is a Dvl2-binding protein that functions in the Wnt/Planar Cell Polarity (PCP) pathway. To examine the role of the Daam proteins in mammalian development, we generated Daam-deficient mice by gene targeting and found that Daam1, but not Daam2, is necessary for fetal survival. Embryonic development of Daam1 mutants was delayed most likely due to functional defects in the labyrinthine layer of the placenta. Examination of Daam2 and Daam1/2 double mutants revealed that Daam1 and Daam2 are functionally redundant during placental development. Of note, neural tube closure defects (NTD), which are observed in several mammalian PCP mutants, are not observed in Wnt5a or Daam1 single mutants, but arise in Daam1;Wnt5a double mutants. These findings demonstrate a unique function for Daam genes in placental development and are consistent with a role for Daam1 in the Wnt/PCP pathway in mammals.


Assuntos
Proteínas dos Microfilamentos/genética , Placentação/genética , Proteínas rho de Ligação ao GTP/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Transporte/metabolismo , Polaridade Celular , Citoesqueleto/metabolismo , Desenvolvimento Embrionário , Feminino , Forminas/genética , Forminas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Camundongos/embriologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Placenta/embriologia , Gravidez , Via de Sinalização Wnt , Proteínas rho de Ligação ao GTP/metabolismo
17.
Proc Natl Acad Sci U S A ; 117(21): 11257-11264, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32404423

RESUMO

Dmc1 recombinases are essential to homologous recombination in meiosis. Here, we studied the kinetics of the nucleoprotein filament assembly of Saccharomyces cerevisiae Dmc1 using single-molecule tethered particle motion experiments and in vitro biochemical assay. ScDmc1 nucleoprotein filaments are less stable than the ScRad51 ones because of the kinetically much reduced nucleation step. The lower nucleation rate of ScDmc1 results from its lower single-stranded DNA (ssDNA) affinity, compared to that of ScRad51. Surprisingly, ScDmc1 nucleates mostly on the DNA structure containing the single-stranded and duplex DNA junction with the allowed extension in the 5'-to-3' polarity, while ScRad51 nucleation depends strongly on ssDNA lengths. This nucleation preference is also conserved for mammalian RAD51 and DMC1. In addition, ScDmc1 nucleation can be stimulated by short ScRad51 patches, but not by EcRecA ones. Pull-down experiments also confirm the physical interactions of ScDmc1 with ScRad51 in solution, but not with EcRecA. Our results are consistent with a model that Dmc1 nucleation can be facilitated by a structural component (such as DNA junction and protein-protein interaction) and DNA polarity. They provide direct evidence of how Rad51 is required for meiotic recombination and highlight a regulation strategy in Dmc1 nucleoprotein filament assembly.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Meiose , Rad51 Recombinase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Citoesqueleto/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Nucleoproteínas/metabolismo , Rad51 Recombinase/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Imagem Individual de Molécula/métodos
18.
Proc Natl Acad Sci U S A ; 117(23): 12817-12825, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32444491

RESUMO

Morphogenesis, tumor formation, and wound healing are regulated by tissue rigidity. Focal adhesion behavior is locally regulated by stiffness; however, how cells globally adapt, detect, and respond to rigidity remains unknown. Here, we studied the interplay between the rheological properties of the cytoskeleton and matrix rigidity. We seeded fibroblasts onto flexible microfabricated pillar arrays with varying stiffness and simultaneously measured the cytoskeleton organization, traction forces, and cell-rigidity responses at both the adhesion and cell scale. Cells adopted a rigidity-dependent phenotype whereby the actin cytoskeleton polarized on stiff substrates but not on soft. We further showed a crucial role of active and passive cross-linkers in rigidity-sensing responses. By reducing myosin II activity or knocking down α-actinin, we found that both promoted cell polarization on soft substrates, whereas α-actinin overexpression prevented polarization on stiff substrates. Atomic force microscopy indentation experiments showed that this polarization response correlated with cell stiffness, whereby cell stiffness decreased when active or passive cross-linking was reduced and softer cells polarized on softer matrices. Theoretical modeling of the actin network as an active gel suggests that adaptation to matrix rigidity is controlled by internal mechanical properties of the cytoskeleton and puts forward a universal scaling between nematic order of the actin cytoskeleton and the substrate-to-cell elastic modulus ratio. Altogether, our study demonstrates the implication of cell-scale mechanosensing through the internal stress within the actomyosin cytoskeleton and its coupling with local rigidity sensing at focal adhesions in the regulation of cell shape changes and polarity.


Assuntos
Citoesqueleto/metabolismo , Módulo de Elasticidade , Mecanotransdução Celular , Tecidos Suporte/química , Actinina/metabolismo , Polaridade Celular , Reagentes para Ligações Cruzadas/química , Citoesqueleto/ultraestrutura , Fibroblastos/metabolismo , Humanos , Modelos Teóricos , Miosinas/metabolismo
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