RESUMO
Forces generated by the actomyosin cytoskeleton are key contributors to many morphogenetic processes. The actomyosin cytoskeleton organises in different types of networks depending on intracellular signals and on cell-cell and cell-extracellular matrix (ECM) interactions. However, actomyosin networks are not static and transitions between them have been proposed to drive morphogenesis. Still, little is known about the mechanisms that regulate the dynamics of actomyosin networks during morphogenesis. This work uses the Drosophila follicular epithelium, real-time imaging, laser ablation and quantitative analysis to study the role of integrins on the regulation of basal actomyosin networks organisation and dynamics and the potential contribution of this role to cell shape. We find that elimination of integrins from follicle cells impairs F-actin recruitment to basal medial actomyosin stress fibers. The available F-actin redistributes to the so-called whip-like structures, present at tricellular junctions, and into a new type of actin-rich protrusions that emanate from the basal cortex and project towards the medial region. These F-actin protrusions are dynamic and changes in total protrusion area correlate with periodic cycles of basal myosin accumulation and constriction pulses of the cell membrane. Finally, we find that follicle cells lacking integrin function show increased membrane tension and reduced basal surface. Furthermore, the actin-rich protrusions are responsible for these phenotypes as their elimination in integrin mutant follicle cells rescues both tension and basal surface defects. We thus propose that the role of integrins as regulators of stress fibers plays a key role on controlling epithelial cell shape, as integrin disruption promotes reorganisation into other types of actomyosin networks, in a manner that interferes with proper expansion of epithelial basal surfaces.
Assuntos
Actomiosina/metabolismo , Forma Celular , Proteínas de Drosophila/metabolismo , Células Epiteliais/metabolismo , Integrinas/metabolismo , Fibras de Estresse/metabolismo , Animais , Membrana Celular/metabolismo , Drosophila , Células Epiteliais/citologia , Fibras de Estresse/ultraestruturaRESUMO
Mechanisms of the assembly of actin stress fibers (SFs) have been extensively studied, while those of the disassembly-particularly cell shortening-induced ones-remain unclear. Here, we show that SFs have helical structures composed of multi-subbundles, and they tend to be delaminated upon cell shortening. Specifically, we observed with atomic force microscopy delamination of helical SFs into their subbundles. We physically caught individual SFs using a pair of glass needles to observe rotational deformations during stretching as well as ATP-driven active contraction, suggesting that they deform in a manner reflecting their intrinsic helical structure. A minimal analytical model was then developed based on the Frenet-Serret formulas with force-strain measurement data to suggest that helical SFs can be delaminated into the constituent subbundles upon axial shortening. Given that SFs are large molecular clusters that bear cellular tension but must promptly disassemble upon loss of the tension, the resulting increase in their surface area due to the shortening-induced delamination may facilitate interaction with surrounding molecules to aid subsequent disintegration. Thus, our results suggest a new mechanism of the disassembly that occurs only in the specific SFs exposed to forced shortening.
Assuntos
Actinas/química , Fibras de Estresse/química , Actinas/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Bovinos , Células Cultivadas , Microscopia de Força Atômica , Modelos Biológicos , Estrutura Secundária de Proteína , Ratos , Fibras de Estresse/metabolismo , Fibras de Estresse/ultraestrutura , Estresse MecânicoRESUMO
Cells remodel their structure in response to mechanical strain. However, how mechanical forces are translated into biochemical signals that coordinate the structural changes observed at the plasma membrane (PM) and the underlying cytoskeleton during mechanoadaptation is unclear. Here, we show that PM mechanoadaptation is controlled by a tension-sensing pathway composed of c-Abl tyrosine kinase and membrane curvature regulator FBP17. FBP17 is recruited to caveolae to induce the formation of caveolar rosettes. FBP17 deficient cells have reduced rosette density, lack PM tension buffering capacity under osmotic shock, and cannot adapt to mechanical strain. Mechanistically, tension is transduced to the FBP17 F-BAR domain by direct phosphorylation mediated by c-Abl, a mechanosensitive molecule. This modification inhibits FBP17 membrane bending activity and releases FBP17-controlled inhibition of mDia1-dependent stress fibers, favoring membrane adaptation to increased tension. This mechanoprotective mechanism adapts the cell to changes in mechanical tension by coupling PM and actin cytoskeleton remodeling.
Assuntos
Cavéolas/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Mecanotransdução Celular , Proteínas Proto-Oncogênicas c-abl/metabolismo , Fibras de Estresse/metabolismo , Cavéolas/ultraestrutura , Proteínas de Ligação a Ácido Graxo/genética , Fibroblastos , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Microscopia Eletrônica , Fosforilação , RNA Interferente Pequeno/metabolismo , Fibras de Estresse/ultraestrutura , Estresse MecânicoRESUMO
Pancreatic cancer has the worst prognosis among major malignancies, largely due to its highly invasive property and difficulty in early detection. Mechanistic insights into cancerous transformation and especially metastatic progression are imperative for developing novel treatment strategies. The actin-regulating protein CAP1 is implicated in human cancers, while the role still remains elusive. In this study, we investigated roles for CAP1 and its phosphor-regulation in pancreatic cancer cells. No evidence supports remarkable up-regulation of CAP1 in the panel of cancer cell lines examined. However, knockdown of CAP1 in cancer cells led to enhanced stress fibers, reduced cell motility and invasion into Matrigel. Phosphorylation of CAP1 at the S308/S310 tandem regulatory site was elevated in cancer cells, consistent with hyper-activated GSK3 reported in pancreatic cancer. Inhibition of GSK3, a kinase for S310, reduced cell motility and invasion. Moreover, phosphor mutants had defects in alleviating actin stress fibers and rescuing the reduced invasiveness in the CAP1-knockdown PANC-1 cells. These results suggest a required role for transient phosphorylation for CAP1 function in controlling cancer cell invasiveness. Depletion of CAP1 also reduced FAK activity and cell adhesion, but did not cause significant alterations in ERK or cell proliferation. CAP1 likely regulates cancer cell invasiveness through effects on both actin filament turnover and cell adhesion. Finally, the growth factor PDGF induced CAP1 dephosphorylation, suggesting CAP1 may mediate extracellular signals to control cancer cell invasiveness. These findings may ultimately help develop strategies targeting CAP1 or its regulatory signals for controlling the invasive cycle of the disease.
Assuntos
Proteínas de Ciclo Celular/genética , Movimento Celular/genética , Transformação Celular Neoplásica/genética , Proteínas do Citoesqueleto/genética , Regulação Neoplásica da Expressão Gênica , Pâncreas/metabolismo , Processamento de Proteína Pós-Traducional , Adesão Celular , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/metabolismo , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mutação , Pâncreas/patologia , Fosforilação/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/metabolismo , Fibras de Estresse/ultraestruturaRESUMO
Contractile actomyosin bundles, stress fibers, contribute to morphogenesis, migration, and mechanosensing of non-muscle cells. In addition to actin and non-muscle myosin II (NMII), stress fibers contain a large array of proteins that control their assembly, turnover, and contractility. Calponin-3 (Cnn3) is an actin-binding protein that associates with stress fibers. However, whether Cnn3 promotes stress fiber assembly, or serves as either a positive or negative regulator of their contractility has remained obscure. Here, we applied U2OS osteosarcoma cells as a model system to study the function of Cnn3. We show that Cnn3 localizes to both NMII-containing contractile ventral stress fibers and transverse arcs, as well as to non-contractile dorsal stress fibers that do not contain NMII. Fluorescence-recovery-after-photobleaching experiments revealed that Cnn3 is a dynamic component of stress fibers. Importantly, CRISPR/Cas9 knockout and RNAi knockdown studies demonstrated that Cnn3 is not essential for stress fiber assembly. However, Cnn3 depletion resulted in increased and uncoordinated contractility of stress fibers that often led to breakage of individual actomyosin bundles within the stress fiber network. Collectively these results provide evidence that Cnn3 is dispensable for the assembly of actomyosin bundles, but that it is required for controlling proper contractility of the stress fiber network.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fibras de Estresse/metabolismo , Proteínas de Ligação ao Cálcio/análise , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular Tumoral , Recuperação de Fluorescência Após Fotodegradação , Inativação Gênica , Humanos , Imageamento Tridimensional , Proteínas dos Microfilamentos/análise , Proteínas dos Microfilamentos/genética , Imagem Óptica , Osteossarcoma/genética , Osteossarcoma/metabolismo , Fibras de Estresse/genética , Fibras de Estresse/ultraestrutura , CalponinasRESUMO
The Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1), which acts on the Rho GTPases that are key regulators of the actin cytoskeleton, is emerging as a potential therapeutic tool against certain neurological diseases characterized by cellular energy homeostasis impairment. In this brief communication, we show explorative results on the toxin's effect on fibroblasts derived from a patient affected by myoclonic epilepsy with ragged-red fibers (MERRF) that carries a mutation in the m.8344A>G gene of mitochondrial DNA. We found that, in the patient's cells, besides rescuing the wild-type-like mitochondrial morphology, CNF1 administration is able to trigger a significant increase in cellular content of ATP and of the mitochondrial outer membrane marker Tom20. These results were accompanied by a profound F-actin reorganization in MERRF fibroblasts, which is a typical CNF1-induced effect on cell cytoskeleton. These results point at a possible role of the actin organization in preventing or limiting the cell damage due to mitochondrial impairment and at CNF1 treatment as a possible novel strategy against mitochondrial diseases still without cure.
Assuntos
Trifosfato de Adenosina/biossíntese , Toxinas Bacterianas/farmacologia , DNA Mitocondrial/genética , Proteínas de Escherichia coli/farmacologia , Fibroblastos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mutação , Toxinas Bacterianas/isolamento & purificação , DNA Mitocondrial/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Fibroblastos/metabolismo , Fibroblastos/patologia , Expressão Gênica , Humanos , Síndrome MERRF/tratamento farmacológico , Síndrome MERRF/genética , Síndrome MERRF/metabolismo , Síndrome MERRF/patologia , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Pessoa de Meia-Idade , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Projetos Piloto , Cultura Primária de Células , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/metabolismo , Fibras de Estresse/ultraestruturaRESUMO
Infantile hemangiomas are the most common benign tumors of infancy, characterized by unregulated angiogenesis and endothelial cells with high mitotic rate. Although spontaneous regression occurs, sometimes treatment is required and alternatives to corticosteroids should be considered to reduce side effects. Imiquimod is an imidazoquinoline, approved for some skin pathologies other than hemangioma. It is proposed that the effectiveness of imiquimod comes from the activation of immune cells at tumor microenvironment. However, the possibility to selectively kill different cell types and to directly impede angiogenesis has been scarcely explored in vitro for endothelial cells. In this work we showed a dramatic cytotoxicity on hemangioma cell, with a significant lower IC50 value in hemangioma compared to normal endothelial cells and melanoma (employed as a non-endothelial tumor cell line). Nuclear morphometric and flow-cytometry assays revealed imiquimod-induced apoptosis on hemangioma and melanoma cells but a small percentage of senescence on normal endothelial cells. At sub-lethal conditions, cell migration, a key step in angiogenesis turned out to be inhibited in a tumor-selective manner along with actin cytoskeleton disorganization on hemangioma cells. Altogether, these findings pointed out the selective cytotoxic effects of imiquimod on transformed endothelial cells, evidencing the potential for imiquimod to be a therapeutic alternative to reduce extensive superficial hemangioma lesions.
Assuntos
Aminoquinolinas/farmacologia , Antineoplásicos/farmacologia , Hemangioma/patologia , Neoplasias Cutâneas/patologia , Aminoquinolinas/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Sobrevivência Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Células Endoteliais/efeitos dos fármacos , Hemangioma/tratamento farmacológico , Humanos , Imiquimode , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Camundongos , Neoplasias Cutâneas/tratamento farmacológico , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/ultraestruturaRESUMO
BACKGROUND: Tumor microenvironment consists of the extracellular matrix (ECM), stromal cells, such as fibroblasts (FBs) and cancer associated fibroblasts (CAFs), and a myriad of soluble factors. In many tumor types, including pancreatic tumors, the interplay between stromal cells and the other tumor microenvironment components leads to desmoplasia, a cancer-specific type of fibrosis that hinders treatment. Transforming growth factor beta (TGF-ß) and CAFs are thought to play a crucial role in this tumor desmoplastic reaction, although the involved mechanisms are unknown. METHODS: Optical/fluorescence microscopy, atomic force microscopy, image processing techniques, invasion assay in 3D collagen I gels and real-time PCR were employed to investigate the effect of TGF-ß on normal pancreatic FBs and CAFs with regard to crucial cellular morphodynamic characteristics and relevant gene expression involved in tumor progression and metastasis. RESULTS: CAFs present specific myofibroblast-like characteristics, such as α-smooth muscle actin expression and cell elongation, they also form more lamellipodia and are softer than FBs. TGF-ß treatment increases cell stiffness (Young's modulus) of both FBs and CAFs and increases CAF's (but not FB's) elongation, cell spreading, lamellipodia formation and spheroid invasion. Gene expression analysis shows that these morphodynamic characteristics are mediated by Rac, RhoA and ROCK expression in CAFs treated with TGF-ß. CONCLUSIONS: TGF-ß modulates CAFs', but not FBs', cell shape, stiffness and invasion. GENERAL SIGNIFICANCE: Our findings elucidate on the effects of TGF-ß on CAFs' behavior and stiffness providing new insights into the mechanisms involved.
Assuntos
Fibroblastos Associados a Câncer/efeitos dos fármacos , Neoplasias Pancreáticas/patologia , Fator de Crescimento Transformador beta/farmacologia , Fibroblastos Associados a Câncer/ultraestrutura , Forma Celular , Módulo de Elasticidade , Fibroblastos/efeitos dos fármacos , Fibroblastos/ultraestrutura , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Invasividade Neoplásica , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Pseudópodes/efeitos dos fármacos , Pseudópodes/ultraestrutura , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/ultraestrutura , Células Tumorais Cultivadas , Microambiente TumoralRESUMO
Contractile actomyosin bundles, stress fibers, are crucial for adhesion, morphogenesis, and mechanosensing in nonmuscle cells. However, the mechanisms by which nonmuscle myosin II (NM-II) is recruited to those structures and assembled into functional bipolar filaments have remained elusive. We report that UNC-45a is a dynamic component of actin stress fibers and functions as a myosin chaperone in vivo. UNC-45a knockout cells display severe defects in stress fiber assembly and consequent abnormalities in cell morphogenesis, polarity, and migration. Experiments combining structured-illumination microscopy, gradient centrifugation, and proteasome inhibition approaches revealed that a large fraction of NM-II and myosin-1c molecules fail to fold in the absence of UNC-45a. The remaining properly folded NM-II molecules display defects in forming functional bipolar filaments. The C-terminal UNC-45/Cro1/She4p domain of UNC-45a is critical for NM-II folding, whereas the N-terminal tetratricopeptide repeat domain contributes to the assembly of functional stress fibers. Thus, UNC-45a promotes generation of contractile actomyosin bundles through synchronized NM-II folding and filament-assembly activities.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Miosina Tipo II/metabolismo , Osteoblastos/metabolismo , Fibras de Estresse/metabolismo , Actomiosina/genética , Actomiosina/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Polaridade Celular , Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Miosina Tipo II/genética , Osteoblastos/ultraestrutura , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fibras de Estresse/ultraestrutura , Repetições de TetratricopeptídeosRESUMO
Melanoma is the leading cause of death from skin disease due to its propensity for metastasis. Studies have shown that integrin-mediated focal adhesion kinase (FAK) signal pathway is implicated in cell proliferation, survival and metastasis of tumor cells. Our previous results indicated that diallyl trisulfide (DATS) provided its antimelanoma activity via inducing cell cycle arrest and apoptosis. The aim of this study was to explore DATS mediated antimetastatic effect and the corresponding mechanism in human melanoma A375 cells. We found that DATS exhibited an inhibitory effect on the abilities of migration and invasion in A375 cells under noncytotoxic concentrations analyzed by wound healing assays and Matrigel invasion chamber system. DATS attenuated invasion of A375 cells with characteristic of decreased activities and protein expressions of matrix metalloproteinase-2 (MMP-2) and MMP-9. Moreover, DATS exerted an inhibitory effect on cell adhesion of A375 cells, which is in correlation with the change in integrin signaling pathway. Results of Western blotting showed that DATS decreased the levels of several integrin subunits, including α4, α5, αv, ß1, ß3 and ß4. Subsequently, DATS induced a strong decrease in total FAK, phosphorylated FAK Tyr-397,-576, -577, and disorganized F-actin stress fibers, resulting in a nonmigratory phenotype. These results suggest that the antimetastatic potential of DATS for human melanoma cells might be due to the disruption of integrin/FAK signaling pathway.
Assuntos
Compostos Alílicos/farmacologia , Antineoplásicos/farmacologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Integrinas/metabolismo , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Sulfetos/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Fosforilação , Transdução de Sinais , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/ultraestruturaRESUMO
BACKGROUND: Podocyte apoptosis is a major mechanism that leads to proteinuria in many kidney diseases. However, the concert mechanisms that cause podocyte apoptosis in these kidney diseases are not fully understood. RhoA is one of Rho GTPases that has been well studied and plays a key role in regulating cytoskeletal architecture. Previous study showed that insufficient RhoA could result in rat aortic smooth muscle cell apoptosis. However, whether RhoA is involved in podocyte apoptosis remains unknown. METHODS: Culture podocytes were treated with LPS, ADR or siRNA for 48 h before harvest. Subcellular immunoblotting, qRT-PCR, immunofluorescence and flow cytometry were used to exam the expression and function of RhoA or YAP in podocytes. RESULTS: We found that the expression of RhoA and its activity were significantly decreased in LPS or ADR-injured podocytes, accompanying loss of stress fibers and increased cell apoptosis. Knocking down RhoA or its downstream effector mDia expression by siRNA also caused loss of stress fibers and podocyte apoptosis. Moreover, our results further demonstrated that RhoA deficiency could reduce the mRNA and protein expression of YAP, which had been regarded as an anti-apoptosis protein in podocyte. Silenced dendrin expression significantly abolished RhoA, mDia or YAP deficiency-induced podocyte apoptosis. CONCLUSION: RhoA deficiency could disrupt podocyte cytoskeleton and induce podocyte apoptosis by inhibiting YAP/dendrin signal. RhoA/mDia/YAP/dendrin signal pathway may potentially play an important role in regulating podocyte apoptosis. Maintaining necessary RhoA would be one potent way to prevent proteinuria kidney diseases.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/metabolismo , Podócitos/fisiologia , Podócitos/ultraestrutura , Proteína rhoA de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , Citoesqueleto/genética , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Doxorrubicina/farmacologia , Forminas , Inativação Gênica , Lipopolissacarídeos/farmacologia , Camundongos , Proteínas do Tecido Nervoso/genética , Fosfoproteínas/genética , Podócitos/efeitos dos fármacos , RNA Mensageiro/metabolismo , Transdução de Sinais , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/ultraestrutura , Proteínas de Sinalização YAP , Proteína rhoA de Ligação ao GTP/deficiência , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Cells respond to fluid shear stress through dynamic processes involving changes in actomyosin and other cytoskeletal stresses, remodeling of cell adhesions, and cytoskeleton reorganization. In this study we simultaneously measured focal adhesion dynamics and cytoskeletal stress and reorganization in MDCK cells under fluid shear stress. The measurements used co-expression of fluorescently labeled paxillin and force sensitive FRET probes of α-actinin. A shear stress of 0.74 dyn/cm(2) for 3 hours caused redistribution of cytoskeletal tension and significant focal adhesion remodeling. The fate of focal adhesions is determined by the stress state and stability of the linked actin stress fibers. In the interior of the cell, the mature focal adhesions disassembled within 35-40 min under flow and stress fibers disintegrated. Near the cell periphery, the focal adhesions anchoring the stress fibers perpendicular to the cell periphery disassembled, while focal adhesions associated with peripheral fibers sustained. The diminishing focal adhesions are coupled with local cytoskeletal stress release and actin stress fiber disassembly whereas sustaining peripheral focal adhesions are coupled with an increase in stress and enhancement of actin bundles. The results show that flow induced formation of peripheral actin bundles provides a favorable environment for focal adhesion remodeling along the cell periphery. Under such condition, new FAs were observed along the cell edge under flow. Our results suggest that the remodeling of FAs in epithelial cells under flow is orchestrated by actin cytoskeletal stress redistribution and structural reorganization.
Assuntos
Adesão Celular/genética , Células Epiteliais/ultraestrutura , Adesões Focais/metabolismo , Estresse Mecânico , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinina/metabolismo , Actinas/metabolismo , Animais , Movimento Celular , Cães , Células Epiteliais/metabolismo , Humanos , Células Madin Darby de Rim Canino , Fibras de Estresse/metabolismo , Fibras de Estresse/ultraestruturaRESUMO
Myopodin is an actin-binding protein that promotes cancer cell migration in response to serum stimulation and is associated with invasive tumor development. To determine whether enhanced migration reflects changes in actin cytoskeleton remodeling, fluorescence confocal microscopy was used to examine the composition and morphology of filamentous actin structures in mock-transduced cells vs. stably transduced PC3 cells expressing human myopodin isoforms, and the chemokinetic response of cells was quantified using transwell assays. The same approaches were used to analyze the effects of external migration stimuli, actin polymerization inhibitors or deletion of the isoform-specific amino- and/or carboxy termini on cell migration and actin bundle formation. Results indicate that the termini of the myopodin isoforms differentially alter the formation of morphologically distinct F-actin networks that also differ in their myosin and myopodin staining patterns. Furthermore, enhanced cell migration was reduced by >50% when actin bundle formation was impaired by myopodin-truncation, low concentrations of an actin polymerization inhibitor, or in the absence of an external migration stimulus. Human myopodin isoforms are therefore potent regulators of stress fiber formation, inducing the formation of biochemically and morphologically distinct F-actin networks in the cell body whose presence directly correlates with increased cell migration.
Assuntos
Movimento Celular , Proteínas dos Microfilamentos/metabolismo , Fibras de Estresse/metabolismo , Linhagem Celular Tumoral , Humanos , Proteínas dos Microfilamentos/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fibras de Estresse/ultraestruturaRESUMO
Cell adhesion to the extracellular matrix is an essential element of various biological processes. TGF-ß cytokines regulate the matrix components and cell-matrix adhesions. The present study investigates the molecular organization of TGF-ß-induced matrix adhesions. The study demonstrates that in various mouse and human epithelial cells TGF-ß induces cellular structures containing 2 matrix adhesions bridged by a stretch of actin fibers. These structures are similar to ventral stress fibers (VSFs). Suppression of integrin-ß5 by RNA interference reduces VSFs in majority of cells (> 75%), while overexpression of integrin-ß5 fragments revealed a critical role of a distinct sequence in the cytoplasmic domain of integrin-ß5 in the VSF structures. In addition, the integrity of actin fibers and Src kinase activity contribute to integrin-ß5-mediated signaling and VSF formation. TGF-ß-Smad signaling upregulates actin-regulatory proteins, such as caldesmon, zyxin, and zyxin-binding protein Csrp1 in mouse and human epithelial cells. Suppression of zyxin markedly inhibits formation of VSFs in response to TGF-ß and integrin-ß5. Zyxin is localized at actin fibers and matrix adhesions of VSFs and might bridge integrin-ß5-mediated adhesions to actin fibers. These findings provide a platform for defining the molecular mechanism regulating the organization and activities of VSFs in response to TGF-ß.
Assuntos
Células Epiteliais/metabolismo , Matriz Extracelular/metabolismo , Adesões Focais/metabolismo , Cadeias beta de Integrinas/metabolismo , Fibras de Estresse/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Zixina/metabolismo , Sequência de Aminoácidos , Animais , Adesão Celular , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/ultraestrutura , Feminino , Adesões Focais/efeitos dos fármacos , Adesões Focais/ultraestrutura , Regulação da Expressão Gênica , Humanos , Cadeias beta de Integrinas/genética , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Proteínas Smad/genética , Proteínas Smad/metabolismo , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/ultraestrutura , Zixina/genéticaRESUMO
The small GTPase RhoA has critical functions in regulating actin dynamics affecting cellular morphogenesis through the RhoA/Rho kinase (ROCK) signaling cascade. RhoA signaling controls stress fiber and focal adhesion formation and cell motility in fibroblasts. RhoA signaling is involved in several aspects of neuronal development, including neuronal migration, growth cone collapse, dendrite branching, and spine growth. Altered RhoA signaling is implicated in cancer and neurodegenerative disease and is linked to inherited intellectual disabilities. Although much is known about factors regulating RhoA activity and/or degradation, little is known about molecular mechanisms regulating RhoA expression and the subsequent effects on RhoA signaling. We hypothesized that posttranscriptional control of RhoA expression may provide a mechanism to regulate RhoA signaling and downstream effects on cell morphology. Here we uncover a cellular function for the mRNA-binding protein heterogeneous nuclear ribonucleoprotein (hnRNP) Q1 in the control of dendritic development and focal adhesion formation that involves the negative regulation of RhoA synthesis and signaling. We show that hnRNP-Q1 represses RhoA translation and knockdown of hnRNP-Q1 induced phenotypes associated with elevated RhoA protein levels and RhoA/ROCK signaling. These morphological changes were rescued by ROCK inhibition and/or RhoA knockdown. These findings further suggest that negative modulation of RhoA mRNA translation can provide control over downstream signaling and cellular morphogenesis.
Assuntos
Dendritos/ultraestrutura , Adesões Focais/ultraestrutura , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Neurônios/citologia , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Regiões 3' não Traduzidas , Actinas/metabolismo , Animais , Linhagem Celular Tumoral , Adesões Focais/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Hipocampo/citologia , Humanos , Camundongos , Morfogênese , Neurônios/metabolismo , Biossíntese de Proteínas , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Transdução de Sinais , Fibras de Estresse/metabolismo , Fibras de Estresse/ultraestrutura , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
It has been proposed that buckling of actin stress fibers (SFs) may be associated with their disassembly. However, much of the detail remains unknown partly because the use of an elastic membrane sheet, conventionally necessary for inducing SF buckling with a mechanical compression to adherent cells, may limit high quality and quick imaging of the dynamic cellular events. Here, we present an alternate approach to induce buckling behavior of SFs on a readily observable glass plate. Actin SFs were extracted from cells, and constituent myosin II (MII) molecules were partially photo-inactivated in contractility. An addition of Mg-ATP allowed actin-myosin cross-bridge cycling and resultant contraction of only thick SFs that still contained active MII in the large volume. Meanwhile, thin SFs with virtually no active motor protein in the small volume had no choice but to buckle with the shortening movement of nearby thick SFs functioning as a compression-inducing element. This novel technique, thus allowing for selective inductions of contraction and buckling of SFs and measurements of the cellular prestress, may be applicable to not only investigations on their disassembly mechanisms but also to measurements of the relative thickness of individual SFs in each cell.
Assuntos
Fibras de Estresse/ultraestrutura , Animais , Linhagem Celular , Miosinas/análise , Fibras de Estresse/química , Fibras de Estresse/fisiologia , Estresse MecânicoRESUMO
OBJECTIVE: To present an algorithm based on Hough transform for recognition and extraction of linear stress fibers formed on exposure to lysophosphatidic acid (LPA). STUDY DESIGN: A ridge set of head points with lower shoulders is calculated, followed by a thinning process shrinking long, narrow regions to regions of single pixel thickness, then converted into a rectangular map whose value is the number of regional points in the path of a straight line at the angle and intercept determined by two coordinates. The location of the maximum in the map is sought, and the corresponding line with an unlimited length is constructed from the paired coordinates. We removed the line before repeating the process for the next longest straight line, continuing until all lines with reasonable lengths are extracted. RESULTS: Application of the algorithm to the stress fiber images of DOV13 cells stained with Texas red-phalloidin on LPA and AG1478 demonstrates close matches between stress fibers in the original images and linear lines. CONCLUSION: An algorithm for recognition of linear stress fibers formed on exposure to LPA is described and applications to stress fiber images using DOV13 cells with Texas red-phalloidin staining are demonstrated.
Assuntos
Algoritmos , Lisofosfolipídeos/química , Fibras de Estresse/fisiologia , Linhagem Celular Tumoral , Humanos , Faloidina/química , Fibras de Estresse/ultraestruturaRESUMO
Cellular mechanics is known to play an important role in many cellular functions including adhesion, migration, proliferation, and differentiation. Human mesenchymal stem cells (hMSCs) demonstrate unique mechanical properties distinct from fully differentiated cells. This observation suggests that the stem cell mechanics may be modulated to regulate the hMSCs' lineage commitment. Specifically, ERM (ezrin, radixin, moesin) proteins are known to mediate the membrane-cytoskeleton adhesion, cell elasticity, actin cytoskeleton organization, and therefore could serve as potential targets for modulation of the cellular mechanics. Combining silencing RNA, atomic force microscopy, and laser optical tweezers, the role of the ERM proteins involved in the regulation of stem cell biomechanics and osteogenic differentiation was quantitatively determined. Transient ERM knockdown by RNAi causes disassembly of actin stress fibers and focal adhesions, a decrease in the cell stiffness, and membrane separation from the cytoskeleton. The silencing RNA treatment not only induced mechanical changes in stem cells but impaired biochemically-directed osteogenic differentiation. The intact actin cytoskeleton and focal adhesions of hMSCs appear critical for the osteogenic induction. Thus, ERM knockdown modulates the dynamics of cell mechanical changes during hMSC differentiation and regulates the expression of tissue specific molecular markers. These findings are of particular interest for modulation of the cellular biomechanics to control hMSCs' activities and fate in tissue engineering, regenerative medicine, and other stem cell-based therapeutic applications.
Assuntos
Diferenciação Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Osteogênese/fisiologia , Antígenos de Diferenciação/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Células Cultivadas , Proteínas do Citoesqueleto/genética , Inativação Gênica , Humanos , Proteínas de Membrana/genética , Células-Tronco Mesenquimais/ultraestrutura , Proteínas dos Microfilamentos/genética , Microscopia de Força Atômica , Fibras de Estresse/genética , Fibras de Estresse/metabolismo , Fibras de Estresse/ultraestruturaRESUMO
Mesenchymal stem cells (MSCs) have been extensively investigated for their applications in regenerative medicine. Successful use of MSCs in cell-based therapies will rely on the ability to effectively identify their properties and functions with a relatively non-destructive methodology. In this study, we measured the surface stiffness and thickness of rat MSCs with atomic force microscopy and clarified their relation at a single-cell level. The role of the perinuclear actin cap in regulating the thickness, stiffness, and proliferative activity of these cells was also determined by using several actin cytoskeleton-modifying reagents. This study has helped elucidate a possible link between the physical properties and the physiological function of the MSCs, and the corresponding regulatory role of the actin cytoskeleton.
Assuntos
Actinas/ultraestrutura , Proliferação de Células , Fenômenos Mecânicos , Células-Tronco Mesenquimais/fisiologia , Células-Tronco Mesenquimais/ultraestrutura , Animais , Células Cultivadas , Citoesqueleto/ultraestrutura , Masculino , Microscopia de Força Atômica , Ratos , Ratos Endogâmicos F344 , Fibras de Estresse/ultraestruturaRESUMO
Viperin is an interferon-inducible protein that is directly induced in cells by human cytomegalovirus (HCMV) infection. Why HCMV would induce viperin, which has antiviral activity, is unknown. We show that HCMV-induced viperin disrupts cellular metabolism to enhance the infectious process. Viperin interaction with the viral protein vMIA resulted in viperin relocalization from the endoplasmic reticulum to the mitochondria. There, viperin interacted with the mitochondrial trifunctional protein that mediates ß-oxidation of fatty acids to generate adenosine triphosphate (ATP). This interaction with viperin, but not with a mutant lacking the viperin iron-sulfur cluster-binding motif, reduced cellular ATP generation, which resulted in actin cytoskeleton disruption and enhancement of infection. This function of viperin, which was previously attributed to vMIA, suggests that HCMV has coopted viperin to facilitate the infectious process.