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The switching of the fibroblast phenotype to myofibroblast is a hallmark of a wide variety of tissue pathologies. This phenotypical switch is known to be influenced not only by humoral factors such as TGF-ß, but also by mechanical and physical cues in the cellular environment, and is accompanied by distinctive changes in cell morphology. However, the causative link between these cues, the concomitant morphological changes, and the resulting phenotypic switch remain elusive. Here, we use protein micropatterning to spatially control dermal fibroblast adhesion without invoking exogenous mechanical changes and demonstrate that varying the spatial configuration of focal adhesions (FAs) is sufficient to direct fibroblast phenotype. We further developed an automated morphometry analysis pipeline, which revealed FA eccentricity as the primary determinant of cell-state positioning along the spectrum of fibroblast phenotype. Moreover, linear fibronectin patterns that constrain the FAs were found to promote a further phenotype transition, characterized by dispersed expression of alpha-smooth muscle actin, pointing to an interesting possibility of controlling fibroblast phenotype beyond the canonical fibroblast-myofibroblast axis. Together, our study reveals that the spatial configuration of adhesion to the cellular microenvironment is a key factor governing fibroblast morphotype and phenotype, shedding new light on fibroblast phenotype regulation.
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Calpain2 is a conventional member of the non-lysosomal calpain protease family that has been shown to affect the dynamics of focal and cell-cell adhesions by proteolyzing the components of adhesion complexes. Here, we inactivated calpain2 using CRISPR/Cas9 in epithelial MDCK cells. We show that depletion of calpain2 has multiple effects on cell morphology and function. Calpain2-depleted cells develop epithelial shape, however, they cover a smaller area, and cell clusters are more compact. Inactivation of calpain2 enhanced restoration of transepithelial electrical resistance after calcium switch, decreased cell migration, and delayed cell scattering induced by HGF/SF. In addition, calpain2 depletion prevented morphological changes induced by ERK2 overexpression. Interestingly, proteolysis of several calpain2 targets, including E-cadherin, ß-catenin, talin, FAK, and paxillin, was not discernibly affected by calpain2 depletion. Taken together, these data suggest that calpain2 regulates the stability of cell-cell and cell-substratum adhesions indirectly without affecting the proteolysis of these adhesion complexes.
Assuntos
Calpaína , Adesão Celular , Células Epiteliais , Calpaína/metabolismo , Animais , Cães , Células Madin Darby de Rim Canino , Células Epiteliais/metabolismo , Células Epiteliais/citologia , Movimento Celular , Caderinas/metabolismo , Proteólise , Fator de Crescimento de Hepatócito/metabolismo , beta Catenina/metabolismo , Cálcio/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Sistemas CRISPR-CasRESUMO
Endothelial cells lining the blood vessel wall communicate intricately with the surrounding extracellular matrix, translating mechanical cues into biochemical signals. Moreover, vessels require the capability to enzymatically degrade the matrix surrounding them, to facilitate vascular expansion. c-Src plays a key role in blood vessel growth, with its loss in the endothelium reducing vessel sprouting and focal adhesion signalling. Here, we show that constitutive activation of c-Src in endothelial cells results in rapid vascular expansion, operating independently of growth factor stimulation or fluid shear stress forces. This is driven by an increase in focal adhesion signalling and size, with enhancement of localised secretion of matrix metalloproteinases responsible for extracellular matrix remodelling. Inhibition of matrix metalloproteinase activity results in a robust rescue of the vascular expansion elicited by heightened c-Src activity. This supports the premise that moderating focal adhesion-related events and matrix degradation can counteract abnormal vascular expansion, with implications for pathologies driven by unusual vascular morphologies.
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Matriz Extracelular , Adesões Focais , Quinases da Família src , Adesões Focais/metabolismo , Matriz Extracelular/metabolismo , Humanos , Quinases da Família src/metabolismo , Quinases da Família src/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Animais , Proteína Tirosina Quinase CSK/metabolismo , Transdução de Sinais , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Metaloproteinases da Matriz/metabolismoRESUMO
Focal adhesions serve as structural and signaling hubs, facilitating bidirectional communication at the cell-extracellular matrix interface. Paxillin and the related Hic-5 (TGFß1i1) are adaptor/scaffold proteins that recruit numerous structural and regulatory proteins to focal adhesions, where they perform both overlapping and discrete functions. In this study, paxillin and Hic-5 were expressed in U2OS osteosarcoma cells as biotin ligase (BioID2) fusion proteins and used as bait proteins for proximity-dependent biotinylation in order to directly compare their respective interactomes. The fusion proteins localized to both focal adhesions and the centrosome, resulting in biotinylation of components of each of these structures. Biotinylated proteins were purified and analyzed by mass spectrometry. The list of proximity interactors for paxillin and Hic-5 comprised numerous shared core focal adhesion proteins that likely contribute to their similar functions in cell adhesion and migration, as well as proteins unique to paxillin and Hic-5 that have been previously localized to focal adhesions, the centrosome, or the nucleus. Western blotting confirmed biotinylation and enrichment of FAK and vinculin, known interactors of Hic-5 and paxillin, as well as several potentially unique proximity interactors of Hic-5 and paxillin, including septin 7 and ponsin, respectively. Further investigation into the functional relationship between the unique interactors and Hic-5 or paxillin may yield novel insights into their distinct roles in cell migration.
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Silver nanoparticles (AgNPs) are increasingly incorporated in diverse products to confer antimicrobial properties. They are released into the environment during manufacture, after disposal, and from the products during use. Because AgNPs bioaccumulate in brain, it is important to understand how they interact with neural cell physiology. We found that the focal adhesion (FA)-associated protein cadherin aggregated in a dose-dependent response to AgNP exposure in differentiating cultured B35 neuroblastoma cells. These aggregates tended to colocalize with F-actin inclusions that form in response to AgNP and also contain ß-catenin. However, using hyperspectral microscopy, we demonstrate that these multi-protein aggregates did not colocalize with the AgNPs themselves. Furthermore, expression and organization of the FA protein vinculin did not change in cells exposed to AgNP. Our findings suggest that AgNPs activate an intermediate mechanism which leads to formation of aggregates via specific protein-protein interactions. Finally, we detail the changes in hyperspectral profiles of AgNPs during different stages of cell culture and immunocytochemistry processing. AgNPs in citrate-stabilized solution present mostly blue with some rainbow spectra and these are maintained upon mounting in Prolong Gold. Exposure to tissue culture medium results in a uniform green spectral shift that is not further altered by fixation and protein block steps of immunocytochemistry.
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Caderinas , Nanopartículas Metálicas , Prata , Nanopartículas Metálicas/toxicidade , Prata/toxicidade , Prata/química , Caderinas/metabolismo , Linhagem Celular Tumoral , Animais , Agregados Proteicos/efeitos dos fármacos , Vinculina/metabolismoRESUMO
Focal adhesions (FAs) are mechanosensory structures that transform physical stimuli into chemical signals guiding cell migration. Comprehensive studies postulate correlation between the FA parameters and cell motility metrics for individual migrating cells. However, which properties of the FAs are critical for epithelial cell motility in a monolayer remains poorly elucidated. We used high-throughput microscopy to describe relationship between the FA parameters and cell migration in immortalized epithelial keratinocytes (HaCaT) and lung carcinoma cells (A549) with depleted or inhibited vinculin and focal adhesion kinase (FAK) FA proteins. To evaluate relationship between the FA morphology and cell migration, we used substrates with varying stiffness in the model of wound healing. Cells cultivated on fibronectin had the highest FA area values, migration rate, and upregulated expression of FAK and vinculin mRNAs, while the smallest FA area and slower migration rate to the wound were specific to cells cultivated on glass. Suppression of vinculin expression in both normal and tumor cells caused decrease of the FA size and fluorescence intensity but did not affect cell migration into the wound. In contrast, downregulation or inactivation of FAK did not affect the FA size but significantly slowed down the wound closure rate by both HaCaT and A549 cell lines. We also showed that the FAK knockdown results in the FA lifetime decrease for the cells cultivated both on glass and fibronectin. Our data indicate that the FA lifetime is the most important parameter defining migration of epithelial cells in a monolayer. The observed change in the cell migration rate in a monolayer caused by changes in expression/activation of FAK kinase makes FAK a promising target for anticancer therapy of lung carcinoma.
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Movimento Celular , Vinculina , Humanos , Vinculina/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células A549 , Quinase 1 de Adesão Focal/metabolismo , Quinase 1 de Adesão Focal/genética , Adesões Focais/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismoRESUMO
Tight junctions are a barrier-forming cell-cell adhesion complex and have been proposed to regulate cell proliferation. However, the underlying mechanisms are not well understood. Here, we used cells deficient in the junction scaffold ZO-1 alone or together with its paralog ZO-2, which disrupts the junctional barrier. We found that ZO-1 knockout increased cell proliferation, induced loss of cell density-dependent proliferation control, and promoted apoptosis and necrosis. These phenotypes were enhanced by double ZO-1/ZO-2 knockout. Increased proliferation was dependent on two transcriptional regulators: YAP and ZONAB. ZO-1 knockout stimulated YAP nuclear translocation and activity without changes in Hippo-dependent phosphorylation. Knockout promoted TANK-binding kinase 1 (TBK1) activation and increased expression of the RhoA activator GEF-H1. Knockdown of ZO-3, another paralog interacting with ZO1, was sufficient to induce GEF-H1 expression and YAP activity. GEF-H1, TBK1, and mechanotransduction at focal adhesions were found to cooperate to activate YAP/TEAD in ZO-1-deficient cells. Thus, ZO-1 controled cell proliferation and Hippo-independent YAP activity by activating a GEF-H1- and TBK1-regulated mechanosensitive signalling network.
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Mecanotransdução Celular , Transdução de Sinais , Proliferação de Células , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fosforilação , Animais , Células Madin Darby de Rim Canino , CãesRESUMO
Mechanotransduction refers to the ability of cells to sense mechanical stimuli and convert them into biochemical signals. In this context, the key players are focal adhesions (FAs): multiprotein complexes that link intracellular actin bundles and the extracellular matrix (ECM). FAs are involved in cellular adhesion, growth, differentiation, gene expression, migration, communication, force transmission, and contractility. Focal adhesion signaling molecules, including Focal Adhesion Kinase (FAK), integrins, vinculin, and paxillin, also play pivotal roles in cardiomyogenesis, impacting cell proliferation and heart tube looping. In fact, cardiomyocytes sense ECM stiffness through integrins, modulating signaling pathways like PI3K/AKT and Wnt/ß-catenin. Moreover, FAK/Src complex activation mediates cardiac hypertrophic growth and survival signaling in response to mechanical loads. This review provides an overview of the molecular and mechanical mechanisms underlying the crosstalk between FAs and cardiac differentiation, as well as the role of FA-mediated mechanotransduction in guiding cardiac muscle responses to mechanical stimuli.
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Adesões Focais , Mecanotransdução Celular , Miócitos Cardíacos , Adesões Focais/metabolismo , Humanos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/citologia , Animais , Diferenciação Celular , Matriz Extracelular/metabolismoRESUMO
Focal adhesions (FAs) form the junction between extracellular matrix (ECM)-bound integrins and the actin cytoskeleton and also transmit signals that regulate cell adhesion, cytoskeletal dynamics, and cell migration. While many of these signals are rooted in reversible tyrosine phosphorylation, phosphorylation of FA proteins on Ser/Thr residues is far more abundant yet its mechanisms and consequences are far less understood. The cAMP-dependent protein kinase (protein kinase A; PKA) has important roles in cell adhesion and cell migration and is both an effector and regulator of integrin-mediated adhesion to the ECM. Importantly, subcellular localization plays a critically important role in specifying PKA function. Here, we show that PKA is present in isolated FA-cytoskeleton complexes and active within FAs in live cells. Furthermore, using kinase-catalyzed biotinylation of isolated FA-cytoskeleton complexes, we identify 53 high-stringency candidate PKA substrates within FAs. From this list, we validate tensin-3 (Tns3)-a well-established molecular scaffold, regulator of cell migration, and a component of focal and fibrillar adhesions-as a novel direct substrate for PKA. These observations identify a new pathway for phospho-regulation of Tns3 and, importantly, establish a new and important niche for localized PKA signaling and thus provide a foundation for further investigation of the role of PKA in the regulation of FA dynamics and signaling.
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Proteínas Quinases Dependentes de AMP Cíclico , Adesões Focais , Tensinas , Animais , Humanos , Adesão Celular , Movimento Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citoesqueleto/metabolismo , Adesões Focais/enzimologia , Fosforilação , Tensinas/metabolismo , Camundongos , Ratos , Linhagem Celular , Transdução de Sinais/genéticaRESUMO
Focal adhesions (FAs) play a crucial role in cell spreading and adhesion, and their autophagic degradation is an emerging area of interest. This study investigates the role of Thrombospondin Type 1 Domain-Containing Protein 1 (THSD1) in regulating autophagy and FA stability in brain endothelial cells, shedding light on its potential implications for cerebrovascular diseases. Our research reveals a physical interaction between THSD1 and FAs. Depletion of THSD1 significantly reduces FA numbers, impairing cell spreading and adhesion. The loss of THSD1 also induces autophagy independently of changes in mTOR and AMPK activation, implying that THSD1 primarily governs FA dynamics rather than serving as a global regulator of nutrient and energy status. Mechanistically, THSD1 negatively regulates Beclin 1, a central autophagy regulator, at FAs through interactions with focal adhesion kinase (FAK). THSD1 inactivation diminishes FAK activity and relieves its inhibitory phosphorylation on Beclin 1. This, in turn, promotes the complex formation between Beclin 1 and ATG14, a critical event for the activation of the autophagy cascade. In summary, our findings identify THSD1 as a novel regulator of autophagy that degrades FAs in brain endothelial cells. This underscores the distinctive nature of THSD1-mediated, cargo-directed autophagy and its potential relevance to vascular diseases due to the loss of endothelial FAs. Investigating the underlying mechanisms of THSD1-mediated pathways holds promise for discovering novel therapeutic targets in vascular diseases.
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Adesões Focais , Trombospondinas , Doenças Vasculares , Humanos , Autofagia , Proteína Beclina-1/metabolismo , Células Endoteliais/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/metabolismo , Fosforilação , Doenças Vasculares/metabolismo , Trombospondinas/metabolismoRESUMO
Inter-alpha-trypsin inhibitor heavy chain 5 (ITIH5) has been identified as a metastasis suppressor gene in pancreatic cancer. Here, we analyzed ITIH5 promoter methylation and protein expression in The Cancer Genome Atlas (TCGA) dataset and three tissue microarray cohorts (n = 618), respectively. Cellular effects, including cell migration, focal adhesion formation and protein tyrosine kinase activity, induced by forced ITIH5 expression in pancreatic cancer cell lines were studied in stable transfectants. ITIH5 promoter hypermethylation was associated with unfavorable prognosis, while immunohistochemistry demonstrated loss of ITIH5 in the metastatic setting and worsened overall survival. Gain-of-function models showed a significant reduction in migration capacity, but no alteration in proliferation. Focal adhesions in cells re-expressing ITIH5 exhibited a smaller and more rounded phenotype, typical for slow-moving cells. An impressive increase of acetylated alpha-tubulin was observed in ITIH5-positive cells, indicating more stable microtubules. In addition, we found significantly decreased activities of kinases related to focal adhesion. Our results indicate that loss of ITIH5 in pancreatic cancer profoundly affects its molecular profile: ITIH5 potentially interferes with a variety of oncogenic signaling pathways, including the PI3K/AKT pathway. This may lead to altered cell migration and focal adhesion formation. These cellular alterations may contribute to the metastasis-inhibiting properties of ITIH5 in pancreatic cancer.
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Adesão Celular , Movimento Celular , Neoplasias Pancreáticas , Transdução de Sinais , Humanos , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Movimento Celular/genética , Adesão Celular/genética , Linhagem Celular Tumoral , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Adesões Focais/metabolismo , Adesões Focais/genética , Metilação de DNA/genética , Regiões Promotoras Genéticas/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Secretadas Inibidoras de ProteinasesRESUMO
To find new molecular targets for triple negative breast cancer (TNBC), we analyzed a large-scale drug screening dataset based on breast cancer subtypes. We discovered that BDP-9066, a specific MRCK inhibitor (MRCKi), may be an effective drug against TNBC. After confirming the efficacy and specificity of BDP-9066 against TNBC in vitro and in vivo, we further analyzed the underlying mechanism of specific activity of BDP-9066 against TNBC. Comparing the transcriptome of BDP-9066-sensitive and -resistant cells, the activation of the focal adhesion and YAP/TAZ pathway were found to play an important role in the sensitive cells. Furthermore, YAP/TAZ is indeed repressed by BDP-9066 in the sensitive cells, and active form of YAP suppresses the effects of BDP-9066. YAP/TAZ expression and activity are high in TNBC, especially the Claudin-low subtype, consistent with the expression of focal adhesion-related genes. Interestingly, NF-κB functions downstream of YAP/TAZ in TNBC cells and is suppressed by BDP-9066. Furthermore, the PI3 kinase pathway adversely affected the effects of BDP-9066 and that alpelisib, a PI3 kinase inhibitor, synergistically increased the effects of BDP-9066, in PIK3CA mutant TNBC cells. Taken together, we have shown for the first time that MRCKi can be new drugs against TNBC, particularly the Claudin-low subtype.
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Proteínas Adaptadoras de Transdução de Sinal , Neoplasias de Mama Triplo Negativas , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Proteínas de Sinalização YAP , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Claudinas/genética , Claudinas/metabolismo , Linhagem Celular TumoralRESUMO
The focal adhesion protein, Hic-5 plays a key role in promoting extracellular matrix deposition and remodeling by cancer associated fibroblasts within the tumor stroma to promote breast tumor cell invasion. However, whether stromal matrix gene expression is regulated by Hic-5 is still unknown. Utilizing a constitutive Hic-5 knockout, Mouse Mammary Tumor Virus-Polyoma Middle T-Antigen spontaneous breast tumor mouse model, bulk RNAseq analysis was performed on cancer associated fibroblasts isolated from Hic-5 knockout mammary tumors. Functional network analysis highlighted a key role for Hic-5 in extracellular matrix organization, with both structural matrix genes, as well as matrix remodeling genes being differentially expressed in relation to Hic-5 expression. The subcellular distribution of the MRTF-A transcription factor and expression of a subset of MRTF-A responsive genes was also impacted by Hic-5 expression. Additionally, cytokine array analysis of conditioned media from the Hic-5 and Hic-5 knockout cancer associated fibroblasts revealed that Hic-5 is important for the secretion of several key factors that are associated with matrix remodeling, angiogenesis and immune evasion. Together, these data provide further evidence of a central role for Hic-5 expression in cancer associated fibroblasts in regulating the composition and organization of the tumor stroma microenvironment to promote breast tumor progression.
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Neoplasias da Mama , Fibroblastos Associados a Câncer , Animais , Feminino , Humanos , Camundongos , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/patologia , Citocinas/genética , Citocinas/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Expressão Gênica , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Fatores de Transcrição/metabolismo , Microambiente Tumoral/genéticaRESUMO
Cellular form and function emerge from complex mechanochemical systems within the cytoplasm. Currently, no systematic strategy exists to infer large-scale physical properties of a cell from its molecular components. This is an obstacle to understanding processes such as cell adhesion and migration. Here, we develop a data-driven modeling pipeline to learn the mechanical behavior of adherent cells. We first train neural networks to predict cellular forces from images of cytoskeletal proteins. Strikingly, experimental images of a single focal adhesion (FA) protein, such as zyxin, are sufficient to predict forces and can generalize to unseen biological regimes. Using this observation, we develop two approaches-one constrained by physics and the other agnostic-to construct data-driven continuum models of cellular forces. Both reveal how cellular forces are encoded by two distinct length scales. Beyond adherent cell mechanics, our work serves as a case study for integrating neural networks into predictive models for cell biology.
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Proteínas do Citoesqueleto , Aprendizado de Máquina , Adesão Celular , Citoplasma/metabolismo , Proteínas do Citoesqueleto/metabolismo , Adesões Focais/metabolismo , Modelos BiológicosRESUMO
The progression of breast cancer is often accompanied by changes in extracellular matrix stiffness and cell adhesion ability, which are closely related to cellular mechanotransduction. However, the underlying regulatory mechanisms remain mysterious. Our study reveals that the macroautophagy/autophagy-inducing kinases, ULK1 and ULK2, inhibit the assembly of focal adhesions and F-actin by phosphorylating the adhesion protein PXN, to prevent breast cancer cell migration in an autophagy-independent fashion. Interestingly, ULK1/ULK2-mediated serine phosphorylation of PXN counteracts PXN phosphorylation at the adjacent tyrosine residues by PTK2 and SRC, to gatekeep cellular mechanotransduction. Our research establishes a new function of ULK1/ULK2 in governing cellular mechanotransduction that might be harnessed for treating breast cancer.
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Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Autofagia , Neoplasias da Mama , Movimento Celular , Peptídeos e Proteínas de Sinalização Intracelular , Mecanotransdução Celular , Proteínas Serina-Treonina Quinases , Humanos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Autofagia/fisiologia , Feminino , Proteínas Serina-Treonina Quinases/metabolismo , Fosforilação , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Adesões Focais/metabolismo , Linhagem Celular Tumoral , Actinas/metabolismoRESUMO
Wnt signaling is essential for embryonic development, influencing processes such as axis formation, cell proliferation and differentiation, cell fate decisions, and axon guidance. It also plays a role in maintaining tissue homeostasis in adult organisms. The loss of normal cell polarity and adhesion caused by Wnt signaling activation is a fundamental step for tumor progression and metastasis. Activating the canonical Wnt pathway is a driving force in many human cancers, especially colorectal, hepatocellular, and mammary carcinomas. Wnt causes the stabilization and nuclear transport of newly synthesized transcriptional regulator ß-catenin. The generally accepted view is that the canonical effects of Wnt growth factors are caused by the transcription of ß-catenin target genes. Here, we review recent findings that indicate Wnt is a regulator of many other cellular physiological activities, such as macropinocytosis, endosome trafficking, protein stability, focal adhesions, and lysosomal activity. Some of these regulatory responses occur within minutes and do not require new protein synthesis, indicating that there is much more to Wnt beyond the well-established transcriptional role of ß-catenin. The main conclusion that emerges from these studies is that in basal cell conditions, the activity of the key protein kinase GSK3, which is inhibited by Wnt pathway activation, normally represses the actin machinery that orchestrates macropinocytosis with implications in cancer. These contributions expand our understanding of the multifaceted roles of Wnt signaling in cellular processes, development, and cancer, providing insights into potential therapeutic targets and strategies.
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Adesão Celular , Neoplasias do Colo , Via de Sinalização Wnt , Humanos , Neoplasias do Colo/patologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/genética , Animais , beta Catenina/metabolismo , beta Catenina/genéticaRESUMO
Cellular response to the topography of their environment, known as contact guidance, is a crucial aspect to many biological processes yet remains poorly understood. A prevailing model to describe cellular contact guidance involves the lateral confinement of focal adhesions (FA) by topography as an underlying mechanism governing how cells can respond to topographical cues. However, it is not clear how this model is consistent with the well-documented depth-dependent contact guidance responses in the literature. To investigate this model, we fabricated a set of contact guidance chips with lateral dimensions capable of confining focal adhesions and relaxing that confinement at various depths. We find at the shallowest depth of 330 nm, the model of focal adhesion confinement is consistent with our observations. However, the cellular response at depths of 725 and 1000 nm is inadequately explained by this model. Instead, we observe a distinct reorganization of F-actin at greater depths in which topographically induced cell membrane deformation alters the structure of the cytoskeleton. These results are consistent with an alternative curvature-hypothesis to explain cellular response to topographical cues. Together, these results indicate a confluence of two molecular mechanisms operating at increased induced membrane curvature that govern how cells sense and respond to topography.
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Adesões Focais , Adesões Focais/metabolismo , Actinas/metabolismo , Humanos , Animais , Citoesqueleto/metabolismoRESUMO
Intercellular communication is critical to the formation and homeostatic function of all tissues. Previous work has shown that cells can communicate mechanically via the transmission of cell-generated forces through their surrounding extracellular matrix, but this process is not well understood. Here, mechanically defined, synthetic electrospun fibrous matrices are utilized in conjunction with a microfabrication-based cell patterning approach to examine mechanical intercellular communication (MIC) between endothelial cells (ECs) during their assembly into interconnected multicellular networks. It is found that cell force-mediated matrix displacements in deformable fibrous matrices underly directional extension and migration of neighboring ECs toward each other prior to the formation of stable cell-cell connections enriched with vascular endothelial cadherin (VE-cadherin). A critical role is also identified for calcium signaling mediated by focal adhesion kinase and mechanosensitive ion channels in MIC that extends to multicellular assembly of 3D vessel-like networks when ECs are embedded within fibrin hydrogels. These results illustrate a role for cell-generated forces and ECM mechanical properties in multicellular assembly of capillary-like EC networks and motivates the design of biomaterials that promote MIC for vascular tissue engineering.
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Comunicação Celular , Células Endoteliais , Matriz Extracelular , Engenharia Tecidual , Materiais BiocompatíveisRESUMO
Organ size is controlled by numerous factors including mechanical forces, which are mediated in part by the Hippo pathway. In growing Drosophila epithelial tissues, cytoskeletal tension influences Hippo signaling by modulating the localization of key pathway proteins to different apical domains. Here, we discovered a Hippo signaling hub at basal spot junctions, which form at the basal-most point of the lateral membranes and resemble adherens junctions in protein composition. Basal spot junctions recruit the central kinase Warts via Ajuba and E-cadherin, which prevent Warts activation by segregating it from upstream Hippo pathway proteins. Basal spot junctions are prominent when tissues undergo morphogenesis and are highly sensitive to fluctuations in cytoskeletal tension. They are distinct from focal adhesions, but the latter profoundly influences basal spot junction abundance by modulating the basal-medial actomyosin network and tension experienced by spot junctions. Thus, basal spot junctions couple morphogenetic forces to Hippo pathway activity and organ growth.
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Proteínas de Drosophila , Verrugas , Animais , Drosophila/metabolismo , Via de Sinalização Hippo , Proteínas de Drosophila/metabolismo , Transdução de Sinais , Junções Aderentes/metabolismo , Verrugas/metabolismo , Morfogênese/fisiologiaRESUMO
Metastasis of breast cancer cells to distant tissue sites is responsible for the majority of deaths associated with breast cancer. Previously we have examined the role of class I myosin motor protein, myosin 1e (myo1e), in cancer metastasis using the Mouse Mammary Tumor Virus-Polyoma Middle T Antigen (MMTV-PyMT) mouse model. Mice deficient in myo1e formed tumors with a more differentiated phenotype relative to the wild-type mice and formed no detectable lung metastases. In the current study, we investigated how the absence of myo1e affects cell migration and invasion in vitro, using the highly invasive and migratory breast cancer cell line, 4T1. 4T1 cells deficient in myo1e exhibited an altered morphology and slower rates of migration in the wound-healing and transwell migration assays compared to the WT 4T1 cells. While integrin trafficking and Golgi reorientation did not appear to be altered upon myo1e loss, we observed lower rates of focal adhesion disassembly in myo1e-deficient cells, which could help explain the cell migration defect.