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Single Cell Genomechanics: Correlating Cancer Cell Mechanics and Gene Expression

ABSTRACT: Single Cell Genomechanics: Correlating Cancer Cell Mechanics and Gene Expression

PROVIDER: PRJNA644902 | ENA |

REPOSITORIES: ENA

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Single Cell Genomechanics: Correlating Cancer Cell Mechanics and Gene Expression

Project description:While cell mechanics and metastatic potential are related, the molecular factors that drive these behaviors remain unknown. Understanding how molecular signaling networks modulate cellular phenotype and mechanotype can help elucidate how metastasis occurs. Therefore, we developed a workflow to measure mechanical properties and gene expression on the single cell level. The process combines atomic force microscopy and optical microscopy to measure the mechanics and morphology of individual ovarian cancer cells, followed by multiplexed RT-qPCR gene expression analysis. Surprisingly, the genes that most strongly correlated with mechanical properties were not cytoskeletal, but rather were markers of epithelial-to-mesenchymal transition and cancer stemness. A dimensionality reduction analysis showed that cells of different metastatic potential were best identified through combining mechanical and gene expression data. Finally, a network analysis revealed master regulators that can predictably stiffen and soften cells while modulating cell migration. The single cell genomechanics methods demonstrate how molecular drivers can disable biophysical processes underpinning metastasis.

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YAP regulates cell mechanics by controlling focal adhesion assembly

Project description:Hippo effectors YAP/TAZ act as on-off mechanosensing switches by sensing modifications in extracellular matrix (ECM) composition and mechanics. The regulation of their activity has been described so far through a hierarchical model in which elements of Hippo pathway are under the control of Focal Adhesions (FAs). Here we unveiled the molecular mechanism by which cell spreading and RhoA GTPase control FA formation through YAP to stabilize the anchorage of actin cytoskeleton to cell membrane. This mechanism required YAP co-transcriptional function and involved the activation of genes encoding for integrins and FA docking proteins. Tuning YAP transcriptional activity led to the modification of cell mechanics, force development, adhesion strength, determined cell shaping, migration and differentiation. These results provide new insights into the mechanism of YAP mechanosensing activity and qualify Hippo effector as the key determinant of cell mechanics in response to ECM cues.

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Correlating single-molecule rupture mechanics with cell population adhesion by yeast display.

Project description:Here, we present a method based on yeast surface display that allows for direct comparison between population-level cell adhesion strength and single-molecule receptor-ligand rupture mechanics. We developed a high-throughput yeast adhesion assay in which yeasts displaying monomeric streptavidin (mSA) or enhanced mutant mSA were adhered to a biotinylated coverglass submerged in fluid. After exposure to shear stress (20-1000 dyn/cm2) by rapid spinning of the coverglass, cells were imaged to quantify the midpoint detachment shear stress for the cell population. We then performed atomic force microscope single-molecule force spectroscopy (SMFS) on purified mSA variants and identified correlations between single-molecule rupture force distributions and cell population adhesion strength. Several features of yeast display were important for successful correlations of adhesion strength to be drawn, including covalent attachment of the receptor to the cell wall, a precisely defined molecular pulling geometry, repression of nonspecific adhesion, and control for multivalency. With these factors properly taken into account, we show that spinning disk cell adhesion assays can be correlated with SMFS and are capable of screening the mechanical strength of receptor-ligand complexes. These workflow enhancements will accelerate research on mechanostable receptor-ligand complexes and receptor-mediated cell adhesion.

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Correlating pancreatic beta cell function with their transcriptional profiles

Project description:Correlating pancreatic beta cell function with their transcriptional profiles

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YAP regulates cell mechanics by controlling focal adhesion assembly

Project description:YAP regulates cell mechanics by controlling focal adhesion assembly

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YAP-TEAD multiphasic activation regulates pluripotent stem cell mechanics to drive cardiac specification and contractility in pathological heart.

Project description:YAP transcriptional regulator controls cell mechanics by activating genes involved in cell-matrix interaction following extracellular matrix (ECM) remodelling and stiffening. YAP is needed for cardiogenesis in mouse but is repressed in adult cardiomyocytes. The protein is reactivated following ischemic insults, although the timing and mechanisms underlying YAP depletion during heart development and the reason for its reactivation are unclear. Here, we combine pluripotent stem cell (PSC) cardiac differentiation, mouse embryo development and human heart tissue analysis to demonstrate that the fine-tuning of cell mechanics, as controlled by YAP multiphasic activation through TEAD transcription, is crucial for mesoderm commitment and cardiac progenitor specification. Finally, by adopting induced PSC models of dilated cardiomyopathy, we prove that YAP-TEAD reactivation in diseased cardiomyocytes empowers calcium handling apparatus and increases cell contractility. Given YAP prompt activation following myocardial infarction, we unveil a novel role for mechanosensing in connecting ECM remodelling to cardiomyocyte function in pathological heart.

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YAP-TEAD multiphasic activation regulates pluripotent stem cell mechanics to drive cardiac specification and contractility in pathological heart.

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