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Force-induced cell polarisation is linked to RhoA-driven microtubule-independent focal-adhesion sliding.

ABSTRACT: Mechanical forces play a crucial role in controlling the integrity and functionality of cells and tissues. External forces are sensed by cells and translated into signals that induce various responses. To increase the detailed understanding of these processes, we investigated cell migration and dynamic cellular reorganisation of focal adhesions and cytoskeleton upon application of cyclic stretching forces. Of particular interest was the role of microtubules and GTPase activation in the course of mechanotransduction. We showed that focal adhesions and the actin cytoskeleton undergo dramatic reorganisation perpendicular to the direction of stretching forces even without microtubules. Rather, we found that microtubule orientation is controlled by the actin cytoskeleton. Using biochemical assays and fluorescence resonance energy transfer (FRET) measurements, we revealed that Rac1 and Cdc42 activities did not change upon stretching, whereas overall RhoA activity increased dramatically, but independently of intact microtubules. In conclusion, we demonstrated that key players in force-induced cellular reorganisation are focal-adhesion sliding, RhoA activation and the actomyosin machinery. In contrast to the importance of microtubules in migration, the force-induced cellular reorganisation, including focal-adhesion sliding, is independent of a dynamic microtubule network. Consequently, the elementary molecular mechanism of cellular reorganisation during migration is different to the one in force-induced cell reorganisation.

SUBMITTER: Goldyn AM

PROVIDER: S-EPMC2758800 | biostudies-other | 2009 Oct

REPOSITORIES: biostudies-other

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Force-induced cell polarisation is linked to RhoA-driven microtubule-independent focal-adhesion sliding.

Goldyn Alexandra M AM Rioja Borja Aragüés BA Spatz Joachim P JP Ballestrem Christoph C Kemkemer Ralf R

Journal of cell science 20091001 Pt 20

Mechanical forces play a crucial role in controlling the integrity and functionality of cells and tissues. External forces are sensed by cells and translated into signals that induce various responses. To increase the detailed understanding of these processes, we investigated cell migration and dynamic cellular reorganisation of focal adhesions and cytoskeleton upon application of cyclic stretching forces. Of particular interest was the role of microtubules and GTPase activation in the course of ...[more]

PMID: 19812308

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Project description:The survival of endothelial cells is dependent on interactions between the matrix and integrins mediated through focal adhesions. Focal adhesion kinase (FAK) is thought to play a key role in maintaining focal adhesion function and cell survival, whereas caspase-mediated FAK proteolysis is implicated in focal adhesion disassembly during apoptosis. We examined the relationship between changes in FAK phosphorylation and proteolysis during apoptosis of primary porcine aortic endothelial cells (PAEC) induced by staurosporine, a widely used apoptogenic agent in diverse cell types. Staurosporine-induced PAEC apoptosis was detected after 1 h and was preceded by disruption and loss of FAK localization to focal adhesions within a few minutes, whereas staurosporine-induced cleavage of FAK occurred only after 8-24 h. Staurosporine induced a very rapid dephosphorylation of FAK at Tyr(861) and Tyr(397) and caused dissociation of phosphorylated FAK from focal adhesions as early as 30 s. The effect of staurosporine was very potent with striking inhibition of Tyr(861) and Tyr(397) phosphorylation and focal adhesion disruption occurring in the range 10-100 nM. Selective inhibition of a known target of staurosporine, protein kinase C, using GF109203X, and of phosphoinositide 3'-kinase using wortmannin, did not reduce FAK tyrosine phosphorylation at Tyr(861) and Tyr(397), or cause disruption of focal adhesions. Cycloheximide, the protein synthesis inhibitor, induced PAEC apoptosis more slowly than staurosporine, but did not induce FAK dephosphorylation or rapid focal adhesion disruption, and instead caused a slower loss of focal adhesions and a marked increase in FAK proteolysis. These studies show that FAK dephosphorylation and focal adhesion disassembly are very early events mediating the onset of staurosporine-induced endothelial cell apoptosis and are dissociated from FAK proteolysis. Cycloheximide induces apoptosis through a pathway involving FAK proteolysis without dephosphorylation.

Project description:Cells interact with the extracellular matrix (ECM) via cell-ECM adhesions. These physical interactions are transduced into biochemical signals inside the cell which influence cell behaviour. Although cell-ECM interactions have been studied extensively, it is not completely understood how immature (nascent) adhesions develop into mature (focal) adhesions and how mechanical forces influence this process. Given the small size, dynamic nature and short lifetimes of nascent adhesions, studying them using conventional microscopic and experimental techniques is challenging. Computational modelling provides a valuable resource for simulating and exploring various "what if?" scenarios in silico and identifying key molecular components and mechanisms for further investigation. Here, we present a simplified mechano-chemical model based on ordinary differential equations with three major proteins involved in adhesions: integrins, talin and vinculin. Additionally, we incorporate a hypothetical signal molecule that influences adhesion (dis)assembly rates. We find that assembly and disassembly rates need to vary dynamically to limit maturation of nascent adhesions. The model predicts biphasic variation of actin retrograde velocity and maturation fraction with substrate stiffness, with maturation fractions between 18-35%, optimal stiffness of ∼1 pN/nm, and a mechanosensitive range of 1-100 pN/nm, all corresponding to key experimental findings. Sensitivity analyses show robustness of outcomes to small changes in parameter values, allowing model tuning to reflect specific cell types and signaling cascades. The model proposes that signal-dependent disassembly rate variations play an underappreciated role in maturation fraction regulation, which should be investigated further. We also provide predictions on the changes in traction force generation under increased/decreased vinculin concentrations, complementing previous vinculin overexpression/knockout experiments in different cell types. In summary, this work proposes a model framework to robustly simulate the mechanochemical processes underlying adhesion maturation and maintenance, thereby enhancing our fundamental knowledge of cell-ECM interactions.

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Force-induced cell polarisation is linked to RhoA-driven microtubule-independent focal-adhesion sliding.

Publications

Force-induced cell polarisation is linked to RhoA-driven microtubule-independent focal-adhesion sliding.

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