Abstract
Focal adhesions are protein complexes that anchor cells to the extracellular matrix. During migration, the growth and disassembly of these structures are spatiotemporally regulated, with new adhesions forming at the leading edge of the cell and mature adhesions disassembling at the rear. Signalling proteins and structural cytoskeletal components tightly regulate adhesion dynamics. Paxillin, an adaptor protein within adhesions, is one of these proteins. Its phosphorylation at serine 273 (S273) is crucial for maintaining fast adhesion assembly and disassembly. Paxillin is known to bind to a GIT1-βPIX-PAK1 complex, which increases the local activation of the small GTPase Rac. To understand quantitatively the behaviour of this system and how it relates to adhesion assembly/disassembly, we developed a mathematical model describing the dynamics of the small GTPases Rac and Rho as determined by paxillin S273 phosphorylation. Our model revealed that the system possesses bistability, where switching between uninduced (active Rho) and induced (active Rac) states can occur through a change in rate of paxillin phosphorylation or PAK1 activation. The bistable switch is characterized by the presence of memory, minimal change in the levels of active Rac and Rho within the induced and uninduced states, respectively, and the limited regime of monostability associated with the uninduced state. These results were validated experimentally by showing the presence of bimodality in adhesion assembly and disassembly rates, and demonstrating that Rac activity increases after treating Chinese Hamster Ovary cells with okadaic acid (a paxillin phosphatase inhibitor), followed by a modest recovery after 20 min washout. Spatial gradients of phosphorylated paxillin in a reaction-diffusion model gave rise to distinct regions of Rac and Rho activities, resembling polarization of a cell into front and rear. Perturbing several parameters of the model also revealed important insights into how signalling components upstream and downstream of paxillin phosphorylation affect dynamics.
Highlights
In multicellular organisms, cell migration is key to proper development and maintenance of physiological processes such as embryogenesis, axonal outgrowth in neurons, and wound healing [1,2,3,4,5]
These adhesions assemble at the leading edge, as the cell extends forward, anchoring the front of the cells to its substrate, while those at the cell rear disassemble, allowing detachment and forward movement. Their dynamics are controlled by a number of regulatory factors, occurring on both cell-wide and adhesion-level scales. The coordination of these regulatory factors is complex, but insights about their dynamics can be gained from the use of mathematical modeling techniques which integrate many of these components together
Bimodality in adhesion dynamics in wild type cells is due to bistability dictated by the initial level of phosphopaxillin
Summary
Cell migration is key to proper development and maintenance of physiological processes such as embryogenesis, axonal outgrowth in neurons, and wound healing [1,2,3,4,5]. To identify key factors that lead to these physiological and pathological functions, a better understanding of the biochemical regulatory pathways governing the dynamics of motility is required. Polarization, for example, has historically been attributed to a cell-wide gradient in the activities of the Rho family of GTPases, including Cdc, Rac (Rac), and RhoA (Rho), and their cycling between the cytoplasm and membrane binding [8,9,12,13,14,15]. The activities of Cdc and Rac, known to promote actin polymerization, membrane protrusion and membrane ruffling [16,17,18,19,20], are thought to be high at the cell front compared to the rear, whereas the activity of RhoA, responsible for actomyosin contraction, is low at the cell front and high at the rear [8,12,13,14]
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