We developed a proteomics approach to profile global FA composition changes in response to inhibition of myosinII activity to better understand myosinII-driven FA maturation. This method provides a powerful approach for dissecting FA composition changes in response to different stimuli or molecular perturbations25
. Our FA proteome contained 905 proteins, supporting the notion that FA are complex organelles. Whether all these proteins participate in FA function is unknown. More importantly, we show that protein abundance of half (459/905) the FA proteome is altered by myosinII activity and half remain constant as core FA components or non-specific contaminants. Most FA compositional change is likely not caused by myosinII ATPase activity acting directly on FA proteins or indicative of protein tension sensitivity per se
, but reflects downstream effects of myosinII-mediated mechanotransduction. Our results define the myosinII-responsive FA proteome, indicating substantial compositional differences between immature and mature FA. Inhibiting myosinII reduced FA abundance of 73% of the myosinII-responsive FA proteome, supporting the established role of myosinII as an enhancer of FA protein recruitment. However, surprisingly, inhibition of myosinII induced FA enrichment of 27% of the myosinII-responsive FA proteome, indicating for the first time that myosinII is an important negative regulator of FA protein recrutiment.
FA composition in myosinII-inhibited cells likely reflects that in nascent FA that form and turn over rapidly during lamellipodial protrusion in the absence of local myosinII activity66,67
, and which are critical to cell migration17
. Though our controls include FA of various maturation states, their FA composition is likely dominated by larger and more abundant matured FA in the lamella and cell center which are disassembling or specialized for ECM remodeling. Whether similar FA compositional changes occur is being investigated.
Our results suggest a model in which myosinII activity regulates FA maturation and turnover by collectively modulating the FA abundance of protein functional modules mediating specialization of FA for cell migration. FA abundance of inside-out integrin activation, (talins, kindlins) was not myosinII-sensitive, supporting the notion that nascent FA formation is contractility-independent17
. Our results suggest that, independent of contractility, activated integrin complexes recruit Rac1 activators (β-PIX, EPS8, MIF, PKA) and Rac1 effectors (IRSP53, N-WASP) and their targets involved in dendritic actin treadmilling (Arp2/3, cofilin-1, CAP-1). This suggests a positive feedback mechanism in which lamellipodial protrusion is coupled to formation of nascent FA that recruit a Rac1 regulatory module which propagates further protrusion and nascent FA formation to drive cell migration.
Nascent FA subjected to myosinII contractility exhibit both dissociation and recruitment of protein functional modules that mediate FA strengthening, stress fiber formation, and mature FA disassembly (). We find that myosinII promotes enrichment of proteins that strengthen the integrin-actin linkage (migfilin, filamins, vinculin). At the same time, contractility drives FA dissociation of the Rac1 regulatory module to terminate the nascent FA turnover loop and allow FA maturation. MyosinII-driven loss of the Rac1 module is mirrored by recruitment of RhoA, its activators (GEF-H1, TRIP6, testin), and downstream targets and stress fiber proteins including actin-bundling proteins (α-actinin, supervillin, formin-2, and synaptopodin-2) and cytoskeletal adapters (PDLI1, PDLI4, PDLI5, PDLI7, zyxin, and FHL2). Contraction also drives FA disassembly15
by recruiting calpain and clathrin-dependent and caveolin-mediated endocytosis protein modules..
To support the role of myosinII in negative regulation of a Rac1 regulatory module in FA, we focused on the Rac GEF β-PIX, and examined the myosinII-dependence of its role in nascent FA turnover and lamellipodial protrusion63,64
. We find that β-PIX concentrates in nascent FA64
, and dissociates from FA as they mature. We show that β-PIX is required for Rac1 activity and lamellipodial protrusion induced by myosinII inhibition, suggesting that β–PIX may enhance Rac1 activity locally in myosinII-free cell regions such as lamellipodia. Finally, we show that β-PIX is required for rapid nascent FA turnover, implicating it in negative regulation of FA maturation and enhancement of cell migration.