Supervillin couples myosin-dependent contractility to podosomes and enables their turnover

Abstract

SummaryPodosomes are actin-rich adhesion and invasion structures. Especially in macrophages, podosomes exist in two subpopulations, large precursors at the cell periphery and smaller podosomes (successors) in the cell interior. To date, the mechanisms that differentially regulate these subpopulations are largely unknown. Here, we show that the membrane-associated protein supervillin localizes preferentially to successor podosomes and becomes enriched at precursors immediately before their dissolution. Consistently, podosome numbers are inversely correlated with supervillin protein levels. Using deletion constructs, we find that the myosin II regulatory Nterminus of supervillin ] is crucial for these effects. Phosphorylated myosin light chain (pMLC) localizes at supervillinpositive podosomes, and time-lapse analyses show that enrichment of GFP-supervillin at podosomes coincides with their coupling to contractile myosin-IIA-positive cables. We also show that supervillin binds only to activated myosin IIA, and a dysregulated N-terminal construct [SV(1-830)] enhances pMLC levels at podosomes. Thus, preferential recruitment of supervillin to podosome subpopulations might both require and induce actomyosin contractility. Using siRNA and pharmacological inhibition, we demonstrate that supervillin and myosin IIA cooperate to regulate podosome lifetime, podosomal matrix degradation and cell polarization. In sum, we show here that podosome subpopulations differ in their molecular composition and identify supervillin, in cooperation with myosin IIA, as a crucial factor in the regulation of podosome turnover and function.

Myosin II Activity Softens Cells in Suspension

“…S6) has been reported in previous studies using magnetic twisting cytometry (58). This effect is not likely due to a low expression level of myosin IIA in macrophages (59,60). Instead, the different expression levels of nesprin 1 and 2, SUN1, vimentin, plectin, and lamin A/C in macrophages compared to neutrophils and monocytes (61,62), as well as a more adherent nature of macrophages that reside normally in the tissue, might play a role.…”

Signaling inputs to invadopodia and podosomes

“…Likewise, we found that ECM rigidity controls the activity, but not the formation of, invadopodia puncta . In addition, several studies have shown that myosin-mediated contractility can induce the turnover of podosomes (Bhuwania et al, 2012;van Helden et al, 2008). Other studies have directly examined the role of adhesion molecules, including integrins and downstream adhesion components.…”

Modulation of T cell signaling by the actin cytoskeleton

“…Actin retrograde motion is likely to be driven by the polymerization of its filaments (Henson et al, 1999). Additionally, the actin-associated molecular motor myosin II has been found to contribute to actin flow by exerting contractile forces on actin filaments (Vicente-Manzanares et al, 2009;Betapudi, 2010;Arii et al, 2010;Bhuwania et al, 2012). Nonmuscle myosin IIA is the dominantly expressed isoform of myosin II in T cells (Jacobelli et al, 2004).…”