Supervillin slows cell spreading by facilitating myosin II activation at the cell periphery

SummaryRecent evidence implicates the endosomal sorting complex required for transport (ESCRT) in the regulation of epithelial polarity in Drosophila melanogaster, but the mechanisms responsible for this action remain unclear. Here we show that ESCRTs determine cell orientation during directed migration in human fibroblasts. We find that endosomal retention of a5b1 integrin and its downstream signaling effector Src in ESCRT-depleted cells is accompanied by the failure to activate myosin light chain kinase (MLCK), which thereby cannot phosphorylate myosin regulatory light chain (MRLC). Using this mechanism, ESCRT-depleted fibroblasts fail to orient their Golgi complex to undergo directional migration and show impaired focal adhesion turnover and increased spreading on fibronectin. Consistent with these findings, expression of a phosphomimetic mutant of MRLC in ESCRT-depleted cells restores normal phenotypes during cell spreading and orientation of the Golgi. These results suggest that, through their role in regulating integrin trafficking, ESCRTs regulate phosphorylation of MRLC and, subsequently, Golgi orientation and cell spreading.

Section: Escrts Positively Regulate Myosin Light Chain Functions Throsupporting

confidence: 70%

The ESCRT machinery mediates polarization of fibroblasts through regulation of myosin light chain

Lobert

Stenmark

2012

“…Previous studies have reported that the association of the Nterminus of SVIL with myosin II can induce the phosphorylation of RLC at Ser19 to activate myosin II for contraction (Takizawa et al, 2007). Activation of myosin II at the cleavage furrow is required for the promotion of constriction of the contractile ring (Asano et al, 2009;Dean and Spudich, 2006;Komatsu et al, 2000); thus, we speculated that the aberrant furrowing in SVILknockdown cells was associated with reduced activity of myosin II.…”

Section: Svil Controls Activation Of Myosin II At the Cleavage Furrowmentioning

confidence: 87%

“…In this screen, we found that an actin/myosin II-binding protein, supervillin (SVIL), was required for the completion of cytokinesis. SVIL consists of an N-terminal F-actin-and myosin-II-binding regions and a C-terminal region, similar to villin/gelsolin proteins, and is involved in cell spreading, cell-substrate attachment and matrix degradation (Bhuwania et al, 2012;Chen et al, 2003;Crowley et al, 2009;Fang et al, 2010;Pestonjamasp et al, 1997;Takizawa et al, 2006;Takizawa et al, 2007). A recent study has shown that SVIL associates with numerous cytokinesis-related proteins and was required for cytokinesis .…”

Section: Introductionmentioning

confidence: 99%

Role of SVIL phosphorylation by PLK1 in myosin II activation and cytokinetic furrowing

Hasegawa

Hyodo

Asano

et al. 2013

SummaryPolo-like kinase 1 (PLK1) is a widely conserved serine/threonine kinase that regulates progression of multiple stages of mitosis. Although extensive studies about PLK1 functions during cell division have been performed, it is still not known how PLK1 regulates myosin II activation at the equatorial cortex and ingression of the cleavage furrow. In this report, we show that an actin/myosin-IIbinding protein, supervillin (SVIL), is a substrate of PLK1. PLK1 phosphorylates Ser238 of SVIL, which can promote the localization of SVIL to the central spindle and association with PRC1. Expression of a PLK1 phosphorylation site mutant, S238A-SVIL, inhibited myosin II activation at the equatorial cortex and induced aberrant furrowing. SVIL has both actin-and myosin-II-binding regions in the N-terminus. Expression of DMyo-SVIL (deleted of the myosin-II-binding region), but not of DAct-SVIL (deleted of actin-binding region), reduced myosin II activation and caused defects in furrowing. Our study indicates a possible role of phosphorylated SVIL as a molecular link between the central spindle and the contractile ring to coordinate the activation of myosin II for the ingression of the cleavage furrow.

“…Supervillin is a member of the villin and gelsolin family, and associates tightly with cholesterol-rich membrane signaling domains (Nebl et al, 2002;Pestonjamasp et al, 1997). A multi-domain protein, supervillin interacts with many cytoskeletal proteins, including Factin, myosin II, the long form of myosin light chain kinase (L-MLCK), and as many as five microtubule-dependent motors (Chen et al, 2003;Smith et al, 2010;Takizawa et al, 2007;Takizawa et al, 2006). Supervillin induces L-MLCK-dependent myosin II contractility through its first N-terminal 171 amino acids, whereas residues 342-571 negatively regulate focal adhesions in COS7 cells (Takizawa et al, 2007;Takizawa et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

“…A multi-domain protein, supervillin interacts with many cytoskeletal proteins, including Factin, myosin II, the long form of myosin light chain kinase (L-MLCK), and as many as five microtubule-dependent motors (Chen et al, 2003;Smith et al, 2010;Takizawa et al, 2007;Takizawa et al, 2006). Supervillin induces L-MLCK-dependent myosin II contractility through its first N-terminal 171 amino acids, whereas residues 342-571 negatively regulate focal adhesions in COS7 cells (Takizawa et al, 2007;Takizawa et al, 2006). Interestingly, supervillin localization at invadopodia is associated with increased matrix degradation by MDB-MB-231 cells (Crowley et al, 2009), and it binds directly to both cortactin and Tks5 (Crowley et al, 2009;Smith et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

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

Bhuwania

Cornfine

Fang

et al. 2012

Self Cite

100

135

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.