Activation of Rac by Asef2 promotes myosin II-dependent contractility to inhibit cell migration on type I collagen

Abstract

SummaryNon-muscle myosin II (MyoII) contractility is central to the regulation of numerous cellular processes, including migration. Rho is a well-characterized modulator of actomyosin contractility, but the function of other GTPases, such as Rac, in regulating contractility is currently not well understood. Here, we show that activation of Rac by the guanine nucleotide exchange factor Asef2 (also known as SPATA13) impairs migration on type I collagen through a MyoII-dependent mechanism that enhances contractility. Knockdown of endogenous Rac or treatment of cells with a Rac-specific inhibitor decreases the amount of active MyoII, as determined by serine 19 (S19) phosphorylation, and negates the Asef2-promoted increase in contractility. Moreover, treatment of cells with blebbistatin, which inhibits MyoII activity, abolishes the Asef2-mediated effect on migration. In addition, Asef2 slows the turnover of adhesions in protrusive regions of cells by promoting large mature adhesions, which has been linked to actomyosin contractility, with increased amounts of active b1 integrin. Hence, our data reveal a new role for Rac activation, promoted by Asef2, in modulating actomyosin contractility, which is important for regulating cell migration and adhesion dynamics.

The Rho family GEF Asef2 regulates cell migration in three dimensional (3D) collagen matrices through myosin II

“…We had previously shown that these Asef2 shRNAs decrease endogenous expression of Asef2 by approximately 65% in HT1080 cells. 12,13 Transfection of Asef2 shRNA 1 or Asef2 shRNA 2 into HT1080 cells caused a significant increase in the migration speeds of these cells in 3D collagen I matrices compared to that observed with a non-targeting shRNA (NT shRNA) (Fig. 2C), indicating that knockdown of endogenous Asef2 promotes migration in 3D collagen I matrices.…”

“…These results collectively show that Asef2 inhibits migration in 3D collagen I matrices, which is consistent with our previous observation on 2D collagen I substrates. 13 We have previously shown that Asef2 hinders migration on 2D collagen I through a mechanism that is dependent on Asef2 augments Rac activity in 3D collagen I matrices. Left, GFP and GFP-Asef2 cells were transfected with FLAG-Rac1 cDNA.…”

“…1). Therefore, we used these devices to examine the migration of HT1080 cells stably expressing GFP or GFP-Asef2 (less than 3-fold over endogenous) 12,13 ( Fig. 2A) in 3D collagen I matrices.…”

“…[12][13][14][15] Our previous work demonstrated that Asef2 impairs cell migration on 2D collagen I by increasing activation of Rac, which subsequently enhances MyoII activity. 13 MyoII is an actin motor protein whose major function in cells is to generate contraction by sliding actin filaments relative to each other. MyoII contractility (activity) is controlled by phosphorylation within its regulatory light chains (RLC).…”

“…17,18 Although MyoII activity is classically thought to be mediated by Rho signaling, 19 our previous data reveal that activation of Rac by Asef2 modulates MyoII activity, and this is an important regulatory mechanism for cell migration on 2D substrates. 13 However, the role of Asef2 and the contribution of Asef2-Rac-MyoII signaling to cell migration in 3D environments is unknown.…”