Liprin-α controls stress fiber formation by binding to mDia and regulating its membrane localization

Abstract

SummaryRegulation of the actin cytoskeleton is crucial for cell morphology and migration. mDia is an actin nucleator that produces unbranched actin filaments downstream of Rho. However, the mechanisms by which mDia activity is regulated in the cell remain unknown. We pulled down Liprin-a as an mDia-binding protein. The binding is mediated through the central region of Liprin-a and through the Nterminal Dia-inhibitory domain (DID) and dimerization domain (DD) of mDia. Liprin-a competes with Dia autoregulatory domain (DAD) for binding to DID, and binds preferably to the open form of mDia. Overexpression of a Liprin-a fragment containing the mDiabinding region decreases localization of mDia to the plasma membrane and attenuates the Rho-mDia-mediated formation of stress fibers in cultured cells. Conversely, depletion of Liprin-a by RNA interference (RNAi) increases the amount of mDia in the membrane fraction and enhances formation of actin stress fibers. Thus, Liprin-a negatively regulates the activity of mDia in the cell by displacing it from the plasma membrane through binding to the DID-DD region.

Structural and Biochemical Basis for the Inhibitory Effect of Liprin-α3 on Mouse Diaphanous 1 (mDia1) Function

“…1A). To thermodynamically characterize the mDia1 liprin-␣3 interaction, we started with the liprin-␣3 fragment 457-737 (Lip(457-737)) defined by Sakamoto et al (29). Lip(457-737) associates with the N-terminal fragment of mDia1, mDia N CC (aa 69 -570), with an affinity of 5.9 M, as shown by ITC ( Table 1).…”

“…This reflects the mechanistic relevance of the N-terminal region contacting the mDia N ID/DD. Notably, Sakamoto et al (29) showed a reduction in cellular F-actin for endogenous full-length liprin-␣3 by siRNA knockdown in HeLa cells. However, we could not observe this overexpressing full-length liprin-␣3 in HeLa cells.…”

“…Recently, Sakamoto et al (29) identified liprin-␣3 as a direct mDia1 binding partner. Liprins can be found in vertebrates, flies, and worms (30).…”

“…The expression of this liprin-␣3 domain in HeLa cells displaces mDia1 from the plasma membrane and reduces actin stress fiber formation (29). Binding of active RhoA is not sufficient to fully activate formins, suggesting that additional events, such as post-translational modifications or binding to proteins and phospholipids, are needed (22,43).…”

Inverted Formin 2 Regulates Actin Dynamics by Antagonizing Rho/Diaphanous-related Formin Signaling

“…46 After starvation in medium containing 0.5% FBS overnight, the cells were scraped and homogenized in cold homogenization buffer (10 mM Tris-HCl, pH 7.5; 1 mM EGTA; 1 mM MgCl 2 ) supplemented with complete protease inhibitor cocktail (Roche LTD). Cells were disrupted by passing through a 22-gauge needle, and lysates were clarified by centrifugation at 4300 g for 10 minutes.…”