Abstract
Chloride intracellular channel 4 (CLIC4) is a cytosolic protein implicated in diverse actin-based processes, including integrin trafficking, cell adhesion, and tubulogenesis. CLIC4 is rapidly recruited to the plasma membrane by RhoA-activating agonists and then partly colocalizes with β1 integrins. Agonist-induced CLIC4 translocation depends on actin polymerization and requires conserved residues that make up a putative binding groove. However, the mechanism and significance of CLIC4 trafficking have been elusive. Here, we show that RhoA activation by either lysophosphatidic acid (LPA) or epidermal growth factor is necessary and sufficient for CLIC4 translocation to the plasma membrane and involves regulation by the RhoA effector mDia2, a driver of actin polymerization and filopodium formation. We found that CLIC4 binds the G-actin–binding protein profilin-1 via the same residues that are required for CLIC4 trafficking. Consistently, shRNA-induced profilin-1 silencing impaired agonist-induced CLIC4 trafficking and the formation of mDia2-dependent filopodia. Conversely, CLIC4 knockdown increased filopodium formation in an integrin-dependent manner, a phenotype rescued by wild-type CLIC4 but not by the trafficking-incompetent mutant CLIC4(C35A). Furthermore, CLIC4 accelerated LPA-induced filopodium retraction. We conclude that through profilin-1 binding, CLIC4 functions in a RhoA–mDia2–regulated signaling network to integrate cortical actin assembly and membrane protrusion. We propose that agonist-induced CLIC4 translocation provides a feedback mechanism that counteracts formin-driven filopodium formation.
Highlights
Chloride intracellular channel 4 (CLIC4) is a cytosolic protein implicated in diverse actin-based processes, including integrin trafficking, cell adhesion, and tubulogenesis
We found that CLIC4 binds the G-actin– binding protein profilin-1 via the same residues that are required for CLIC4 trafficking
Using HeLa cells, we found that CLIC4 is rapidly recruited to the plasma membrane by G12/13–RhoA-coupled receptor agonists such as lysophosphatidic acid (LPA) and, somewhat unexpectedly, by a prototypic receptor tyrosine kinase ligand, notably epidermal growth factor (EGF) (Fig. 1A and supporting Movies S1 and S2)
Summary
Rapid but transient translocation of CLIC4 to the plasma membrane induced by LPA and EGF. LPA- and EGF-induced CLIC4 translocation to the plasma membrane was strongly reduced upon profilin-1 depletion (Fig. 5, B and C). The filopodia induced by mDia were decreased in these cells (Fig. S5) These results support the view that profilin-1 binding connects CLIC4 to RhoA–mDia regulated actin dynamics. LPA treatment reduced filopodium length in both control and CLIC4-depleted cells (Fig. 7G) This is consistent with LPA promoting actomyosin tension, which has a net negative effect on formins [33] and boosts the actin retrograde flow [37, 38]. Given that the activities of Cdc (Fig. 3C) and RhoA (Fig. 2, A–C), key regulators of actomyosin-based tension and F-actin retrograde flow [37,38,39], are independent of CLIC4 in LPA-treated cells, we conclude that CLIC4 primarily controls formins at the tip of filopodia. Our findings suggest that recruitment of CLIC4 to the plasma membrane serves to suppress the formation of integrin-regulated filopodia, along with promoting integrin trafficking [10]
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