Abstract
Heterodimeric capping protein (CP) binds the rapidly growing barbed ends of actin filaments and prevents the addition (or loss) of subunits. Capping activity is generally considered to be essential for actin-based motility induced by Arp2/3 complex nucleation. By stopping barbed end growth, CP favors nucleation of daughter filaments at the functionalized surface where the Arp2/3 complex is activated, thus creating polarized network growth, which is necessary for movement. However, here using an in vitro assay where Arp2/3 complex-based actin polymerization is induced on bead surfaces in the absence of CP, we produce robust polarized actin growth and motility. This is achieved either by adding the actin polymerase Ena/VASP or by boosting Arp2/3 complex activity at the surface. Another actin polymerase, the formin FMNL2, cannot substitute for CP, showing that polymerase activity alone is not enough to override the need for CP. Interfering with the polymerase activity of Ena/VASP, its surface recruitment or its bundling activity all reduce Ena/VASP's ability to maintain polarized network growth in the absence of CP. Taken together, our findings show that CP is dispensable for polarized actin growth and motility in situations where surface-directed polymerization is favored by whatever means over the growth of barbed ends in the network.
Highlights
The discovery two decades ago of a mix of purified proteins capable of sustaining actin-based motility in the test tube was a breakthrough for understanding how actin dynamics drove movement [1]
When formin is immobilized on a surface, formin’s mechanism of action means that filament growth is coupled to the surface, actin polymerization is polarized, and propulsion occurs without the need for capping protein (CP), as opposed to motility based on nucleation by the Arp2/3 complex
Employing an in vitro system to address this question, we fortuitously observed that the presence of Ena/VASP made CP unnecessary for polarized actin growth and movement, even though actin polymerization nucleation was occurring via the Arp2/3 complex
Summary
Our in vitro system consisted of profilin-actin, CP, the Arp2/ 3 complex and 1-mm diameter beads, coated with the pVCA domain of the human WASP protein, an activator of the Arp2/ 3 complex. We used the mouse Ena/VASP protein VASP, which increased barbed end elongation by about 35% as measured by TIRF microscopy: at 1.5 mM profilin-actin, elongation speeds were 1.3 6 0.4 mm/min without VASP and 1.8 6 0.4 mm/min in the presence of VASP (p = 0.002) (Fig. S1) This is in the same range as the 20% enhancement reported for human VASP under comparable salt conditions, using a similar TIRF method [18] much smaller than the 7-fold enhancement observed for Dictyostelium VASP [17]. Ena/VASP proteins in the presence of CP have been studied extensively using bead or bead-type systems, and found to enhance Arp2/3 complex-based bead motility [20, 28,29,30] In these cases Ena/VASP was recruited to the surface, where it exercised its barbed end elongation enhancement activity on freshly-nucleated barbed ends since all other barbed ends were capped by CP. In the absence of CP, VASP would enhance barbed end elongation everywhere in the network, not just at the surface, so it was perplexing as to how VASP in the absence of CP could rescue polarized actin growth and movement
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