Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly.
Vasodilator‐stimulated phosphoprotein (VASP) family proteins play a crucial role in mediating the actin network architecture in the cytoskeleton. The Ena/VASP homology 2 (EVH2) domain in each of the four identical arms of the tetrameric VASP consists of a loading poly‐Pro region, a G‐actin‐binding domain (GAB), and an F‐actin‐binding domain (FAB). Together, the poly‐Pro, GAB, and FAB domains allow VASP to bind to sides of actin filaments in a bundle, and recruit profilin–G‐actin to processively elongate the filaments. The atomic resolution structure of the ternary complex, consisting of the loading poly‐Pro region and GAB domain of VASP with profilin–actin, has been solved over a decade ago; however, a detailed structure of the FAB‐F‐actin complex has not been resolved to date. Experimental insights, based on homology of the FAB domain with the C region of WASP, have been used to hypothesize that the FAB domain binds to the cleft between subdomains 1 and 3 of F‐actin. Here, in order to develop our understanding of the VASP–actin complex, we first augment known structural information about the GAB domain binding to actin with the missing FAB domain‐actin structure, which we predict using homology modeling and docking simulations. In earlier work, we used mutagenesis and kinetic modeling to study the role of domain‐level binding–unbinding kinetics of Ena/VASP on actin filaments in a bundle, specifically on the side of actin filaments. We further look at the nature of the side‐binding of the FAB domain of VASP at the atomistic level using our predicted structure, and tabulate effective mutation sites on the FAB domain that would disrupt the VASP–actin complex. We test the binding affinity of Ena with mutated FAB domain using total internal reflection fluorescence microscopy experiments. The binding affinity of VASP is affected significantly for the mutant, providing additional support for our predicted structure.
more » « less- PAR ID:
- 10501795
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Cytoskeleton
- Volume:
- 81
- Issue:
- 8
- ISSN:
- 1949-3584
- Format(s):
- Medium: X Size: p. 382-392
- Size(s):
- p. 382-392
- Sponsoring Org:
- National Science Foundation
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