Distinct VASP tetramers synergize in the processive elongation of individual actin filaments from clustered arrays

Abstract

Ena/VASP proteins act as actin polymerases that drive the processive elongation of filament barbed ends in membrane protrusions or at the surface of bacterial pathogens. Based on previous analyses of fast and slow elongating VASP proteins by in vitro total internal reflection fluorescence microscopy (TIRFM) and kinetic and thermodynamic measurements, we established a kinetic model of Ena/VASP-mediated actin filament elongation. At steady state, it entails that tetrameric VASP uses one of its arms to processively track growing filament barbed ends while three G-actin-binding sites (GABs) on other arms are available to recruit and deliver monomers to the filament tip, suggesting that VASP operates as a single tetramer in solution or when clustered on a surface, albeit processivity and resistance toward capping protein (CP) differ dramatically between both conditions. Here, we tested the model by variation of the oligomerization state and by increase of the number of GABs on individual polypeptide chains. In excellent agreement with model predictions, we show that in solution the rates of filament elongation directly correlate with the number of free GABs. Strikingly, however, irrespective of the oligomerization state or presence of additional GABs, filament elongation on a surface invariably proceeded with the same rate as with the VASP tetramer, demonstrating that adjacent VASP molecules synergize in the elongation of a single filament. Additionally, we reveal that actin ATP hydrolysis is not required for VASP-mediated filament assembly. Finally, we show evidence for the requirement of VASP to form tetramers and provide an amended model of processive VASPmediated actin assembly in clustered arrays.actin | Ena/VASP | TIRF | cluster | formin

Computational modeling highlights the role of the disordered Formin Homology 1 domain in profilin‐actin transfer

“…The latter gives a reference for the effect of intermolecular attractive or repulsive interactions. We show results using profilin II but similar results apply for the more highly charged profilin I, consistent with there being no difference between the two in mDia1-mediated actin polymerization [55]. To ensure sufficient sampling of bound molecule distributions, data were collected for at least 5 ls simulation time for occupancy greater than 2, and at least 10 ls simulation time for other simulations.…”

Computational modeling highlights disordered Formin Homology 1 domain’s role in profilin-actin transfer

“…The latter gives a reference for the effect of intermolecular attractive or repulsive interactions. We show results using profilin II but similar results apply for the more highly charged profilin I, consistent with there being no difference between the two in mDia1-mediated actin polymerization [12]. To ensure sufficient sampling of bound molecule distributions, data were collected for at least 5ms simulation time for occupancy greater than 2, and at least 10ms simulation time for other simulations.…”

Single molecule dynamics of Dishevelled at the plasma membrane and Wnt pathway activation

“…Can we say something about whether association of Dvl subunits itself is sufficient to activate the pathway? To answer this question, we generated a series of artificial Dvl oligomers by replacing the DIX domain of Dvl2 with a set of constructs containing unrelated oligomerization motifs, artificially designed to generate specific oligomer states (36). The set of Dvl oligomeric constructs are named Dimer, Trimer, Tetramer, and Hexamer which correspondingly contain 2, 3, 4, and 6 Dvl molecules in stable complexes (Fig.…”

“…Dvl2 mutants was made by using Agilent QuickChange II mutagenesis kit (#200523) on the basis of Dvl2-mEGFP plasmid. For the Dvl2 oligomer mutations, all oligomer sequences are from table S1 of (36). SAM domain sequence are from Arabidopsis thaliana (SAM1) and Ginkgo biloba (SAM2) (37).…”