MachActAssembly

Molecular machines must be built by assembly factors before they perform their function. Assembly intermediates are degraded by quality control mechanisms when they cannot reach the fully assembled state due to the absence of a subunit or an assembly factor. Here, we propose to systematically identify assembly factors of three molecular machines that polymerize branched actin: the WAVE, WASH and Arp2/3 complexes, which jointly control the persistence of cell migration. To this end, we will first employ large-scale unbiased interactomics screens with their subunits using yeast two-hybrid (Y2H) screens and proteomics following tandem affinity purification (TAP). To identify direct partners from our hits, we will streamline AlphaFold2-mediated structural predictions by delineating evolutionary conserved domains to increase the speed and accuracy of predictions. The structural models provided by AlphaFold2 will be experimentally validated in Y2H by mutating the bait residues predicted to be critical for the interaction and selecting compensatory mutations using another Y2H screening after random mutagenesis of the prey. Candidate assembly factors will then be identified by functional genomics using a reporter cell line that expresses a subunit of each of these three complexes tagged with a different fluorescent protein. The siRNAs that destabilize one complex, but not all three, are likely to correspond to a specific assembly factor. The expected property of an assembly factor is to produce defects in cell migration (WAVE, Arp2/3) and intracellular trafficking (WASH, Arp2/3) upon knock-down and knock-out, depending on the function of the molecular machinery whose assembly it promotes. In a final section, the interaction and and functional data will be integrated into a structural scenario. Intermediate complexes in the assembly pathway will be characterized using several types of pulse-chase experiments. In the cell, knock-out phenotypes should be rescued by the wild type assembly factor, but not by the mutated versions. Using this combination of global, unbiased approaches, we hope to dissect the assembly pathways of these three molecular machines that control the polymerization of branched actin. This fundamental knowledge could probably be exploited as new ways of controlling these critical molecular machines involved in several human pathologies.