Dissecting the role of I-BAR proteins in VASP clustering and actin assembly

• Applicant: Professor Dr. Jan Faix
• Subject Area: Cell Biology ; Biochemistry
• Term: from 2013 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 234826310

Ena/VASP proteins act as actin polymerases that drive the processive elongation of filament barbed ends in membrane protrusions such as filopodia or lamellipodia. Based on biochemical data and in vitro total internal reflection fluorescence microscopy (TIRFM) measurements, we have previously shown 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, formally suggesting that VASP operates as a single tetramer in solution or when firmly clustered on a bead surface, albeit processivity and resistance toward capping protein (CP) differ dramatically between both conditions. Consistently, by variation of the oligomerization state and by increase of the number of GABs on individual polypeptide chains, we recently confirmed that the molecular mechanisms of VASP-mediated actin assembly in bulk solution as compared in static surface-tethered clusters are markedly different. Of note, in the physiological context Ena/VASP proteins operate exclusively in dynamic multi-protein clusters beneath the plasma membrane. Since clustering is central to understand VASP-mediated actin assembly in cells we now intend to advance a significant step further in order to mimic physiological conditions. Thus, in this follow-up proposal we aim to reconstitute and analyze VASP-mediated actin assembly and dynamics of VASP clustering by SH3-domain containing I-BAR proteins of the IRSp53 family in supported lipid bilayers by multicolor TIRF imaging. The I-BAR protein IRSp53 is currently the major candidate to drive clustering of VASP downstream of Cdc42 signaling, but the two other IRSp53-related proteins IRTKS and Pinkbar or other and as yet uncharacterized proteins might also drive or at least assist VASP clustering in actin-assembly complexes at the membrane-cytoskeleton interface. Thus, by the use of pulldowns with suitable Ena/VASP fragments from cell extracts followed by proteomics we additionally aim to identify and characterize novel factors that mediate Ena/VASP-clustering at the membrane-cytosol interface. Comparable to Ena/VASP, IRSp53 has been also implicated in filopodium formation and is thought to contribute to clustering, membrane deformation and filopodial actin filament assembly although a clear molecular understanding of its specific functions in this process remains elusive. The final major objective is therefore the CRISPR/Cas9-mediated knockout of IRSp53-related I-BAR proteins and comprehensive analyses of B16-F1 derived mouse melanoma mutant cells to assess the precise physiological roles of these I-BAR proteins in VASP clustering, actin assembly, formation of migratory cell protrusion and motility.

• DFG Programme: Research Grants