Deciphering the molecular mechanism regulating the subcellular targeting of Ena/VASP proteins

• Applicant: Professor Dr. Jan Faix
• Subject Area: Cell Biology ; Biochemistry
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 560246590

Ena/VASP proteins act as actin polymerases that drive the processive elongation of filament barbed ends in membrane protrusions such as filopodia and lamellipodia or in focal adhesions. Given their role in promoting cell migration, it is therefore not surprising that Ena/VASP proteins have also been implicated in various cancers. Thus, it is instrumental to understand the function and regulation of these intriguing proteins at the molecular level. Although, we have previously dissected the molecular mechanisms of VASP-mediated actin assembly and recently found that unconventional myosin X mediates Ena/VASP clustering, which is instrumental for processive actin assembly in the presence of heterodimeric capping protein, the regulation of the subcellular targeting of Ena/VASP proteins by phosphorylation has remained poorly understood. This gap in knowledge likely arises from previous challenges, such as the use of wild-type or partially-depleted cells as transfection hosts, and the tendency of Ena/VASP proteins to hetero-oligomerize. In our preliminary work using authentic Ena/VASP-deficient B16-F1 cells, we found that phosphorylation at Y16, but not of the predicted residues Y39 or S157, regulates the accumulation of VASP and Evl at lamellipodia tips and focal adhesions. To sketch a resilient working model of the underlying molecular mechanism regulating the subcellular targeting of Ena/VASP proteins, we will extend our analysis to Mena, followed by a detailed investigation of VASP phosphorylation in processes like 2D-cell migration, cell protrusion, actin turnover, and microspike formation. Furthermore, we aim to determine the affinities of Listeria ActA for wild-type, non-phosphorylatable and phosphomimetic VASP mutants by analytical centrifugation, and explore the recruitment and VASP-mediated actin assembly on ActA-derivatized beads by TIRF imaging in vitro. In parallel, we will also test the effects of these mutants on actin assembly on the surface of Listeria in vivo. Subsequently, we aim to determine the contribution of distinct kinases for Ena/VASP phosphorylation at specific tyrosine residues. Finally, we will use the C. elegans model and show that the mechanism is conserved across species. We are confident that this work will have significant and far-reaching implications for our understanding of the molecular mechanism underlying Ena/VASP recruitment to sites of active actin assembly, which will not only be valuable for basic research, but could also be pivotal in advancing our ability to develop novel therapeutic compounds, and target cancer cells in translational science.

• DFG Programme: Research Grants