Ena/VASP proteins in cell motility and adhesion

• Applicant: Professor Dr. Jan Faix
• Subject Area: Cell Biology
• Term: from 2014 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 264240917

Cells exploit the power of actin polymerization for the formation of protruding membrane sheets filled with a dense actin filament network at the leading edge referred to as lamellipodia to drive cell migration. According to our current knowledge, actin nucleation in lamellipodia and ruffles is mainly accomplished by the Arp2/3-complex, while subsequent elongation of actin filaments beneath the plasma membrane is driven by actin filament-elongating proteins such as formins or Enabled/vasodilator-stimulated phosphoproteins (Ena/VASP). Ena/VASP proteins are a structurally conserved family, which are highly expressed in motile cells. Vertebrates contain three VASP-related proteins Mena (mouse Ena), VASP and EVL (Ena-VASP-like), which all were shown to localize in sites of active actin assembly such as focal adhesions, the tips of lamellipodia or filopodia. They are implicated in a variety of fundamental cellular processes including axon guidance, cell migration and dissemination of pathogens. Seminal studies carried out with mouse embryonic fibroblasts (MEF) devoid of Mena and VASP (MVD7), additionally assumed to lack detectable levels of EVL, found no significant changes in cell adhesion, but surprisingly reported faster protrusion rates and increased cell motility. However, this view has been challenged by our recent work, which contrary to expectations, revealed substantial expression of EVL in this cell line. In light of these finding and based on the comparable biochemical activities of all three Ena/VASP isoforms catalyzing tethered and processive actin filament elongation in vitro, we assume to observe marked defects in the absence of Ena/VASP proteins on cell migration and the formation of adhesion sites. For that reason, the physiological functions of Ena/VASP must be revisited and carefully addressed in cells lacking all three isoforms. We therefore intend to disrupt the EVL gene in MVD7 cells through genome editing employing zinc finger nucleases to obtain bona fide Ena/VASP-null cells. This technology will also be employed to disrupt all three Ena/VASP members in the highly motile and widely used B16-F1 mouse melanoma cells. The objective of this work is to assess the precise physiological roles of Ena/VASP proteins in lamellipodium architecture and protrusion as well as in cell adhesion, presumably linked to their tight interaction with the focal adhesion protein vinculin, to better understand the molecular mechanisms underlying cell migration.

• DFG Programme: Research Grants