Arp2/3 complex in mammalian cell motility

Suraneni, Praveen K (2012). Arp2/3 complex in mammalian cell motility. Doctor of Philosophy (PhD) thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000ffb5

Abstract

The Arp2/3 complex is the key component of the actin polymerization engine that drives amoeboid cell motility. ARPC3, a component of the Arp2/3 complex, plays a critical role in actin nucleation to generate Y-shaped branches from existing actin filaments. Although the biochemical activity of the Arp2/3 complex has been well characterized, it remains unclear whether the Arp2/3 complex is truly important for leading edge extension in amoeboid form of cell motility. Gene targeting in mouse provides an opportunity to assess these functions in vivo. We have generated ARPC3^+/- mice using a gene-trap ES cell line. Consistent with a published study, ARPC3^-/- mutant embryos survive only to the blastocyst stage. To further investigate the function of the Arp2/3 complex in different motile cells we developed an approach to derive ARPC3^+/+, ARPC3^+/- and ARPC3^-/- ES cells from blastocysts of ARPC3^+/- crosses and differentiate them into fibroblasts cells. Here we address the question, is Arp2/3 complex necessary for lamellipodia extension and fibroblast cell motility? The fibroblast cells differentiated from ARPC3^-/- ES cells looks morphologically different from the ARPC3^+/+ cells with filopodia like structures at the leading edge. Hence, Arp2/3 complex is required for lamellipodia formation in differentiated mouse fibroblasts. We further examined their migration behavior in 2D cultures. ARPC3^-/- fibroblasts display no difference in motility from wild-type fibroblasts and vigorous protrusive activity using filopodia-like structures. However they failed to sustain directional movement, leading to reduced scratch healing efficiency. The mutant fibroblast cells also failed to undergo directional migration towards a chemotaxis gradient. Immunofluorescence studies suggest that the mDia1 formin protein may play a role in nucleating actin assembly in the absence of Arp2/3 to form these filopodia-like protrusions. These results provide strong evidence that the Arp2/3 complex is critical for lamellipodia formation and directional cell migration in mammalian fibroblast cells.