Galloni, C; (2016) Arp3 isoforms differentially regulate the activity of the Arp2/3 complex. Doctoral thesis , UCL (University College London).

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Abstract

Branched actin networks generated by the Arp2/3 complex are essential for many different cellular processes, including cell motility and invadopodium formation. In addition, some intracellular pathogens, such as vaccinia virus, use Arp2/3-driven actin nucleation to enhance their spread. The Arp2/3 complex consists of seven evolutionary conserved proteins (Arp2, Arp3 and ARPC1-5). In higher eukaryotes, ARPC1 and ARPC5 are represented by two isoforms that are ~67% identical. Recent studies in HeLa cells revealed that ARPC1 and ARPC5 isoforms differentially regulate the actin-based motility of vaccinia virus by directly impacting on the nucleation activity of the Arp2/3 complex and the disassembly of the branched actin network. These studies revealed that the Arp2/3 complex can assemble in four different combinations with distinct properties. This situation is likely to be more complex, as Arp3 also has three isoforms, referred as Arp3, Arp3B and Arp3C. My studies have sought to investigate whether Arp3 isoforms interact with the other subunits and isoforms of the complex and impact on Arp2/3-dependent vaccinia virus motility. Pulldown experiments revealed that Arp3 and Arp3B, but not Arp3C, assemble into Arp2/3 complexes. Depletion of Arp3 leads to short actin tails, while loss of Arp3B induces to long tails. In addition, viral particles move faster when Arp3B is depleted. The amino acid differences between Arp3 and Arp3B are equally distributed throughout both proteins. By constructing hybrids, I have mapped the residues responsible for the different activities of Arp3 and Arp3B. Taking into account the two ARPC1 and ARPC5 isoforms, my studies demonstrate that eight different combinations of Arp2/3 complexes with unique ability to regulate actin-based motility of vaccinia virus exist. Finally, to further interrogate whether Arp2/3 complex combinations are better suited to regulate specific cellular processes, I examined the impact of Arp3 and Arp3B depletion on invadopodium formation and two-dimensional cell migration.

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