Analysis of the cellular properties of Arp3B containing Arp2/3 complexes

Abstract

Branched actin networks generated by the Arp2/3 complex are essential for many different cellular processes, including cell motility, endocytosis and phagocytosis. In addition, a number of intracellular pathogens such as Listeria, Shigella and Vaccinia virus hijack the power of Arp2/3-driven actin polymerization to enhance their cell-to-cell spread. The Arp2/3 complex is composed of seven subunits (Arp2, Arp3, ArpC1-5). Interestingly, in humans, Arp3, ArpC1 and ArpC5 exist as two different isoforms (Arp3/Arp3B, ArpC1A/ArpC1B and ArpC5/ArpC5L). This raises the possibility that Arp2/3 is actually a family of 8 complexes with different interactions and properties. Using Arp2/3-driven motility of Vaccinia as a model system, the Way Lab demonstrated that Arp3 and Arp3B impart different properties to the Arp2/3 complex, despite the proteins being 91% identical. Depletion of Arp3B in HeLa cells enhances branched network production whereas its over-expression suppresses it. By analysing a series of Arp3:Arp3B hybrids, this suppressive behaviour was shown to be dependent on methionine 293 (Met293) of Arp3B. In my thesis, I have examined how Arp3 and Arp3B impact on the stability of the branched actin networks they generate and the role of Arp3B Met293 in the underlying molecular mechanism. Using photoactivatable variants of Arp3 and Arp3B, I demonstrated that Arp3B containing complexes have a significantly faster turnover within the branched actin network. This could be due to oxidation of Met293 by the MICAL monooxygenases, which are known for oxidising methionine 44 and 47 in actin to promote filament disassembly. Consistent with this, depletion of MICAL2 slows down Arp3B turnover and abolishes its suppressive action towards branched network formation. Moreover, mimicking constitutive oxidation by mutating residue 293 to glutamine in either Arp3 or Arp3B enhances disassembly of branched actin networks. In addition, GFP-tagged MICAL2, but not MICAL1, localises to the branched network and this localisation requires Coronin 1C. My collective observations are consistent with a model in which Arp3B-containing Arp2/3 complexes promote disassembly of branched actin networks via a MICAL2/Coronin 1C-dependent mechanism. To explore the physiological function of Arp3B, Arp3B knock-out mice were generated and analysed for potential phenotypes.

Date of Award	1 Aug 2021
Original language	English
Awarding Institution	King's College London
Supervisor	Mathias Gautel (Supervisor) & Michael Way (Supervisor)