Understanding How the Crowded Interior of Cells Stabilises DNA/DNA and DNA/RNA Hybrids - in Silico Predictions and in vitro Proof

Abstract

The actin cytoskeleton is intimately involved in most cellular functions, including cell motility, endo/exocytosis and intracellular trafficking. These processes are characterized by rapid oscillations of actin polymerization/depolymerization under tight temporal and spatial regulation. Hundreds of G-and F-actinbinding proteins, along with signaling and scaffolding proteins regulate the assembly of actin networks. Among these proteins, filament nucleators play a critical role by determining the time and location for actin polymerization, as well as the specific structures of the actin networks that they generate. Eukaryotic cells and certain pathogens use filament nucleators to stabilize actin nuclei (small oligomers of 2-4 actin subunits), whose formation is rate-limiting. Known filament nucleators include the Arp2/3 complex and its large family of Nucleation Promoting Factors (NPFs), Formins, Spire, Cobl, Lmod, VopL/VopF and TARP. Structural and functional studies are beginning to shed light on the diverse mechanisms used by these molecules to stabilize actin nuclei. Thus, with the exception of Formins known filament nucleators use the WASP-Homology 2 domain (WH2 or W), a small and versatile actin-binding motif, for interaction with actin. A common architecture, found in Spire, Cobl and VopL/VopF, consists of tandem W domains that bind three to four actin subunits to form a nucleus. Structural considerations suggest that NPFs-Arp2/3 complex can also be viewed as a specialized form of tandem W-based nucleator. The nucleation activities of these proteins vary significantly, and the most effective nucleators are not necessarily those with the largest number of W domains. We show that the inter-W linkers play a critical role in determining the nucleation activities of filament nucleators and the structures of the actin nuclei that they generate. Furthermore, we present evidence that a previously neglected factor, oligomerization, is a major determinant of filament nucleation activity and nuclei structure.