Actin Nucleation By ARP-WASP Complex For many cell types, the ability to move across a solid surface is fundamental to their biological function. Certain aspects of cell locomotion, such as the protrusion of the plasma membrane in lamellipodia and filopodia, are driven by the polymerization of actin
cytoskeleton. The actin cytoskeleton is a dynamic filament network that is essential for cell movement during embryo development, polarization, morphogenesis, cell division, and immune system function and in the metastasis of cancer cells. To engage in these complex behaviors, cells must direct actin assembly with a high degree of spatial and temporal resolution in response to extracellular signals (Ref.1). To coordinate these behaviors, tight spatial and temporal control is exerted over several aspects of the polymerization cycle, including the nucleation of new actin filaments and the elongation of existing ones. Members of the WASP (Wiskott-Aldrich Syndrome Protein) family, including WASP, N-WASP (Neuronal homologue of WASP), and at least three variants of SCAR1 (Suppressor of cAMP Receptor-1)/WAVE (WASP-family Verprolin-Homologous Protein); seem to play a central role in regulating these processes (Ref.2).
Nucleation of new actin filaments is spatially controlled by the ARP2/3
(Actin-Related Proteins) complex, which is a complex of seven evolutionarily conserved proteins. This complex is important for the initiation of actin assembly in several cellular processes and it can nucleate a branched network of actin filaments at the leading edges of cells, which push the plasma membrane forward and cause protrusion (Ref.1). ARP2/3 complex is stimulated by extracellular signals transmitted through Growth Factors, RTKs (Receptor Tyrosine Kinase), GPCR (G Protein-Coupled Receptors), Integrins involving phosphoinositides, adaptor proteins like Nck and GRB2, IRSp53 (Insulin Receptor Substrate Protein), and the Rho-family GTPases— Rho, CDC42 and Rac. Rho activates ROCK (Rho-associated coiled-coil containing protein kinase) to activate Myosin via direct phosphorylation of the light chain and by inhibition of Myosin PPtase (Myosin phosphatase); Rho also interacts with GDIA to modulate actin filament formation. The joint action of GDIA and Rho Kinase leads to stress fiber formation. Activation of PAK
(p21-Activated Kinase) by Rac results in LIMK (LIM kinase) activation and inhibition of its target, the actin-severing protein Cofilin, thus promoting actin filament growth. CDC42 and Rac are implicated in the formation of filopodia and lamellipodia by regulating the activity of WASP-family members. The WASP family in humans comprises WASP, N-WASP and three SCAR/WAVE molecules (WAVE1, WAVE2 and WAVE3). WASP is expressed solely in haematopoietic cells, whereas N-WASP and SCAR/WAVE are expressed more generally. Upon binding to active CDC42 and Rac, WASP proteins induce ARP2/3-dependent actin assembly, leading to the generation of specific actin-containing structures (Ref.3). Binding of CDC42 to N-WASP causes a conformational change in N-WASP, which allows the VCA (Verprolin-homology-Cofilin-homology-Acidic) domain to interact with the ARP2/3
complex and initiate actin polymerization. This is further enhanced by PIP2 (Phosphatidylinositol-4, 5-Bisphosphate). Over expression of WASP in cells induces clusters of F-actin, whereas inducible recruitment of WASP to a cell-surface receptor triggers both actin polymerization and filopodium formation. Although interaction of N-WASP with CDC42 is sufficient to induce actin polymerization, other proteins are also required for spatial organization of actin into filopodia. WIP (WASP-Interacting Protein) binds to N-WASP and actin, regulates N-WASP-mediated actin nucleation by the ARP2/3 complex, and has an important role in induction of filopodia by Bradykinin and CDC42 (Ref.4). IRSp53 binds to CDC42 and induces formation of filopodia. In addition, IRSp53 interacts with two proteins that promote actin assembly. It binds SCAR, a WASP family protein capable of recruiting and activating the ARP2/3
complex, and Mena, a member of the Ena/VASP (Vasodilator-Stimulated Phosphoprotein) family of proteins (Ref.6). WASP and N-WASP also contains a CRIB (CDC42/Rac Interactive-Binding) motif that interacts with activated CDC42, an upstream, N-terminal located EVH1 (Ena-VASP Homology-1) domain that binds WIP and a downstream proline-rich region, which associates with Nck, Fyn, GRB2, and other SH3 (Src Homology 3) domain-containing proteins. These interactions facilitate the recruitment of WASP into antigen and other receptor-evoked signaling pathways and the concomitant induction of WASP relocalization to the immunologic synapse, endocytic vesicles, and other sites of dynamic actin remodeling within the cell. WASP association with activated CDC42 also disrupts an intramolecular interaction that suppresses VCA domain induction of ARP2/3
activity and is thus critical to the capacity for WASP to evoke actin polymerization after cell stimulation (Ref.5).
WASP is also a key regulator of lymphocyte cytoskeletal organization and has been implicated in the coupling of TCR (T-Cell Antigen Receptor) engagement to induction of both actin polymerization and transcriptional activation. The WASP protein is only expressed in haematopoietic cells, which is why the immune system is affected. Mutations in the gene encoding WASP results in Wiskott-Aldrich Syndrome, an immune system disorder, where affected individuals die unless they receive a successful bone marrow transplant in early childhood. Other related disorders like thrombocytopenia, eczema, and immunodeficiency in humans, which are less severe, also exist due to specific mutations in WASP, which alter its activity (Ref.5).