Actin Nucleation and Branching
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Actin Nucleation and Branching
The Actin family is a diverse and evolutionarily ancient group of proteins that provide the supportive framework to the three-dimensional structure of eukaryotic cells. It provides the forces that enable the cell to adopt a variety of shapes and to undertake directed movements. Certain cell types, such as polymorphonuclear leukocytes, monocyte/macrophages, and metastatic cells, are able to move rapidly through tissues and these movements are mediated by the Actin cytoskeleton (Ref.1). An important property of Actin is its ability to produce movement in the absence of motor proteins. At the cell membrane Actin microfilament assembly protrudes the membrane forward producing the ruffling membranes in actively moving cells. These microfilaments also play a passive structural role by providing the internal stiffening rods in microvilli, maintaining cell shape, and anchoring cytoskeletal proteins (Ref.2). Moreover, Actin microfilaments provide the "rails" along which myosin "motors" perform work in a variety of cellular functions. Microfilaments cooperate with microtubules via MAPs (Microtubule-Associated Proteins) during the transport of vesicles and organelles. Actin filaments interact with other intermediate filaments, a function that has an important role in enabling extracellular stimuli to be transmitted to key targets like ribosome and chromosomes deep within the cell (Ref.2).

Actin is only found in eukaryotes and comprises a highly conserved family of proteins that fall into three broad classes: Alpha-, Beta-, and Gamma-isoforms. It is mainly located in the cytoplasm, but it is also present in the nucleus where it may or may not have motor-associated functions. The highest concentrations of Actin are in striated muscles; however, significant quantities of actin are present in nonmuscle cells where it plays a variety of roles including myosin-independent changes of cells shape, motor-based organelle transport, regulation of ion transport, and receptor-mediated responses of the cell to external signals. Actin binds a substantial number of proteins collectively called ABPs (Actin-Binding Proteins), which is classified into seven groups: 1) Monomer-binding proteins that sequester G-actin and prevent its polymerization. 2) Filament-depolymerizing proteins that induce the conversion of F-actin to G-actin (e.g., CapZ and Cofilin). 3) Filament end-binding proteins, which cap the ends of the Actin filament preventing the exchange of monomers at the pointed end (e.g., tropomodulin) and at the barbed end (e.g., CapZ). 4) Filament severing proteins that shorten the average length of filaments by binding to the side of F-actin and cutting it into two pieces (e.g.gelsolin, ADF/Cofilin) 5) Cross-linking proteins that facilitate the formation of filament bundles, branching filaments, and three-dimensional networks (e.g., ARP2/3 (Actin Related Protein), Alpha-actinin, fascin, Filamin). 6) Stabilizing proteins that bind to the sides of Actin filaments and prevent depolymerization (e.g., tropomyosin). 7) Motor proteins that use F-actin as a track upon which to move (e.g., the myosin family of motors) (Ref.2).

Extracellular signals are transmitted through Growth factors, RTK (Receptor Tyrosine Kinase), GPCR (G Protein-Coupled Receptors) and Integrins to WASP (Wiskott-Aldrich Syndrome Protein), N-WASP (Neuronal homologue of WASP), or SCAR1 (Suppressor of cAMP Receptor-1), whereas chemotactic factors bind to plasma membrane receptors, activating intracellular signaling molecules of Rho family GTPases including CDC42, Rac and Rho. These GTPases bind to and activate WASP/ SCAR1 family proteins by freeing them from autoinhibition. Active WASP/ SCAR1 proteins bring together an Actin monomer and ARP2/3 complex. ARP2/3 complex then choreographs the formation of branched actin networks at the leading edge of migrating cells (Ref.3) by changing its conformation so that it can nucleate the assembly of new Actin filaments and assemble a "daughter" filament at an angle of ~70° to the "mother" filament. The branch grows rapidly at its barbed end by addition of Actin-profilin complexes. As it grows, it pushes the plasma membrane forward (Ref.4). The new filament elongates, binding the pointed end of a new (uncapped) daughter filament to the mother filament thus leaving the barbed (growing) end available for filament elongation. Several different proteins can bind to the barbed end of filaments like gelsolin (and its close relatives adseverin and fragmin), profilin, Villin, CapG and tensin (Ref.2). The CDC42 and Rac signal transduction pathways lead to Actin rearrangements in the form of lamellipodia (membrane ruffles) and filopodia (micro spikes) (Ref.1). Activation by WAVE1 (WASP-family Verprolin-Homologous Protein), another member of the WASP family, also induces Actin alterations in response to Rac1 signals upstream. WAVE1 is held in an inactive complex along with HSP90 (Heat Shock 90KD Protein) in the cytosol that is activated by interaction with Rac1 and Nck to allow WAVE1 to associate with ARP2/3 and activate actin branching and polymerization. Other major regulators of Actin polymerization are Ena/VASP (Enabled Vasodilator-Stimulated Phosphoprotein) and PIP2 (Phosphatidylinositol-4,5-Bisphosphate).

Rho family GTPases also activates PAK (p21-Activated Protein Kinase), which stimulates LIMK (LIM kinase) to phosphorylate ADF/Cofilin. Phosphorylation inactivates ADF/Cofilin and slows down the rate of filament disassembly. The incorporation of ATP-actin into a filament promotes hydrolysis of the bound ATP. Phosphate dissociates slowly from polymerized ADP-P-actin subunits, which promotes further dissociation of branches from ARP2/3 complex and binding of ADF/Cofilin to ADP-actin subunits. ADF/Cofilin bound to filaments promotes severing of the filaments and dissociation of ADP-actin bound to ADF/Cofilin. profilin is the nucleotide-exchange factor for Actin and promotes the exchange of ADP for ATP by tightly binding to ATP-actin monomers, refilling the Actin monomer pool (Ref.4). profilin also maintains a pool of Mg-ATP-actin that is ready to elongate any available Actin filament barbed ends. Calcium regulates Actin filament binding and severing by gelsolin (Ref.5).

Dynamic reorganization of the Actin cytoskeleton transforms cell shapes and generates the forces necessary for cell locomotion, cell division, chemotaxis, phagocytosis, macropinocytosis, and cytokinesis, as well as for cell polarity and differentiation processes (Ref.6). Structures based on Actin filaments range from parallel bundles to gel networks. The bundles provide tensile strength; serve as cables for strong contractile activity and as tracks for organelle transport. Three-dimensional orthogonal networks provide elasticity, accommodate internal diffusion of water, solutes and small organelles, resist weak osmotic and hydrostatic fluid flows and serve as scaffolds for weak contractions. They are barriers to spontaneous movement of large organelles and they localize signal transduction and other intracellular reactions (Ref.7). Assembly of Actin filaments drives the locomotion of many cells including nerve growth cones, fibroblasts, and leukocytes. Actin polymerization also moves some cytoplasmic particles including endosomes, pathogenic bacteria, and viruses, as well as driving particle engulfment during phagocytosis (Ref.8). In general the important function of the Actin cytoskeleton in multicellular organisms is to drive many different processes depending on cell locomotion, including morphogenetic movements during embryonic development, movement of neurites during development and remodeling of the nervous system, chemotactic movements of immune cells, and fibroblast migration during wound healing (Ref.5).