Actin Cytoskeleton

The actin cytoskeleton has major functions in many cellular processes such as cell division, regulation of the cell shape and migration. Furthermore, it is required for the formation and internalization of endosomes, phagocytic vesicles and macropinosomes (Disanza et al., 2005; Etienne-Manneville, 2006; Glotzer, 2001; Glotzer, 2003; Kaksonen et al., 2006; May and Machesky, 2001; Mercer and Helenius, 2012; Piekny et al., 2005; Small et al., 2002;

Vicente-Manzanares et al., 2009). The actin cytoskeleton is composed of different stable as well as highly dynamic structures (Ladwein and Rottner, 2008; Ridley, 2011). Underlying the plasma membrane, a dense cortical actin meshwork forms a membrane skeleton ensuring both, the structural integrity and the shape of cells (Heuser and Kirschner, 1980; Hirokawa and Heuser, 1981; Ladwein and Rottner, 2008; Medalia et al., 2002; Morone et al., 2006).

Actin is first synthesized as a globular protein (G-actin) of 43 kDa that binds adenosine triphosphate (ATP) in its central binding cleft (Disanza et al., 2005). The polymerization of G-actin into filamentous G-actin (F-G-actin) is initialized by the association of 2 or 3 G-G-actin monomers into a nucleation core (Disanza et al., 2005). Subsequently ATP-bound G-actin molecules bind to the plus-end of the filaments and subsequently hydrolyze the ATP to ADP.

Since each actin monomer binds to the next monomer opposite to the ATP binding site, actin filaments have a distinct polarity with a fast growing plus-end and a slow growing minus-end.

The interactions between actin monomers are non-covalent and weak which enables both, fast assembly as well as disassembly of actin filaments. The spatial distribution and dynamic behavior of the actin filaments are strictly regulated by actin binding and accessory proteins to form different actin structures. The actin cytoskeleton contributes to a wide range of cellular processes. The formation of the different actin structures contributing to these processes is mainly regulated by a family of small G proteins that operate as molecular switches (Fig. 2).

10 containing a lattice-like network of filamentous actin. Lamellipodia that detach from the extracellular matrix and fold up and back towards the cell are called ruffles. Ruffles can also form on the cell surface and are called dorsal ruffles. Filopodia are extended beyond the cell periphery and are composed of parallel actin bundles. Regions of highly dynamic F-actin are shown in red (Figure taken from Ladwein & Rottner, 2008).

Such Rho GTPases are active in their GTP bound state but inactive in the GDP bound state (Pollard & Cooper, 2009; Heasman & Ridley, 2008; Pertz, 2010; Curtis & Meldolesi, 2012).

Among the 20 known Rho GTPases, Cdc42, Rac1 and RhoA are most important for the reorganization of the cortical actin cytoskeleton; their respective activation leads to the formation of filopodia, lamellipodia, or stress fibers (Bisi et al., 2013; Etienne-Manneville and Hall, 2002; Heasman and Ridley, 2008; Ridley, 2011).

Filopodia are rod-like protrusions of the plasma membrane containing parallel bundles of actin filaments that are cross-linked by fascin (Aratyn et al., 2007; Kureishy et al., 2002). The elongation of the F-actin bundles occurs by actin polymerization at the plus-ends that face towards the filopodia tips. Filopodia are cellular sensors mediating path finding of moving cells or screening the substrate for suitable adhesion sites (Faix et al., 2009; Gupton and Gertler, 2007; Heckman and Plummer, 2013). The formation of filopodia is independent of the formation of lamellipodia ((Vidali et al., 2006) see below). The strongest known inductor of filopodia formation is Cdc42-GTP. Active Cdc42 mediates its function by directly binding and activating formins such as mDia2, a class of actin nucleators which act independent of the Arp2/3 complex (Block et al., 2008; Yang and Zheng, 2007).

11

However, other Rho GTPases have also been suggested to induce filopodia formation (Abe et al., 2003; Aspenstrom et al., 2004; Ellis and Mellor, 2000; Murphy et al., 1999; Neudauer et al., 1998; Tao et al., 2001; Vignal et al., 2000). A constitutively-active form of the Rho GTPase Rif is able to mediate filopodia formation independently of Cdc42 (Ellis and Mellor, 2000).

Lamellipodia are sheet-like protrusions of the plasma membrane of stimulated cells or at the leading edge of migrating cells. Lamellipodia consist of a highly dynamic lattice-like network of actin filaments. Through the elongation of the actin fibers at the plus-ends facing the plasma membrane, they are continuously pushed forward. During this process, lamellipodia adhere to the substratum and then often focal adhesions are formed that connect the plasma membrane with the extra-cellular matrix (Ladwein and Rottner, 2008; Ridley, 2011).

Lamellipodia that detach from the substratum and bend upwards are called membrane ruffles. In addition, circular ruffles with a similar appearance can be found on the dorsal surface of cells. Due to their shape, circular ruffles allow the enclosure and uptake of extracellular particles, ligands or fluids in a process that has been named macropinocytosis (Mercer and Helenius, 2012; Orth and McNiven, 2006). Among the Rac family of the Rho GTPases, Rac1 regulates the extension of lamellipodia, migration as well as membrane ruffling (Ridley, 2001) by activation of the Arp2/3 complex via the Rac1 effector WAVE-complex (Innocenti et al., 2004; Kunda et al., 2003; Rogers et al., 2003; Steffen et al., 2004).

Stress fibers are contractile actin-myosin structures of cultured epithelial cells and fibroblasts (Cramer et al., 1997; Tojkander et al., 2012). Stress fibers consist of short cables of 10 to 30 actin filaments that are connected with each other by non-muscle myosin II. Such actin bundles are cross-linked by actinin, fascin and epsin (Adams, 1995; Chen et al., 1999;

Cramer et al., 1997; Lazarides and Burridge, 1975; Tojkander et al., 2012). The ends of most stress fibers are connected to the extracellular matrix via focal adhesions (Izzard and Lochner, 1976). The connecting myosin II patches are able to contract and thereby exert tension relative to the extracellular substrate. This allows the regulation of the cell shape and the retraction of the rear of the cell during migration (Vicente-Manzanares et al., 2009). The formation of stress fibers is regulated by three Rho GTPases: RhoA, RhoB and RhoC in response to extracellular stimuli such as lysophosphatidic acid or the drug calyculin A (Naumanen et al., 2008; Ridley, 2006). Among the downstream effectors of RhoA are Rho-kinase (ROCK) and LIM-Rho-kinase. ROCK activates the myosin II motor function in two ways; it directly phosphorylates and activates the myosin regulatory light chain and prevents its dephosphorylation by inhibiting the myosin light chain phosphatase. LIM-kinase inhibits the destabilization of stress fibers by blocking the depolymerisation factor ADF/cofilin (Amano et al., 2001; Maekawa et al., 1999; Pritchard et al., 2004).

12