Actin is one of the most abundant proteins in the cell. It exists as globular monomers called G-Actin (Globular) that can assemble in filaments called F-Actin (filamentous). Actin filaments
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provide a framework that supports the plasma membrane and thus determine cell shape. Two distinct patterns of filaments can be distinguished: bundles, which are composed by parallel, straight filaments, and networks, made by branched filaments. Branched actin filaments are found for example in the lamellipodia, whereas actin bundles sustain fingerlike structures like microvilli. Two major types of proteins are needed in order to assemble actin in bundles or networks. Diaphanous Related Formins (DRFs, a group comprising Dia1, 2 and 3, also called mDia1, 2 and 3) are responsible for nucleating parallel filaments, whereas the Arp2/3 complex leads to formation of a new branch of actin with a 70° angle relative to the original filament, producing branched actin filaments typically referred to as barbed ends 35. Rho GTPases control actin by localizing and activating those actin nucleators. RhoA binds to Diaphanous related formins causing a conformational change that activates them, thus promoting nucleation of parallel filaments of actin that will assemble in bundles to form stress fibers 36. RhoA can also induce stress fiber formation via a cascade involving activation of Rho kinase (ROCK) which activates LIM kinase (LIMK), which in turn phosphorylates and inactivates Cofilin
37. ROCK also phosphorylates MLC (myosin light chain) 38 and at the same time phosphorylates MLC phosphatase 39, thereby increasing myosin contractility, resulting finally in stress fibers formation 40.
RhoA signals to Rac1 via two pathways that antagonize each other. mDia binds to Src, promoting phosphorylation of the kinase Cas that will recruit the Rac GEF Dock180, resulting in activation of Rac 41. At the same time, however, ROCK antagonizes Rac1 activation via mechanism that is not completely clear 41. Rac and Cdc42 also signal to LIM kinase via their shared effector PAK1, thus they promote actin polymerization by inhibiting cofilin 42, 43. In addition, Rac1 controls the Arp2/3 complex by localizing and activating it. It was shown that Rac1 activates the WAVE/Scar complex, a direct activator of Arp2/3, by causing the dissociation of WAVE from the complex that keeps it inactive 44. An important effector of Rac is IRSp53, an SH3 domain-containing scaffold protein that, together with Rac1, forms a ternary complex with The Arp2/3 activator WAVE2. Recruitment of IRSp53 by Rac1-GTP promotes local activation of Arp2/3 and results in actin branching and lamellipodia formation 45 46. However, a study by Krugmann et al. found that IRSp53 interacts mainly with Cdc42 and it is responsible for filopodia, but not lamellipodia formation 47. In this model, the complex Cdc42-IRSp53 recruits MENA, a component of the ENA/Vasp complex. The ENA/Vasp complex promotes actin dynamics by interfering with capping proteins, a class of proteins that blocks
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monomer addition at the barbed ends of actin filaments, inhibiting the elongation of such filaments 48. ENA/Vasp and capping proteins must both be properly regulated at the leading edge in order to promote migration. When ENA/Vasp activity is too high, capping proteins are totally inhibited and as result there will be very long filaments with few branches that will translate into faster speed, but decreased persistence. Conversely, when capping proteins are highly active, the leading edge will be characterized by highly branched actin filaments and migration will be more persistent 49. With this in mind, it is not surprising that, in addition to recruiting MENA, Cdc42 promotes binding of IRSp53 to the capping protein Esp8 and this protein is also necessary for filopodia formation 50. IRSp53 could therefore play a role in lamellipodia or in filopodia formation and the data on this appear to be contradictory.
Probably, an explanation that reconciles all observations is that there are two pools of IRSp53, one that interacts with Rac and activates WAVE, thus promoting lamellipodia, and another pool that binds to Cdc42, leading to the recruitment of MENA and Esp8 and promotion of filopodia. There is also the possibility that those two pools signal to each other, as it was shown that Esp8 indirectly activates Rac by recruiting the Rac GEF Sos1 45, 51, 52. Cdc42 can activate the Arp2/3 activators N-WASP and WASP and thus it can activate the Arp2/3 complex itself 53. Surprisingly, however, depletion of Arp2/3, WAVE and WASP causes loss of lamellipodia, but not of filopodia 54, 55, indicating that networks of branched actin are not critical for the formation of filopodia. If branched actin is not sustaining filopodia, then it is probably parallel actin fibers that provide the structure to grow filopodia. If this is the case, then Cdc42 should also be able to control DRFs. As a matter of fact, Peng et al. showed that Cdc42 interacts directly with DRF3 (mDia2), this interaction being sufficient for filopodia formation 56. Filopodia can be induced also by other small Rho GTPases, namely RIF (RhoF), RhoD and WRCH1 32 and mDia2 was also found downstream of RIF 57.
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Figure 1 – Schematic of the main signaling routes downstream of Cdc42, Rac1 and RhoA. Arrows are of the same color of the GTPase from which they depend and if more GTPases control the same pathway, the overlaid color is used.