Regulation of Actin-based Motility by Rho


Pathway Description

The actin filament network immediately under the plasma membrane in regions of rapid cellular protrusion consists of short, branched filaments while those deeper in the cortex, as well as at focal adhesions, stress fibers and in microvilli, are much longer and rarely branched. The dynamic organization of the actin cytoskeleton provides the force for cell motility and is regulated by small GTPases of the Rho family, in particular Rac1, RhoA and CDC42. The microtubule cytoskeleton is also polarized in a migrating cell, and in addition to organizing the actin cytoskeleton; Rho GTPases also influence the organization and dynamics of these microtubules.

Rho family proteins regulate a broad diversity of cellular functions including cytoskeletal organization, membrane trafficking, cytokinesis, cell proliferation, cell motility and transcriptional regulation. These G-Proteins function as molecular switches in signal transduction pathways by cycling between an active GTP-bound and an inactive GDP-bound state. GEFs (Guanine Nucleotide Exchange Factors) catalyze the exchange bound GDP for GTP, whereas GAPs (GTP Activating Proteins) increase their intrinsic GTPase activity and GDIs (GDP Dissociation Inhibitors) prevent release of bound GDP. In fibroblasts, these proteins regulate various cytoskeletal rearrangements: RhoA controls stress fiber formation and the attachment of contractile bundles of actin and myosin filaments to the cell membrane at points of focal adhesion, where integrin clusters are present. Rac regulates the polymerization to drive lamellipodial protrusion and the formation of membrane ruffles, whereas CDC42 generates polarity and induces formation of filopodia and microspikes. These GTPases function sequentially: CDC42 stimulates Rac activity, which then activates Rho. Activated CDC42, Rac and Rho bind to and specifically activate their downstream effectors, which are either kinases such as ROCK, PAK and PI5K or scaffolding proteins such as GDIA, WASP and IRSp53. GDIA mediates force-induced contact formation, even if the entire ROCK-activated pathway, including Myosin-II activation, is eliminated. Constitutively active GDIA lacking Rho-binding domains cooperate with activated ROCK to form stress fibers. PAK activates LIM-kinases (LIMK1 and LIMK2) to phosphorylate ADF/cofilins. This allows signals flowing through Rho family GTPases to coordinate the initiation of new filaments through WASP and ARP2/3 complex. Both LIMK1 and LIMK2 are downstream effectors of the Rho GTPases. GTP-bound Rho also activates an enzyme known as Rho-kinase, which phosphorylates the myosin-binding subunit of MLCP, inactivating it and thereby preventing MLC dephosphorylation. As a result, Rho activation leads to an accumulation of the phosphorylated MLC and, subsequently, to the stimulation of actomyosin ATPase activity. Activation by WAVE1, another member of the WASP family, also induces actin alterations in response to Rac1 signals upstream. Activated Rac, which is known to bind and activate PI5K, stimulate biosynthesis of PIP2, leading to promotion of actin assembly from profilin and gelsolin.

The Rho family of GTPases comprises some 21 genes in humans, encoding at least 23 signaling proteins. Although these proteins control an amazingly diverse range of cellular functions, one general role is in the establishment of polarity and of polarised structures through dynamic regulation of the actin cytoskeleton. Rho GTPases control the polymerization, branching and bundling of actin, allowing them to regulate the remodeling of the actin cytoskeleton into distinct architectural elements.