Role of Actin Polymerization and Adhesion to Extracellular Matrix in Rac- and Rho-induced Cytoskeletal Reorganization

Most animal cells use a combination of actin-myosin–based contraction and actin polymerization– based protrusion to control their shape and motility. The small GTPase Rho triggers the formation of contractile stress fibers and focal adhesion complexes (Ridley, A.J., and A. Hall. 1992. Cell. 70:389–399) while a close relative, Rac, induces lamellipodial protrusions and focal complexes in the lamellipodium (Nobes, C.D., and A. Hall. 1995. Cell. 81:53–62; Ridley, A.J., H.F. Paterson, C.L. Johnston, D. Diekmann, and A. Hall. 1992. Cell. 70:401–410); the Rho family of small GTPases may thus play an important role in regulating cell movement. Here we explore the roles of actin polymerization and extracellular matrix in Rho- and Rac-stimulated cytoskeletal changes. To examine the underlying mechanisms through which these GTPases control F-actin assembly, fluorescently labeled monomeric actin, Cy3-actin, was introduced into serum-starved Swiss 3T3 fibroblasts. Incorporation of Cy3- actin into lamellipodial protrusions is concomitant with F-actin assembly after activation of Rac, but Cy3-actin is not incorporated into stress fibers formed immediately after Rho activation. We conclude that Rac induces rapid actin polymerization in ruffles near the plasma membrane, whereas Rho induces stress fiber assembly primarily by the bundling of actin filaments. Activation of Rho or Rac also leads to the formation of integrin adhesion complexes. Integrin clustering is not required for the Rho-induced assembly of actin-myosin filament bundles, or for vinculin association with actin bundles, but is required for stress fiber formation. Integrin-dependent focal complex assembly is not required for the Rac-induced formation of lamellipodia or membrane ruffles. It appears, therefore, that the assembly of large integrin complexes is not required for most of the actin reorganization or cell morphology changes induced by Rac or Rho activation in Swiss 3T3 fibroblasts.     

Spatio-Temporal Regulation of Rac1 Mobility by Actin Islands

Rho GTPases play important roles in many aspects of cell migration, including polarity establishment and organizing actin cytoskeleton. In particular, the Rho GTPase Rac1 has been associated with the generation of protrusions at leading edge of migrating cells. Previously we showed the mobility of Rac1 molecules is not uniform throughout a migrating cell (Hinde E et. al. PNAS 2013). Specifically, the closer a Rac1 molecule is to the leading edge, the slower the molecule diffuses. Because actin-bound Rac1 diffuses slower than unbound Rac1, we hypothesized that regions of high actin concentration, called “actin islands”, act as diffusive traps and are responsible for the non-uniform diffusion observed in vivo. Here, in silico model simulations demonstrate that equally spaced actin islands can regulate the time scale for Rac1 diffusion in a manner consistent with data from live-cell imaging experiments. Additionally, we find this mechanism is robust; different patterns of Rac1 mobility can be achieved by changing the actin islands’ positions or their affinity for Rac1.     

The Dictyostelium discoideum family of Rho-related proteins

Taking advantage of the ongoing Dictyostelium genome sequencing project, we have assembled >73 kb of genomic DNA in 15 contigs harbouring 15 genes and one pseudogene of Rho-related proteins. Comparison with EST sequences revealed that every gene is interrupted by at least one and up to four introns. For racC extensive alternative splicing was identified. Northern blot analysis showed that mRNAs for racA, racE, racG, racH and racI were present at all stages of development, whereas racJ and racL were expressed only at late stages. Amino acid sequences have been analysed in the context of Rho-related proteins of other organisms. Rac1a/1b/1c, RacF1/F2 and to a lesser extent RacB and the GTPase domain of RacA can be grouped in the Rac subfamily. None of the additional Dictyostelium Rho-related proteins belongs to any of the well-defined subfamilies, like Rac, Cdc42 or Rho. RacD and RacA are unique in that they lack the prenylation motif characteristic of Rho proteins. RacD possesses a 50 residue C-terminal extension and RacA a 400 residue C-terminal extension that contains a proline-rich region, two BTB domains and a novel C-terminal domain. We have also identified homologues for RacA in Drosophila and mammals, thus defining a new subfamily of Rho proteins, RhoBTB.     

Isolation and sequencing of the 5′ end of the rat microtubule-associated protein (MAP1B)-encoding cDNA

We have isolated and sequenced the 5′ end of the cDNA encoding the rat microtubule-associated protein 1B (MAP1B). We found that this region is highly homologous to the corresponding regions of the human [Lien et al., 22 (1994) 273–280] and mouse [Noble et al., J. Cell Biol. 109 (1989) 3367–3376] MAPIB genes. The combination of the sequence that we are presenting with the previously published sequence [Zauner et al., Eur. J. Cell Biol. 57 (1992) 66–74], represents the complete rat MAP1B cDNA coding sequence.     

Rac1 function during fucoid development

Morphogenesis in fucoid algae begins with adhesive secretion and rhizoid germination, developmental events that secure the alga within the intertidal zone. The importance of the actin cytoskeleton during these processes has been well established; but in general, little is known about actin regulation within the stramenopile lineage. Based on conserved strategies for regulation of actin in other lineages, co-localization of the Arp2/3 complex with actin structures that are essential for rhizoid formation may implicate members of the Rho family of small GTPases in the signaling pathway(s) regulating actin polymerization during fucoid development. Our lab recently demonstrated Rac1 dependent regulation of endomembrane polarization, polarization of adhesive secretion, germination and tip growth in the fucoid brown alga Silvetia compressa. We also present new evidence revealing Rac1 localization during germination in S. compressa, and show that membrane localization is essential for proper Rac1 function.Key words: actin, Arp2/3 complex, manumycin A, NSC23766, Rac1, Rho GTPase, Scar/WAVE, Silvetia compressa     

p190RhoGAP has cellular RacGAP activity regulated by a polybasic region

p190RhoGAP is a GTPase-activating protein (GAP) known to regulate actin cytoskeleton dynamics by decreasing RhoGTP levels through activation of the intrinsic GTPase activity of Rho. Although the GAP domain of p190RhoGAP stimulates the intrinsic' GTPase activity of several Rho family members (Rho, Rac, Cdc42) under in vitro conditions, p190RhoGAP is generally regarded as a GAP for RhoA in the cell. The cellular RacGAP activity of the protein has not been proven directly. We have previously shown that the in vitro RacGAP and RhoGAP activity of p190RhoGAP was inversely regulated through a polybasic region of the protein. Here we provide evidence that p190RhoGAP shows remarkable GAP activity toward Rac also in the cell. The cellular RacGAP activity of p190RhoGAP requires an intact polybasic region adjacent to the GAP domain whereas the RhoGAP activity is inhibited by the same domain. Our data indicate that through its alternating RacGAP and RhoGAP activity, p190RhoGAP plays a more complex role in the Rac–Rho antagonism than it was realized earlier.     

FERM Domain Containing Protein 7 Interacts with the Rho GDP Dissociation Inhibitor and Specifically Activates Rac1 Signaling

The FERM domain containing protein 7 gene (FRMD7) associated with the X-linked disorder idiopathic congenital nystagmus (ICN) is involved in the regulation of neurite elongation during neuronal development. Members of the Rho family of small G-proteins (Rho GTPases) are key regulators of the actin cytoskeleton and are implicated in the control of neuronal morphology. The Rho GDP dissociation inhibitor alpha, RhoGDIα, the main regulator of Rho GTPases, can form a complex with the GDP-bound form of Rho GTPases and inhibit their activation. Here, we demonstrate that the full length of the mouse FRMD7, rather than the N-terminus or the C-terminus alone, directly interacts with RhoGDIα and specifically initiates Rac1 signaling in mouse neuroblastoma cell line (neuro-2a). Moreover, we show that wild-type human FRMD7 protein is able to activate Rac1 signaling by interacting with RhoGDIα and releasing Rac1 from Rac1-RhoGDIα complex. However, two missense mutations (c.781C>G and c.886G>C) of human FRMD7 proteins weaken the ability to interact with RhoGDIα and release less Rac1, that induce the activation of Rac1 to a lesser degree; while an additional mutant, c.1003C>T, which results in a C-terminal truncated protein, almost fails to interact with RhoGDIα and to activate Rac1 signaling. Collectively, these results suggest that FRMD7 interacts with one of the Rho GTPase regulators, RhoGDIα, and activates the Rho subfamily member Rac1, which regulates reorganization of actin filaments and controls neuronal outgrowth. We predict that human mutant FRMD7 thus influences Rac1 signaling activation, which can lead to abnormal neuronal outgrowth and cause the X-linked ICN.     

Point-spread sensitivity analysis for computational optical-sectioning microscopy

Experimentally determined point-spread functions (PSF) have been used routinely for reconstructions of three-dimensional (3-D) microscopic objects from optical sections (Agard et al., 1989, Meth. Cell Biol., 30: 353–377; Fay et al., 1986, Opt. Meth. Cell Physiol., 40: 51–63). The microscope's PSF is usually measured by imaging a small fluorescent bead. There is a tradeoff in this measurement: very small beads are dim and bleach rapidly, while larger beads are a poorer approximation to a point source.We have simulated the effect of the bead's size on the shape of the PSF by convolving a theoretically determined PSF (of a 40 × 1.0 N.A. oil-immersion lens) with spheres of varying diameters. Simulated data were generated with a 3-D phantom and the theoretical PSF, which is defined to be the ‘true’ PSF for the simulation. Reconstructions of the phantom were obtained with each of the theoretical PSFs obtained from the beads using a regularized linear least-squares method (Preza et al., 1992, J. Opt. Soc. Am., 9: 219–228). Results show a significant drop (more than 50%) in the signal-to-noise ratio of the reconstructions for beads with diameter large than 0.22 μm. These results suggest that the bead used in the PSF measurement should have a diameter less than 30% of the diameter of the first dark ring of the infocus two-dimensional (2-D) PSF. This study quantifies the tradeoff between the quality of the reconstructions and the bead size used in the PSF measurement.     

A role for Rho and Rac in secretagogue-induced amylase release by pancreatic acini

The actin cytoskeleton has long been implicated in protein secretion. We investigated whether Rho and Rac, known regulators of the cytoskeleton, are involved in amylase secretion by mouse pancreatic acini. Secretagogues, including cholecystokinin (CCK) and the acetylcholine analog carbachol, increased the amount of GTP-bound RhoA and Rac1 and induced translocation from cytosol to a membrane fraction. Immunocytochemistry revealed the translocation of Rho and Rac within the apical region of the cell. Expression by means of adenoviral vectors of dominant-negative Rho (RhoN19), dominant-negative Rac (RacN17), and Clostridium Botulinum C3 exotoxin, which ADP ribosylates and inactivates Rho, significantly inhibited amylase secretion by CCK and carbachol; inhibiting both Rho and Rac resulted in a greater reduction. This inhibitory effect of RhoN19 on CCK-induced amylase secretion was apparent in both the early and late phases of secretion, whereas RacN17 was more potent on the late phase of secretion. None of these three affected the basal Ca²⁺ or the peak intracellular Ca²⁺ concentration stimulated by CCK. Latrunculin, a marine toxin that sequesters actin monomers, time-dependently decreased the total amount of filamentous actin (F-actin) and dose-dependently decreased secretion by secretagogues without affecting Ca²⁺ signaling. These data suggest that Rho and Rac are both involved in CCK-induced amylase release in pancreatic acinar cell possibly through an effect on the actin cytoskeleton. cholecystokinin; carbachol; pancreas; cytoskeleton     

Adenovirus Endocytosis Requires Actin Cytoskeleton Reorganization Mediated by Rho Family GTPases

Adenovirus (Ad) endocytosis via α_v integrins requires activation of the lipid kinase phosphatidylinositol-3-OH kinase (PI3K). Previous studies have linked PI3K activity to both the Ras and Rho signaling cascades, each of which has the capacity to alter the host cell actin cytoskeleton. Ad interaction with cells also stimulates reorganization of cortical actin filaments and the formation of membrane ruffles (lamellipodia). We demonstrate here that members of the Rho family of small GTP binding proteins, Rac and CDC42, act downstream of PI3K to promote Ad endocytosis. Ad internalization was significantly reduced in cells treated with Clostridium difficile toxin B and in cells expressing a dominant-negative Rac or CDC42 but not a H-Ras protein. Viral endocytosis was also inhibited by cytochalasin D as well as by expression of effector domain mutants of Rac or CDC42 that impair cytoskeletal function but not JNK/MAP kinase pathway activation. Thus, Ad endocytosis requires assembly of the actin cytoskeleton, an event initiated by activation of PI3K and, subsequently, Rac and CDC42.     

Stuck in the middle: Rac, adhesion, and cytokinesis

Rho family small GTPases (Rac, RhoA, and Cdc42) function at the core of cytokinesis, the physical division of one cell into two. In this issue, Bastos et al. (2012. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201204107) identify a new role for Rac inhibition: to release cell adhesion at the division plane and allow efficient constriction of the contractile ring. They show that the GTPase-activating protein, CYK4, suppresses equatorial cell substrate adhesion by inhibiting Rac and therefore its effectors ARFGEF7 and PAK1/2.     

Neural crest cell-specific deletion of Rac1 results in defective cell-matrix interactions and severe craniofacial and cardiovascular malformations

The small GTP-binding protein Rac1, a member of the Rho family of small GTPases, has been implicated in regulation of many cellular processes including adhesion, migration and cytokinesis. These functions have largely been attributed to its ability to reorganize cytoskeleton. While the function of Rac1 is relatively well known in vitro, its role in vivo has been poorly understood. It has previously been shown that in neural crest cells (NCCs) Rac1 is required in a stage-specific manner to acquire responsiveness to mitogenic EGF signals. Here we demonstrate that mouse embryos lacking Rac1 in neural crest cells (Rac1/Wnt1-Cre) showed abnormal craniofacial development including regional ectodermal detachment associated with mesenchymal acellularity culminating in cleft face at E12. Rac1/Wnt1-Cre mutants also displayed inappropriate remodelling of pharyngeal arch arteries and defective outflow tract septation resulting in the formation of a common arterial trunk (‘persistent truncus arteriosus’ or PTA). The mesenchyme around the aortic sac also developed acellular regions, and the distal aortic sac became grossly dysmorphic, forming a pair of bilateral, highly dilated arterial structures connecting to the dorsal aortas. Smooth muscle cells lacking Rac1 failed to differentiate appropriately, and subpopulations of post-migratory NCCs demonstrated aberrant cell death and attenuated proliferation. These novel data demonstrate that while Rac1 is not required for normal NCC migration in vivo, it plays a critical cell-autonomous role in post-migratory NCCs during craniofacial and cardiac development by regulating the integrity of the craniofacial and pharyngeal mesenchyme.     

A Drosophila homolog of the Rac- and Cdc42-activated serine/threonine kinase PAK is a potential focal adhesion and focal complex protein that colocalizes with dynamic actin structures.

Changes in cell morphology are essential in the development of a multicellular organism. The regulation of the cytoskeleton by the Rho subfamily of small GTP-binding proteins is an important determinant of cell shape. The Rho subfamily has been shown to participate in a variety of morphogenetic processes during Drosophila melanogaster development. We describe here a Drosophila homolog, DPAK, of the serine/threonine kinase PAK, a protein which is a target of the Rho subfamily proteins Rac and Cdc42. Rac, Cdc42, and PAK have previously been implicated in signaling by c-Jun amino-terminal kinases. DPAK bound to activated (GTP-bound) Drosophila Rac (DRacA) and Drosophila Cdc42. Similarities in the distributions of DPAK, integrin, and phosphotyrosine suggested an association of DPAK with focal adhesions and Cdc42- and Rac-induced focal adhesion-like focal complexes. DPAK was elevated in the leading edge of epidermal cells, whose morphological changes drive dorsal closure of the embryo. We have previously shown that the accumulation of cytoskeletal elements initiating cell shape changes in these cells could be inhibited by expression of a dominant-negative DRacA transgene. We show that leading-edge epidermal cells flanking segment borders, which express particularly large amounts of DPAK, undergo transient losses of cytoskeletal structures during dorsal closure. We propose that DPAK may be regulating the cytoskeleton through its association with focal adhesions and focal complexes and may be participating with DRacA in a c-Jun amino-terminal kinase signaling pathway recently demonstrated to be required for dorsal closure.     

Another job for the talented p120-catenin

Given the complexity of signaling pathways in the cell, it is a mystery how these pathways communicate with one other. In this issue of Cell, Wildenberg et al. (2006) reveal that the key effector molecule p120-catenin can mediate crosstalk between the Rac and Rho signaling pathways.     

Identification of P-Rex1 as a novel Rac1-guanine nucleotide exchange factor (GEF) that promotes actin remodeling and GLUT4 protein trafficking in adipocytes

Phosphoinositide 3-kinase (PI3K) signaling promotes the translocation of the glucose transporter, GLUT4, to the plasma membrane in insulin-sensitive tissues to facilitate glucose uptake. In adipocytes, insulin-stimulated reorganization of the actin cytoskeleton has been proposed to play a role in promoting GLUT4 translocation and glucose uptake, in a PI3K-dependent manner. However, the PI3K effectors that promote GLUT4 translocation via regulation of the actin cytoskeleton in adipocytes remain to be fully elucidated. Here we demonstrate that the PI3K-dependent Rac exchange factor, P-Rex1, enhances membrane ruffling in 3T3-L1 adipocytes and promotes GLUT4 trafficking to the plasma membrane at submaximal insulin concentrations. P-Rex1-facilitated GLUT4 trafficking requires a functional actin network and membrane ruffle formation and occurs in a PI3K- and Rac1-dependent manner. In contrast, expression of other Rho GTPases, such as Cdc42 or Rho, did not affect insulin-stimulated P-Rex1-mediated GLUT4 trafficking. P-Rex1 siRNA knockdown or expression of a P-Rex1 dominant negative mutant reduced but did not completely inhibit glucose uptake in response to insulin. Collectively, these studies identify a novel RacGEF in adipocytes as P-Rex1 that, at physiological insulin concentrations, functions as an insulin-dependent regulator of the actin cytoskeleton that contributes to GLUT4 trafficking to the plasma membrane.     

Clp1p and Mid1p form links between cell cycle progression and gene expression at cytokinesis in fission yeast

Comment on: Papadopoulou K, et al. J Cell Sci 2010; 123:4374–81 and Agarwal M, et al. J Cell Sci 2010; 123:4366–73     

Curcumin inhibits lung cancer cell migration and invasion through Rac1-dependent signaling pathway

Curcumin, a natural and crystalline compound isolated from the plant Curcuma longa with low toxicity in normal cells, has been shown to protect against carcinogenesis and prevent tumor development. However, little is known about antimetastasis effects and mechanism of curcumin in lung cancer. Rac1 is an important small Rho GTPases family protein and has been widely implicated in cytoskeleton rearrangements and cancer cell migration, invasion and metastasis. In this study, we examined the influence of curcumin on in vitro invasiveness of human lung cancer cells and the expressions of Rac1. The results indicate that curcumin at 10 μM slightly reduced the proliferation of 801D lung cancer cells but showed an obvious inhibitory effect on epidermal growth factor or transforming growth factor β1-induced lung cancer cell migration and invasion. Meanwhile, we demonstrated that the suppression of invasiveness correlated with inhibition of Rac1/PAK1 signaling pathways and matrix metalloproteinase (MMP) 2 and 9 protein expression by combining curcumin treatment with the methods of Rac1 gene silence and overexpression in lung cancer cells. Laser confocal microscope also showed that Rac1-regulated actin cytoskeleton rearrangement may be involved in anti-invasion effect of curcumin on lung cancer cell. At last, through xenograft experiments, we confirmed the connection between Rac1 and the growth and metastasis inhibitory effect of curcumin in vivo. In summary, these data demonstrated that low-toxic levels of curcumin could efficiently inhibit migration and invasion of lung cancer cells through inhibition of Rac1/PAK1 signaling pathway and MMP-2 and MMP-9 expression, which provided a novel insight into the molecular mechanism of curcumin against lung cancer.     

Method for isolation of intact titin (connectin) molecules from mammalian cardiac muscle

Cardiac titin was isolated from rabbit and ground squirrel ventricular muscles by a method that was used earlier to obtain myofibrils with intact minor proteins located in A-bands of sarcomeres (Podlubnaya, Z. A., et al. (1989) J. Mol. Biol., 210, 655–658). Small pieces of cardiac muscle were incubated for 2–3 weeks at 4°C in Ca²⁺-depleting solution before their homogenization to decrease activity of Ca²⁺-dependent proteases. Then the muscle was homogenized, and titin was isolated by the method of Soteriou, A., et al. (1993) J. Cell Sci., 14, 119–123. In control experiments, titin was isolated from cardiac muscle without its preincubation in Ca²⁺-depleting solution. Sometimes control titin preparations contained only T2-fragment, but generally they contained ～5–20% N2B-isoform of titin along with its T2-fragment. Preparations of titin obtained from rabbit cardiac muscle by our method contained ～30–50% of N2BA- and N2B-titin isoforms along with its T2-fragment. The content of α-structures in titin isolated by our method was increased. Actomyosin ATPase activity in vitro increased in the presence of titin preparations containing more intact molecules. This result confirms the significant role of titin in the regulation of actin-myosin interaction in muscles. The method used by us to preserve titin might be used for isolation of other proteins that are substrates of Ca²⁺-dependent proteases.