Rho, Rac and Cdc42 regulate actin organization and cell adhesion in macrophages

1997 ◽  
Vol 110 (6) ◽  
pp. 707-720 ◽  
Author(s):  
W.E. Allen ◽  
G.E. Jones ◽  
J.W. Pollard ◽  
A.J. Ridley
Keyword(s):  
Actin Reorganization ◽  
Membrane Ruffling ◽  
Actin Organization ◽  
Phosphorylated Proteins ◽  
Adhesion Sites ◽  
Actin Cable ◽  
Cable Assembly ◽  
Beta1 Integrin ◽  
Actin Cables ◽  
In Cells

Rho family proteins are known to regulate actin organization in fibroblasts, but their functions in cells of haematopoietic origin have not been studied in detail. Bac1.2F5 cells are a colony-stimulating factor-1 (CSF-1)-dependent murine macrophage cell line; CSF-1 stimulates their proliferation and motility, and acts as a chemoattractant. CSF-1 rapidly induced actin reorganization in Bac1 cells: it stimulated the formation of filopodia, lamellipodia and membrane ruffles at the plasma membrane, as well as the appearance of fine actin cables within the cell interior. Microinjection of constitutively activated (V12)Rac1 stimulated lamellipodium formation and membrane ruffling. The dominant inhibitory Rac mutant, N17Rac1, inhibited CSF-1-induced lamellipodium formation, and also induced cell rounding. V12Cdc42 induced the formation of long filopodia, while the dominant inhibitory mutant N17Cdc42 prevented CSF-1-induced formation of filopodia but not lamellipodia. V14RhoA stimulated actin cable assembly and cell contraction, while the Rho inhibitor, C3 transferase, induced the loss of actin cables. Bac1 cells had cell-to-substratum adhesion sites containing beta1 integrin, pp125FAK, paxillin, vinculin, and tyrosine phosphorylated proteins. These ‘focal complexes’ were present in growing and CSF-1-starved cells, but were disassembled in cells injected with N17Cdc42 or N17Rac1. Interestingly, beta1 integrin did not disperse until long after focal phosphotyrosine and vinculin staining had disappeared. We conclude that in Bac1 macrophages Cdc42, Rac and Rho regulate the formation of distinct actin filament-based structures, and that Cdc42 and Rac are also required for the assembly of adhesion sites to the extracellular matrix.

scholarly journals Mechanism and cellular function of Bud6 as an actin nucleation–promoting factor

2011 ◽  
Vol 22 (21) ◽  
pp. 4016-4028 ◽  
Author(s):  
Brian R. Graziano ◽  
Amy Grace DuPage ◽  
Alphee Michelot ◽  
Dennis Breitsprecher ◽  
James B. Moseley ◽  
...  
Keyword(s):  
Actin Assembly ◽  
Actin Cable ◽  
Polarized Cell Growth ◽  
Cable Assembly ◽  
Nucleation Promoting Factor ◽  
Actin Cables ◽  
Elongation Phase

Formins are a conserved family of actin assembly–promoting factors with diverse biological roles, but how their activities are regulated in vivo is not well understood. In Saccharomyces cerevisiae, the formins Bni1 and Bnr1 are required for the assembly of actin cables and polarized cell growth. Proper cable assembly further requires Bud6. Previously it was shown that Bud6 enhances Bni1-mediated actin assembly in vitro, but the biochemical mechanism and in vivo role of this activity were left unclear. Here we demonstrate that Bud6 specifically stimulates the nucleation rather than the elongation phase of Bni1-mediated actin assembly, defining Bud6 as a nucleation-promoting factor (NPF) and distinguishing its effects from those of profilin. We generated alleles of Bud6 that uncouple its interactions with Bni1 and G-actin and found that both interactions are critical for NPF activity. Our data indicate that Bud6 promotes filament nucleation by recruiting actin monomers to Bni1. Genetic analysis of the same alleles showed that Bud6 regulation of formin activity is critical for normal levels of actin cable assembly in vivo. Our results raise important mechanistic parallels between Bud6 and WASP, as well as between Bud6 and other NPFs that interact with formins such as Spire.

scholarly journals Emerin organizes actin flow for nuclear movement and centrosome orientation in migrating fibroblasts

2013 ◽  
Vol 24 (24) ◽  
pp. 3869-3880 ◽  
Author(s):  
Wakam Chang ◽  
Eric S. Folker ◽  
Howard J. Worman ◽  
Gregg G. Gundersen
Keyword(s):  
Membrane Protein ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Myosin Ii ◽  
Leading Edge ◽  
Nuclear Movement ◽  
Linear Arrays ◽  
Actin Cable ◽  
Actin Cables ◽  
In Cells

In migrating fibroblasts, rearward movement of the nucleus orients the centrosome toward the leading edge. Nuclear movement results from coupling rearward-moving, dorsal actin cables to the nucleus by linear arrays of nesprin-2G and SUN2, termed transmembrane actin-associated nuclear (TAN) lines. A-type lamins anchor TAN lines, prompting us to test whether emerin, a nuclear membrane protein that interacts with lamins and TAN line proteins, contributes to nuclear movement. In fibroblasts depleted of emerin, nuclei moved nondirectionally or completely failed to move. Consistent with these nuclear movement defects, dorsal actin cable flow was nondirectional in cells lacking emerin. TAN lines formed normally in cells lacking emerin and were coordinated with the erratic nuclear movements, although in 20% of the cases, TAN lines slipped over immobile nuclei. Myosin II drives actin flow, and depletion of myosin IIB, but not myosin IIA, showed similar nondirectional nuclear movement and actin flow as in emerin-depleted cells. Myosin IIB specifically coimmunoprecipitated with emerin, and emerin depletion prevented myosin IIB localization near nuclei. These results show that emerin functions with myosin IIB to polarize actin flow and nuclear movement in fibroblasts, suggesting a novel function for the nuclear envelope in organizing directional actin flow and cytoplasmic polarity.

scholarly journals Pob1 Participates in the Cdc42 Regulation of Fission Yeast Actin Cytoskeleton

2009 ◽  
Vol 20 (20) ◽  
pp. 4390-4399 ◽  
Author(s):  
Sergio A. Rincón ◽  
Yanfang Ye ◽  
M. Antonia Villar-Tajadura ◽  
Beatriz Santos ◽  
Sophie G. Martin ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Fission Yeast ◽  
Mutant Strain ◽  
Rho Gtpases ◽  
Ph Domain ◽  
Actin Organization ◽  
Multicopy Suppressor ◽  
Sam Domain ◽  
Actin Cable ◽  
Actin Cables

Rho GTPases regulate the actin cytoskeleton in all eukaryotes. Fission yeast Cdc42 is involved in actin cable assembly and formin For3 regulation. We isolated cdc42-879 as a thermosensitive strain with actin cable and For3 localization defects. In a multicopy suppressor screening, we identified pob1+as suppressor of cdc42-879 thermosensitivity. Pob1 overexpression also partially restores actin cables and localization of For3 in the mutant strain. Pob1 interacts with Cdc42 and this GTPase regulates Pob1 localization and/or stability. The C-terminal pleckstrin homology (PH) domain of Pob1 is required for Cdc42 binding. Pob1 also binds to For3 through its N-terminal sterile alpha motif (SAM) domain and contributes to the formin localization at the cell tips. The previously described pob1-664 mutant strain (Mol. Biol. Cell. 10, 2745–2757, 1999), which carries a mutation in the PH domain, as well as pob1 mutant strains in which Pob1 lacks the N-terminal region (pob1ΔN) or the SAM domain (pob1ΔSAM), have cytoskeletal defects similar to that of cdc42-879 cells. Expression of constitutively active For3DAD* partially restores actin organization in cdc42-879, pob1-664, pob1ΔN, and pob1ΔSAM. Therefore, we propose that Pob1 is required for For3 localization to the tips and facilitates Cdc42-mediated relief of For3 autoinhibition to stimulate actin cable formation.

scholarly journals The C-terminal domain of EFA6A interacts directly with F-actin and assembles F-actin bundles

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Eric Macia ◽  
Mariagrazia Partisani ◽  
Hong Wang ◽  
Sandra Lacas-Gervais ◽  
Christophe Le Clainche ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Neuronal Morphogenesis ◽  
Actin Reorganization ◽  
Actin Bundles ◽  
Actin Organization ◽  
C Terminus ◽  
Membrane Protrusions ◽  
Actin Cables

AbstractThe Arf6-specific exchange factor EFA6 is involved in the endocytic/recycling pathway for different cargos. In addition EFA6 acts as a powerful actin cytoskeleton organizer, a function required for its role in the establishment of the epithelial cell polarity and in neuronal morphogenesis. We previously showed that the C-terminus of EFA6 (EFA6-Ct) is the main domain which contributes to actin reorganization. Here, by in vitro and in vivo experiments, we sought to decipher, at the molecular level, how EFA6 controls the dynamic and structuring of actin filaments. We showed that EFA6-Ct interferes with actin polymerization by interacting with and capping actin filament barbed ends. Further, in the presence of actin mono-filaments, the addition of EFA6-Ct triggered the formation of actin bundles. In cells, when the EFA6-Ct was directed to the plasma membrane, as is the case for the full-length protein, its expression induced the formation of membrane protrusions enriched in actin cables. Collectively our data explain, at least in part, how EFA6 plays an essential role in actin organization by interacting with and bundling F-actin.

scholarly journals Regulation of the Formin for3p by cdc42p and bud6p

2007 ◽  
Vol 18 (10) ◽  
pp. 4155-4167 ◽  
Author(s):  
Sophie G. Martin ◽  
Sergio A. Rincón ◽  
Roshni Basu ◽  
Pilar Pérez ◽  
Fred Chang
Keyword(s):  
Cell Growth ◽  
Fission Yeast ◽  
Rho Gtpase ◽  
Intramolecular Interaction ◽  
Actin Cable ◽  
Polarized Cell Growth ◽  
N Terminus ◽  
Cable Assembly ◽  
Actin Cables

Formins are conserved actin nucleators responsible for the assembly of diverse actin structures. Many formins are controlled through an autoinhibitory mechanism involving the interaction of a C-terminal DAD sequence with an N-terminal DID sequence. Here, we show that the fission yeast formin for3p, which mediates actin cable assembly and polarized cell growth, is regulated by a similar autoinhibitory mechanism in vivo. Multiple sites govern for3p localization to cell tips. The localization and activity of for3p are inhibited by an intramolecular interaction of divergent DAD and DID-like sequences. A for3p DAD mutant expressed at endogenous levels produces more robust actin cables, which appear to have normal organization and dynamics. We identify cdc42p as the primary Rho GTPase involved in actin cable assembly and for3p regulation. Both cdc42p, which binds at the N terminus of for3p, and bud6p, which binds near the C-terminal DAD-like sequence, are needed for for3p localization and full activity, but a mutation in the for3p DAD restores for3p localization and other phenotypes of cdc42 and bud6 mutants. In particular, the for3p DAD mutation suppresses the bipolar growth (NETO) defect of bud6Δ cells. These findings suggest that cdc42p and bud6p activate for3p by relieving autoinhibition.

scholarly journals Yeast Formins Bni1 and Bnr1 Utilize Different Modes of Cortical Interaction during the Assembly of Actin Cables

2007 ◽  
Vol 18 (5) ◽  
pp. 1826-1838 ◽  
Author(s):  
Shawnna M. Buttery ◽  
Satoshi Yoshida ◽  
David Pellman
Keyword(s):  
Mother Cell ◽  
Fluorescence Recovery After Photobleaching ◽  
Actin Assembly ◽  
Actin Cable ◽  
Bud Neck ◽  
Cable Assembly ◽  
Different Populations ◽  
Actin Cables ◽  
Assembly Activity

The budding yeast formins Bni1 and Bnr1 control the assembly of actin cables. These formins exhibit distinct patterns of localization and polymerize two different populations of cables: Bni1 in the bud and Bnr1 in the mother cell. We generated a functional Bni1-3GFP that improved the visualization of Bni1 in vivo at endogenous levels. Bni1 exists as speckles in the cytoplasm, some of which colocalize on actin cables. These Bni1 speckles display linear, retrograde-directed movements. Loss of polymerized actin or specifically actin cables abolished retrograde movement, and resulted in depletion of Bni1 speckles from the cytoplasm, with enhanced targeting of Bni1 to the bud tip. Mutations that impair the actin assembly activity of Bni1 abolished the movement of Bni1 speckles, even when actin cables were present. In contrast, Bnr1-GFP or 3GFP-Bnr1 did not detectably associate with actin cables and was not observed as cytoplasmic speckles. Finally, fluorescence recovery after photobleaching demonstrated that Bni1 was very dynamic, exchanging between polarized sites and the cytoplasm, whereas Bnr1 was confined to the bud neck and did not exchange with a cytoplasmic pool. In summary, our results indicate that formins can have distinct modes of cortical interaction during actin cable assembly.

scholarly journals Regulation of a formin complex by the microtubule plus end protein tea1p

2004 ◽  
Vol 165 (5) ◽  
pp. 697-707 ◽  
Author(s):  
Becket Feierbach ◽  
Fulvia Verde ◽  
Fred Chang
Keyword(s):  
Cell Growth ◽  
Cell Polarization ◽  
Actin Assembly ◽  
Triple Mutant ◽  
Spatial Regulation ◽  
Actin Cable ◽  
Polarized Cell Growth ◽  
Cable Assembly ◽  
Actin Cables ◽  
Mutant Cells

The plus ends of microtubules have been speculated to regulate the actin cytoskeleton for the proper positioning of sites of cell polarization and cytokinesis. In the fission yeast Schizosaccharomyces pombe, interphase microtubules and the kelch repeat protein tea1p regulate polarized cell growth. Here, we show that tea1p is directly deposited at cell tips by microtubule plus ends. Tea1p associates in large “polarisome” complexes with bud6p and for3p, a formin that assembles actin cables. Tea1p also interacts in a separate complex with the CLIP-170 protein tip1p, a microtubule plus end–binding protein that anchors tea1p to the microtubule plus end. Localization experiments suggest that tea1p and bud6p regulate formin distribution and actin cable assembly. Although single mutants still polarize, for3Δbud6Δtea1Δ triple-mutant cells lack polarity, indicating that these proteins contribute overlapping functions in cell polarization. Thus, these experiments begin to elucidate how microtubules contribute to the proper spatial regulation of actin assembly and polarized cell growth.

Actin reorganization in airway smooth muscle cells involves Gq and Gi-2 activation of Rho

1999 ◽  
Vol 277 (3) ◽  
pp. L653-L661 ◽  
Author(s):  
Carol A. Hirshman ◽  
Charles W. Emala
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Airway Smooth Muscle ◽  
Actin Polymerization ◽  
Muscle Cells ◽  
Fiber Formation ◽  
Airway Smooth Muscle Cells ◽  
Actin Reorganization ◽  
Endothelin 1 ◽  
In Cells

Extracellular stimuli induce cytoskeleton reorganization (stress-fiber formation) in cells and Ca2+ sensitization in intact smooth muscle preparations by activating signaling pathways that involve Rho proteins, a subfamily of the Ras superfamily of monomeric G proteins. In airway smooth muscle, the agonists responsible for cytoskeletal reorganization via actin polymerization are poorly understood. Carbachol-, lysophosphatidic acid (LPA)-, and endothelin-1-induced increases in filamentous actin staining are indicative of actin reorganization (filamentous-to-globular actin ratios of 2.4 ± 0.3 in control cells, 6.7 ± 0.8 with carbachol, 7.2 ± 0.8 with LPA, and 7.4 ± 0.9 with endothelin-1; P < 0.001; n = 14 experiments). Although the effect of all agonists was blocked by C3 exoenzyme (inactivator of Rho), only carbachol was blocked by pertussis toxin. Although carbachol-induced actin reorganization was blocked in cells pretreated with antisense oligonucleotides directed against Gαi-2 alone, LPA- and endothelin-1-induced actin reorganization were only blocked when both Gαi-2 and Gqα were depleted. These data indicate that in human airway smooth muscle cells, carbachol induces actin reorganization via a Gαi-2pathway, whereas LPA or endothelin-1 induce actin reorganization via either a Gαi-2 or a Gqα pathway.

scholarly journals From Function to Shape: A Novel Role of a Formin in Morphogenesis of the Fungus Ashbya gossypii

2006 ◽  
Vol 17 (1) ◽  
pp. 130-145 ◽  
Author(s):  
Hans-Peter Schmitz ◽  
Andreas Kaufmann ◽  
Michael Köhli ◽  
Pierre Philippe Laissue ◽  
Peter Philippsen
Keyword(s):  
Giant Cells ◽  
Polar Growth ◽  
Ashbya Gossypii ◽  
Essential Factor ◽  
Wild Type ◽  
Actin Cable ◽  
Lateral Branches ◽  
Actin Cables ◽  
Two Hybrid

Morphogenesis of filamentous ascomycetes includes continuously elongating hyphae, frequently emerging lateral branches, and, under certain circumstances, symmetrically dividing hyphal tips. We identified the formin AgBni1p of the model fungus Ashbya gossypii as an essential factor in these processes. AgBni1p is an essential protein apparently lacking functional overlaps with the two additional A. gossypii formins that are nonessential. Agbni1 null mutants fail to develop hyphae and instead expand to potato-shaped giant cells, which lack actin cables and thus tip-directed transport of secretory vesicles. Consistent with the essential role in hyphal development, AgBni1p locates to tips, but not to septa. The presence of a diaphanous autoregulatory domain (DAD) indicates that the activation of AgBni1p depends on Rho-type GTPases. Deletion of this domain, which should render AgBni1p constitutively active, completely changes the branching pattern of young hyphae. New axes of polarity are no longer established subapically (lateral branching) but by symmetric divisions of hyphal tips (tip splitting). In wild-type hyphae, tip splitting is induced much later and only at much higher elongation speed. When GTP-locked Rho-type GTPases were tested, only the young hyphae with mutated AgCdc42p split at their tips, similar to the DAD deletion mutant. Two-hybrid experiments confirmed that AgBni1p interacts with GTP-bound AgCdc42p. These data suggest a pathway for transforming one axis into two new axes of polar growth, in which an increased activation of AgBni1p by a pulse of activated AgCdc42p stimulates additional actin cable formation and tip-directed vesicle transport, thus enlarging and ultimately splitting the polarity site.

scholarly journals Cdc42 and Phosphoinositide 3-Kinase Drive Rac-Mediated Actin Polymerization Downstream of c-Met in Distinct and Common Pathways

2007 ◽  
Vol 27 (19) ◽  
pp. 6615-6628 ◽  
Author(s):  
Tanja Bosse ◽  
Julia Ehinger ◽  
Aleksandra Czuchra ◽  
Stefanie Benesch ◽  
Anika Steffen ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Actin Polymerization ◽  
Pi3 Kinase ◽  
Actin Assembly ◽  
Actin Reorganization ◽  
Membrane Ruffling ◽  
Internalin B ◽  
Phosphoinositide 3 Kinase ◽  
Hepatocyte Growth ◽  
Wave Complex

ABSTRACT Activation of c-Met, the hepatocyte growth factor (HGF)/scatter factor receptor induces reorganization of the actin cytoskeleton, which drives epithelial cell scattering and motility and is exploited by pathogenic Listeria monocytogenes to invade nonepithelial cells. However, the precise contributions of distinct Rho-GTPases, the phosphatidylinositol 3-kinases, and actin assembly regulators to c-Met-mediated actin reorganization are still elusive. Here we report that HGF-induced membrane ruffling and Listeria invasion mediated by the bacterial c-Met ligand internalin B (InlB) were significantly impaired but not abrogated upon genetic removal of either Cdc42 or pharmacological inhibition of phosphoinositide 3-kinase (PI3-kinase). While loss of Cdc42 or PI3-kinase function correlated with reduced HGF- and InlB-triggered Rac activation, complete abolishment of actin reorganization and Rac activation required the simultaneous inactivation of both Cdc42 and PI3-kinase signaling. Moreover, Cdc42 activation was fully independent of PI3-kinase activity, whereas the latter partly depended on Cdc42. Finally, Cdc42 function did not require its interaction with the actin nucleation-promoting factor N-WASP. Instead, actin polymerization was driven by Arp2/3 complex activation through the WAVE complex downstream of Rac. Together, our data establish an intricate signaling network comprising as key molecules Cdc42 and PI3-kinase, which converge on Rac-mediated actin reorganization essential for Listeria invasion and membrane ruffling downstream of c-Met.

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