scholarly journals Formin-dependent actin assembly is regulated by distinct modes of Rho signaling in yeast

2003 ◽  
Vol 161 (6) ◽  
pp. 1081-1092 ◽  
Author(s):  
Yuqing Dong ◽  
David Pruyne ◽  
Anthony Bretscher
Keyword(s):  
Rho Gtpases ◽  
Elevated Temperatures ◽  
Mapk Pathway ◽  
Actin Assembly ◽  
Rho Proteins ◽  
Polarized Secretion ◽  
Cable Assembly ◽  
Bud Growth ◽  
Essential Function ◽  
Actin Cables

Formins are actin filament nucleators regulated by Rho-GTPases. In budding yeast, the formins Bni1p and Bnr1p direct the assembly of actin cables, which guide polarized secretion and growth. From the six yeast Rho proteins (Cdc42p and Rho1–5p), we have determined that four participate in the regulation of formin activity. We show that the essential function of Rho3p and Rho4p is to activate the formins Bni1p and Bnr1p, and that activated alleles of either formin are able to bypass the requirement for these Rho proteins. Through a separate signaling pathway, Rho1p is necessary for formin activation at elevated temperatures, acting through protein kinase C (Pkc1p), the major effector for Rho1p signaling to the actin cytoskeleton. Although Pkc1p also activates a MAPK pathway, this pathway does not function in formin activation. Formin-dependent cable assembly does not require Cdc42p, but in the absence of Cdc42p function, cable assembly is not properly organized during initiation of bud growth. These results show that formin function is under the control of three distinct, essential Rho signaling pathways.

scholarly journals Stable and Dynamic Axes of Polarity Use Distinct Formin Isoforms in Budding Yeast

2004 ◽  
Vol 15 (11) ◽  
pp. 4971-4989 ◽  
Author(s):  
David Pruyne ◽  
Lina Gao ◽  
Erfei Bi ◽  
Anthony Bretscher
Keyword(s):  
Mother Cell ◽  
Feedback Signal ◽  
Secretory Vesicles ◽  
Rho Proteins ◽  
Polarized Secretion ◽  
Bud Neck ◽  
Morphological Defects ◽  
Bud Growth ◽  
Actin Cables ◽  
Cytoskeletal Elements

Bud growth in yeast is guided by myosin-driven delivery of secretory vesicles from the mother cell to the bud. We find transport occurs along two sets of actin cables assembled by two formin isoforms. The Bnr1p formin assembles cables that radiate from the bud neck into the mother, providing a stable mother-bud axis. These cables also depend on septins at the neck and are required for efficient transport from the mother to the bud. The Bni1p formin assembles cables that line the bud cortex and target vesicles to varying locations in the bud. Loss of these cables results in morphological defects as vesicles accumulate at the neck. Assembly of these cables depends on continued polarized secretion, suggesting vesicular transport provides a positive feedback signal for Bni1p activation, possibly by rho-proteins. By coupling different formin isoforms to unique cortical landmarks, yeast uses common cytoskeletal elements to maintain stable and dynamic axes in the same cell.

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 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.

scholarly journals Rho GTPases as Key Molecular Players within Intestinal Mucosa and GI Diseases

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 66
Author(s):  
Rashmita Pradhan ◽  
Phuong A. Ngo ◽  
Luz d. C. Martínez-Sánchez ◽  
Markus F. Neurath ◽  
Rocío López-Posadas
Keyword(s):  
Intestinal Mucosa ◽  
Rho Gtpases ◽  
Current Knowledge ◽  
Cell Types ◽  
Effector Proteins ◽  
Special Focus ◽  
Specific Cell ◽  
Rho Proteins

Rho proteins operate as key regulators of the cytoskeleton, cell morphology and trafficking. Acting as molecular switches, the function of Rho GTPases is determined by guanosine triphosphate (GTP)/guanosine diphosphate (GDP) exchange and their lipidation via prenylation, allowing their binding to cellular membranes and the interaction with downstream effector proteins in close proximity to the membrane. A plethora of in vitro studies demonstrate the indispensable function of Rho proteins for cytoskeleton dynamics within different cell types. However, only in the last decades we have got access to genetically modified mouse models to decipher the intricate regulation between members of the Rho family within specific cell types in the complex in vivo situation. Translationally, alterations of the expression and/or function of Rho GTPases have been associated with several pathological conditions, such as inflammation and cancer. In the context of the GI tract, the continuous crosstalk between the host and the intestinal microbiota requires a tight regulation of the complex interaction between cellular components within the intestinal tissue. Recent studies demonstrate that Rho GTPases play important roles for the maintenance of tissue homeostasis in the gut. We will summarize the current knowledge on Rho protein function within individual cell types in the intestinal mucosa in vivo, with special focus on intestinal epithelial cells and T cells.

scholarly journals Postprenylation CAAX Processing Is Required for Proper Localization of Ras but Not Rho GTPases

2005 ◽  
Vol 16 (4) ◽  
pp. 1606-1616 ◽  
Author(s):  
David Michaelson ◽  
Wasif Ali ◽  
Vi K. Chiu ◽  
Martin Bergo ◽  
Joseph Silletti ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Protein Trafficking ◽  
Rho Gtpases ◽  
Ras Proteins ◽  
Rho Proteins ◽  
Carboxyl Methylation ◽  
C Terminus ◽  
Individual Contributions ◽  
The Individual ◽  
And Function

The CAAX motif at the C terminus of most monomeric GTPases is required for membrane targeting because it signals for a series of three posttranslational modifications that include isoprenylation, endoproteolytic release of the C-terminal– AAX amino acids, and carboxyl methylation of the newly exposed isoprenylcysteine. The individual contributions of these modifications to protein trafficking and function are unknown. To address this issue, we performed a series of experiments with mouse embryonic fibroblasts (MEFs) lacking Rce1 (responsible for removal of the –AAX sequence) or Icmt (responsible for carboxyl methylation of the isoprenylcysteine). In MEFs lacking Rce1 or Icmt, farnesylated Ras proteins were mislocalized. In contrast, the intracellular localizations of geranylgeranylated Rho GTPases were not perturbed. Consistent with the latter finding, RhoGDI binding and actin remodeling were normal in Rce1- and Icmt-deficient cells. Swapping geranylgeranylation for farnesylation on Ras proteins or vice versa on Rho proteins reversed the differential sensitivities to Rce1 and Icmt deficiency. These results suggest that postprenylation CAAX processing is required for proper localization of farnesylated Ras but not geranygeranylated Rho proteins.

scholarly journals The Crossroads between RAS and RHO Signaling Pathways in Cellular Transformation, Motility and Contraction

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 819
Author(s):  
Olga Soriano ◽  
Marta Alcón-Pérez ◽  
Miguel Vicente-Manzanares ◽  
Esther Castellano
Keyword(s):  
Signaling Pathways ◽  
Actin Polymerization ◽  
Mapk Pathway ◽  
Human Cancer ◽  
Cellular Transformation ◽  
Dependent Manner ◽  
Rho Proteins ◽  
Filament Assembly ◽  
Multiple Signaling ◽  
Mechanical Feedback

Ras and Rho proteins are GTP-regulated molecular switches that control multiple signaling pathways in eukaryotic cells. Ras was among the first identified oncogenes, and it appears mutated in many forms of human cancer. It mainly promotes proliferation and survival through the MAPK pathway and the PI3K/AKT pathways, respectively. However, the myriad proteins close to the plasma membrane that activate or inhibit Ras make it a major regulator of many apparently unrelated pathways. On the other hand, Rho is weakly oncogenic by itself, but it critically regulates microfilament dynamics; that is, actin polymerization, disassembly and contraction. Polymerization is driven mainly by the Arp2/3 complex and formins, whereas contraction depends on myosin mini-filament assembly and activity. These two pathways intersect at numerous points: from Ras-dependent triggering of Rho activators, some of which act through PI3K, to mechanical feedback driven by actomyosin action. Here, we describe the main points of connection between the Ras and Rho pathways as they coordinately drive oncogenic transformation. We emphasize the biochemical crosstalk that drives actomyosin contraction driven by Ras in a Rho-dependent manner. We also describe possible routes of mechanical feedback through which myosin II activation may control Ras/Rho activation.

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 Regulation and Functions of ROP GTPases in Plant–Microbe Interactions

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2016
Author(s):  
Stefan Engelhardt ◽  
Adriana Trutzenberg ◽  
Ralph Hückelhoven
Keyword(s):  
Cell Invasion ◽  
Rho Gtpases ◽  
Fungal Pathogens ◽  
Plant Immunity ◽  
Pathogen Resistance ◽  
Research Field ◽  
Rho Proteins ◽  
Downstream Signaling ◽  
Microbe Interactions ◽  
Developmental Reprogramming

Rho proteins of plants (ROPs) form a specific clade of Rho GTPases, which are involved in either plant immunity or susceptibility to diseases. They are intensively studied in grass host plants, in which ROPs are signaling hubs downstream of both cell surface immune receptor kinases and intracellular nucleotide-binding leucine-rich repeat receptors, which activate major branches of plant immune signaling. Additionally, invasive fungal pathogens may co-opt the function of ROPs for manipulation of the cytoskeleton, cell invasion and host cell developmental reprogramming, which promote pathogenic colonization. Strikingly, mammalian bacterial pathogens also initiate both effector-triggered susceptibility for cell invasion and effector-triggered immunity via Rho GTPases. In this review, we summarize central concepts of Rho signaling in disease and immunity of plants and briefly compare them to important findings in the mammalian research field. We focus on Rho activation, downstream signaling and cellular reorganization under control of Rho proteins involved in disease progression and pathogen resistance.

scholarly journals Differential Localization of Rho Gtpases in Live Cells

2001 ◽  
Vol 152 (1) ◽  
pp. 111-126 ◽  
Author(s):  
David Michaelson ◽  
Joseph Silletti ◽  
Gretchen Murphy ◽  
Peter D'Eustachio ◽  
Mark Rush ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Fluorescent Protein ◽  
Essential Feature ◽  
Hypervariable Region ◽  
Dominant Negative ◽  
Live Cells ◽  
Rho Proteins ◽  
Guanine Nucleotide Dissociation Inhibitor ◽  
Membrane Compartment ◽  
Green Fluorescent

Determinants of membrane targeting of Rho proteins were investigated in live cells with green fluorescent fusion proteins expressed with or without Rho-guanine nucleotide dissociation inhibitor (GDI)α. The hypervariable region determined to which membrane compartment each protein was targeted. Targeting was regulated by binding to RhoGDIα in the case of RhoA, Rac1, Rac2, and Cdc42hs but not RhoB or TC10. Although RhoB localized to the plasma membrane (PM), Golgi, and motile peri-Golgi vesicles, TC10 localized to PMs and endosomes. Inhibition of palmitoylation mislocalized H-Ras, RhoB, and TC10 to the endoplasmic reticulum. Although overexpressed Cdc42hs and Rac2 were observed predominantly on endomembrane, Rac1 was predominantly at the PM. RhoA was cytosolic even when expressed at levels in vast excess of RhoGDIα. Oncogenic Dbl stimulated translocation of green fluorescent protein (GFP)-Rac1, GFP-Cdc42hs, and GFP-RhoA to lamellipodia. RhoGDI binding to GFP-Cdc42hs was not affected by substituting farnesylation for geranylgeranylation. A palmitoylation site inserted into RhoA blocked RhoGDIα binding. Mutations that render RhoA, Cdc42hs, or Rac1, either constitutively active or dominant negative abrogated binding to RhoGDIα and redirected expression to both PMs and internal membranes. Thus, despite the common essential feature of the CAAX (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) motif, the subcellular localizations of Rho GTPases, like their functions, are diverse and dynamic.

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