scholarly journals Arf6 Can Trigger Wave Regulatory Complex-Dependent Actin Assembly Independent of Arno

2020 ◽  
Vol 21 (7) ◽  
pp. 2457 ◽  
Author(s):  
Vikash Singh ◽  
Anthony C. Davidson ◽  
Peter J. Hume ◽  
Vassilis Koronakis
Keyword(s):  
Lipid Bilayers ◽  
Membrane Trafficking ◽  
Actin Polymerization ◽  
Small Gtpase ◽  
Actin Dynamics ◽  
Actin Assembly ◽  
Nucleotide Exchange ◽  
Cortical Actin ◽  
Nucleotide Exchange Factor ◽  
Regulatory Complex

The small GTPase ADP-ribosylation factor 6 (Arf6) anchors at the plasma membrane to orchestrate key functions, such as membrane trafficking and regulating cortical actin cytoskeleton rearrangement. A number of studies have identified key players that interact with Arf6 to regulate actin dynamics in diverse cell processes, yet it is still unknown whether Arf6 can directly signal to the wave regulatory complex to mediate actin assembly. By reconstituting actin dynamics on supported lipid bilayers, we found that Arf6 in co-ordination with Rac1(Ras-related C3 botulinum toxin substrate 1) can directly trigger actin polymerization by recruiting wave regulatory complex components. Interestingly, we demonstrated that Arf6 triggers actin assembly at the membrane directly without recruiting the Arf guanine nucleotide exchange factor (GEF) ARNO (ARF nucleotide-binding site opener), which is able to activate Arf1 to enable WRC-dependent actin assembly. Furthermore, using labelled E. coli, we demonstrated that actin assembly by Arf6 also contributes towards efficient phagocytosis in THP-1 macrophages. Taken together, this study reveals a mechanism for Arf6-driven actin polymerization.

Integrin-Independent Role of CalDAG-GEFI in Neutrophil Chemotaxis.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1266-1266 ◽  
Author(s):  
Carla Carbo ◽  
Tobias Goerge ◽  
Hidenori Hattori ◽  
Daniel Duerschmied ◽  
Stephen M. Cifuni ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Neutrophil Migration ◽  
Small Gtpase ◽  
Calcium Flux ◽  
Migration Speed ◽  
Neutrophil Chemotaxis ◽  
Nucleotide Exchange ◽  
Transwell Assay ◽  
Nucleotide Exchange Factor

Abstract Neutrophil chemotaxis and transmigration towards a source of inflammation are two crucial processes for host defense against infection that rely on integrin function. Recently, integrin-independent migration of dendritic cells to the lymph node has been brought to light, although neutrophil migration in the presence of EDTA was reported many years ago. Ca2+ and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), is a small signaling protein that plays a key role in the activation of beta-1, beta-2, and beta-3 integrins in platelets and neutrophils by activating the small GTPase Rap1. We explored the role of CalDAG-GEFI in integrin-independent chemotaxis in neutrophils. Here we report that CalDAG-GEFI−/− neutrophils have impaired chemotaxis that is independent of integrin function. In a chemotaxis transwell assay towards LTB4 and in the presence of 10mM EDTA, CalDAG-GEFI−/− neutrophils had a 50% reduction in transmigration over 60 minutes compared to wild-type (WT) neutrophils (p<0.05). In separate experiments we confirmed that the transwell assay is independent of integrins using either CD18−/− neutrophils or WT neutrophils plus a blocking anti-CD18 monoclonal antibody. We previously showed that LTB4 signaling upstream of CalDAG-GEFI was not affected in CalDAG-GEFI−/− neutrophils, as assessed by intracellular calcium flux measurements. Using videomicroscopy to visualize the live migrating neutrophils in a horizontal plate in the presence of 10mM EDTA, we found that the reason CalDAG-GEFI−/− neutrophils fail to reach the chemotactic stimulus (10 pg/mL LTB4) is because they have a significantly reduced migration speed compared to WT neutrophils (16 um/sec vs. 23 um/sec, p<0.05), and also because they have an abnormal chemotactic directionality, with a directionality index (the distance between the start and finish points of a migrating neutrophil/total distance covered by the migrating neutrophil) of 0.84 vs 0.94 in WT neutrophils, p<0.05. We investigated whether the observed differences in chemotaxis between CalDAG-GEFI−/− and WT neutrophils could be explained by differences in F-actin polymerization. Using fluorescence microscopy, we found that the percentage of CalDAG-GEFI−/− neutrophils with F-actin pseudopodia after LTB4 stimulation was significantly lower compared to WT neutrophils (22% vs. 56.7%, p<0.05), suggesting that CalDAG-GEFI−/− neutrophils have a defect in F-actin polymerization. Overall, our studies suggest that CalDAG-GEFI plays a role in the mechanisms that regulate both the migration speed and direction of neutrophils during chemotaxis, independent of its established role in integrin activation.

scholarly journals Structure of CYRI-B (FAM49B), a key regulator of cellular actin assembly

2020 ◽  
Vol 76 (10) ◽  
pp. 1015-1024 ◽  
Author(s):  
Elise Kaplan ◽  
Rachael Stone ◽  
Peter J. Hume ◽  
Nicholas P. Greene ◽  
Vassilis Koronakis
Keyword(s):  
Actin Polymerization ◽  
Small Gtpase ◽  
Actin Assembly ◽  
X Ray ◽  
Rhincodon Typus ◽  
X Ray Crystallography ◽  
Conserved Domain ◽  
Rac1 Activity ◽  
Regulatory Complex ◽  
Lamellipodia Formation

In eukaryotes, numerous fundamental processes are controlled by the WAVE regulatory complex (WRC) that regulates cellular actin polymerization, crucial for cell motility, cell–cell adhesion and epithelial differentiation. Actin assembly is triggered by interaction of the small GTPase Rac1 with CYFIP1, a key component of the WRC. Previously known as FAM49B, CYRI-B is a protein that is highly conserved across the Eukaryota and has recently been revealed to be a key regulator of Rac1 activity. Mutation of CYRI-B or alteration of its expression therefore leads to altered actin nucleation dynamics, with impacts on lamellipodia formation, cell migration and infection by intracellular pathogens. In addition, knockdown of CYRI-B expression in cancer cell lines results in accelerated cell proliferation and invasiveness. Here, the structure of Rhincodon typus (whale shark) CYRI-B is presented, which is the first to be reported of any CYRI family member. Solved by X-ray crystallography, the structure reveals that CYRI-B comprises three distinct α-helical subdomains and is highly structurally related to a conserved domain present in CYFIP proteins. The work presented here establishes a template towards a better understanding of CYRI-B biological function.

scholarly journals The Diaphanous-related Formin mDia1 Controls Serum Response Factor Activity through its Effects on Actin Polymerization

2002 ◽  
Vol 13 (11) ◽  
pp. 4088-4099 ◽  
Author(s):  
John W. Copeland ◽  
Richard Treisman
Keyword(s):  
Actin Polymerization ◽  
Serum Response Factor ◽  
Signal Pathway ◽  
Small Gtpase ◽  
Actin Dynamics ◽  
Actin Assembly ◽  
Extracellular Signals ◽  
Activation Assay ◽  
The Relationship ◽  
Serum Response

SRF-dependent transcription is regulated by the small GTPase RhoA via its effects on actin dynamics. The diaphanous-related formin (DRF) proteins have been identified as candidate RhoA effectors mediating signaling to SRF. Here we investigate the relationship between SRF activation and actin polymerization by the DRF mDia1. We show that the ability of mDia1 to potentiate SRF activity is strictly correlated with its ability to promote F-actin assembly. Both processes can occur independently of the mDia1 FH1 domain but require sequences in an extended C-terminal region encompassing the conserved FH2 domain. mDia-mediated SRF activation, but not F-actin assembly, can be blocked by a nonpolymerizable actin mutant, placing actin downstream of mDia in the signal pathway. The SRF activation assay was used to identify inactive mDia1 derivatives that inhibit serum- and LPA-induced signaling to SRF. We show that these interfering mutants also block F-actin assembly, whether induced by mDia proteins or extracellular signals. These results identify novel functional elements of mDia1 and show that it regulates SRF activity by inducing depletion of the cellular pool of G-actin.

scholarly journals A functional family of fluorescent nucleotide analogues to investigate actin dynamics and energetics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jessica Colombo ◽  
Adrien Antkowiak ◽  
Konstantin Kogan ◽  
Tommi Kotila ◽  
Jenna Elliott ◽  
...  
Keyword(s):  
Single Molecule ◽  
Actin Polymerization ◽  
Eukaryotic Cell ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Assembly ◽  
Nucleotide Exchange ◽  
Nucleotide Analogues ◽  
Highly Sensitive ◽  
Fluorescent Nucleotides

AbstractActin polymerization provides force for vital processes of the eukaryotic cell, but our understanding of actin dynamics and energetics remains limited due to the lack of high-quality probes. Most current probes affect dynamics of actin or its interactions with actin-binding proteins (ABPs), and cannot track the bound nucleotide. Here, we identify a family of highly sensitive fluorescent nucleotide analogues structurally compatible with actin. We demonstrate that these fluorescent nucleotides bind to actin, maintain functional interactions with a number of essential ABPs, are hydrolyzed within actin filaments, and provide energy to power actin-based processes. These probes also enable monitoring actin assembly and nucleotide exchange with single-molecule microscopy and fluorescence anisotropy kinetics, therefore providing robust and highly versatile tools to study actin dynamics and functions of ABPs.

scholarly journals PLEKHG3 enhances polarized cell migration by activating actin filaments at the cell front

2016 ◽  
Vol 113 (36) ◽  
pp. 10091-10096 ◽  
Author(s):  
Trang Thi Thu Nguyen ◽  
Wei Sun Park ◽  
Byung Ouk Park ◽  
Cha Yeon Kim ◽  
Yohan Oh ◽  
...  
Keyword(s):  
Positive Feedback ◽  
Actin Polymerization ◽  
Leading Edge ◽  
Cell Polarization ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Cell Front ◽  
Control Protein

Cells migrate by directing Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) activities and by polymerizing actin toward the leading edge of the cell. Previous studies have proposed that this polarization process requires a local positive feedback in the leading edge involving Rac small GTPase and actin polymerization with PI3K likely playing a coordinating role. Here, we show that the pleckstrin homology and RhoGEF domain containing G3 (PLEKHG3) is a PI3K-regulated Rho guanine nucleotide exchange factor (RhoGEF) for Rac1 and Cdc42 that selectively binds to newly polymerized actin at the leading edge of migrating fibroblasts. Optogenetic inactivation of PLEKHG3 showed that PLEKHG3 is indispensable both for inducing and for maintaining cell polarity. By selectively binding to newly polymerized actin, PLEKHG3 promotes local Rac1/Cdc42 activation to induce more local actin polymerization, which in turn promotes the recruitment of more PLEKHG3 to induce and maintain cell front. Thus, autocatalytic reinforcement of PLEKHG3 localization to the leading edge of the cell provides a molecular basis for the proposed positive feedback loop that is required for cell polarization and directed migration.

Isolation and Characterization of Nrf1p, a Novel Negative Regulator of the Cdc42p GTPase in Schizosaccharomyces pombe

Genetics ◽  
2000 ◽  
Vol 154 (1) ◽  
pp. 155-165 ◽  
Author(s):  
Janet M Murray ◽  
Douglas I Johnson
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fluorescent Protein ◽  
Amino Acid Identity ◽  
Negative Regulator ◽  
Nucleotide Exchange ◽  
Cortical Actin ◽  
Isolation And Characterization ◽  
Nucleotide Exchange Factor ◽  
Green Fluorescent ◽  
Vacuolar Membranes

Abstract The Cdc42p GTPase and its regulators, such as the Saccharomyces cerevisiae Cdc24p guanine-nucleotide exchange factor, control signal-transduction pathways in eukaryotic cells leading to actin rearrangements. A cross-species genetic screen was initiated based on the ability of negative regulators of Cdc42p to reverse the Schizosaccharomyces pombe Cdc42p suppression of a S. cerevisiae cdc24ts mutant. A total of 32 S. pombe nrf (negative regulator of Cdc forty two) cDNAs were isolated that reversed the suppression. One cDNA, nrf1+, encoded an ~15 kD protein with three potential transmembrane domains and 78% amino-acid identity to a S. cerevisiae gene, designated NRF1. A S. pombe Δnrf1 mutant was viable but overexpression of nrf1+ in S. pombe resulted in dose-dependent lethality, with cells exhibiting an ellipsoidal morphology indicative of loss of polarized cell growth along with partially delocalized cortical actin and large vacuoles. nrf1+ also displayed synthetic overdose phenotypes with cdc42 and pak1 alleles. Green fluorescent protein (GFP)-Cdc42p and GFP-Nrf1p colocalized to intracellular membranes, including vacuolar membranes, and to sites of septum formation during cytokinesis. GFP-Nrf1p vacuolar localization depended on the S. pombe Cdc24p homolog Scd1p. Taken together, these data are consistent with Nrf1p functioning as a negative regulator of Cdc42p within the cell polarity pathway.

scholarly journals Activation of Cdc42 GTPase upon CRY2-Induced Cortical Recruitment Is Antagonized by GAPs in Fission Yeast

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2089 ◽  
Author(s):  
Iker Lamas ◽  
Nathalie Weber ◽  
Sophie G. Martin
Keyword(s):  
Fission Yeast ◽  
Positive Feedback ◽  
Antagonistic Activity ◽  
Cell Polarization ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Gtpase Activating Protein ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Cell Architecture

The small GTPase Cdc42 is critical for cell polarization in eukaryotic cells. In rod-shaped fission yeast Schizosaccharomyces pombe cells, active GTP-bound Cdc42 promotes polarized growth at cell poles, while inactive Cdc42-GDP localizes ubiquitously also along cell sides. Zones of Cdc42 activity are maintained by positive feedback amplification involving the formation of a complex between Cdc42-GTP, the scaffold Scd2, and the guanine nucleotide exchange factor (GEF) Scd1, which promotes the activation of more Cdc42. Here, we use the CRY2-CIB1 optogenetic system to recruit and cluster a cytosolic Cdc42 variant at the plasma membrane and show that this leads to its moderate activation also on cell sides. Surprisingly, Scd2, which binds Cdc42-GTP, is still recruited to CRY2-Cdc42 clusters at cell sides in individual deletion of the GEFs Scd1 or Gef1. We show that activated Cdc42 clusters at cell sides are able to recruit Scd1, dependent on the scaffold Scd2. However, Cdc42 activity is not amplified by positive feedback and does not lead to morphogenetic changes, due to antagonistic activity of the GTPase activating protein Rga4. Thus, the cell architecture is robust to moderate activation of Cdc42 at cell sides.

scholarly journals Insulin Stimulates Phosphatidylinositol 3-Phosphate Production via the Activation of Rab5

2008 ◽  
Vol 19 (7) ◽  
pp. 2718-2728 ◽  
Author(s):  
Irfan J. Lodhi ◽  
Dave Bridges ◽  
Shian-Huey Chiang ◽  
Yanling Zhang ◽  
Alan Cheng ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Critical Role ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Glut4 Translocation ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Nucleotide Exchange Factor ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-phosphate (PI(3)P) plays an important role in insulin-stimulated glucose uptake. Insulin promotes the production of PI(3)P at the plasma membrane by a process dependent on TC10 activation. Here, we report that insulin-stimulated PI(3)P production requires the activation of Rab5, a small GTPase that plays a critical role in phosphoinositide synthesis and turnover. This activation occurs at the plasma membrane and is downstream of TC10. TC10 stimulates Rab5 activity via the recruitment of GAPEX-5, a VPS9 domain–containing guanyl nucleotide exchange factor that forms a complex with TC10. Although overexpression of plasma membrane-localized GAPEX-5 or constitutively active Rab5 promotes PI(3)P formation, knockdown of GAPEX-5 or overexpression of a dominant negative Rab5 mutant blocks the effects of insulin or TC10 on this process. Concomitant with its effect on PI(3)P levels, the knockdown of GAPEX-5 blocks insulin-stimulated Glut4 translocation and glucose uptake. Together, these studies suggest that the TC10/GAPEX-5/Rab5 axis mediates insulin-stimulated production of PI(3)P, which regulates trafficking of Glut4 vesicles.

scholarly journals Myoblast Migration and Directional Persistence Affected by Syndecan-4-Mediated Tiam-1 Expression and Distribution

2020 ◽  
Vol 21 (3) ◽  
pp. 823 ◽  
Author(s):  
Daniel Becsky ◽  
Szuzina Gyulai-Nagy ◽  
Arpad Balind ◽  
Peter Horvath ◽  
Laszlo Dux ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Underlying Mechanism ◽  
Small Gtpase ◽  
Asymmetric Distribution ◽  
Nucleotide Exchange ◽  
Muscle Stem Cells ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
T Lymphoma ◽  
Migration Capacity

Skeletal muscle is constantly renewed in response to injury, exercise, or muscle diseases. Muscle stem cells, also known as satellite cells, are stimulated by local damage to proliferate extensively and form myoblasts that then migrate, differentiate, and fuse to form muscle fibers. The transmembrane heparan sulfate proteoglycan syndecan-4 plays multiple roles in signal transduction processes, such as regulating the activity of the small GTPase Rac1 (Ras-related C3 botulinum toxin substrate 1) by binding and inhibiting the activity of Tiam1 (T-lymphoma invasion and metastasis-1), a guanine nucleotide exchange factor for Rac1. The Rac1-mediated actin remodeling is required for cell migration. Syndecan-4 knockout mice cannot regenerate injured muscle; however, the detailed underlying mechanism is unknown. Here, we demonstrate that shRNA-mediated knockdown of syndecan-4 decreases the random migration of mouse myoblasts during live-cell microscopy. Treatment with the Rac1 inhibitor NSC23766 did not restore the migration capacity of syndecan-4 silenced cells; in fact, it was further reduced. Syndecan-4 knockdown decreased the directional persistence of migration, abrogated the polarized, asymmetric distribution of Tiam1, and reduced the total Tiam1 level of the cells. Syndecan-4 affects myoblast migration via its role in expression and localization of Tiam1; this finding may facilitate greater understanding of the essential role of syndecan-4 in the development and regeneration of skeletal muscle.

scholarly journals Actin filament dynamics are dominated by rapid growth and severing activity in the Arabidopsis cortical array

2009 ◽  
Vol 184 (2) ◽  
pp. 269-280 ◽  
Author(s):  
Christopher J. Staiger ◽  
Michael B. Sheahan ◽  
Parul Khurana ◽  
Xia Wang ◽  
David W. McCurdy ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Stochastic Dynamics ◽  
Actin Dynamics ◽  
Epifluorescence Microscopy ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Filament Dynamics ◽  
Cortical Array

Metazoan cells harness the power of actin dynamics to create cytoskeletal arrays that stimulate protrusions and drive intracellular organelle movements. In plant cells, the actin cytoskeleton is understood to participate in cell elongation; however, a detailed description and molecular mechanism(s) underpinning filament nucleation, growth, and turnover are lacking. Here, we use variable-angle epifluorescence microscopy (VAEM) to examine the organization and dynamics of the cortical cytoskeleton in growing and nongrowing epidermal cells. One population of filaments in the cortical array, which most likely represent single actin filaments, is randomly oriented and highly dynamic. These filaments grow at rates of 1.7 µm/s, but are generally short-lived. Instead of depolymerization at their ends, actin filaments are disassembled by severing activity. Remodeling of the cortical actin array also features filament buckling and straightening events. These observations indicate a mechanism inconsistent with treadmilling. Instead, cortical actin filament dynamics resemble the stochastic dynamics of an in vitro biomimetic system for actin assembly.

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