The formin FHOD1 and the small GTPase Rac1 promote vaccinia virus actin–based motility

Vaccinia virus dissemination relies on the N-WASP–ARP2/3 pathway, which mediates actin tail formation underneath cell-associated extracellular viruses (CEVs). Here, we uncover a previously unappreciated role for the formin FHOD1 and the small GTPase Rac1 in vaccinia actin tail formation. FHOD1 depletion decreased the number of CEVs forming actin tails and impaired the elongation rate of the formed actin tails. Recruitment of FHOD1 to actin tails relied on its GTPase binding domain in addition to its FH2 domain. In agreement with previous studies showing that FHOD1 is activated by the small GTPase Rac1, Rac1 was enriched and activated at the membrane surrounding actin tails. Rac1 depletion or expression of dominant-negative Rac1 phenocopied the effects of FHOD1 depletion and impaired the recruitment of FHOD1 to actin tails. FHOD1 overexpression rescued the actin tail formation defects observed in cells overexpressing dominant-negative Rac1. Altogether, our results indicate that, to display robust actin-based motility, vaccinia virus integrates the activity of the N-WASP–ARP2/3 and Rac1–FHOD1 pathways.

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RBD11, a bioengineered Rab11-binding module for visualizing and analyzing endogenous Rab11

10.1101/2021.01.25.428185 ◽

2021 ◽

Author(s):

Futaba Osaki ◽

Takahide Matsui ◽

Shu Hiragi ◽

Yuta Homma ◽

Mitsunori Fukuda

Keyword(s):

Membrane Proteins ◽

Neurite Outgrowth ◽

Membrane Trafficking ◽

Specific Binding ◽

Binding Specificity ◽

Small Gtpase ◽

Binding Domain ◽

Dominant Negative ◽

Epithelial Morphogenesis ◽

Phenotype Characteristic

ABSTRACTThe small GTPase Rab11 plays pivotal roles in diverse physiological phenomena, including the recycling of membrane proteins, cytokinesis, neurite outgrowth, and epithelial morphogenesis. One effective method of analyzing the function of endogenous Rab11 is to overexpress a Rab11-binding domain of one of its effectors, e.g., the C-terminal domain of Rab11-FIP2 (Rab11-FIP2-C), as a dominant-negative construct. However, the drawback of this method is the broader Rab binding specificity of the effector domain, because Rab11-FIP2-C binds to Rabs other than Rab11, e.g., to Rab14 and Rab25. In this study, we bioengineered an artificial Rab11-specific binding domain, named RBD11. Expression of RBD11 visualized endogenous Rab11 without affecting its localization or function, whereas expression of a tandem RBD11, named 2×RBD11, inhibited epithelial morphogenesis and induced a multi-lumen phenotype characteristic of Rab11-deficient cysts. We also developed two tools for temporally and reversibly analyzing Rab11-dependent membrane trafficking: tetracycline-inducible 2×RBD11 and an artificially oligomerized domain (FM)-tagged RBD11.

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EphA4 Signaling Regulates Blastomere Adhesion in the Xenopus Embryo by Recruiting Pak1 to Suppress Cdc42 Function

Molecular Biology of the Cell ◽

10.1091/mbc.e06-04-0294 ◽

2007 ◽

Vol 18 (3) ◽

pp. 1030-1043 ◽

Cited By ~ 19

Author(s):

Nicolas Bisson ◽

Luc Poitras ◽

Alexander Mikryukov ◽

Michel Tremblay ◽

Tom Moss

Keyword(s):

Catalytic Activity ◽

Cell Adhesion ◽

Cell Sorting ◽

Ectopic Expression ◽

Binding Domain ◽

Dominant Negative ◽

Membrane Targeting ◽

Dependent Manner ◽

Eph Receptors ◽

Gtpase Binding Domain

The control of cell adhesion is an important mechanism by which Eph receptors regulate cell sorting during development. Activation of EphA4 in Xenopus blastulae induces a reversible, cell autonomous loss-of-adhesion and disruption of the blastocoel roof. We show this phenotype is rescued by Nckβ (Grb4) dependent on its interaction with EphA4. Xenopus p21Cdc42/Rac-activated kinase xPAK1 interacts with Nck, is activated in embryo by EphA4 in an Nck-dependent manner, and is required for EphA4-induced loss-of-adhesion. Ectopic expression of xPAK1 phenocopies EphA4 activation. This does not require the catalytic activity of xPAK1, but it does require its GTPase binding domain and is enhanced by membrane targeting. Indeed, membrane targeting of the GTPase binding domain (GBD) of xPAK1 alone is sufficient to phenocopy EphA4 loss-of-adhesion. Both EphA4 and the xPAK1-GBD down-regulate RhoA-GTP levels, and consistent with this, loss-of-adhesion can be rescued by activated Cdc42, Rac, and RhoA and can be epistatically induced by dominant-negative RhoA. Despite this, neither Cdc42 nor Rac activities are down-regulated by EphA4 activation or by the xPAK1-GBD. Together, the data suggest that EphA4 activation sequesters active Cdc42 and in this way down-regulates cell–cell adhesion. This novel signaling pathway suggests a mechanism for EphA4-guided migration.

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RBD11, a bioengineered Rab11-binding module for visualizing and analyzing endogenous Rab11

Journal of Cell Science ◽

10.1242/jcs.257311 ◽

2021 ◽

pp. jcs.257311

Author(s):

Futaba Osaki ◽

Takahide Matsui ◽

Shu Hiragi ◽

Yuta Homma ◽

Mitsunori Fukuda

Keyword(s):

Membrane Proteins ◽

Neurite Outgrowth ◽

Membrane Trafficking ◽

Specific Binding ◽

Binding Specificity ◽

Small Gtpase ◽

Binding Domain ◽

Dominant Negative ◽

Epithelial Morphogenesis ◽

Phenotype Characteristic

The small GTPase Rab11 plays pivotal roles in diverse physiological phenomena, including the recycling of membrane proteins, cytokinesis, neurite outgrowth, and epithelial morphogenesis. One effective method of analyzing the function of endogenous Rab11 is to overexpress a Rab11-binding domain of one of its effectors, e.g., the C-terminal domain of Rab11-FIP2 (Rab11-FIP2-C), as a dominant-negative construct. However, the drawback of this method is the broader Rab binding specificity of the effector domain, because Rab11-FIP2-C binds to Rabs other than Rab11, e.g., to Rab14 and Rab25. In this study, we bioengineered an artificial Rab11-specific binding domain, named RBD11. Expression of RBD11 visualized endogenous Rab11 without affecting its localization or function, whereas expression of a tandem RBD11, named 2×RBD11, inhibited epithelial morphogenesis and induced a multi-lumen phenotype characteristic of Rab11-deficient cysts. We also developed two tools for temporally and reversibly analyzing Rab11-dependent membrane trafficking: tetracycline-inducible 2×RBD11 and an artificially oligomerized domain (FM)-tagged RBD11.

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Truncation of TRIM5 in the Feliformia Explains the Absence of Retroviral Restriction in Cells of the Domestic Cat

Journal of Virology ◽

10.1128/jvi.00670-09 ◽

2009 ◽

Vol 83 (16) ◽

pp. 8270-8275 ◽

Cited By ~ 42

Author(s):

William A. McEwan ◽

Torsten Schaller ◽

Laura M. Ylinen ◽

Margaret J. Hosie ◽

Greg J. Towers ◽

...

Keyword(s):

Mammalian Species ◽

Ectopic Expression ◽

Binding Domain ◽

Dominant Negative ◽

Domestic Cat ◽

Dominant Negative Effect ◽

Negative Effect ◽

In Cells

ABSTRACT TRIM5α mediates a potent retroviral restriction phenotype in diverse mammalian species. Here, we identify a TRIM5 transcript in cat cells with a truncated B30.2 capsid binding domain and ablated restrictive function which, remarkably, is conserved across the Feliformia. Cat TRIM5 displayed no restriction activity, but ectopic expression conferred a dominant negative effect against human TRIM5α. Our findings explain the absence of retroviral restriction in cat cells and suggest that disruption of the TRIM5 locus has arisen independently at least twice in the Carnivora, with implications concerning the evolution of the host and pathogen in this taxon.

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Poxviral Regulation of the Host NF-κB Response: the Vaccinia Virus M2L Protein Inhibits Induction of NF-κB Activation via an ERK2 Pathway in Virus-Infected Human Embryonic Kidney Cells

Journal of Virology ◽

10.1128/jvi.00935-06 ◽

2006 ◽

Vol 80 (17) ◽

pp. 8676-8685 ◽

Cited By ~ 73

Author(s):

Roderick Gedey ◽

Xiao-Lu Jin ◽

Olivia Hinthong ◽

Joanna L. Shisler

Keyword(s):

Vaccinia Virus ◽

Nuclear Translocation ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Wild Type ◽

Cell Nuclei ◽

Human Embryonic Kidney Cells ◽

Reading Frame ◽

Regulated Gene Expression ◽

In Cells

ABSTRACT Exposure of eukaryotic cells to viruses will activate the host NF-κB transcription factor, resulting in proinflammatory and immune protein production. Vaccinia virus (VV), the prototypic orthopoxvirus, expresses products that inhibit this antiviral event. To identify novel mechanisms responsible for this effect, we made use of a VV deletion mutant (MVA) that stimulates NF-κΒ activation in infected 293T cells. In this virus-host system, the extents of NF-κΒ-regulated gene expression and nuclear translocation were reduced in the presence of either PD 98059 or U0126, two compounds capable of blocking ERK1 and ERK2 phosphorylation. A similar repression was also observed in cells that contained a dominant, nonactive form of ERK2 but not in cells where ERK1 phosphorylation was inhibited via overexpression of a dominant-negative mutant MEK1 protein. Presumably, proteins expressed from a wild-type VV that block ERK2 activity would also inhibit MVA-induced NF-κB activation. Indeed, the expression of one such open reading frame, M2L, supported this prediction. First, ectopic M2L expression hampered ERK2 phosphorylation induced by exposure to phorbol myristate acetate. Second, viral M2L expression via infection of cells with a recombinant MVA construct that stably expressed M2L decreased the phosphorylation of ERK2 compared to that in cells infected with the parental MVA strain. Finally, the recombinant M2L-expressing virus restored the “wild-type” NF-κB-inhibitory phenotype, as indicated by decreased NF-κB migration to infected cell nuclei and interference in transcription. Thus, in 293T cells, VV apparently utilizes its M2L protein to interfere with a step(s) that would otherwise enable ERK2 phosphorylation and the consequential activation of an NF-κΒ response.

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RhoA Controls Wnt Upregulation on Microstructured Titanium Surfaces

BioMed Research International ◽

10.1155/2014/401859 ◽

2014 ◽

Vol 2014 ◽

pp. 1-9 ◽

Cited By ~ 11

Author(s):

Simone Lumetti ◽

Silvia Mazzotta ◽

Sara Ferrillo ◽

Maddalena Piergianni ◽

Marilina Piemontese ◽

...

Keyword(s):

Wnt Signaling ◽

Molecular Mechanisms ◽

Myosin Ii ◽

Small Gtpase ◽

Dominant Negative ◽

C2c12 Cells ◽

Differentiation Markers ◽

Rhoa Activation ◽

Titanium Surfaces ◽

In Cells

Rough topography enhances the activation of Wnt canonical signaling in vitro, and this mediates its effects on cell differentiation. However, the molecular mechanisms underlying topography-dependent control of Wnt signaling are still poorly understood. As the small GTPase RhoA controls cytoskeletal reorganization and actomyosin-induced tensional forces, we hypothesized that RhoA could affect the activation of Wnt signaling in cells on micropatterned titanium surfaces. G-LISA assay revealed that RhoA activation was higher in C2C12 cells on rough (SLA) surfaces under basal conditions than on smooth (Polished) titanium. Transfection with dominant negative RhoA decreased Wnt activation by normalized TCF-Luc activity on SLA, whilst transfection with constitutively active RhoA increased TCF-Luc activation on Polished titanium. One mM Myosin II inhibitor Blebbistatin increased RhoA activation but decreased Wnt activation on SLA surfaces, indicating that tension-generating structures are required for canonical Wnt modulation on titanium surfaces. Actin inhibitor Cytochalasin markedly enhanced RhoA and TCF-Luc activation on both surfaces and increased the expression of differentiation markers in murine osteoblastic MC3T3 cells. Taken together, these data show that RhoA is upregulated in cells on rough surfaces and it affects the activation of Wnt canonical signaling through Myosin II modulation.

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A short splicing isoform of afadin suppresses the cortical axon branching in a dominant-negative manner

Molecular Biology of the Cell ◽

10.1091/mbc.e15-01-0039 ◽

2015 ◽

Vol 26 (10) ◽

pp. 1957-1970 ◽

Cited By ~ 6

Author(s):

Kentaro Umeda ◽

Nariaki Iwasawa ◽

Manabu Negishi ◽

Izumi Oinuma

Keyword(s):

Neuronal Development ◽

Ectopic Expression ◽

Small Gtpase ◽

Binding Domain ◽

Dominant Negative ◽

Actin Binding ◽

Axon Branching ◽

Axonal Development ◽

Actin Binding Domain ◽

Ras Family

Precise wiring patterns of axons are among the remarkable features of neuronal circuit formation, and establishment of the proper neuronal network requires control of outgrowth, branching, and guidance of axons. R-Ras is a Ras-family small GTPase that has essential roles in multiple phases of axonal development. We recently identified afadin, an F-actin–binding protein, as an effector of R-Ras mediating axon branching through F-actin reorganization. Afadin comprises two isoforms—l-afadin, having the F-actin–binding domain, and s-afadin, lacking the F-actin–binding domain. Compared with l-afadin, s-afadin, the short splicing variant of l-afadin, contains RA domains but lacks the F-actin–binding domain. Neurons express both isoforms; however, the function of s-afadin in brain remains unknown. Here we identify s-afadin as an endogenous inhibitor of cortical axon branching. In contrast to the abundant and constant expression of l-afadin throughout neuronal development, the expression of s-afadin is relatively low when cortical axons branch actively. Ectopic expression and knockdown of s-afadin suppress and promote branching, respectively. s-Afadin blocks the R-Ras–mediated membrane translocation of l-afadin and axon branching by inhibiting the binding of l-afadin to R-Ras. Thus s-afadin acts as a dominant-negative isoform in R-Ras-afadin–regulated axon branching.

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MiR-485-5p Promotes Neuron Survival through Mediating Rac1/Notch2 Signaling Pathway after Cerebral Ischemia/Reperfusion

Current Neurovascular Research ◽

10.2174/1567202617666200415154822 ◽

2020 ◽

Vol 17 (3) ◽

pp. 259-266 ◽

Cited By ~ 2

Author(s):

Xuan Chen ◽

Sumei Zhang ◽

Peipei Shi ◽

Yangli Su ◽

Dong Zhang ◽

...

Keyword(s):

Oxidative Stress ◽

Cell Viability ◽

Therapeutic Option ◽

Ischemia Reperfusion ◽

Glucose Deprivation ◽

Small Gtpase ◽

Luciferase Reporter ◽

Pathological Feature ◽

In Cells

Objective: Ischemia-reperfusion (I/R) injury is a pathological feature of ischemic stroke. This study investigated the regulatory role of miR-485-5p in I/R injury. Methods: SH-SY5Y cells were induced with oxygen and glucose deprivation/reoxygenation (OGD/R) to mimic I/R injury in vitro. Cells were transfected with designated constructs (miR-485- 5p mimics, miR-485-5p inhibitor, lentiviral vectors overexpressing Rac1 or their corresponding controls). Cell viability was evaluated using the MTT assay. The concentrations of lactate dehydrogenase, malondialdehyde, and reactive oxygen species were detected to indicate the degree of oxidative stress. Flow cytometry and caspase-3 activity assay were used for apoptosis assessment. Dual-luciferase reporter assay was performed to confirm that Rac family small GTPase 1 (Rac1) was a downstream gene of miR-485-5p. Results: OGD/R resulted in decreased cell viability, elevated oxidative stress, increased apoptosis, and downregulated miR-485-5p expression in SH-SY5Y cells. MiR-485-5p upregulation alleviated I/R injury, evidenced by improved cell viability, decreased oxidative markers, and reduced apoptotic rate. OGD/R increased the levels of Rac1 and neurogenic locus notch homolog protein 2 (Notch2) signaling-related proteins in cells with normal miR-485-5p expression, whereas miR- 485-5p overexpression successfully suppressed OGD/R-induced upregulation of these proteins. Furthermore, the delivery of vectors overexpressing Rac1 in miR-485-5p mimics-transfected cells reversed the protective effect of miR-485-5p in cells with OGD/R-induced injury. Conclusion: This study showed that miR-485-5p protected cells following I/R injury via targeting Rac1/Notch2 signaling suggest that targeted upregulation of miR-485-5p might be a promising therapeutic option for the protection against I/R injury.

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