scholarly journals Baculovirus actin-rearrangement-inducing factor ARIF-1 induces the formation of dynamic clusters of invadosome-like structures

2020 ◽  
Author(s):  
Domokos I. Lauko ◽  
Taro Ohkawa ◽  
Sergio E. Mares ◽  
Matthew D. Welch
Keyword(s):  
Structure Formation ◽  
Mammalian Cells ◽  
Early Stage ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Lepidopteran Insects ◽  
Associated Proteins ◽  
Infected Cells ◽  
Necessary And Sufficient ◽  
Actin Rearrangement

AbstractThe baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a pathogen of lepidopteran insects, has a striking dependence on the host cell actin cytoskeleton. During the delayed-early stage of infection, AcMNPV was shown to induce the accumulation of actin at the cortex of infected cells. However, the dynamics and molecular mechanism of cortical actin assembly remained unknown. Here, we show that AcMNPV induces dynamic cortical clusters of dot-like actin structures that resemble clusters of invadosomes in mammalian cells. Furthermore, we find that the AcMNPV protein actin-rearrangement-inducing factor-1 (ARIF-1), which was previously shown to be necessary and sufficient for cortical actin assembly and efficient viral infection in insect hosts, is both necessary and sufficient for invadosome-like structure formation. We mapped the sequences within the C-terminal cytoplasmic region of ARIF-1 that are required for invadosome-like structure formation and identified individual tyrosine and proline residues that are required for organizing these structures. Additionally, we found that ARIF-1 and the invadosome-associated proteins cortactin and the Arp2/3 complex localize to invadosome-like structures, and Arp2/3 complex is required for their formation. These ARIF-1-induced invadosome-like structures may be important for the function of ARIF-1 in systemic virus spread.

scholarly journals Baculovirus actin-rearrangement-inducing factor ARIF-1 induces the formation of dynamic invadosome clusters

2021 ◽  
pp. mbc.E20-11-0705
Author(s):  
Domokos I. Lauko ◽  
Taro Ohkawa ◽  
Sergio E. Mares ◽  
Matthew D. Welch
Keyword(s):  
Mammalian Cells ◽  
Early Stage ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Lepidopteran Insects ◽  
Associated Proteins ◽  
Infected Cells ◽  
Necessary And Sufficient ◽  
Autographa Californica Multiple Nucleopolyhedrovirus ◽  
Actin Rearrangement

The baculovirus  Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a pathogen of lepidopteran insects, has a striking dependence on the host cell actin cytoskeleton. During the delayed-early stage of infection, AcMNPV was shown to induce the accumulation of actin at the cortex of infected cells. However, the dynamics and molecular mechanism of cortical actin assembly remained unknown. Here, we show that AcMNPV induces dynamic cortical clusters of dot-like actin structures that mediate degradation of underlying extracellular matrix and therefore function similarly to clusters of invadosomes in mammalian cells. Furthermore, we find that the AcMNPV protein actin-rearrangement-inducing factor-1 (ARIF-1), which was previously shown to be necessary and sufficient for cortical actin assembly and efficient viral infection in insect hosts, is both necessary and sufficient for invadosome formation. We mapped the sequences within the C-terminal cytoplasmic region of ARIF-1 that are required for invadosome formation and identified individual tyrosine and proline residues that are required for organizing these structures. Additionally, we found that ARIF-1 and the invadosome-associated proteins cortactin and the Arp2/3 complex localize to invadosomes, and Arp2/3 complex is required for their formation. These ARIF-1-induced invadosomes may be important for the function of ARIF-1 in systemic virus spread. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

scholarly journals CLIC4 is regulated by RhoA-mDia2 signaling through Profilin-1 binding to modulate filopodium length

2018 ◽  
Author(s):  
Elisabetta Argenzio ◽  
Katarzyna M. Kedziora ◽  
Leila Nahidiazar ◽  
Tadamoto Isogai ◽  
Anastassis Perrakis ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Binding Protein ◽  
Feedback Mechanism ◽  
Actin Binding ◽  
Actin Assembly ◽  
Wild Type ◽  
Cortical Actin ◽  
Conserved Residues ◽  
Rhoa Activation ◽  
Necessary And Sufficient

AbstractCLIC4 is a cytosolic protein implicated in diverse actin-based processes, including integrin trafficking, cell adhesion and tubulogenesis. CLIC4 is rapidly recruited to the plasma membrane by G12/13-coupled receptor agonists and then partly co-localizes with β1 integrins. Receptor-mediated CLIC4 translocation depends on actin polymerization, but the mechanism and functional significance of CLIC4 trafficking are unknown. Here we show that RhoA activation by either LPA or EGF is necessary and sufficient for CLIC4 translocation, with a regulatory role for the RhoA effector mDia2, an inducer of actin polymerization. We find that CLIC4 directly interacts with the G-actin-binding protein Profilin-1 via conserved residues that are required for CLIC4 trafficking and lie in a concave surface. Consistently, silencing of Profilin-1 impaired CLIC4 trafficking induced by either LPA or EGF. CLIC4 knockdown promoted the formation of long integrin-dependent filopodia, a phenotype rescued by wild-type CLIC4 but not by trafficking-incompetent CLIC4(C35A). Our results establish CLIC4 as a Profilin-1-binding protein and suggest that CLIC4 translocation provides a feedback mechanism to modulate mDia2/Profilin-1-driven cortical actin assembly and membrane protrusion.

scholarly journals Scanning Electron-Assisted Dielectric Microscopy Reveals Autophagosome Formation by LC3 and ATG12 in Cultured Mammalian Cells

2021 ◽  
Vol 22 (4) ◽  
pp. 1834
Author(s):  
Tomoko Okada ◽  
Toshihiko Ogura
Keyword(s):  
Mammalian Cells ◽  
Culture Medium ◽  
Related Gene ◽  
Autophagosome Formation ◽  
Intact Cells ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
The Difference ◽  
Related Proteins ◽  
Scanning Electron

Autophagy is an intracellular self-devouring system that plays a central role in cellular recycling. The formation of functional autophagosomes depends on several autophagy-related proteins, including the microtubule-associated proteins 1A/1B light chain 3 (LC3) and the conserved autophagy-related gene 12 (Atg12). We have recently developed a novel scanning electron-assisted dielectric microscope (SE-ADM) for nanoscale observations of intact cells. Here, we used the SE-ADM system to observe LC3- and Atg12-containing autophagosomes in cells labelled in the culture medium with antibodies conjugated to colloidal gold particles. We observed that, during autophagosome formation, Atg12 localized along the actin meshwork structure, whereas LC3 formed arcuate or circular alignments. Our system also showed a difference in the distribution of LC3 and Atg12; Atg12 was broadly distributed while LC3 was more localized. The difference in the spatial distribution demonstrated by our system explains the difference in the size of fluorescent spots due to the fluorescently labelled antibodies observed using optical microscopy. The direct SE-ADM observation of cells should thus be effective in analyses of autophagosome formation.

scholarly journals Dicer is involved in protection against influenza A virus infection

2007 ◽  
Vol 88 (10) ◽  
pp. 2627-2635 ◽  
Author(s):  
Alexey A. Matskevich ◽  
Karin Moelling
Keyword(s):  
Virus Infection ◽  
Influenza A Virus ◽  
Mammalian Cells ◽  
Influenza A ◽  
Virus Production ◽  
Vero Cells ◽  
Antiviral Response ◽  
Protein Levels ◽  
Infected Cells ◽  
Influenza A Virus Infection

In mammals the interferon (IFN) system is a central innate antiviral defence mechanism, while the involvement of RNA interference (RNAi) in antiviral response against RNA viruses is uncertain. Here, we tested whether RNAi is involved in the antiviral response in mammalian cells. To investigate the role of RNAi in influenza A virus-infected cells in the absence of IFN, we used Vero cells that lack IFN-α and IFN-β genes. Our results demonstrate that knockdown of a key RNAi component, Dicer, led to a modest increase of virus production and accelerated apoptosis of influenza A virus-infected cells. These effects were much weaker in the presence of IFN. The results also show that in both Vero cells and the IFN-producing alveolar epithelial A549 cell line influenza A virus targets Dicer at mRNA and protein levels. Thus, RNAi is involved in antiviral response, and Dicer is important for protection against influenza A virus infection.

scholarly journals The polymerization of actin: II. how nonfilamentous actin becomes nonrandomly distributed in sperm: evidence for the association of this actin with membranes

1976 ◽  
Vol 69 (1) ◽  
pp. 51-72 ◽  
Author(s):  
LG Tilney
Keyword(s):  
Plasma Membrane ◽  
Nuclear Envelope ◽  
Thin Section ◽  
Actin Filaments ◽  
Early Stage ◽  
Mature Sperm ◽  
Vacuole Membrane ◽  
Other Substances ◽  
Associated Proteins ◽  
Basal Half

At an early stage in spermiogenesis the acrosomal vacuole and other organelles including ribosomes are located at the basal end of the cell. From here actin must be transported to its future location at the anterior end of the cell. At no stage in the accumulation of actin in the periacrosomal region is the actin sequestered in a membrane-bounded compartment such as a vacuole or vesicle. Since filaments are not present in the periacrosomal region during the accumulation of the actin even though the fixation of these cells is sufficiently good to distinguish actin filaments in thin section, the actin must accumulate in the nonfilamentous state. The membranes in the periacrosomal region, specifically a portion of the nuclear envelope and the basal half of the acrosomal vacuole membrane, become specialized morphologically in advance of the accumulation of actin in this region. My working hypothesis is that the actin in combination with other substances binds to these specialized membranes and to itself and thus can accumulate in the periacrosmoal region by being trapped on these specialized membranes. Diffusion would then be sufficient to move these substances to this region. In support of this hypothesis are experiments in which I treated mature sperm with detergents, glycols, and hypotonic media, which solubilize or lift away the plasma membrane. The actin and its associated proteins remain attached to these specialized membranes. Thus actin can be nonrandomly distributed in cells in a nonfilamentous state presumably by its association with specialized membranes.

scholarly journals Specific release of membrane-bound annexin II and cortical cytoskeletal elements by sequestration of membrane cholesterol.

1997 ◽  
Vol 8 (3) ◽  
pp. 533-545 ◽  
Author(s):  
T Harder ◽  
R Kellner ◽  
R G Parton ◽  
J Gruenberg
Keyword(s):  
Actin Cytoskeleton ◽  
Cytoskeletal Proteins ◽  
Annexin Ii ◽  
Dependent Manner ◽  
Cortical Actin ◽  
Independent Manner ◽  
Low Concentrations ◽  
Early Endosomes ◽  
Associated Proteins ◽  
Cytoskeletal Elements

Annexin II is an abundant protein which is present in the cytosol and on the cytoplasmic face of plasma membrane and early endosomes. It is generally believed that this association occurs via Ca(2+)-dependent binding to lipids, a mechanism typical for the annexin protein family. Although previous studies have shown that annexin II is involved in early endosome dynamics and organization, the precise biological role of the protein is unknown. In this study, we found that approximately 50% of the total cellular annexin was associated with membranes in a Ca(2+)-independent manner. This binding was extremely tight, since it resisted high salt and, to some extent, high pH treatments. We found, however, that membrane-associated annexin II could be quantitatively released by low concentrations of the cholesterol-sequestering agents filipin and digitonin. Both treatments released an identical and limited set of proteins but had no effects on other membrane-associated proteins. Among the released proteins, we identified, in addition to annexin II itself, the cortical cytoskeletal proteins alpha-actinin, ezrin and moesin, and membrane-associated actin. Our biochemical and immunological observations indicate that these proteins are part of a complex containing annexin II and that stability of the complex is sensitive to cholesterol sequestering agents. Since annexin II is tightly membrane-associated in a cholesterol-dependent manner, and since it seems to interact physically with elements of the cortical actin cytoskeleton, we propose that the protein serves as interface between membranes containing high amounts of cholesterol and the actin cytoskeleton.

scholarly journals Visualization and Molecular Analysis of Actin Assembly in Living Cells

1998 ◽  
Vol 143 (7) ◽  
pp. 1919-1930 ◽  
Author(s):  
Dorothy A. Schafer ◽  
Matthew D. Welch ◽  
Laura M. Machesky ◽  
Paul C. Bridgman ◽  
Shelley M. Meyer ◽  
...  
Keyword(s):  
Actin Filament ◽  
Eukaryotic Cell ◽  
Cell Periphery ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Lim Kinase ◽  
Permeabilized Cells ◽  
Capping Protein ◽  
Filament Assembly

Actin filament assembly is critical for eukaryotic cell motility. Arp2/3 complex and capping protein (CP) regulate actin assembly in vitro. To understand how these proteins regulate the dynamics of actin filament assembly in a motile cell, we visualized their distribution in living fibroblasts using green flourescent protein (GFP) tagging. Both proteins were concentrated in motile regions at the cell periphery and at dynamic spots within the lamella. Actin assembly was required for the motility and dynamics of spots and for motility at the cell periphery. In permeabilized cells, rhodamine-actin assembled at the cell periphery and at spots, indicating that actin filament barbed ends were present at these locations. Inhibition of the Rho family GTPase rac1, and to a lesser extent cdc42 and RhoA, blocked motility at the cell periphery and the formation of spots. Increased expression of phosphatidylinositol 5-kinase promoted the movement of spots. Increased expression of LIM–kinase-1, which likely inactivates cofilin, decreased the frequency of moving spots and led to the formation of aggregates of GFP–CP. We conclude that spots, which appear as small projections on the surface by whole mount electron microscopy, represent sites of actin assembly where local and transient changes in the cortical actin cytoskeleton take place.

scholarly journals Molecular cloning of cellular genes encoding retinoblastoma-associated proteins: identification of a gene with properties of the transcription factor E2F.

1992 ◽  
Vol 12 (12) ◽  
pp. 5620-5631 ◽  
Author(s):  
B Shan ◽  
X Zhu ◽  
P L Chen ◽  
T Durfee ◽  
Y Yang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Mammalian Cells ◽  
Leucine Zipper ◽  
T Antigen ◽  
Transactivation Domain ◽  
Recognition Sequence ◽  
Cellular Genes ◽  
Genes Encoding ◽  
Associated Proteins ◽  
Transcription Factor E2f

The retinoblastoma protein interacts with a number of cellular proteins to form complexes which are probably crucial for its normal physiological function. To identify these proteins, we isolated nine distinct clones by direct screening of cDNA expression libraries using purified RB protein as a probe. One of these clones, Ap12, is expressed predominantly at the G1-S boundary and in the S phase of the cell cycle. The nucleotide sequence of Ap12 has features characteristic of transcription factors. The C-terminal region binds to unphosphorylated RB in regions similar to those to which T antigen binds and contains a transactivation domain. A region containing a potential leucine zipper flanked by basic residues is able to bind an E2F recognition sequence specifically. Expression of Ap12 in mammalian cells significantly enhances E2F-dependent transcriptional activity. These results suggest that Ap12 encodes a protein with properties known to be characteristic of transcription factor E2F.

scholarly journals DjA1 maintains Golgi integrity via interaction with GRASP65

2019 ◽  
Vol 30 (4) ◽  
pp. 478-490 ◽  
Author(s):  
Jie Li ◽  
Danming Tang ◽  
Stephen C. Ireland ◽  
Yanzhuang Wang
Keyword(s):  
Heat Shock ◽  
Membrane Fusion ◽  
Structure Formation ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Golgi Membrane ◽  
Biochemical Methods ◽  
Golgi Fragmentation ◽  
Golgi Structure

In mammalian cells, the Golgi reassembly stacking protein of 65 kDa (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers. To better understand its function and regulation, we used biochemical methods to identify the DnaJ homolog subfamily A member 1 (DjA1) as a novel GRASP65-binding protein. In cells, depletion of DjA1 resulted in Golgi fragmentation, short and improperly aligned cisternae, and delayed Golgi reassembly after nocodazole washout. In vitro, immunodepletion of DjA1 from interphase cytosol reduced its activity to enhance GRASP65 oligomerization and Golgi membrane fusion, while adding purified DjA1 enhanced GRASP65 oligomerization. DjA1 is a cochaperone of Heat shock cognate 71-kDa protein (Hsc70), but the activity of DjA1 in Golgi structure formation is independent of its cochaperone activity or Hsc70, rather, through DjA1-GRASP65 interaction to promote GRASP65 oligomerization. Thus, DjA1 interacts with GRASP65 to enhance Golgi structure formation through the promotion of GRASP65 trans-oligomerization.

scholarly journals Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Edward JD Greenwood ◽  
Nicholas J Matheson ◽  
Kim Wals ◽  
Dick JH van den Boomen ◽  
Robin Antrobus ◽  
...  
Keyword(s):  
Hiv Infection ◽  
Time Course ◽  
Cellular Proteins ◽  
Temporal Expression ◽  
Aurora Kinases ◽  
Regulatory Subunits ◽  
Tandem Mass Tag ◽  
Infected Cells ◽  
Necessary And Sufficient ◽  
Viral Accessory Protein

Viruses manipulate host factors to enhance their replication and evade cellular restriction. We used multiplex tandem mass tag (TMT)-based whole cell proteomics to perform a comprehensive time course analysis of >6500 viral and cellular proteins during HIV infection. To enable specific functional predictions, we categorized cellular proteins regulated by HIV according to their patterns of temporal expression. We focussed on proteins depleted with similar kinetics to APOBEC3C, and found the viral accessory protein Vif to be necessary and sufficient for CUL5-dependent proteasomal degradation of all members of the B56 family of regulatory subunits of the key cellular phosphatase PP2A (PPP2R5A-E). Quantitative phosphoproteomic analysis of HIV-infected cells confirmed Vif-dependent hyperphosphorylation of >200 cellular proteins, particularly substrates of the aurora kinases. The ability of Vif to target PPP2R5 subunits is found in primate and non-primate lentiviral lineages, and remodeling of the cellular phosphoproteome is therefore a second ancient and conserved Vif function.

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