scholarly journals The Human Arp2/3 Complex Is Composed of Evolutionarily Conserved Subunits and Is Localized to Cellular Regions of Dynamic Actin Filament Assembly

1997 ◽  
Vol 138 (2) ◽  
pp. 375-384 ◽  
Author(s):  
Matthew D. Welch ◽  
Angela H. DePace ◽  
Suzie Verma ◽  
Akihiro Iwamatsu ◽  
Timothy J. Mitchison
Keyword(s):  
Actin Polymerization ◽  
Predicted Amino Acid Sequence ◽  
Actin Assembly ◽  
Filament Assembly ◽  
Evolutionarily Conserved ◽  
And Function ◽  
Related Proteins ◽  
Human Complex ◽  
Novel Protein ◽  
In Cells

The Arp2/3 protein complex has been implicated in the control of actin polymerization in cells. The human complex consists of seven subunits which include the actin related proteins Arp2 and Arp3, and five others referred to as p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc (Arp complex). We have determined the predicted amino acid sequence of all seven subunits. Each has homologues in diverse eukaryotes, implying that the structure and function of the complex has been conserved through evolution. Human Arp2 and Arp3 are very similar to family members from other species. p41-Arc is a new member of the Sop2 family of WD (tryptophan and aspartate) repeat–containing proteins and may be posttranslationally modified, suggesting that it may be involved in regulating the activity and/or localization of the complex. p34-Arc, p21-Arc, p20-Arc, and p16-Arc define novel protein families. We sought to evaluate the function of the Arp2/3 complex in cells by determining its intracellular distribution. Arp3, p34-Arc, and p21-Arc were localized to the lamellipodia of stationary and locomoting fibroblasts, as well to Listeria monocytogenes assembled actin tails. They were not detected in cellular bundles of actin filaments. Taken together with the ability of the Arp2/3 complex to induce actin polymerization, these observations suggest that the complex promotes actin assembly in lamellipodia and may participate in lamellipodial protrusion.

scholarly journals WASP and SCAR are evolutionarily conserved in actin-filled pseudopod-based motility

2017 ◽  
Vol 216 (6) ◽  
pp. 1673-1688 ◽  
Author(s):  
Lillian K. Fritz-Laylin ◽  
Samuel J. Lord ◽  
R. Dyche Mullins
Keyword(s):  
Actin Polymerization ◽  
Eukaryotic Cells ◽  
Human Neutrophils ◽  
Evolutionary Relationships ◽  
Actin Assembly ◽  
Complex Environments ◽  
Clear Trend ◽  
Cell Crawling ◽  
Evolutionarily Conserved ◽  
Underlying Mechanisms

Diverse eukaryotic cells crawl through complex environments using distinct modes of migration. To understand the underlying mechanisms and their evolutionary relationships, we must define each mode and identify its phenotypic and molecular markers. In this study, we focus on a widely dispersed migration mode characterized by dynamic actin-filled pseudopods that we call “α-motility.” Mining genomic data reveals a clear trend: only organisms with both WASP and SCAR/WAVE—activators of branched actin assembly—make actin-filled pseudopods. Although SCAR has been shown to drive pseudopod formation, WASP’s role in this process is controversial. We hypothesize that these genes collectively represent a genetic signature of α-motility because both are used for pseudopod formation. WASP depletion from human neutrophils confirms that both proteins are involved in explosive actin polymerization, pseudopod formation, and cell migration. WASP and WAVE also colocalize to dynamic signaling structures. Moreover, retention of WASP together with SCAR correctly predicts α-motility in disease-causing chytrid fungi, which we show crawl at >30 µm/min with actin-filled pseudopods. By focusing on one migration mode in many eukaryotes, we identify a genetic marker of pseudopod formation, the morphological feature of α-motility, providing evidence for a widely distributed mode of cell crawling with a single evolutionary origin.

scholarly journals Baculovirus AC102 is a nucleocapsid protein that is crucial for nuclear actin polymerization and nucleocapsid morphogenesis

2018 ◽  
Author(s):  
Susan E. Hepp ◽  
Gina M. Borgo ◽  
Simina Ticau ◽  
Taro Ohkawa ◽  
Matthew D. Welch
Keyword(s):  
Protein Expression ◽  
Viral Replication ◽  
Actin Cytoskeleton ◽  
Nucleocapsid Protein ◽  
Actin Polymerization ◽  
Autographa Californica ◽  
Nuclear Actin ◽  
Actin Assembly ◽  
Late Infection ◽  
And Function

ABSTRACTThe baculovirusAutographa californicamultiple nucleopolyhedrovirus (AcMNPV), the type species of alphabaculoviruses, is an enveloped DNA virus that infects lepidopteran insects and is commonly known as a vector for protein expression and cell transduction. AcMNPV belongs to a diverse group of viral and bacterial pathogens that target the host cell actin cytoskeleton during infection. AcMNPV is unusual, however, in that it absolutely requires actin translocation into the nucleus early in infection, and actin polymerization within the nucleus late in infection coincident with viral replication. Of the six viral factors that are sufficient, when coexpressed, to induce the nuclear localization of actin, only AC102 is essential for viral replication and the nuclear accumulation of actin. We therefore sought to better understand the role of AC102 in actin mobilization in the nucleus early and late in infection. Although AC102 was thought to function early in infection, we found that AC102 is predominantly expressed as a late protein. In addition, we observed that AC102 is required for F-actin assembly in the nucleus during late infection, as well as for proper formation of viral replication structures and nucleocapsid morphogenesis. Finally, we found that AC102 is a nucleocapsid protein and a newly recognized member of a complex consisting of the viral proteins EC27, C42, and the actin polymerization protein P78/83. Taken together, our findings suggest that AC102 is necessary for nucleocapsid morphogenesis and actin assembly during late infection through its role as a component of the P78/83-C42-EC27-AC102 protein complex.IMPORTANCEThe baculovirusAutographa californicamultiple nucleopolyhedrovirus (AcMNPV) is an important biotechnological tool for protein expression and cell transduction, and related nucleopolyhedroviruses are also employed as environmentally benign insecticides. One impact of our work is to better understand the fundamental mechanisms through which AcMNPV exploits the cellular machinery of the host for replication, which may aid in the development of improved baculovirus-based research and industrial tools. Moreover, AcMNPV’s ability to mobilize the host actin cytoskeleton within the cell’s nucleus during infection makes it a powerful cell biological tool. It is becoming increasingly clear that actin plays important roles in the cell’s nucleus, yet the regulation and function of nuclear actin is poorly understood. Our work to better understand how AcMNPV relocalizes and polymerizes actin within the nucleus may reveal fundamental mechanisms that govern nuclear actin regulation and function, even in the absence of viral infection.

scholarly journals WASP and SCAR are evolutionarily conserved in actin-filled pseudopod-based motility

2016 ◽  
Author(s):  
Lillian K. Fritz-Laylin ◽  
Samuel J. Lord ◽  
R. Dyche Mullins
Keyword(s):  
Actin Polymerization ◽  
Eukaryotic Cells ◽  
Human Neutrophils ◽  
Evolutionary Relationships ◽  
Actin Assembly ◽  
Complex Environments ◽  
Clear Trend ◽  
Cell Crawling ◽  
Evolutionarily Conserved ◽  
Underlying Mechanisms

AbstractDiverse eukaryotic cells crawl through complex environments using distinct modes of migration. To understand the underlying mechanisms and their evolutionary relationships, we must define each mode, and identify its phenotypic and molecular markers. Here, we focus on a widely dispersed migration mode characterized by dynamic, actin-filled pseudopods that we call “α-motility.” Mining genomic data reveals a clear trend: only organisms with both WASP and SCAR/WAVE—activators of branched actin assembly—make actin-filled pseudopods. While SCAR has been shown to drive pseudopod formation, WASP’s role in this process is controversial. We hypothesize that these genes together represent a genetic signature of α-motility, because both are used for pseudopod formation. WASP depletion from human neutrophils confirms that both proteins are involved in explosive actin polymerization, pseudopod formation, and cell migration, and colocalize to dynamic signaling structures. Moreover, retention of WASP together with SCAR correctly predicts α-motility in disease-causing chytrid fungi, which we show crawl at >30 μm/min with actin-filled pseudopods. By focusing on one migration mode in many eukaryotes, we identify a genetic marker of pseudopod formation, the morphological feature of α-motility, providing evidence for a widely distributed mode of cell crawling with a single evolutionary origin.

scholarly journals The Actin-Family Protein Arp4 Is a Novel Suppressor for the Formation and Functions of Nuclear F-Actin

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 758 ◽  
Author(s):  
Shota Yamazaki ◽  
Christian Gerhold ◽  
Koji Yamamoto ◽  
Yuya Ueno ◽  
Robert Grosse ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Critical Role ◽  
Strand Break ◽  
Xenopus Laevis Oocytes ◽  
Actin Assembly ◽  
Nih3t3 Cells ◽  
Damage Repair ◽  
Family Protein ◽  
Related Proteins ◽  
Inducible Gene

The crosstalk between actin and actin-related proteins (Arps), namely Arp2 and Arp3, plays a central role in facilitating actin polymerization in the cytoplasm and also in the nucleus. Nuclear F-actin is required for transcriptional regulation, double-strand break repair, and nuclear organization. The formation of nuclear F-actin is highly dynamic, suggesting the involvement of positive and negative regulators for nuclear actin polymerization. While actin assembly factors for nuclear F-actin have been recently described, information about inhibitory factors is still limited. The actin-related protein Arp4 which is predominantly localized in the nucleus, has been previously identified as an integral subunit of multiple chromatin modulation complexes, where it forms a heterodimer with monomeric actin. Therefore, we tested whether Arp4 functions as a suppressor of nuclear F-actin formation. The knockdown of Arp4 (Arp4 KD) led to an increase in nuclear F-actin formation in NIH3T3 cells, and purified Arp4 potently inhibited F-actin formation in mouse nuclei transplanted into Xenopus laevis oocytes. Consistently, Arp4 KD facilitated F-actin-inducible gene expression (e.g., OCT4) and DNA damage repair. Our results suggest that Arp4 has a critical role in the formation and functions of nuclear F-actin.

scholarly journals Scd5p Mediates Phosphoregulation of Actin and Endocytosis by the Type 1 Phosphatase Glc7p in Yeast

2007 ◽  
Vol 18 (12) ◽  
pp. 4885-4898 ◽  
Author(s):  
Guisheng Zeng ◽  
Bo Huang ◽  
Suat Peng Neo ◽  
Junxia Wang ◽  
Mingjie Cai
Keyword(s):  
Cell Growth ◽  
Actin Polymerization ◽  
Binding Protein ◽  
Actin Assembly ◽  
Endocytic Vesicle ◽  
Actin Organization ◽  
Related Proteins ◽  
Type 1 Phosphatase ◽  
Endocytic Proteins

Pan1p plays essential roles in both actin and endocytosis in yeast. It interacts with, and regulates the function of, multiple endocytic proteins and actin assembly machinery. Phosphorylation of Pan1p by the kinase Prk1p down-regulates its activity, resulting in disassembly of the endocytic vesicle coat complex and termination of vesicle-associated actin polymerization. In this study, we focus on the mechanism that acts to release Pan1p from phosphorylation inhibition. We show that Pan1p is dephosphorylated by the phosphatase Glc7p, and the dephosphorylation is dependent on the Glc7p-targeting protein Scd5p, which itself is a phosphorylation target of Prk1p. Scd5p links Glc7p to Pan1p in two ways: directly by interacting with Pan1p and indirectly by interacting with the Pan1p-binding protein End3p. Depletion of Glc7p from the cells causes defects in cell growth, actin organization, and endocytosis, all of which can be partially suppressed by deletion of the PRK1 gene. These results suggest that Glc7p antagonizes the activity of the Prk1p kinase in regulating the functions of Pan1p and possibly other actin- and endocytosis-related proteins.

scholarly journals Baculovirus AC102 Is a Nucleocapsid Protein That Is Crucial for Nuclear Actin Polymerization and Nucleocapsid Morphogenesis

2018 ◽  
Vol 92 (11) ◽  
Author(s):  
Susan E. Hepp ◽  
Gina M. Borgo ◽  
Simina Ticau ◽  
Taro Ohkawa ◽  
Matthew D. Welch
Keyword(s):  
Protein Expression ◽  
Viral Replication ◽  
Nucleocapsid Protein ◽  
Actin Polymerization ◽  
Autographa Californica ◽  
Nuclear Actin ◽  
Actin Assembly ◽  
Late Infection ◽  
And Function ◽  
Autographa Californica Multiple Nucleopolyhedrovirus

ABSTRACTThe baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type species of alphabaculoviruses, is an enveloped DNA virus that infects lepidopteran insects and is commonly known as a vector for protein expression and cell transduction. AcMNPV belongs to a diverse group of viral and bacterial pathogens that target the host cell actin cytoskeleton during infection. AcMNPV is unusual, however, in that it absolutely requires actin translocation into the nucleus early in infection and actin polymerization within the nucleus late in infection coincident with viral replication. Of the six viral factors that are sufficient, when coexpressed, to induce the nuclear localization of actin, only AC102 is essential for viral replication and the nuclear accumulation of actin. We therefore sought to better understand the role of AC102 in actin mobilization in the nucleus early and late in infection. Although AC102 was proposed to function early in infection, we found that AC102 is predominantly expressed as a late protein. In addition, we observed that AC102 is required for F-actin assembly in the nucleus during late infection, as well as for proper formation of viral replication structures and nucleocapsid morphogenesis. Finally, we found that AC102 is a nucleocapsid protein and a newly recognized member of a complex consisting of the viral proteins EC27, C42, and the actin polymerization protein P78/83. Taken together, our findings suggest that AC102 is necessary for nucleocapsid morphogenesis and actin assembly during late infection through its role as a component of the P78/83-C42-EC27-AC102 protein complex.IMPORTANCEThe baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an important biotechnological tool for protein expression and cell transduction, and related nucleopolyhedroviruses are also used as environmentally benign insecticides. One impact of our work is to better understand the fundamental mechanisms through which AcMNPV exploits the cellular machinery of the host for replication, which may aid in the development of improved baculovirus-based research and industrial tools. Moreover, AcMNPV's ability to mobilize the host actin cytoskeleton within the cell's nucleus during infection makes it a powerful cell biological tool. It is becoming increasingly clear that actin plays important roles in the cell's nucleus, and yet the regulation and function of nuclear actin is poorly understood. Our work to better understand how AcMNPV relocalizes and polymerizes actin within the nucleus may reveal fundamental mechanisms that govern nuclear actin regulation and function, even in the absence of viral infection.

scholarly journals Neuronal drebrin A directly interacts with mDia2 formin to inhibit actin assembly

2019 ◽  
Vol 30 (5) ◽  
pp. 646-657 ◽  
Author(s):  
Anush A. Ginosyan ◽  
Elena E. Grintsevich ◽  
Emil Reisler
Keyword(s):  
Dendritic Spines ◽  
Actin Polymerization ◽  
Higher Order ◽  
Actin Binding ◽  
Actin Assembly ◽  
Truncated Protein ◽  
Brain Functions ◽  
And Function ◽  
Protein Constructs

Dendritic spines (DS) are actin-rich postsynaptic terminals of neurons that are critical for higher-order brain functions. Maturation of DS is accompanied by a change in actin architecture from linear to branched filamentous structures. Presumably, the underlying cause of this is a switch in a mode of actin assembly from formin-driven to Arp2/3-mediated via an undefined mechanism. Here we present data suggesting that neuron-specific actin-binding drebrin A may be a part of such a switch. It is well documented that DS are highly enriched in drebrin A, which is critical for their plasticity and function. At the same time, mDia2 is known to mediate the formation of filopodia-type (immature) spines. We found that neuronal drebrin A directly interacts with mDia2 formin. Drebrin inhibits formin-mediated nucleation of actin and abolishes mDia2-induced actin bundling. Using truncated protein constructs we identified the domain requirements for drebrin–mDia2 interaction. We hypothesize that accumulation of drebrin A in DS (that coincides with spine maturation) leads to inhibition of mDia2-driven actin polymerization and, therefore, may contribute to a change in actin architecture from linear to branched filaments.

scholarly journals The state of actin assembly regulates actin and vinculin expression by a feedback loop

1995 ◽  
Vol 108 (3) ◽  
pp. 1183-1193 ◽  
Author(s):  
A.D. Bershadsky ◽  
U. Gluck ◽  
O.N. Denisenko ◽  
T.V. Sklyarova ◽  
I. Spector ◽  
...  
Keyword(s):  
Cell Shape ◽  
Actin Polymerization ◽  
The State ◽  
Cell Junctions ◽  
Actin Assembly ◽  
3T3 Cells ◽  
Clostridium Botulinum C2 Toxin ◽  
Actin Mrna ◽  
C2 Toxin ◽  
In Cells

Actin filaments are major determinants of cell shape, motility and adhesion, which control important biological processes including embryonic development and wound healing. These processes are associated with changes in actin assembly, which is regulated by controlling the balance between polymerized and non-polymerized actin. To maintain a significant pool of non-polymerized actin, mechanism(s) linking actin synthesis to its state of polymerization were proposed. We have studied this relationship between actin synthesis and organization by modulating actin assembly using different drugs. Unassembled actin was increased in 3T3 cells using either the Clostridium botulinum C2 toxin, which ADP-ribosylates actin, or by latrunculin A, a Red Sea sponge product, which binds monomeric actin. The synthesis of actin was dramatically reduced in these cells owing to a concomitant decrease in actin RNA level. Similar results were obtained with HeLa cells grown in both monolayer and in suspension, suggesting that cell shape changes associated with drug treatment are not the primary cause for the effect on actin synthesis. In contrast, the scrape-loading of 3T3 cells with phalloidin, a stabilizer of polymerized actin that increased the level of assembled actin, resulted in elevated actin synthesis and RNA content. The expression of vinculin, a major component of adhesion plaques and cell-cell junctions, which is involved in actin-membrane associations, was altered in parallel with that of actin in cells treated with these drugs. The decrease in actin RNA resulted from destabilization of actin mRNA in cells where unassembled actin level was elevated. This is suggested by the unchanged transcription of actin in isolated nuclei from drug-treated cells, and by demonstrating that actin mRNA was degraded faster in cells after C2 toxin treatment than in control cells. This feedback control mechanism is mainly confined to the cytoplasm, as it remained active in enucleated cells. The results suggest the existence of an autoregulatory pathway for the expression of actin and other microfilament-associated proteins which is linked to the state of actin polymerization in the cell.

scholarly journals Actin and dynamin2 dynamics and interplay during clathrin-mediated endocytosis

2014 ◽  
Vol 205 (5) ◽  
pp. 721-735 ◽  
Author(s):  
Alexandre Grassart ◽  
Aaron T. Cheng ◽  
Sun Hae Hong ◽  
Fan Zhang ◽  
Nathan Zenzer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Fusion Proteins ◽  
Live Cell Imaging ◽  
Actin Polymerization ◽  
Cell Imaging ◽  
Actin Assembly ◽  
Specific Proteins ◽  
Quantitative Analyses ◽  
And Function

Clathrin-mediated endocytosis (CME) involves the recruitment of numerous proteins to sites on the plasma membrane with prescribed timing to mediate specific stages of the process. However, how choreographed recruitment and function of specific proteins during CME is achieved remains unclear. Using genome editing to express fluorescent fusion proteins at native levels and live-cell imaging with single-molecule sensitivity, we explored dynamin2 stoichiometry, dynamics, and functional interdependency with actin. Our quantitative analyses revealed heterogeneity in the timing of the early phase of CME, with transient recruitment of 2–4 molecules of dynamin2. In contrast, considerable regularity characterized the final 20 s of CME, during which ∼26 molecules of dynamin2, sufficient to make one ring around the vesicle neck, were typically recruited. Actin assembly generally preceded dynamin2 recruitment during the late phases of CME, and promoted dynamin recruitment. Collectively, our results demonstrate precise temporal and quantitative regulation of the dynamin2 recruitment influenced by actin polymerization.

scholarly journals Profilin promotes formin-mediated actin filament assembly and vesicle transport during polarity formation in pollen

2021 ◽  
Author(s):  
Chang Liu ◽  
Yi Zhang ◽  
Haiyun Ren
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Pollen Germination ◽  
Actin Polymerization ◽  
Vesicle Trafficking ◽  
Pollen Grains ◽  
Actin Assembly ◽  
Filament Assembly ◽  
Polarity Establishment

Abstract Pollen germination is critical for the reproduction of flowering plants. Formin-dependent actin polymerization plays vital roles in vesicle trafficking and polarity establishment during this process. However, how formin-mediated actin assembly is regulated in vivo remains poorly understood. Here, we investigated the function of reproductive profilin 4 and 5 (PRF4 and PRF5) in polarity establishment during pollen germination in Arabidopsis thaliana. Our data showed that the actin filament content was reduced in the prf4 prf5 double mutant and substantially increased in both PRF4- and PRF5-overexpressing pollen grains. By contrast, the positive effect of profilin in promoting actin polymerization was abolished in a formin mutant, atfh5. In addition, the interaction between Arabidopsis formin homology 5 (AtFH5) and actin filaments was attenuated and the trafficking of AtFH5-labeled vesicles was slowed in prf4 prf5 pollen grains. Formation of the collar-like structure at the germination pore was also defective in prf4 prf5 pollen grains as the fast assembly of actin filaments was impaired. Together, our results suggest that PRF4 and PRF5 regulate vesicle trafficking and polarity establishment during pollen germination by promoting AtFH5-mediated actin polymerization and enhancing the interaction between AtFH5 and actin filaments.

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