scholarly journals Mechanism of synergistic actin filament pointed end depolymerization by cyclase-associated protein and cofilin

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Tommi Kotila ◽  
Hugo Wioland ◽  
Giray Enkavi ◽  
Konstantin Kogan ◽  
Ilpo Vattulainen ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Actin Filament ◽  
Actin Filaments ◽  
Monomeric Form ◽  
Actin Monomer ◽  
Filament Assembly ◽  
Structural Work ◽  
Cytoskeletal Dynamics ◽  
Pointed End ◽  
Dependent Processes

AbstractThe ability of cells to generate forces through actin filament turnover was an early adaptation in evolution. While much is known about how actin filaments grow, mechanisms of their disassembly are incompletely understood. The best-characterized actin disassembly factors are the cofilin family proteins, which increase cytoskeletal dynamics by severing actin filaments. However, the mechanism by which severed actin filaments are recycled back to monomeric form has remained enigmatic. We report that cyclase-associated-protein (CAP) works in synergy with cofilin to accelerate actin filament depolymerization by nearly 100-fold. Structural work uncovers the molecular mechanism by which CAP interacts with actin filament pointed end to destabilize the interface between terminal actin subunits, and subsequently recycles the newly-depolymerized actin monomer for the next round of filament assembly. These findings establish CAP as a molecular machine promoting rapid actin filament depolymerization and monomer recycling, and explain why CAP is critical for actin-dependent processes in all eukaryotes.

scholarly journals Twinfilin, a molecular mailman for actin monomers

2002 ◽  
Vol 115 (5) ◽  
pp. 881-886 ◽  
Author(s):  
Sandra Palmgren ◽  
Maria Vartiainen ◽  
Pekka Lappalainen
Keyword(s):  
Drosophila Melanogaster ◽  
Actin Filament ◽  
Binding Sites ◽  
Actin Filaments ◽  
Actin Binding ◽  
Actin Monomer ◽  
Nucleotide Exchange ◽  
Capping Protein ◽  
Filament Assembly

Twinfilin is a ubiquitous actin-monomer-binding protein that is composed of two ADF-homology domains. It forms a 1:1 complex with ADP-actin-monomers,inhibits nucleotide exchange on actin monomers and prevents assembly of the monomer into filaments. The two ADF-H domains in twinfilin probably have 3D structures similar to those of the ADF/cofilin proteins and overlapping actin-binding sites. Twinfilin also interacts with PtdIns(4,5)P2, which inhibits its actin-monomer-sequestering activity in vitro. Mutations in the twinfilin gene result in defects in the bipolar budding pattern in S. cerevisiae and in a rough eye phenotype and aberrant bristle morphology in Drosophila melanogaster. These phenotypes are caused by the uncontrolled polymerization of actin filaments in the absence of twinfilin. Studies on budding yeast suggest that twinfilin contributes to actin filament turnover by localizing actin monomers, in their `inactive'ADP-form, to the sites of rapid filament assembly. This is mediated through direct interactions between twinfilin and capping protein. Therefore,twinfilin might serve as a link between rapid actin filament depolymerization and assembly in cells.

scholarly journals Actin filament assembly by bacterial factors VopL/F: Which end is up?

2017 ◽  
Vol 216 (5) ◽  
pp. 1211-1213
Author(s):  
Christina L. Vizcarra ◽  
Margot E. Quinlan
Keyword(s):  
Actin Filament ◽  
Direct Observation ◽  
Actin Filaments ◽  
Bacterial Proteins ◽  
Filament Assembly ◽  
Cell Biol ◽  
Competing Models ◽  
Pointed End

Competing models have been proposed for actin filament nucleation by the bacterial proteins VopL/F. In this issue, Burke et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201608104) use direct observation to demonstrate that VopL/F bind the barbed and pointed ends of actin filaments but only nucleate new filaments from the pointed end.

scholarly journals Actin-depolymerizing Factor and Cofilin-1 Play Overlapping Roles in Promoting Rapid F-Actin Depolymerization in Mammalian Nonmuscle Cells

2005 ◽  
Vol 16 (2) ◽  
pp. 649-664 ◽  
Author(s):  
Pirta Hotulainen ◽  
Eija Paunola ◽  
Maria K. Vartiainen ◽  
Pekka Lappalainen
Keyword(s):  
Cell Motility ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Depolymerizing Factor ◽  
Cytoskeletal Dynamics ◽  
Nonmuscle Cells ◽  
In Cells

Actin-depolymerizing factor (ADF)/cofilins are small actin-binding proteins found in all eukaryotes. In vitro, ADF/cofilins promote actin dynamics by depolymerizing and severing actin filaments. However, whether ADF/cofilins contribute to actin dynamics in cells by disassembling “old” actin filaments or by promoting actin filament assembly through their severing activity is a matter of controversy. Analysis of mammalian ADF/cofilins is further complicated by the presence of multiple isoforms, which may contribute to actin dynamics by different mechanisms. We show that two isoforms, ADF and cofilin-1, are expressed in mouse NIH 3T3, B16F1, and Neuro 2A cells. Depleting cofilin-1 and/or ADF by siRNA leads to an accumulation of F-actin and to an increase in cell size. Cofilin-1 and ADF seem to play overlapping roles in cells, because the knockdown phenotype of either protein could be rescued by overexpression of the other one. Cofilin-1 and ADF knockdown cells also had defects in cell motility and cytokinesis, and these defects were most pronounced when both ADF and cofilin-1 were depleted. Fluorescence recovery after photobleaching analysis and studies with an actin monomer-sequestering drug, latrunculin-A, demonstrated that these phenotypes arose from diminished actin filament depolymerization rates. These data suggest that mammalian ADF and cofilin-1 promote cytoskeletal dynamics by depolymerizing actin filaments and that this activity is critical for several processes such as cytokinesis and cell motility.

scholarly journals Dimer arrangement and monomer flattening determine actin filament formation

2018 ◽  
Author(s):  
Maria Hoyer ◽  
Jose Rafael Cabral Correia ◽  
Don C. Lamb ◽  
Alvaro H. Crevenna
Keyword(s):  
Actin Filament ◽  
Zero Mode ◽  
Filament Formation ◽  
Cellular Processes ◽  
The Past ◽  
Filament Dynamics ◽  
Filament Assembly ◽  
Initial Monomer ◽  
Pointed End ◽  
Two Populations

ABSTRACTActin filament dynamics underlie key cellular processes, such as cell motility. Although actin filament elongation has been extensively studied under the past decades, the mechanism of filament nucleation remains unclear. Here, we immobilized gelsolin, a pointed-end nucleator, at the bottom of zero-mode waveguides to directly monitor the early steps of filament assembly. Our data revealed extensive dynamics and that only one, of two populations, elongates. Annalysis of the kinetics revealed a more stable trimer but a less stable tetramer in the elongating population compared to the non-elongating one. Furthermore, blocking flattening, the conformational change associated with filament formation, prevented the formation of both types of assemblies. Thus, flattening and the initial monomer arrangement determine gelsolin-mediated filament initiation.

scholarly journals Inhibition of CapZ during myofibrillogenesis alters assembly of actin filaments.

1995 ◽  
Vol 128 (1) ◽  
pp. 61-70 ◽  
Author(s):  
D A Schafer ◽  
C Hug ◽  
J A Cooper
Keyword(s):  
Monoclonal Antibody ◽  
Actin Filament ◽  
Actin Filaments ◽  
Binding Activity ◽  
Actin Binding ◽  
Mutant Form ◽  
Actin Binding Protein ◽  
Filament Assembly ◽  
Skeletal Myotubes ◽  
Aligned Array

The actin filaments of myofibrils are highly organized; they are of a uniform length and polarity and are situated in the sarcomere in an aligned array. We hypothesized that the barbed-end actin-binding protein, CapZ, directs the process of actin filament assembly during myofibrillogenesis. We tested this hypothesis by inhibiting the actin-binding activity of CapZ in developing myotubes in culture using two different methods. First, injection of a monoclonal antibody that prevents the interaction of CapZ and actin disrupts the non-striated bundles of actin filaments formed during the early stages of myofibril formation in skeletal myotubes in culture. The antibody, when injected at concentrations lower than that required for disrupting the actin filaments, binds at nascent Z-disks. Since the interaction of CapZ and the monoclonal antibody are mutually exclusive, this result indicates that CapZ binds nascent Z-disks independent of an interaction with actin filaments. In a second approach, expression in myotubes of a mutant form of CapZ that does not bind actin results in a delay in the appearance of actin in a striated pattern in myofibrils. The organization of alpha-actinin at Z-disks also is delayed, but the organization of titin and myosin in sarcomeres is not significantly altered. We conclude that the interaction of CapZ and actin is important for the organization of actin filaments of the sarcomere.

scholarly journals Pointed-end capping by tropomodulin3 negatively regulates endothelial cell motility

2003 ◽  
Vol 161 (2) ◽  
pp. 371-380 ◽  
Author(s):  
Robert S. Fischer ◽  
Kimberly L. Fritz-Six ◽  
Velia M. Fowler
Keyword(s):  
Cell Migration ◽  
Cell Motility ◽  
Actin Filament ◽  
Actin Filaments ◽  
Critical Role ◽  
Leading Edge ◽  
Average Cell ◽  
Transient Overexpression ◽  
End Capping ◽  
Pointed End

Actin filament pointed-end dynamics are thought to play a critical role in cell motility, yet regulation of this process remains poorly understood. We describe here a previously uncharacterized tropomodulin (Tmod) isoform, Tmod3, which is widely expressed in human tissues and is present in human microvascular endothelial cells (HMEC-1). Tmod3 is present in sufficient quantity to cap pointed ends of actin filaments, localizes to actin filament structures in HMEC-1 cells, and appears enriched in leading edge ruffles and lamellipodia. Transient overexpression of GFP–Tmod3 leads to a depolarized cell morphology and decreased cell motility. A fivefold increase in Tmod3 results in an equivalent decrease in free pointed ends in the cells. Unexpectedly, a decrease in the relative amounts of F-actin, free barbed ends, and actin-related protein 2/3 (Arp2/3) complex in lamellipodia are also observed. Conversely, decreased expression of Tmod3 by RNA interference leads to faster average cell migration, along with increases in free pointed and barbed ends in lamellipodial actin filaments. These data collectively demonstrate that capping of actin filament pointed ends by Tmod3 inhibits cell migration and reveal a novel control mechanism for regulation of actin filaments in lamellipodia.

scholarly journals How Listeria exploits host cell actin to form its own cytoskeleton. II. Nucleation, actin filament polarity, filament assembly, and evidence for a pointed end capper.

1992 ◽  
Vol 118 (1) ◽  
pp. 83-93 ◽  
Author(s):  
L G Tilney ◽  
D J DeRosier ◽  
A Weber ◽  
M S Tilney
Keyword(s):  
Host Cell ◽  
Actin Filaments ◽  
Critical Concentration ◽  
Filament Assembly ◽  
Tightly Coupled ◽  
Pointed End ◽  
Phagosomal Membrane ◽  
Simple Picture

After Listeria, a bacterium, is phagocytosed by a macrophage, it dissolves the phagosomal membrane and enters the cytoplasm. The Listeria than nucleates actin filaments from its surface. These newly assembled actin filaments show unidirectional polarity with their barbed ends associated with the surface of the Listeria. Using actin concentrations below the pointed end critical concentration we find that filament elongation must be occurring by monomers adding to the barbed ends, the ends associated with the Listerial surface. If Listeria with tails are incubated in G actin under polymerizing conditions, the Listeria is translocated away from its preformed tail by the elongation of filaments attached to the Listeria. This experiment and others tell us that in vivo filament assembly must be tightly coupled to filament capping and cross-bridging so that if one process outstrips another, chaos ensues. We also show that the actin filaments in the tail are capped on their pointed ends which inhibits further elongation and/or disassembly in vitro. From these results we suggest a simple picture of how Listeria competes effectively for host cell actin. When Listeria secretes a nucleator, the host's actin subunits polymerize into a filament. Host cell machinery terminate the assembly leaving a short filament. Listeria overcomes the host control by nucleating new filaments and thus many short filaments assemble. The newest filaments push existing ones into a growing tail. Thus the competition is between nucleation of filaments caused by Listeria and the filament terminators produced by the host.

scholarly journals Cofilin promotes stimulus-induced lamellipodium formation by generating an abundant supply of actin monomers

2007 ◽  
Vol 177 (3) ◽  
pp. 465-476 ◽  
Author(s):  
Tai Kiuchi ◽  
Kazumasa Ohashi ◽  
Souichi Kurita ◽  
Kensaku Mizuno
Keyword(s):  
Actin Filament ◽  
Cell Periphery ◽  
Actin Monomer ◽  
Cytoplasmic Actin ◽  
Filament Assembly ◽  
Abundant Supply

Cofilin stimulates actin filament disassembly and accelerates actin filament turnover. Cofilin is also involved in stimulus-induced actin filament assembly during lamellipodium formation. However, it is not clear whether this occurs by replenishing the actin monomer pool, through filament disassembly, or by creating free barbed ends, through its severing activity. Using photoactivatable Dronpa-actin, we show that cofilin is involved in producing more than half of all cytoplasmic actin monomers and that the rate of actin monomer incorporation into the tip of the lamellipodium is dependent on the size of this actin monomer pool. Finally, in cofilin-depleted cells, stimulus-induced actin monomer incorporation at the cell periphery is attenuated, but the incorporation of microinjected actin monomers is not. We propose that cofilin contributes to stimulus-induced actin filament assembly and lamellipodium extension by supplying an abundant pool of cytoplasmic actin monomers.

scholarly journals Identification of critical functional and regulatory domains in gelsolin.

1989 ◽  
Vol 108 (5) ◽  
pp. 1717-1726 ◽  
Author(s):  
D J Kwiatkowski ◽  
P A Janmey ◽  
H L Yin
Keyword(s):  
Actin Filament ◽  
Binding Sites ◽  
Actin Filaments ◽  
Dna Transfection ◽  
Plasma Gelsolin ◽  
Cos Cells ◽  
Regulatory Domains ◽  
Filament Assembly ◽  
Potential Binding ◽  
Major Loss

Gelsolin can sever actin filaments, nucleate actin filament assembly, and cap the fast-growing end of actin filaments. These functions are activated by Ca2+ and inhibited by polyphosphoinositides (PPI). We report here studies designed to delineate critical domains within gelsolin by deletional mutagenesis, using COS cells to secrete truncated plasma gelsolin after DNA transfection. Deletion of 11% of gelsolin from the COOH terminus resulted in a major loss of its ability to promote the nucleation step in actin filament assembly, suggesting that a COOH-terminal domain is important in this function. In contrast, derivatives with deletion of 79% of the gelsolin sequence exhibited normal PPI-regulated actin filament-severing activity. Combined with previous results using proteolytic fragments, we deduce that an 11-amino acid sequence in the COOH terminus of the smallest severing gelsolin derivative identified here mediates PPI-regulated binding of gelsolin to the sides of actin filaments before severing. Deletion of only 3% of gelsolin at the COOH terminus, including a dicarboxylic acid sequence similar to that found on the NH2 terminus of actin, resulted in a loss of Ca2+-requirement for filament severing and monomer binding. Since these residues in actin have been implicated as potential binding sites for gelsolin, our results raise the possibility that the analogous sequence at the COOH terminus of gelsolin may act as a Ca2+-regulated pseudosubstrate. However, derivatives with deletion of 69-79% of the COOH-terminal residues of gelsolin exhibited normal Ca2+ regulation of severing activity, establishing the intrinsic Ca2+ regulation of the NH2-terminal region. One or both mechanisms of Ca2+ regulation may occur in members of the gelsolin family of actin-severing proteins.

scholarly journals Erythroid differentiation in mouse erythroleukemia cells is driven via actin filament-tropomodulin3-tropomyosin networks

2021 ◽  
Author(s):  
Arit Ghosh ◽  
Megan Coffin ◽  
Richard West ◽  
Velia M Fowler
Keyword(s):  
Actin Filament ◽  
Terminal Differentiation ◽  
Erythroid Differentiation ◽  
Erythroid Terminal Differentiation ◽  
Capping Proteins ◽  
Filament Assembly ◽  
Maturation Stages ◽  
Pointed End ◽  
Triton X ◽  
Mouse Erythroleukemia

Erythroid differentiation (ED) is a complex cellular process entailing morphologically distinct maturation stages of erythroblasts during terminal differentiation. Studies of actin filament assembly and organization during terminal ED have revealed essential roles for the pointed-end actin filament capping proteins, tropomodulins (Tmod1 and Tmod3). Additionally, tropomyosin (Tpm) binding to Tmods is a key feature promoting Tmod-mediated actin filament capping. Global deletion of Tmod3 leads to embryonic lethality in mice with impaired ED. To test a cell autonomous function for Tmod3 and further decipher its biochemical function during ED, we generated a Tmod3 knockout in a mouse erythroleukemia cell line (Mel ds19). Tmod3 knockout cells appeared normal prior to ED, but showed defects during progression of ED, characterized by a marked failure to reduce cell and nuclear size, reduced viability and increased apoptosis. In Mel ds19 cells, both Tpms and actin were preferentially associated with the Triton-X 100 insoluble cytoskeleton during ED, indicating Tpm-coated actin filament assembly during ED. While loss of Tmod3 did not lead to a change in total actin levels, it led to a severe reduction in the proportion of Tpms and actin associated with the Triton-X 100 insoluble cytoskeleton during ED. We conclude that Tmod3-regulation of actin cytoskeleton assembly via Tpms is integral to morphological maturation and cell survival during normal erythroid terminal differentiation.

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