scholarly journals Requirements for Hirano Body Formation

2014 ◽  
Vol 13 (5) ◽  
pp. 625-634 ◽  
Author(s):  
Paul Griffin ◽  
Ruth Furukawa ◽  
Cleveland Piggott ◽  
Andrew Maselli ◽  
Marcus Fechheimer
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Myosin Ii ◽  
Actin Binding ◽  
Hirano Bodies ◽  
Latrunculin B ◽  
Content Type ◽  
Inhibitory Domain

ABSTRACT Hirano bodies are paracrystalline F-actin-rich structures associated with diverse conditions, including neurodegeneration and aging. Generation of model Hirano bodies using altered forms of Dictyostelium 34-kDa actin-bundling protein allows studies of their physiological function and mechanism of formation. We describe a novel 34-kDa protein mutant, E60K, with a point mutation within the inhibitory domain of the 34-kDa protein. Expression of E60K in Dictyostelium induces the formation of model Hirano bodies. The E60K protein has activated actin binding and is calcium regulated, unlike other forms of the 34-kDa protein that induce Hirano bodies and that have activated actin binding but lack calcium regulation. Actin filaments in the presence of E60K in vitro show enhanced resistance to disassembly induced by latrunculin B. Actin filaments in model Hirano bodies are also protected from latrunculin-induced depolymerization. We used nocodazole and blebbistatin to probe the role of the microtubules and myosin II, respectively, in the formation of model Hirano bodies. In the presence of these inhibitors, model Hirano bodies can form but are smaller than controls at early times of formation. The ultrastructure of model Hirano bodies did not reveal any major difference in structure and organization in the presence of inhibitors. In summary, these results support the conclusion that formation of model Hirano bodies is promoted by gain-of-function actin filament bundling, which enhances actin filament stabilization. Microtubules and myosin II contribute to but are not required for formation of model Hirano bodies.

scholarly journals Myosin and gelsolin cooperate in actin filament severing and actomyosin motor activity

2020 ◽  
pp. jbc.RA120.015863
Author(s):  
Venukumar Vemula ◽  
Tamás Huber ◽  
Marko Ušaj ◽  
Beáta Bugyi ◽  
Alf Mansson
Keyword(s):  
Motor Activity ◽  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
Actin Binding ◽  
In Vitro Motility Assay ◽  
Cooperative Effects ◽  
Myosin Motor ◽  
Filament Dynamics

Actin is a major intracellular protein with key functions in cellular motility, signaling and structural rearrangements. Its dynamic behavior, such as polymerisation and depolymerisation of actin filaments in response to intra- and extracellular cues, is regulated by an abundance of actin binding proteins. Out of these, gelsolin is one of the most potent for filament severing. However, myosin motor activity also fragments actin filaments through motor induced forces, suggesting that these two proteins could cooperate to regulate filament dynamics and motility. To test this idea, we used an in vitro motility assay, where actin filaments are propelled by surface-adsorbed heavy meromyosin (HMM) motor fragments. This allows studies of both motility and filament dynamics using isolated proteins. Gelsolin, at both nanomolar and micromolar Ca2+ concentration, appreciably enhanced actin filament severing caused by HMM-induced forces at 1 mM MgATP, an effect that was increased at higher HMM motor density. This finding is consistent with cooperativity between actin filament severing by myosin-induced forces and by gelsolin. We also observed reduced sliding velocity of the HMM-propelled filaments in the presence of gelsolin, providing further support of myosin-gelsolin cooperativity. Total internal reflection fluorescence microscopy based single molecule studies corroborated that the velocity reduction was a direct effect of gelsolin-binding to the filament and revealed different filament severing pattern of stationary and HMM propelled filaments. Overall, the results corroborate cooperative effects between gelsolin-induced alterations in the actin filaments and changes due to myosin motor activity leading to enhanced F-actin severing of possible physiological relevance.

scholarly journals Dynamics of Tpm1.8 domains on actin filaments with single molecule resolution

2020 ◽  
Author(s):  
Ilina Bareja ◽  
Hugo Wioland ◽  
Miro Janco ◽  
Philip R. Nicovich ◽  
Antoine Jégou ◽  
...  
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
High Probability ◽  
Actin Binding ◽  
Single Molecule Level ◽  
Filament Dynamics ◽  
Kinetics Of ◽  
Insight Into

ABSTRACTTropomyosins regulate dynamics and functions of the actin cytoskeleton by forming long chains along the two strands of actin filaments that act as gatekeepers for the binding of other actin-binding proteins. The fundamental molecular interactions underlying the binding of tropomyosin to actin are still poorly understood. Using microfluidics and fluorescence microscopy, we observed the binding of fluorescently labelled tropomyosin isoform Tpm1.8 to unlabelled actin filaments in real time. This approach in conjunction with mathematical modeling enabled us to quantify the nucleation, assembly and disassembly kinetics of Tpm1.8 on single filaments and at the single molecule level. Our analysis suggests that Tpm1.8 decorates the two strands of the actin filament independently. Nucleation of a growing tropomyosin domain proceeds with high probability as soon as the first Tpm1.8 molecule is stabilised by the addition of a second molecule, ultimately leading to full decoration of the actin filament. In addition, Tpm1.8 domains are asymmetrical, with enhanced dynamics at the edge oriented towards the barbed end of the actin filament. The complete description of Tpm1.8 kinetics on actin filaments presented here provides molecular insight into actin-tropomyosin filament formation and the role of tropomyosins in regulating actin filament dynamics.

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 Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii

2011 ◽  
Vol 22 (8) ◽  
pp. 1290-1299 ◽  
Author(s):  
Simren Mehta ◽  
L. David Sibley
Keyword(s):  
Toxoplasma Gondii ◽  
Actin Filaments ◽  
Gliding Motility ◽  
Conditional Knockout ◽  
Host Cells ◽  
Actin Binding ◽  
Actin Depolymerizing Factor

Apicomplexan parasites rely on actin-based gliding motility to move across the substratum, cross biological barriers, and invade their host cells. Gliding motility depends on polymerization of parasite actin filaments, yet ∼98% of actin is nonfilamentous in resting parasites. Previous studies suggest that the lack of actin filaments in the parasite is due to inherent instability, leaving uncertain the role of actin-binding proteins in controlling dynamics. We have previously shown that the single allele of Toxoplasma gondii actin depolymerizing factor (TgADF) has strong actin monomer–sequestering and weak filament-severing activities in vitro. Here we used a conditional knockout strategy to investigate the role of TgADF in vivo. Suppression of TgADF led to accumulation of actin-rich filaments that were detected by immunofluorescence and electron microscopy. Parasites deficient in TgADF showed reduced speed of motility, increased aberrant patterns of motion, and inhibition of sustained helical gliding. Lack of TgADF also led to severe defects in entry and egress from host cells, thus blocking infection in vitro. These studies establish that the absence of stable actin structures in the parasite are not simply the result of intrinsic instability, but that TgADF is required for the rapid turnover of parasite actin filaments, gliding motility, and cell invasion.

scholarly journals Actin turnover maintains actin filament homeostasis during cytokinetic ring contraction

2017 ◽  
Vol 216 (9) ◽  
pp. 2657-2667 ◽  
Author(s):  
Ting Gang Chew ◽  
Junqi Huang ◽  
Saravanan Palani ◽  
Ruth Sommese ◽  
Anton Kamnev ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Contractile Ring ◽  
Ring Contraction ◽  
Actin Turnover ◽  
Exogenous Addition ◽  
Turn Over

Cytokinesis in many eukaryotes involves a tension-generating actomyosin-based contractile ring. Many components of actomyosin rings turn over during contraction, although the significance of this turnover has remained enigmatic. Here, using Schizosaccharomyces japonicus, we investigate the role of turnover of actin and myosin II in its contraction. Actomyosin ring components self-organize into ∼1-µm-spaced clusters instead of undergoing full-ring contraction in the absence of continuous actin polymerization. This effect is reversed when actin filaments are stabilized. We tested the idea that the function of turnover is to ensure actin filament homeostasis in a synthetic system, in which we abolished turnover by fixing rings in cell ghosts with formaldehyde. We found that these rings contracted fully upon exogenous addition of a vertebrate myosin. We conclude that actin turnover is required to maintain actin filament homeostasis during ring contraction and that the requirement for turnover can be bypassed if homeostasis is achieved artificially.

scholarly journals Biased localization of actin binding proteins by actin filament conformation

2020 ◽  
Author(s):  
Andrew R Harris ◽  
Pamela Jreij ◽  
Brian Belardi ◽  
Andreas Bausch ◽  
Daniel A Fletcher
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Conformational Changes ◽  
Actin Filaments ◽  
Binding Proteins ◽  
Critical Role ◽  
Actin Binding ◽  
Cell Physiology ◽  
Physical Constraints

ABSTRACTThe assembly of actin filaments into distinct cytoskeletal structures plays a critical role in cell physiology, but how proteins localize differentially to these structures within a shared cytoplasm remains unclear. Here, we show that the actin-binding domains of accessory proteins can be sensitive to filament conformational changes. Using a combination of live cell imaging and in vitro single molecule binding measurements, we show that tandem calponin homology domains (CH1-CH2) can be mutated to preferentially bind actin networks at the front or rear of motile cells, and we demonstrate that the affinity of CH1-CH2 domain mutants varies as actin filament conformation is altered by perturbations that include stabilizing drugs, physical constraints, and other binding proteins. These findings suggest that conformational heterogeneity of actin filaments in cells could help to direct accessory binding proteins to different actin cytoskeletal structures through a biophysical feedback loop.

scholarly journals Biased localization of actin binding proteins by actin filament conformation

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Andrew R. Harris ◽  
Pamela Jreij ◽  
Brian Belardi ◽  
Aaron M. Joffe ◽  
Andreas R. Bausch ◽  
...  
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Conformational Changes ◽  
Actin Filaments ◽  
Binding Proteins ◽  
Critical Role ◽  
Actin Binding ◽  
Cell Physiology ◽  
Binding Domains

AbstractThe assembly of actin filaments into distinct cytoskeletal structures plays a critical role in cell physiology, but how proteins localize differentially to these structures within a shared cytoplasm remains unclear. Here, we show that the actin-binding domains of accessory proteins can be sensitive to filament conformational changes. Using a combination of live cell imaging and in vitro single molecule binding measurements, we show that tandem calponin homology domains (CH1–CH2) can be mutated to preferentially bind actin networks at the front or rear of motile cells. We demonstrate that the binding kinetics of CH1–CH2 domain mutants varies as actin filament conformation is altered by perturbations that include stabilizing drugs and other binding proteins. These findings suggest that conformational changes of actin filaments in cells could help to direct accessory binding proteins to different actin cytoskeletal structures through a biophysical feedback loop.

scholarly journals The structure of the actin filament uncapping complex mediated by twinfilin

2021 ◽  
Vol 7 (5) ◽  
pp. eabd5271
Author(s):  
Dennis M. Mwangangi ◽  
Edward Manser ◽  
Robert C. Robinson
Keyword(s):  
Actin Filament ◽  
Protein Interactions ◽  
Protein Complex ◽  
Actin Filaments ◽  
Actin Binding ◽  
Capping Protein ◽  
Binding Surface ◽  
Actin Capping Protein ◽  
Actin Capping

Uncapping of actin filaments is essential for driving polymerization and depolymerization dynamics from capping protein–associated filaments; however, the mechanisms of uncapping leading to rapid disassembly are unknown. Here, we elucidated the x-ray crystal structure of the actin/twinfilin/capping protein complex to address the mechanisms of twinfilin uncapping of actin filaments. The twinfilin/capping protein complex binds to two G-actin subunits in an orientation that resembles the actin filament barbed end. This suggests an unanticipated mechanism by which twinfilin disrupts the stable capping of actin filaments by inducing a G-actin conformation in the two terminal actin subunits. Furthermore, twinfilin disorders critical actin-capping protein interactions, which will assist in the dissociation of capping protein, and may promote filament uncapping through a second mechanism involving V-1 competition for an actin-binding surface on capping protein. The extensive interactions with capping protein indicate that the evolutionary conserved role of twinfilin is to uncap actin filaments.

scholarly journals Myosin-gelsolin cooperativity in actin filament severing and actomyosin motor activity

2020 ◽  
Author(s):  
Venukumar Vemula ◽  
Tamas Huber ◽  
Marko Usaj ◽  
Beáta Bugyi ◽  
Alf Mansson
Keyword(s):  
Motor Activity ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Binding ◽  
Cellular Motility ◽  
In Vitro Motility Assay ◽  
Cooperative Effects ◽  
In Vitro Motility ◽  
Filament Dynamics

AbstractActin is a major intracellular protein with key functions in cellular motility, signalling and structural rearrangements. Its dynamic behavior with actin filaments (F-actin) polymerising and depolymerising in response to intracellular changes, is controlled by actin-binding proteins (ABPs). Gelsolin is one of the most potent filament severing ABPs. However, myosin motors that interact with actin in the presence of ATP also produce actin filament fragmentation through motor induced shearing forces. To test the idea that gelsolin and myosin cooperate in these processes we used the in vitro motility assay, where actin filaments are propelled by surface-adsorbed heavy meromyosin (HMM) motor fragments. This allows studies of both motility and filament dynamics using isolated proteins. Gelsolin (5 nM) at very low [Ca2+] (free [Ca2+] ∼6.8 nM) appreciably enhanced actin filament severing caused by HMM-induced forces at 1 mM [MgATP], an effect that was increased at increased HMM motor density. This finding is consistent with cooperativity between actin filament severing by myosin-induced forces and by gelsolin. As further support of myosin-gelsolin cooperativity we observed reduced sliding velocity of the HMM propelled filaments in the presence of gelsolin. Overall, the results corroborate ideas for cooperative effects between gelsolin-induced alterations in the actin filaments and changes due to myosin motor activity, leading among other effects to enhanced F-actin severing of possible physiological relevance.

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.

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