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

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.

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.015863 ◽

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.

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Cofilin is a pH sensor for actin free barbed end formation: role of phosphoinositide binding

The Journal of Cell Biology ◽

10.1083/jcb.200804161 ◽

2008 ◽

Vol 183 (5) ◽

pp. 865-879 ◽

Cited By ~ 122

Author(s):

Christian Frantz ◽

Gabriela Barreiro ◽

Laura Dominguez ◽

Xiaoming Chen ◽

Robert Eddy ◽

...

Keyword(s):

Actin Filament ◽

Structural Changes ◽

Ph Sensor ◽

Ph Sensitive ◽

Actin Binding ◽

Filamentous Actin ◽

Filament Dynamics ◽

Ph Dependent ◽

Dynamics Simulations

Newly generated actin free barbed ends at the front of motile cells provide sites for actin filament assembly driving membrane protrusion. Growth factors induce a rapid biphasic increase in actin free barbed ends, and we found both phases absent in fibroblasts lacking H+ efflux by the Na-H exchanger NHE1. The first phase is restored by expression of mutant cofilin-H133A but not unphosphorylated cofilin-S3A. Constant pH molecular dynamics simulations and nuclear magnetic resonance (NMR) reveal pH-sensitive structural changes in the cofilin C-terminal filamentous actin binding site dependent on His133. However, cofilin-H133A retains pH-sensitive changes in NMR spectra and severing activity in vitro, which suggests that it has a more complex behavior in cells. Cofilin activity is inhibited by phosphoinositide binding, and we found that phosphoinositide binding is pH-dependent for wild-type cofilin, with decreased binding at a higher pH. In contrast, phosphoinositide binding by cofilin-H133A is attenuated and pH insensitive. These data suggest a molecular mechanism whereby cofilin acts as a pH sensor to mediate a pH-dependent actin filament dynamics.

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

10.1101/2020.06.15.152033 ◽

2020 ◽

Cited By ~ 1

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.

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Effects of aging on actin sliding speed on myosin from single skeletal muscle cells of mice, rats, and humans

AJP Cell Physiology ◽

10.1152/ajpcell.2001.280.4.c782 ◽

2001 ◽

Vol 280 (4) ◽

pp. C782-C788 ◽

Cited By ~ 105

Author(s):

Peter Höök ◽

Vidyasagar Sriramoju ◽

Lars Larsson

Keyword(s):

Posttranslational Modifications ◽

Actin Filaments ◽

Mammalian Species ◽

Type I ◽

In Vitro Motility Assay ◽

Sliding Speed ◽

Myosin Heavy Chain Isoform ◽

In Vitro Motility ◽

Effects Of Aging

The effects of aging on the mechanical properties of myosin were measured in 87 fibers from muscles of humans ( n = 40), rats ( n = 21), and mice ( n = 26) using a single fiber in vitro motility assay. Irrespective of species, an 18–25% aging-related slowing in the speed of actin filaments was observed from 62 single fibers expressing the slow (type I) β-myosin heavy chain isoform. The mechanisms underlying the aging-related slowing of motility speed remain unknown, but it is suggested that posttranslational modifications of myosin by oxidative stress, glycation, or nitration play an important role. The aging-related slowing in the speed of actin filaments propelled by the type I myosin was confirmed in three mammalian species with an ∼3,400-fold difference in body size. Motility speed from human myosin was 3-fold slower than from myosin of the ∼3,400-fold smaller mouse and approximately twofold slower when compared with the ∼130-fold smaller rat, irrespective of age. A strong correlation was observed between the log values of actin sliding speed and body mass, suggesting that the effects of scaling is, at least in part, due to altered functional properties of the motor protein itself.

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Spreading of perturbations in myosin group kinetics along actin filaments

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1904164116 ◽

2019 ◽

Vol 116 (35) ◽

pp. 17336-17344 ◽

Cited By ~ 1

Author(s):

Zsombor Balassy ◽

Anne-Marie Lauzon ◽

Lennart Hilbert

Keyword(s):

Actin Filament ◽

Actin Filaments ◽

Nearest Neighbor ◽

Global Changes ◽

Chain Model ◽

Mechanical Coupling ◽

In Vitro Motility ◽

Local Perturbations ◽

Nearest Neighbor Coupling

Global changes in the state of spatially distributed systems can often be traced back to perturbations that arise locally. Whether such local perturbations grow into global changes depends on the system geometry and the spatial spreading of these perturbations. Here, we investigate how different spreading behaviors of local perturbations determine their global impact in 1-dimensional systems of different size. Specifically, we assessed sliding arrest events in in vitro motility assays where myosins propel actin, and simulated the underlying mechanochemistry of myosins that bind along the actin filament. We observed spontaneous sliding arrest events that occurred more frequently for shorter actin filaments. This observation could be explained by spontaneous local arrest of myosin kinetics that stabilizes once it spreads throughout an entire actin filament. When we introduced intermediate concentrations of the actin cross-linker filamin, longer actin was arrested more frequently. This observation was reproduced by simulations where filamin binding induces persistent local arrest of myosin kinetics, which subsequently spreads throughout the actin filament. A spin chain model with nearest-neighbor coupling reproduced key features of our experiments and simulations, thus extending to other linear systems with nearest-neighbor coupling the following conclusions: 1) perturbations that are persistent only once they spread throughout the system are more effective in smaller systems, and 2) perturbations that are persistent upon their establishment are more effective in larger systems. Beyond these general conclusions, our work also provides a theoretical model of collective myosin kinetics with a finite range of mechanical coupling along the actin filament.

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Path Reconstruction as a Tool for Actin Filament Speed Determination in the in Vitro Motility Assay

Analytical Biochemistry ◽

10.1006/abio.1999.4178 ◽

1999 ◽

Vol 273 (1) ◽

pp. 12-19 ◽

Cited By ~ 13

Author(s):

W. Hamelink ◽

J.G. Zegers ◽

B.W. Treijtel ◽

T. Blangé

Keyword(s):

Actin Filament ◽

Motility Assay ◽

In Vitro Motility Assay ◽

In Vitro Motility

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

Molecular Biology of the Cell ◽

10.1091/mbc.e04-07-0555 ◽

2005 ◽

Vol 16 (2) ◽

pp. 649-664 ◽

Cited By ~ 240

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.

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Actin filament dynamics are dominated by rapid growth and severing activity in the Arabidopsis cortical array

The Journal of Cell Biology ◽

10.1083/jcb.200806185 ◽

2009 ◽

Vol 184 (2) ◽

pp. 269-280 ◽

Cited By ~ 130

Author(s):

Christopher J. Staiger ◽

Michael B. Sheahan ◽

Parul Khurana ◽

Xia Wang ◽

David W. McCurdy ◽

...

Keyword(s):

Actin Filament ◽

Actin Filaments ◽

Stochastic Dynamics ◽

Actin Dynamics ◽

Epifluorescence Microscopy ◽

Actin Assembly ◽

Cortical Actin ◽

Filament Dynamics ◽

Cortical Array

Metazoan cells harness the power of actin dynamics to create cytoskeletal arrays that stimulate protrusions and drive intracellular organelle movements. In plant cells, the actin cytoskeleton is understood to participate in cell elongation; however, a detailed description and molecular mechanism(s) underpinning filament nucleation, growth, and turnover are lacking. Here, we use variable-angle epifluorescence microscopy (VAEM) to examine the organization and dynamics of the cortical cytoskeleton in growing and nongrowing epidermal cells. One population of filaments in the cortical array, which most likely represent single actin filaments, is randomly oriented and highly dynamic. These filaments grow at rates of 1.7 µm/s, but are generally short-lived. Instead of depolymerization at their ends, actin filaments are disassembled by severing activity. Remodeling of the cortical actin array also features filament buckling and straightening events. These observations indicate a mechanism inconsistent with treadmilling. Instead, cortical actin filament dynamics resemble the stochastic dynamics of an in vitro biomimetic system for actin assembly.

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