Actin Dynamics, Architecture, and Mechanics in Cell Motility

Tight coupling between biochemical and mechanical properties of the actin cytoskeleton drives a large range of cellular processes including polarity establishment, morphogenesis, and motility. This is possible because actin filaments are semi-flexible polymers that, in conjunction with the molecular motor myosin, can act as biological active springs or “dashpots” (in laymen's terms, shock absorbers or fluidizers) able to exert or resist against force in a cellular environment. To modulate their mechanical properties, actin filaments can organize into a variety of architectures generating a diversity of cellular organizations including branched or crosslinked networks in the lamellipodium, parallel bundles in filopodia, and antiparallel structures in contractile fibers. In this review we describe the feedback loop between biochemical and mechanical properties of actin organization at the molecular level in vitro, then we integrate this knowledge into our current understanding of cellular actin organization and its physiological roles.

Download Full-text

Tropomyosin inhibits ADF/cofilin-dependent actin filament dynamics

The Journal of Cell Biology ◽

10.1083/jcb.200110013 ◽

2002 ◽

Vol 156 (6) ◽

pp. 1065-1076 ◽

Cited By ~ 180

Author(s):

Shoichiro Ono ◽

Kanako Ono

Keyword(s):

Actin Filament ◽

Actin Filaments ◽

Actin Polymerization ◽

Biological Properties ◽

Actin Dynamics ◽

Background Suppression ◽

Thin Filaments ◽

Actin Organization ◽

C Elegans ◽

Filament Dynamics

Tropomyosin binds to actin filaments and is implicated in stabilization of actin cytoskeleton. We examined biochemical and cell biological properties of Caenorhabditis elegans tropomyosin (CeTM) and obtained evidence that CeTM is antagonistic to ADF/cofilin-dependent actin filament dynamics. We purified CeTM, actin, and UNC-60B (a muscle-specific ADF/cofilin isoform), all of which are derived from C. elegans, and showed that CeTM and UNC-60B bound to F-actin in a mutually exclusive manner. CeTM inhibited UNC-60B–induced actin depolymerization and enhancement of actin polymerization. Within isolated native thin filaments, actin and CeTM were detected as major components, whereas UNC-60B was present at a trace amount. Purified UNC-60B was unable to interact with the native thin filaments unless CeTM and other associated proteins were removed by high-salt extraction. Purified CeTM was sufficient to restore the resistance of the salt-extracted filaments from UNC-60B. In muscle cells, CeTM and UNC-60B were localized in different patterns. Suppression of CeTM by RNA interference resulted in disorganized actin filaments and paralyzed worms in wild-type background. However, in an ADF/cofilin mutant background, suppression of CeTM did not worsen actin organization and worm motility. These results suggest that tropomyosin is a physiological inhibitor of ADF/cofilin-dependent actin dynamics.

Download Full-text

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.

Download Full-text

Vascular disease-causing mutation R258C in ACTA2 disrupts actin dynamics and interaction with myosin

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1507587112 ◽

2015 ◽

Vol 112 (31) ◽

pp. E4168-E4177 ◽

Cited By ~ 28

Author(s):

Hailong Lu ◽

Patricia M. Fagnant ◽

Carol S. Bookwalter ◽

Peteranne Joel ◽

Kathleen M. Trybus

Keyword(s):

Smooth Muscle ◽

Actin Filaments ◽

Contractile Function ◽

Smooth Muscles ◽

Aortic Aneurysms ◽

Actin Dynamics ◽

In Vitro Motility Assay ◽

Subcortical Region ◽

Muscle Tropomyosin

Point mutations in vascular smooth muscle α-actin (SM α-actin), encoded by the gene ACTA2, are the most prevalent cause of familial thoracic aortic aneurysms and dissections (TAAD). Here, we provide the first molecular characterization, to our knowledge, of the effect of the R258C mutation in SM α-actin, expressed with the baculovirus system. Smooth muscles are unique in that force generation requires both interaction of stable actin filaments with myosin and polymerization of actin in the subcortical region. Both aspects of R258C function therefore need investigation. Total internal reflection fluorescence (TIRF) microscopy was used to quantify the growth of single actin filaments as a function of time. R258C filaments are less stable than WT and more susceptible to severing by cofilin. Smooth muscle tropomyosin offers little protection from cofilin cleavage, unlike its effect on WT actin. Unexpectedly, profilin binds tighter to the R258C monomer, which will increase the pool of globular actin (G-actin). In an in vitro motility assay, smooth muscle myosin moves R258C filaments more slowly than WT, and the slowing is exacerbated by smooth muscle tropomyosin. Under loaded conditions, small ensembles of myosin are unable to produce force on R258C actin-tropomyosin filaments, suggesting that tropomyosin occupies an inhibitory position on actin. Many of the observed defects cannot be explained by a direct interaction with the mutated residue, and thus the mutation allosterically affects multiple regions of the monomer. Our results align with the hypothesis that defective contractile function contributes to the pathogenesis of TAAD.

Download Full-text

Small-Molecule Screen Identifies ROS as Key Regulators of Actin Dynamics in Neutrophils

Blood ◽

10.1182/blood.v112.11.4641.4641 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4641-4641

Author(s):

Hidenori Hattori ◽

Kulandayan K Subramanian ◽

Hongbo R. Luo

Keyword(s):

Small Molecule ◽

Actin Polymerization ◽

Neutrophil Migration ◽

Physiological Role ◽

Leading Edge ◽

Actin Dynamics ◽

Functional Screening ◽

Cellular Processes

Abstract Precise spatial and temporal control of actin polymerization and depolymerization is essential for mediating various cellular processes such as migration, phagocytosis, vesicle trafficking and adhesion. In this study, we used a small-molecule functional screening approach to identify novel regulators of actin dynamics during neutrophil migration. Here we show that NADPH-oxidase dependent Reactive Oxygen Species act as negative regulators of actin polymerization. Neutrophils with pharmacologically inhibited oxidase or isolated from Chronic Granulomatous Disease (CGD) patient and mice displayed enhanced F-actin polymerization, multiple pseudopods formation and impaired chemotaxis. ROS localized to pseudopodia and inhibited actin polymerization by driving actin glutathionylation at the leading edge of migrating cells. Consistent with these in vitro results, adoptively transferred CGD murine neutrophils also showed impaired in vivo recruitment to sites of inflammation. Together, these results present a novel physiological role for ROS in regulation of action polymerization and shed new light on the pathogenesis of CGD.

Download Full-text

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.

Download Full-text

Radial F-actin Organization During Early Neuronal Development

10.1101/372813 ◽

2018 ◽

Author(s):

Durga Praveen Meka ◽

Robin Scharrenberg ◽

Bing Zhao ◽

Theresa König ◽

Irina Schaefer ◽

...

Keyword(s):

Neuronal Development ◽

Super Resolution ◽

Actin Dynamics ◽

Cell Periphery ◽

Microtubule Organizing Center ◽

Actin Organization ◽

Organizing Center ◽

Super Resolution Microscopy ◽

Radial Organization

AbstractThe centrosome is thought to be the major neuronal microtubule-organizing center (MTOC) in early neuronal development, producing microtubules with a radial organization. In addition, albeit in vitro, recent work showed that isolated centrosomes could serve as an actin-organizing center (Farina et al., 2016), raising the possibility that neuronal development may, in addition, require a centrosome-based actin radial organization. Here we report, using super-resolution microscopy and live-cell imaging, F-actin organization around the centrosome with dynamic F-actin aster-like structures with F-actin fibers extending and retracting actively. Photoconversion/photoactivation experiments and molecular manipulations of F-actin stability reveal a robust flux of somatic F-actin towards the cell periphery. Finally, we show that somatic F-actin intermingles with centrosomal PCM-1 satellites. Knockdown of PCM-1 and disruption of centrosomal activity not only affect F-actin dynamics near the centrosome but also in distal growth cones. Collectively the data show a radial F-actin organization during early neuronal development, which might be a cellular mechanism for providing peripheral regions with a fast and continuous source of actin polymers; hence sustaining initial neuronal development.

Download Full-text

Basic Characterization of Plant Actin Depolymerizing Factors: A Simplified, Streamlined Guide

10.21203/rs.2.16466/v1 ◽

2019 ◽

Author(s):

Sonali Sengupta ◽

Kanniah Rajasekaran ◽

Niranjan Baisakh

Keyword(s):

Ionic Strength ◽

Actin Filaments ◽

Actin Dynamics ◽

Actin Binding ◽

Actin Monomer ◽

Oligomeric State ◽

Binding Partners ◽

Helical Twist

Abstract Actin depolymerizing factors (ADFs) are small monomeric actin-binding proteins that alter the oligomeric state of cellular actin. Members of the ADF family can bind both the G-actin and F-actin in plants, and their functions are regulated by cellular pH, ionic strength and availability of other binding partners. Actin depolymerization activity is reportedly essential for plant viability. By binding to the ADP-bound form of actin, ADFs severe actin filaments and thereby provide more barbed filament ends for polymerization. They also increase the rate of dissociation of F-actin monomer by changing the helical twist of the actin filament. These two activities together make ADF the major regulator of actin dynamics in plant cell. Therefore, it is essential to measure the binding and depolymerization activity of the plant ADFs. Here, we present a simplified, streamlined step-by-step protocol to quickly measure these important functions of the ADF proteins in vitro.

Download Full-text

Myosin 1b Flattens and Prunes Branched Actin Filaments

10.1101/2020.02.26.966663 ◽

2020 ◽

Author(s):

Julien Pernier ◽

Antoine Morchain ◽

Valentina Caorsi ◽

Aurélie Bertin ◽

Hugo Bousquet ◽

...

Keyword(s):

Total Internal Reflection ◽

Molecular Motor ◽

Internal Reflection ◽

Total Internal Reflection Fluorescence ◽

Actin Network ◽

Tirf Microscopy ◽

Actin Networks ◽

Cellular Processes

AbstractMotile and morphological cellular processes require a spatially and temporally coordinated branched actin network that is controlled by the activity of various regulatory proteins including the Arp2/3 complex, profilin, cofilin and tropomyosin. We have previously reported that myosin 1b regulates the density of the actin network in the growth cone. Using in vitro F-actin gliding assays and total internal reflection fluorescence (TIRF) microscopy we show in this report that this molecular motor flattens the Arp2/3-dependent actin branches up to breaking them and reduces the probability to form new branches. This experiment reveals that myosin 1b can produce force sufficient enough to break up the Arp2/3-mediated actin junction. Together with the former in vivo studies, this work emphasizes the essential role played by myosins in the architecture and in the dynamics of actin networks in different cellular regions.Short summaryUsing in vitro F-actin gliding assays and total internal reflection fluorescence (TIRF) microscopy we show that myosin flattens the Arp2/3-dependent actin branches up to breaking them and reduces the probability to form new branches

Download Full-text

Synergy between Cyclase-associated protein and Cofilin accelerates actin filament depolymerization by two orders of magnitude

Nature Communications ◽

10.1038/s41467-019-13268-1 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 15

Author(s):

Shashank Shekhar ◽

Johnson Chung ◽

Jane Kondev ◽

Jeff Gelles ◽

Bruce L. Goode

Keyword(s):

Actin Filament ◽

Single Molecule ◽

Actin Filaments ◽

Binding Protein ◽

Actin Dynamics ◽

Actin Network ◽

Cellular Mechanisms ◽

Actin Networks ◽

Binding Event

AbstractCellular actin networks can be rapidly disassembled and remodeled in a few seconds, yet in vitro actin filaments depolymerize slowly over minutes. The cellular mechanisms enabling actin to depolymerize this fast have so far remained obscure. Using microfluidics-assisted TIRF, we show that Cyclase-associated protein (CAP) and Cofilin synergize to processively depolymerize actin filament pointed ends at a rate 330-fold faster than spontaneous depolymerization. Single molecule imaging further reveals that hexameric CAP molecules interact with the pointed ends of Cofilin-decorated filaments for several seconds at a time, removing approximately 100 actin subunits per binding event. These findings establish a paradigm, in which a filament end-binding protein and a side-binding protein work in concert to control actin dynamics, and help explain how rapid actin network depolymerization is achieved in cells.

Download Full-text

A barbed end interference mechanism reveals how capping protein promotes nucleation in branched actin networks

Nature Communications ◽

10.1038/s41467-021-25682-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Johanna Funk ◽

Felipe Merino ◽

Matthias Schaks ◽

Klemens Rottner ◽

Stefan Raunser ◽

...

Keyword(s):

Structural Biology ◽

Actin Filaments ◽

Actin Network ◽

Essential Factor ◽

Actin Networks ◽

Capping Protein ◽

Cellular Processes ◽

In Vitro Reconstitution ◽

Interference Mechanism

AbstractHeterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside from terminating filament growth, CP potentiates the nucleation of actin filaments by the Arp2/3 complex in branched actin networks through an unclear mechanism. Here, we combine structural biology with in vitro reconstitution to demonstrate that CP not only terminates filament elongation, but indirectly stimulates the activity of Arp2/3 activating nucleation promoting factors (NPFs) by preventing their association to filament barbed ends. Key to this function is one of CP’s C-terminal “tentacle” extensions, which sterically masks the main interaction site of the terminal actin protomer. Deletion of the β tentacle only modestly impairs capping. However, in the context of a growing branched actin network, its removal potently inhibits nucleation promoting factors by tethering them to capped filament ends. End tethering of NPFs prevents their loading with actin monomers required for activation of the Arp2/3 complex and thus strongly inhibits branched network assembly both in cells and reconstituted motility assays. Our results mechanistically explain how CP couples two opposed processes—capping and nucleation—in branched actin network assembly.

Download Full-text