Two distinct actin networks drive the protrusion of migrating cells
17 Sep 2004-Science (American Association for the Advancement of Science)-Vol. 305, Iss: 5691, pp 1782-1786
TL;DR: Computational analysis of fluorescent speckle microscopy movies of migrating epithelial cells revealed this process is mediated by two spatially colocalized but kinematically, kinetically, molecularly, and functionally distinct actin networks.
Abstract: Cell migration initiates by extension of the actin cytoskeleton at the leading edge. Computational analysis of fluorescent speckle microscopy movies of migrating epithelial cells revealed this process is mediated by two spatially colocalized but kinematically, kinetically, molecularly, and functionally distinct actin networks. A lamellipodium network assembled at the leading edge but completely disassembled within 1 to 3 micrometers. It was weakly coupled to the rest of the cytoskeleton and promoted the random protrusion and retraction of the leading edge. Productive cell advance was a function of the second colocalized network, the lamella, where actomyosin contraction was integrated with substrate adhesion.
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TL;DR: A new method for fluorescence imaging has been developed that can obtain spatial distributions of large numbers of fluorescent molecules on length scales shorter than the classical diffraction limit, and suggests a means to address a significant number of biological questions that had previously been limited by microscope resolution.
3,437 citations
Cites methods from "Two distinct actin networks drive t..."
...The procedures for velocity and position analysis of large numbers of single molecules (;10(5)) already developed for use with FSM and TIR-FSM (21,22) could be used to analyze FPALM images as well....
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...The procedures for velocity and position analysis of large numbers of single molecules (;105) already developed for use with FSM and TIR-FSM (21,22) could be used to analyze FPALM images as well....
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TL;DR: The mechanisms of such environmental sensing are discussed, based on the finely tuned crosstalk between the assembly of one type of integrin-based adhesion complex, namely focal adhesions, and the forces that are at work in the associated cytoskeletal network owing to actin polymerization and actomyosin contraction.
Abstract: Recent progress in the design and application of artificial cellular microenvironments and nanoenvironments has revealed the extraordinary ability of cells to adjust their cytoskeletal organization, and hence their shape and motility, to minute changes in their immediate surroundings. Integrin-based adhesion complexes, which are tightly associated with the actin cytoskeleton, comprise the cellular machinery that recognizes not only the biochemical diversity of the extracellular neighbourhood, but also its physical and topographical characteristics, such as pliability, dimensionality and ligand spacing. Here, we discuss the mechanisms of such environmental sensing, based on the finely tuned crosstalk between the assembly of one type of integrin-based adhesion complex, namely focal adhesions, and the forces that are at work in the associated cytoskeletal network owing to actin polymerization and actomyosin contraction.
2,322 citations
Cites background from "Two distinct actin networks drive t..."
...As a rule, the formation of the focal complexes occurs underneath the lamellipodia...
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TL;DR: Adhesion formation and disassembly drive the migration cycle by activating Rho GTPases, which in turn regulate actin polymerization and myosin II activity, and therefore adhesion dynamics.
Abstract: Cell migration affects all morphogenetic processes and contributes to numerous diseases, including cancer and cardiovascular disease. For most cells in most environments, movement begins with protrusion of the cell membrane followed by the formation of new adhesions at the cell front that link the actin cytoskeleton to the substratum, generation of traction forces that move the cell forwards and disassembly of adhesions at the cell rear. Adhesion formation and disassembly drive the migration cycle by activating Rho GTPases, which in turn regulate actin polymerization and myosin II activity, and therefore adhesion dynamics.
1,775 citations
Cites background from "Two distinct actin networks drive t..."
...The initial step in the migration cycle, protrusion of a leading edge, is driven by actin polymerization in filaments organized into two distinct zones, the lamellipodium and the lamellu...
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TL;DR: Non-muscle myosin II is an actin-binding protein that has actin cross-linking and contractile properties and is regulated by the phosphorylation of its light and heavy chains.
Abstract: Non-muscle myosin II (NM II) is an actin-binding protein that has actin cross-linking and contractile properties and is regulated by the phosphorylation of its light and heavy chains. The three mammalian NM II isoforms have both overlapping and unique properties. Owing to its position downstream of convergent signalling pathways, NM II is central in the control of cell adhesion, cell migration and tissue architecture. Recent insight into the role of NM II in these processes has been gained from loss-of-function and mutant approaches, methods that quantitatively measure actin and adhesion dynamics and the discovery of NM II mutations that cause monogenic diseases.
1,745 citations
Cites background from "Two distinct actin networks drive t..."
...It was recently proposed that these two actin networks overlap at the leading edge, with the lamellipodium superimposed onto the lamellu...
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TL;DR: Progress towards understanding the molecular, cellular and tissue-level effects that promote mechanical homeostasis has helped to identify key questions for future research.
Abstract: Soft connective tissues at steady state are dynamic; resident cells continually read environmental cues and respond to them to promote homeostasis, including maintenance of the mechanical properties of the extracellular matrix (ECM) that are fundamental to cellular and tissue health. The mechanosensing process involves assessment of the mechanics of the ECM by the cells through integrins and the actomyosin cytoskeleton, and is followed by a mechanoregulation process, which includes the deposition, rearrangement or removal of the ECM to maintain overall form and function. Progress towards understanding the molecular, cellular and tissue-level effects that promote mechanical homeostasis has helped to identify key questions for future research.
1,449 citations
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TL;DR: The authors are grateful for financial support from the National Institutes of Health (grants GM23244 and GM53905), and to very helpful comments on the manuscript from Elliot Elson, Vlodya Gelfand, Paul Matsudaira, Julie Theriot, and Sally Zigmond.
3,973 citations
TL;DR: A core set of proteins including actin, Arp2/3 complex, profilin, capping protein, and ADF/cofilin can reconstitute the process in vitro, and mathematical models of the constituent reactions predict the rate of motion.
3,793 citations
TL;DR: It is shown that Arp2/3 complex purified from Acanthamoeba caps the pointed ends of actin filaments with high affinity and increases the critical concentration for polymerization at the pointed end from 0.6 to 1.0 microM.
Abstract: The Arp2/3 complex is a stable assembly of seven protein subunits including two actin-related proteins (Arp2 and Arp3) and five novel proteins. Previous work showed that this complex binds to the sides of actin filaments and is concentrated at the leading edges of motile cells. Here, we show that Arp2/3 complex purified from Acanthamoeba caps the pointed ends of actin filaments with high affinity. Arp2/3 complex inhibits both monomer addition and dissociation at the pointed ends of actin filaments with apparent nanomolar affinity and increases the critical concentration for polymerization at the pointed end from 0.6 to 1.0 μM. The high affinity of Arp2/3 complex for pointed ends and its abundance in amoebae suggest that in vivo all actin filament pointed ends are capped by Arp2/3 complex. Arp2/3 complex also nucleates formation of actin filaments that elongate only from their barbed ends. From kinetic analysis, the nucleation mechanism appears to involve stabilization of polymerization intermediates (probably actin dimers). In electron micrographs of quick-frozen, deep-etched samples, we see Arp2/3 bound to sides and pointed ends of actin filaments and examples of Arp2/3 complex attaching pointed ends of filaments to sides of other filaments. In these cases, the angle of attachment is a remarkably constant 70 ± 7°. From these in vitro biochemical properties, we propose a model for how Arp2/3 complex controls the assembly of a branching network of actin filaments at the leading edge of motile cells.
1,432 citations
TL;DR: It is shown that exit from the cytokinetic phase of the cell cycle depends on ubiquitin-mediated proteolysis and continuous signals from microtubules are required to maintain the position of the cleavage furrow, and these signals control the localization of myosin II independently of other furrow components.
Abstract: Completion of cell division during cytokinesis requires temporally and spatially regulated communication from the microtubule cytoskeleton to the actin cytoskeleton and the cell membrane. We identified a specific inhibitor of nonmuscle myosin II, blebbistatin, that inhibited contraction of the cleavage furrow without disrupting mitosis or contractile ring assembly. Using blebbistatin and other drugs, we showed that exit from the cytokinetic phase of the cell cycle depends on ubiquitin-mediated proteolysis. Continuous signals from microtubules are required to maintain the position of the cleavage furrow, and these signals control the localization of myosin II independently of other furrow components.
1,359 citations
TL;DR: Results suggest that actin normally polymerizes at the leading edge and then flows rearward at a rate between 3-6 microns/min, which is consistent with their being secondary to effects of CB on lamellar F-actin.
Abstract: Actions of cytochalasin B (CB) on cytoskeletons and motility of growth cones from cultured Aplysia neurons were studied using a rapid flow perfusion chamber and digital video light microscopy. Living growth cones were observed using differential interference contrast optics and were also fixed at various time points to assay actin filament (F-actin) and microtubule distributions. Treatment with CB reversibly blocked motility and eliminated most of the phalloidin-stainable F-actin from the leading lamella. The loss of F-actin was nearly complete within 2-3 min of CB application and was largely reversed within 5-6 min of CB removal. The loss and recovery of F-actin were found to occur with a very distinctive spatial organization. Within 20-30 s of CB application, F-actin networks receded from the entire peripheral margin of the lamella forming a band devoid of F-actin. This band widened as F-actin receded at rates of 3-6 microns/min. Upon removal of CB, F-actin began to reappear within 20-30 s. The initial reappearance of F-actin took two forms: a coarse isotropic matrix of F-actin bundles throughout the lamella, and a denser matrix along the peripheral margin. The denser peripheral matrix then expanded in width, extending centrally to replace the coarse matrix at rates again between 3-6 microns/min. These results suggest that actin normally polymerizes at the leading edge and then flows rearward at a rate between 3-6 microns/min. CB treatment was also observed to alter the distribution of microtubules, assayed by antitubulin antibody staining. Normally, microtubules are restricted to the neurite shaft and a central growth cone domain. Within approximately 5 min after CB application, however, microtubules began extending into the lamellar region, often reaching the peripheral margin. Upon removal of CB, the microtubules were restored to their former central localization. The timing of these microtubule redistributions is consistent with their being secondary to effects of CB on lamellar F-actin.
850 citations