scholarly journals Arp2/3 complex–dependent actin networks constrain myosin II function in driving retrograde actin flow

2012 ◽  
Vol 197 (7) ◽  
pp. 939-956 ◽  
Author(s):  
Qing Yang ◽  
Xiao-Feng Zhang ◽  
Thomas D. Pollard ◽  
Paul Forscher
Keyword(s):  
Growth Cone ◽  
Myosin Ii ◽  
Rho Kinase ◽  
Leading Edge ◽  
Actin Dynamics ◽  
Actin Networks ◽  
Motile Cells ◽  
Nonmuscle Myosin Ii ◽  
Neuronal Growth Cone ◽  
Contractile Forces

The Arp2/3 complex nucleates actin filaments to generate networks at the leading edge of motile cells. Nonmuscle myosin II produces contractile forces involved in driving actin network translocation. We inhibited the Arp2/3 complex and/or myosin II with small molecules to investigate their respective functions in neuronal growth cone actin dynamics. Inhibition of the Arp2/3 complex with CK666 reduced barbed end actin assembly site density at the leading edge, disrupted actin veils, and resulted in veil retraction. Strikingly, retrograde actin flow rates increased with Arp2/3 complex inhibition; however, when myosin II activity was blocked, Arp2/3 complex inhibition now resulted in slowing of retrograde actin flow and veils no longer retracted. Retrograde flow rate increases induced by Arp2/3 complex inhibition were independent of Rho kinase activity. These results provide evidence that, although the Arp2/3 complex and myosin II are spatially segregated, actin networks assembled by the Arp2/3 complex can restrict myosin II–dependent contractility with consequent effects on growth cone motility.

scholarly journals Contribution of Filopodia to Cell Migration: A Mechanical Link between Protrusion and Contraction

2010 ◽  
Vol 2010 ◽  
pp. 1-13 ◽  
Author(s):  
Fei Xue ◽  
Deanna M. Janzen ◽  
David A. Knecht
Keyword(s):  
Cell Migration ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Temporal Distribution ◽  
Leading Edge ◽  
Distal Portion ◽  
Actin Binding ◽  
Actin Networks ◽  
Motile Cells ◽  
A Cell

Numerous F-actin containing structures are involved in regulating protrusion of membrane at the leading edge of motile cells. We have investigated the structure and dynamics of filopodia as they relate to events at the leading edge and the function of the trailing actin networks. We have found that although filopodia contain parallel bundles of actin, they contain a surprisingly nonuniform spatial and temporal distribution of actin binding proteins. Along the length of the actin filaments in a single filopodium, the most distal portion contains primarily T-plastin, while the proximal portion is primarily bound byα-actinin and coronin. Some filopodia are stationary, but lateral filopodia move with respect to the leading edge. They appear to form a mechanical link between the actin polymerization network at the front of the cell and the myosin motor activity in the cell body. The direction of lateral filopodial movement is associated with the direction of cell migration. When lateral filopodia initiate from and move toward only one side of a cell, the cell will turn opposite to the direction of filopodial flow. Therefore, this filopodia-myosin II system allows actin polymerization driven protrusion forces and myosin II mediated contractile force to be mechanically coordinated.

scholarly journals Actions of cytochalasins on the organization of actin filaments and microtubules in a neuronal growth cone.

1988 ◽  
Vol 107 (4) ◽  
pp. 1505-1516 ◽  
Author(s):  
P Forscher ◽  
S J Smith
Keyword(s):  
Growth Cone ◽  
Spatial Organization ◽  
Leading Edge ◽  
Growth Cones ◽  
Actin Networks ◽  
Antibody Staining ◽  
Flow Perfusion ◽  
Rapid Flow ◽  
Lamellar Region ◽  
Neuronal Growth Cone

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.

Drosophila myosin phosphatase and its role in dorsal closure

Development ◽  
2002 ◽  
Vol 129 (5) ◽  
pp. 1215-1223 ◽  
Author(s):  
Tomoaki Mizuno ◽  
Kyoko Tsutsui ◽  
Yasuyoshi Nishida
Keyword(s):  
Genetic Interaction ◽  
Shape Change ◽  
Myosin Ii ◽  
Rho Kinase ◽  
Leading Edge ◽  
Subcellular Location ◽  
Dorsal Closure ◽  
Nonmuscle Myosin ◽  
Myosin Phosphatase ◽  
Nonmuscle Myosin Ii

Myosin phosphatase negatively regulates nonmuscle myosin II through dephosphorylation of the myosin regulatory light chain (MRLC). Its regulatory myosin-binding subunit, MBS, is responsible for regulating the catalytic subunit in response to upstream signals and for determining the substrate specificity. DMBS, the Drosophila homolog of MBS, was identified to study the roles of myosin phosphatase in morphogenesis. The embryos defective for both maternal and zygotic DMBS demonstrated a failure in dorsal closure. In the mutant embryos, the defects were mainly confined to the leading edge cells which failed to fully elongate. Ectopic accumulation of phosphorylated MRLC was detected in lateral region of the leading edge cells, suggesting that the role of DMBS is to repress the activation of nonmuscle myosin II at the subcellular location for coordinated cell shape change. Aberrant accumulation of F-actin within the leading edge cells may correspond to the morphological aberrations of such cells. Similar defects were seen in embryos overexpressing Rho-kinase, suggesting that myosin phosphatase and Rho-kinase function antagonistically. The genetic interaction of DMBS with mutations in the components of the Rho signaling cascade also indicates that DMBS functions antagonistically to the Rho signal transduction pathway. The results indicate an important role for myosin phosphatase in morphogenesis.

scholarly journals Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks

2019 ◽  
Author(s):  
Markku Hakala ◽  
Hugo Wioland ◽  
Mari Tolonen ◽  
Antoine Jegou ◽  
Guillaume Romet-Lemonne ◽  
...  
Keyword(s):  
Leading Edge ◽  
Underlying Mechanism ◽  
Actin Dynamics ◽  
Dissociation Kinetics ◽  
Single Filament ◽  
Actin Networks ◽  
Capping Protein ◽  
Specific Localization ◽  
In Cells

AbstractCoordinated polymerization of actin filaments provides force for cell migration, morphogenesis, and endocytosis. Capping Protein (CP) is central regulator of actin dynamics in all eukaryotes. It binds actin filament (F-actin) barbed ends with high affinity and slow dissociation kinetics to prevent filament polymerization and depolymerization. In cells, however, CP displays remarkably rapid dynamics within F-actin networks, but the underlying mechanism has remained enigmatic. We report that a conserved cytoskeletal regulator, twinfilin, is responsible for CP’s rapid dynamics and specific localization in cells. Depletion of twinfilin led to stable association of CP with cellular F-actin arrays and its treadmilling throughout leading-edge lamellipodium. These were accompanied by diminished F-actin disassembly rates. In vitro single filament imaging approaches revealed that twinfilin directly promotes dissociation of CP from filament barbed ends, while allowing subsequent filament depolymerization. These results uncover an evolutionary conserved bipartite mechanism that controls how actin cytoskeleton-mediated forces are generated in cells.

scholarly journals Expansion and concatenation of nonmuscle myosin IIA filaments drive cellular contractile system formation during interphase and mitosis

2016 ◽  
Vol 27 (9) ◽  
pp. 1465-1478 ◽  
Author(s):  
Aidan M. Fenix ◽  
Nilay Taneja ◽  
Carmen A. Buttler ◽  
John Lewis ◽  
Schuyler B. Van Engelenburg ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Molecular Motor ◽  
Myosin Ii ◽  
Cell Movement ◽  
Nonmuscle Myosin ◽  
Contractile Ring ◽  
Contractile System ◽  
Nonmuscle Myosin Ii ◽  
Dividing Cells ◽  
Contractile Forces

Cell movement and cytokinesis are facilitated by contractile forces generated by the molecular motor, nonmuscle myosin II (NMII). NMII molecules form a filament (NMII-F) through interactions of their C-terminal rod domains, positioning groups of N-terminal motor domains on opposite sides. The NMII motors then bind and pull actin filaments toward the NMII-F, thus driving contraction. Inside of crawling cells, NMIIA-Fs form large macromolecular ensembles (i.e., NMIIA-F stacks), but how this occurs is unknown. Here we show NMIIA-F stacks are formed through two non–mutually exclusive mechanisms: expansion and concatenation. During expansion, NMIIA molecules within the NMIIA-F spread out concurrent with addition of new NMIIA molecules. Concatenation occurs when multiple NMIIA-Fs/NMIIA-F stacks move together and align. We found that NMIIA-F stack formation was regulated by both motor activity and the availability of surrounding actin filaments. Furthermore, our data showed expansion and concatenation also formed the contractile ring in dividing cells. Thus interphase and mitotic cells share similar mechanisms for creating large contractile units, and these are likely to underlie how other myosin II–based contractile systems are assembled.

scholarly journals Rho Kinase Differentially Regulates Phosphorylation of Nonmuscle Myosin II Isoforms A and B during Cell Rounding and Migration

2006 ◽  
Vol 281 (47) ◽  
pp. 35873-35883 ◽  
Author(s):  
Joshua C. Sandquist ◽  
Katherine I. Swenson ◽  
Kris A. DeMali ◽  
Keith Burridge ◽  
Anthony R. Means
Keyword(s):  
Myosin Ii ◽  
Rho Kinase ◽  
Nonmuscle Myosin ◽  
Cell Rounding ◽  
Nonmuscle Myosin Ii ◽  
And Migration

scholarly journals A Myosin IK-Abp1-PakB Circuit Acts as a Switch to Regulate Phagocytosis Efficiency

2010 ◽  
Vol 21 (9) ◽  
pp. 1505-1518 ◽  
Author(s):  
Régis Dieckmann ◽  
Yosuke von Heyden ◽  
Claudia Kistler ◽  
Navin Gopaldass ◽  
Stéphanie Hausherr ◽  
...  
Keyword(s):  
Myosin Ii ◽  
Interaction Network ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Binding Affinities ◽  
Membrane Anchor ◽  
Large Particles ◽  
Contractile Forces ◽  
Phagocytic Uptake ◽  
Mutation Analyses

Actin dynamics and myosin (Myo) contractile forces are necessary for formation and closure of the phagocytic cup. In Dictyostelium, the actin-binding protein Abp1 and myosin IK are enriched in the closing cup and especially at an actin-dense constriction furrow formed around the neck of engulfed budded yeasts. This phagocytic furrow consists of concentric overlapping rings of MyoK, Abp1, Arp3, coronin, and myosin II, following an order strikingly reminiscent of the overall organization of the lamellipodium of migrating cells. Mutation analyses of MyoK revealed that both a C-terminal farnesylation membrane anchor and a Gly-Pro-Arg domain that interacts with profilin and Abp1 were necessary for proper localization in the furrow and efficient phagocytosis. Consequently, we measured the binding affinities of these interactions and unraveled further interactions with profilins, dynamin A, and PakB. Due to the redundancy of the interaction network, we hypothesize that MyoK and Abp1 are restricted to regulatory roles and might affect the dynamic of cup progression. Indeed, phagocytic uptake was regulated antagonistically by MyoK and Abp1. MyoK is phosphorylated by PakB and positively regulates phagocytosis, whereas binding of Abp1 negatively regulates PakB and MyoK. We conclude that a MyoK-Abp1-PakB circuit acts as a switch regulating phagocytosis efficiency of large particles.

Modulation of epithelial tubule formation by Rho kinase

2004 ◽  
Vol 286 (4) ◽  
pp. C857-C866 ◽  
Author(s):  
Randi Eisen ◽  
Don R. Ratcliffe ◽  
George K. Ojakian
Keyword(s):  
Mdck Cells ◽  
Myosin Ii ◽  
Rho Kinase ◽  
Signal Transduction Pathway ◽  
Collagen Gel ◽  
Leading Edge ◽  
Transduction Pathway ◽  
Tubule Formation ◽  
Epithelial Remodeling ◽  
Lamellipodia Formation

We have developed a model system for studying integrin regulation of mammalian epithelial tubule formation. Application of collagen gel overlays to Madin-Darby canine kidney (MDCK) cells induced coordinated disassembly of junctional complexes that was accompanied by lamellipodia formation and cell rearrangement (termed epithelial remodeling). In this study, we present evidence that the Rho signal transduction pathway regulates epithelial remodeling and tubule formation. Incubation of MDCK cells with collagen gel overlays facilitated formation of migrating lamellipodia with membrane-associated actin. Inhibitors of myosin II and actin prevented lamellipodia formation, which suggests that actomyosin function was involved in regulation of epithelial remodeling. To determine this, changes in myosin II distribution, function, and phosphorylation were studied during epithelial tubule biogenesis. Myosin II colocalized with actin at the leading edge of lamellipodia thereby providing evidence that myosin is important in epithelial remodeling. This possibility is supported by observations that inhibition of Rho kinase, a regulator of myosin II function, alters formation of lamellipodia and results in attenuated epithelial tubule development. These data and those demonstrating myosin regulatory light-chain phosphorylation at the leading edge of lamellipodia strongly suggest that Rho kinase and myosin II are important modulators of epithelial remodeling. They support a hypothesis that the Rho signal transduction pathway plays a significant role in regulation of epithelial tubule formation.

scholarly journals Filopodia formation and Disabled degradation downstream of Reelin

2004 ◽  
Vol 384 (1) ◽  
Author(s):  
Steven J. WINDER
Keyword(s):  
Actin Polymerization ◽  
Leading Edge ◽  
Negative Control ◽  
Actin Dynamics ◽  
Abelson Tyrosine Kinase ◽  
Motile Cells ◽  
Biochemical Journal ◽  
Subtle Effect ◽  
Important Regulator ◽  
Syndrome Protein

During Drosophila embryogenesis, Abl (Abelson tyrosine kinase) is localized in the axons of the CNS (central nervous system). Mutations in Abl have a subtle effect on the morphology of the embryonic CNS, and the mutant animals survive to the pupal and adult stages. However, genetic screens have identified several genes that, when mutated along with the Abl gene, modified the phenotypes. Two prominent genes that arose from these screens were enabled (Ena) and disabled (Dab). It has been known for some time that Enabled and its mammalian homologues are involved in the regulation of actin dynamics, and promote actin polymerization at the leading edge of motile cells. It was a defect in actin polymerization in migrating neurons in particular that resulted in the identification of Enabled as an important regulator of neuronal migration. Defects in Disabled, in both Drosophila and mammals, also gave rise to neuronal defects which, in mice, were indistinguishable from phenotypes observed in the reeler mouse. These observations suggested that mDab1 (mammalian Disabled homologue 1) acted in a pathway downstream of Reelin, the product of the reelin gene found to be defective in reeler mice. Now, in this issue of the Biochemical Journal, Takenawa and colleagues have demonstrated that Disabled also acts in a pathway to regulate actin dynamics through the direct activation of N-WASP (neuronal Wiskott–Aldrich syndrome protein). Furthermore, they were also able to link several lines of investigation from other groups to show that the ability of mDab1 to regulate actin dynamics during cell motility was under the negative control of tyrosine phosphorylation, leading to ubiquitin-mediated degradation of mDab1.

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