scholarly journals Two Components of Actin-based Retrograde Flow in Sea Urchin Coelomocytes

1999 ◽  
Vol 10 (12) ◽  
pp. 4075-4090 ◽  
Author(s):  
John H. Henson ◽  
Tatyana M. Svitkina ◽  
Andrew R. Burns ◽  
Heather E. Hughes ◽  
Kenneth J. MacPartland ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Sea Urchin ◽  
Actin Polymerization ◽  
Kinase Inhibitor ◽  
Wide Spectrum ◽  
Myosin Ii ◽  
Specific Inhibitor ◽  
Supramolecular Organization ◽  
Cell Periphery ◽  
Retrograde Flow

Sea urchin coelomocytes represent an excellent experimental model system for studying retrograde flow. Their extreme flatness allows for excellent microscopic visualization. Their discoid shape provides a radially symmetric geometry, which simplifies analysis of the flow pattern. Finally, the nonmotile nature of the cells allows for the retrograde flow to be analyzed in the absence of cell translocation. In this study we have begun an analysis of the retrograde flow mechanism by characterizing its kinetic and structural properties. The supramolecular organization of actin and myosin II was investigated using light and electron microscopic methods. Light microscopic immunolocalization was performed with anti-actin and anti-sea urchin egg myosin II antibodies, whereas transmission electron microscopy was performed on platinum replicas of critical point-dried and rotary-shadowed cytoskeletons. Coelomocytes contain a dense cortical actin network, which feeds into an extensive array of radial bundles in the interior. These actin bundles terminate in a perinuclear region, which contains a ring of myosin II bipolar minifilaments. Retrograde flow was arrested either by interfering with actin polymerization or by inhibiting myosin II function, but the pathway by which the flow was blocked was different for the two kinds of inhibitory treatments. Inhibition of actin polymerization with cytochalasin D caused the actin cytoskeleton to separate from the cell margin and undergo a finite retrograde retraction. In contrast, inhibition of myosin II function either with the wide-spectrum protein kinase inhibitor staurosporine or the myosin light chain kinase–specific inhibitor KT5926 stopped flow in the cell center, whereas normal retrograde flow continued at the cell periphery. These differential results suggest that the mechanism of retrograde flow has two, spatially segregated components. We propose a “push–pull” mechanism in which actin polymerization drives flow at the cell periphery, whereas myosin II provides the tension on the actin cytoskeleton necessary for flow in the cell interior.

scholarly journals Analysis of the Actin–Myosin II System in Fish Epidermal Keratocytes: Mechanism of Cell Body Translocation

1997 ◽  
Vol 139 (2) ◽  
pp. 397-415 ◽  
Author(s):  
Tatyana M. Svitkina ◽  
Alexander B. Verkhovsky ◽  
Kyle M. McQuade ◽  
Gary G. Borisy
Keyword(s):  
Cell Body ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Leading Edge ◽  
Time Lapse ◽  
Supramolecular Organization ◽  
Retrograde Flow ◽  
Body Boundary ◽  
Filament Bundles

While the protrusive event of cell locomotion is thought to be driven by actin polymerization, the mechanism of forward translocation of the cell body is unclear. To elucidate the mechanism of cell body translocation, we analyzed the supramolecular organization of the actin–myosin II system and the dynamics of myosin II in fish epidermal keratocytes. In lamellipodia, long actin filaments formed dense networks with numerous free ends in a brushlike manner near the leading edge. Shorter actin filaments often formed T junctions with longer filaments in the brushlike area, suggesting that new filaments could be nucleated at sides of preexisting filaments or linked to them immediately after nucleation. The polarity of actin filaments was almost uniform, with barbed ends forward throughout most of the lamellipodia but mixed in arc-shaped filament bundles at the lamellipodial/cell body boundary. Myosin II formed discrete clusters of bipolar minifilaments in lamellipodia that increased in size and density towards the cell body boundary and colocalized with actin in boundary bundles. Time-lapse observation demonstrated that myosin clusters appeared in the lamellipodia and remained stationary with respect to the substratum in locomoting cells, but they exhibited retrograde flow in cells tethered in epithelioid colonies. Consequently, both in locomoting and stationary cells, myosin clusters approached the cell body boundary, where they became compressed and aligned, resulting in the formation of boundary bundles. In locomoting cells, the compression was associated with forward displacement of myosin features. These data are not consistent with either sarcomeric or polarized transport mechanisms of cell body translocation. We propose that the forward translocation of the cell body and retrograde flow in the lamellipodia are both driven by contraction of an actin–myosin network in the lamellipodial/cell body transition zone.

scholarly journals Nonmuscle myosin II is responsible for maintaining endothelial cell basal tone and stress fiber integrity

2008 ◽  
Vol 295 (4) ◽  
pp. C994-C1006 ◽  
Author(s):  
Zoe M. Goeckeler ◽  
Paul C. Bridgman ◽  
Robert B. Wysolmerski
Keyword(s):  
Endothelial Cell ◽  
Light Chain ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Rho Kinase ◽  
Specific Inhibitor ◽  
Isometric Tension ◽  
Stress Fiber ◽  
Tension Development ◽  
Basal Tone

Cultured confluent endothelial cells exhibit stable basal isometric tone associated with constitutive myosin II regulatory light chain (RLC) phosphorylation. Thrombin treatment causes a rapid increase in isometric tension concomitant with myosin II RLC phosphorylation, actin polymerization, and stress fiber reorganization while inhibitors of myosin light chain kinase (MLCK) and Rho-kinase prevent these responses. These findings suggest a central role for myosin II in the regulation of endothelial cell tension. The present studies examine the effects of blebbistatin, a specific inhibitor of myosin II activity, on basal tone and thrombin-induced tension development. Although blebbistatin treatment abolished basal tension, this was accompanied by an increase in myosin II RLC phosphorylation. The increase in RLC phosphorylation was Ca2+ dependent and mediated by MLCK. Similarly, blebbistatin inhibited thrombin-induced tension without interfering with the increase in RLC phosphorylation or in F-actin polymerization. Blebbistatin did prevent myosin II filament incorporation and association with polymerizing or reorganized actin filaments leading to the disappearance of stress fibers. Thus the inhibitory effects of blebbistatin on basal tone and induced tension are consistent with a requirement for myosin II activity to maintain stress fiber integrity.

Endothelial cell retraction is induced by PAK2 monophosphorylation of myosin II

2000 ◽  
Vol 113 (3) ◽  
pp. 471-482 ◽  
Author(s):  
Q. Zeng ◽  
D. Lagunoff ◽  
R. Masaracchia ◽  
Z. Goeckeler ◽  
G. Cote ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Actin Cytoskeleton ◽  
Light Chain ◽  
Myosin Ii ◽  
Specific Inhibitor ◽  
Regulatory Light Chain ◽  
Atpase Inhibitor ◽  
Cytoskeletal Rearrangements ◽  
Cell Retraction

The p21-activated kinase (PAK) family includes several enzyme isoforms regulated by the GTPases Rac1 and Cdc42. PAK1, found in brain, muscle and spleen, has been implicated in triggering cytoskeletal rearrangements such as the dissolution of stress fibers and reorganization of focal complexes. The role of the more widely distributed PAK2 in controlling the cytoskeleton has been less well studied. Previous work has demonstrated that PAK2 can monophosphorylate the myosin II regulatory light chain and induce retraction of permeabilized endothelial cells. In this report we characterize PAK2's morphological and biochemical effect on intact endothelial cells utilizing microinjection of constitutively active PAK2. Under these conditions we observed a modification of the actin cytoskeleton with retraction of endothelial cell margins accompanied by an increase in monophosphorylation of myosin II. Selective inhibitors were used to analyze the mechanism of action of PAK2. Staurosporine, a direct inhibitor of PAK2, largely prevented the action of microinjected PAK2 in endothelial cells. Butanedione monoxime, a non-specific myosin ATPase inhibitor, also inhibited the effects of PAK2 implicating myosin in the changes in cytoskeletal reorganization. In contrast, KT5926, a specific inhibitor of myosin light chain kinase was ineffective in preventing the changes in morphology and the actin cytoskeleton. The additional finding that endogenous PAK2 associates with myosin II is consistent with the proposal that cell retraction and cytoskeletal rearrangements induced by microinjected PAK2 depend on the direct activation of myosin II by PAK2 monophosphorylation of the regulatory light chain.

Myosin II-dependent cylindrical protrusions induced by quinine inDictyostelium: antagonizing effects of actin polymerization at the leading edge

2001 ◽  
Vol 114 (11) ◽  
pp. 2155-2165
Author(s):  
Kunito Yoshida ◽  
Kei Inouye
Keyword(s):  
Cell Membrane ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Cell Movement ◽  
Leading Edge ◽  
Contractile Vacuole ◽  
Cell Periphery ◽  
Slowing Down ◽  
On Line ◽  
Cylindrical Protrusion

We found that amoeboid cells of Dictyostelium are induced by a millimolar concentration of quinine to form a rapidly elongating, cylindrical protrusion, which often led to sustained locomotion of the cells. Formation of the protrusion was initiated by fusion of a contractile vacuole with the cell membrane. During protrusion extension, a patch of the contractile vacuole membrane stayed undiffused on the leading edge of the protrusion for over 30 seconds. Protrusion formation was not inhibited by high osmolarity of the external medium (at least up to 400 mosM). By contrast, mutant cells lacking myosin II (mhc− cells) failed to extend protrusions upon exposure to quinine. When GFP-myosin-expressing cells were exposed to quinine, GFP-myosin was accumulated in the cell periphery forming a layer under the cell membrane, but a newly formed protrusion was initially devoid of a GFP-myosin layer, which gradually formed and extended from the base of the protrusion. F-actin was absent in the leading front of the protrusion during the period of its rapid elongation, and the formation of a layer of F-actin in the front was closely correlated with its slowing-down or retraction. Periodical or continuous detachment of the F-actin layer from the apical membrane of the protrusion, accompanied by a transient increase in the elongation speed at the site of detachment, was observed in some of the protrusions. The detached F-actin layers, which formed a spiral layer of F-actin in the case of continuous detachment, moved in the opposite direction of protrusion elongation. In the presence of both cytochalasin A and quinine, the protrusions formed were not cylindrical but spherical, which swallowed up the entire cellular contents. The estimated bulk flux into the expanding spherical protrusions of such cells was four-times higher than the flux into the elongating cylindrical protrusions of the cells treated with quinine alone. These results indicate that the force responsible for the quinine-induced protrusion is mainly due to contraction of the cell body, which requires normal myosin II functions, while actin polymerization is important in restricting the direction of its expansion. We will discuss the possible significance of tail contraction in cell movement in the multicellular phase of Dictyostelium development, where cell locomotion similar to that induced by quinine is often observed without quinine treatment, and in protrusion elongation in general.Movies available on-line

Live dynamics of GFP-calponin: isoform-specific modulation of the actin cytoskeleton and autoregulation by C-terminal sequences

2000 ◽  
Vol 113 (21) ◽  
pp. 3725-3736 ◽  
Author(s):  
C. Danninger ◽  
M. Gimona
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Muscle Cells ◽  
Protein Kinase Inhibitors

The calponin family of F-actin-, tropomyosin- and calmodulin-binding proteins currently comprises three genetic variants. Their functional roles implicated from in vitro studies include the regulation of actomyosin interactions in smooth muscle cells (h1 calponin), cytoskeletal organisation in non-muscle cells (h2 calponin) and the control of neurite outgrowth (acidic calponin). We have now investigated the effects of calponin (CaP) isoforms and their C-terminal deletion mutants on the actin cytoskeleton by time lapse video microscopy of GFP fusion proteins in living smooth muscle cells and fibroblasts. It is shown that h1 CaP associates with the actin stress fibers in the more central part of the cell, whereas h2 CaP localizes to the ends of stress fibres and in the motile lamellipodial protrusions of spreading cells. Cells expressing h2 CaP spread more efficiently than those expressing h1 CaP and expression of GFP h1 CaP resulted in reduced cell motility in wound healing experiments. Notably, expression of GFP h1 CaP, but not GFP h2 CaP, conferred increased resistance of the actin cytoskeleton to the actin polymerization antagonists cytochalasin B and latrunculin B, as well as to the protein kinase inhibitors H7-dihydrochloride and rho-kinase inhibitor Y-27632. These data point towards a dual role of CaP in the stabilization and regulation of the actin cytoskeleton in vivo. Deletion studies further identify an autoregulatory role for the unique C-terminal tail sequences in the respective CaP isoforms.

scholarly journals Tyrosine kinases activate store-mediated Ca2+ entry in human platelets through the reorganization of the actin cytoskeleton

2000 ◽  
Vol 351 (2) ◽  
pp. 429-437 ◽  
Author(s):  
Juan A. ROSADO ◽  
Darren GRAVES ◽  
Stewart O. SAGE
Keyword(s):  
Tyrosine Kinase ◽  
Actin Cytoskeleton ◽  
Tyrosine Kinases ◽  
Actin Polymerization ◽  
Kinase Inhibitor ◽  
Coupling Model ◽  
Human Platelets ◽  
Ras Proteins ◽  
Model Treatment ◽  
Independent Effects

We have recently reported that store-mediated Ca2+ entry in platelets is likely to be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, a model termed ‘secretion-like coupling’. In this model the actin cytoskeleton plays a key regulatory role. Since tyrosine kinases have been shown to be important for Ca2+ entry in platelets and other cells, we have now investigated the possible involvement of tyrosine kinases in the secretion-like-coupling model. Treatment of platelets with thrombin or thapsigargin induced actin polymerization by a calcium-independent pathway. Methyl 2,5-dihydroxycinnamate, a tyrosine kinase inhibitor, prevented thrombin- or thapsigargin-induced actin polymerization. The effects of tyrosine kinases in store-mediated Ca2+ entry were found to be entirely dependent on the actin cytoskeleton. PP1, an inhibitor of the Src family of proteins, partially inhibited store-mediated Ca2+ entry. In addition, depletion of intracellular Ca2+ stores stimulated cytoskeletal association of the cytoplasmic tyrosine kinase pp60src, a process that was sensitive to treatment with cytochalasin D and PP1, but not to inhibition of Ras proteins using prenylcysteine analogues. Finally, combined inhibition of both Ras proteins and tyrosine kinases resulted in complete inhibition of Ca2+ entry, suggesting that these two families of proteins have independent effects in the activation of store-mediated Ca2+ entry in human platelets.

scholarly journals Supervillin Reorganizes the Actin Cytoskeleton and Increases Invadopodial Efficiency

2009 ◽  
Vol 20 (3) ◽  
pp. 948-962 ◽  
Author(s):  
Jessica L. Crowley ◽  
Tara C. Smith ◽  
Zhiyou Fang ◽  
Norio Takizawa ◽  
Elizabeth J. Luna
Keyword(s):  
Extracellular Matrix ◽  
Actin Cytoskeleton ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Myosin Ii ◽  
Cell Periphery ◽  
Related Protein ◽  
Peripheral Membrane Protein ◽  
Green Fluorescent

Tumor cells use actin-rich protrusions called invadopodia to degrade extracellular matrix (ECM) and invade tissues; related structures, termed podosomes, are sites of dynamic ECM interaction. We show here that supervillin (SV), a peripheral membrane protein that binds F-actin and myosin II, reorganizes the actin cytoskeleton and potentiates invadopodial function. Overexpressed SV induces redistribution of lamellipodial cortactin and lamellipodin/RAPH1/PREL1 away from the cell periphery to internal sites and concomitantly increases the numbers of F-actin punctae. Most punctae are highly dynamic and colocalize with the podosome/invadopodial proteins, cortactin, Tks5, and cdc42. Cortactin binds SV sequences in vitro and contributes to the formation of enhanced green fluorescent protein (EGFP)-SV induced punctae. SV localizes to the cores of Src-generated podosomes in COS-7 cells and with invadopodia in MDA-MB-231 cells. EGFP-SV overexpression increases average numbers of ECM holes per cell; RNA interference-mediated knockdown of SV decreases these numbers. Although SV knockdown alone has no effect, simultaneous down-regulation of SV and the closely related protein gelsolin reduces invasion through ECM. Together, our results show that SV is a component of podosomes and invadopodia and that SV plays a role in invadopodial function, perhaps as a mediator of cortactin localization, activation state, and/or dynamics of metalloproteinases at the ventral cell surface.

scholarly journals Pathogenic Puppetry: Manipulation of the Host Actin Cytoskeleton by Chlamydia trachomatis

2019 ◽  
Vol 21 (1) ◽  
pp. 90 ◽  
Author(s):  
Liam Caven ◽  
Rey A. Carabeo
Keyword(s):  
Chlamydia Trachomatis ◽  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Host Cells ◽  
Surface Receptors ◽  
Obligate Intracellular ◽  
Membrane Bound ◽  
Experimental Findings ◽  
Chlamydial Inclusion

The actin cytoskeleton is crucially important to maintenance of the cellular structure, cell motility, and endocytosis. Accordingly, bacterial pathogens often co-opt the actin-restructuring machinery of host cells to access or create a favorable environment for their own replication. The obligate intracellular organism Chlamydia trachomatis and related species exemplify this dynamic: by inducing actin polymerization at the site of pathogen-host attachment, Chlamydiae induce their own uptake by the typically non-phagocytic epithelium they infect. The interaction of chlamydial adhesins with host surface receptors has been implicated in this effect, as has the activity of the chlamydial effector TarP (translocated actin recruitment protein). Following invasion, C. trachomatis dynamically assembles and maintains an actin-rich cage around the pathogen’s membrane-bound replicative niche, known as the chlamydial inclusion. Through further induction of actin polymerization and modulation of the actin-crosslinking protein myosin II, C. trachomatis promotes egress from the host via extrusion of the inclusion. In this review, we present the experimental findings that can inform our understanding of actin-dependent chlamydial pathogenesis, discuss lingering questions, and identify potential avenues of future study.

scholarly journals Wound Closure in the Lamellipodia of Single Cells: Mediation by Actin Polymerization in the Absence of an Actomyosin Purse String

2002 ◽  
Vol 13 (3) ◽  
pp. 1001-1014 ◽  
Author(s):  
John H. Henson ◽  
Ronniel Nazarian ◽  
Katrina L. Schulberg ◽  
Valerie A. Trabosh ◽  
Sarah E. Kolnik ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Wound Closure ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Single Cells ◽  
Myosin Ii ◽  
General Mechanism ◽  
Purse String ◽  
Dramatic Form ◽  
Wound Margin

The actomyosin purse string is an evolutionarily conserved contractile structure that is involved in cytokinesis, morphogenesis, and wound healing. Recent studies suggested that an actomyosin purse string is crucial for the closure of wounds in single cells. In the present study, morphological and pharmacological methods were used to investigate the role of this structure in the closure of wounds in the peripheral cytoplasm of sea urchin coelomocytes. These discoidal shaped cells underwent a dramatic form of actin-based centripetal/retrograde flow and occasionally opened and closed spontaneous wounds in their lamellipodia. Fluorescent phalloidin staining indicated that a well defined fringe of actin filaments assembles from the margin of these holes, and drug studies with cytochalasin D and latrunculin A indicated that actin polymerization is required for wound closure. Additional evidence that actin polymerization is involved in wound closure was provided by the localization of components of the Arp2/3 complex to the wound margin. Significantly, myosin II immunolocalization demonstrated that it is not associated with wound margins despite being present in the perinuclear region. Pharmacological evidence for the lack of myosin II involvement in wound closure comes from experiments in which a microneedle was used to produce wounds in cells in which actomyosin contraction was inhibited by treatment with kinase inhibitors. Wounds produced in kinase inhibitor-treated cells closed in a manner similar to that seen with control cells. Taken together, our results suggest that an actomyosin purse string mechanism is not responsible for the closure of lamellar wounds in coelomocytes. We hypothesize that the wounds heal by means of a combination of the force produced by actin polymerization alone and centripetal flow. Interestingly, these cells did assemble an actomyosin structure around the margin of phagosome-like membrane invaginations, indicating that myosin is not simply excluded from the periphery by some general mechanism. The results indicate that the actomyosin purse string is not the only mechanism that can mediate wound closure in single cells.

Stability of actin cytoskeleton and PKC-δ binding to actin regulate NKCC1 function in airway epithelial cells

2003 ◽  
Vol 284 (2) ◽  
pp. C487-C496 ◽  
Author(s):  
Carole M. Liedtke ◽  
Melinda Hubbard ◽  
Xiangyun Wang
Keyword(s):  
Actin Cytoskeleton ◽  
Protein Interactions ◽  
Actin Polymerization ◽  
Direct Interaction ◽  
Airway Epithelial Cells ◽  
Cell Periphery ◽  
Airway Epithelial ◽  
Protein Protein Interactions ◽  
Signaling Mechanism ◽  
Increased Activity

Activation of airway epithelial Na-K-2Cl cotransporter (NKCC)1 requires increased activity of protein kinase C (PKC)-δ, which localizes predominantly to the actin cytoskeleton. Prompted by reports of a role for actin in NKCC1 function, we studied a signaling mechanism linking NKCC1 and PKC. Stabilization of actin polymerization with jasplakinolide increased activity of NKCC1, whereas inhibition of actin polymerization with latrunculin B prevented hormonal activation of NKCC1. Protein-protein interactions among NKCC1, actin, and PKC-δ were verified by Western blot analysis of immunoprecipitated proteins. PKC-δ was detected in immunoprecipitates of NKCC1 and vice versa. Actin was also detected in immunoprecipitates of NKCC1 and PKC-δ. Pulldown of endogenous actin revealed the presence of NKCC1 and PKC-δ. Binding of recombinant PKC-δ to NKCC1 was not detected in overlay assays. Rather, activated PKC-δ bound to actin, and this interaction was prevented by a peptide encoding δC2, a C2-like domain based on the amino acid sequence of PKC-δ. δC2 also blocked stimulation of NKCC1 function by methoxamine. Immunofluorescence and confocal microscopy revealed PKC-δ in the cytosol and cell periphery. Merged images of cells stained for actin and PKC-δ indicated colocalization of PKC-δ and actin at the cell periphery. The results indicate that actin is critical for the activation of NKCC1 through a direct interaction with PKC-δ.

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