Shape oscillations of human neutrophil leukocytes: characterization and relationship to cell motility.

When neutrophil leukocytes are stimulated by chemotactic factors or by substratum contact, they change their shape. Shape changes are a prerequisite for cellular migration and typically involve the extrusion of thin, veil-like lamellipods and the development of morphological polarity. Stimulation also leads to changes in the neutrophil content of filamentous actin (F-actin), which is the major cytoskeletal component. Suspensions of human neutrophils stimulated with chemoattractants exhibit sinusoidal light-scattering oscillations with a period of approximately 8 s at 37 degrees C. These oscillations arise from periodic fluctuations in the cell body size caused by lamellipod extension and retraction cycles. The light-scattering oscillations are paralleled by corresponding oscillations in F-actin content. This raises the interesting possibility that cyclic actin polymerization constitutes the driving force for shape oscillations of suspended neutrophils. Similar periodic shape changes are present in neutrophils crawling on a surface, suggesting that shape oscillations are important for neutrophil motion. This review summarizes our present knowledge about shape oscillations in suspended and crawling neutrophils and discusses a possible role for these oscillations in neutrophil motility.

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The protein kinase C inhibitor H-7 activates human neutrophils: effect on shape, actin polymerization, fluid pinocytosis and locomotion

Journal of Cell Science ◽

10.1242/jcs.96.1.99 ◽

1990 ◽

Vol 96 (1) ◽

pp. 99-106

Author(s):

H.U. Keller ◽

V. Niggli ◽

A. Zimmermann ◽

R. Portmann

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Actin Polymerization ◽

Kinase Inhibitor ◽

Human Neutrophils ◽

Kinase C ◽

Chemotactic Peptides ◽

Protein Kinase C Inhibitor ◽

Shape Changes ◽

Stimulation Of

The present study demonstrates new properties of H-7. The protein kinase inhibitor H-7 is a potent activator of several neutrophil functions. Stimulation of initially spherical nonmotile neutrophils elicits vigorous shape changes within a few seconds, increases in cytoskeletal actin, altered F-actin distribution, increased adhesiveness and a relatively small increase in pinocytic activity. H-7 has also chemokinetic activities. Depending on the experimental condition, H-7 may elicit or inhibit neutrophil locomotion. It failed to induce chemotaxis. Thus, the response pattern elicited by H-7 is different from that of other leukocyte activators such as chemotactic peptides, PMA or diacylglycerols. The finding that H-7 can elicit shape changes, actin polymerization and pinocytosis suggests that these events can occur without activation of protein kinase C (PKC). PMA-induced shape changes and stimulation of pinocytosis were not inhibited by H-7.

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ADP-ribosylation of Rho enhances actin polymerization-coupled shape oscillations in human neutrophils

FEBS Letters ◽

10.1016/0014-5793(95)00880-i ◽

1995 ◽

Vol 372 (2-3) ◽

pp. 161-164 ◽

Cited By ~ 15

Author(s):

Markus U. Ehrengruber ◽

Patrice Boquet ◽

Thomas D. Coates ◽

David A. Deranleau

Keyword(s):

Actin Polymerization ◽

Human Neutrophils ◽

Adp Ribosylation ◽

Shape Oscillations

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Beta 2 integrin engagement triggers actin polymerization and phosphatidylinositol trisphosphate formation in non-adherent human neutrophils.

The Journal of Cell Biology ◽

10.1083/jcb.123.6.1597 ◽

1993 ◽

Vol 123 (6) ◽

pp. 1597-1605 ◽

Cited By ~ 43

Author(s):

R Löfgren ◽

J Ng-Sikorski ◽

A Sjölander ◽

T Andersson

Keyword(s):

Actin Polymerization ◽

Cellular Level ◽

Human Neutrophils ◽

Induced Response ◽

Actin Assembly ◽

Filamentous Actin ◽

Integrin Receptors ◽

Absolute Requirement ◽

Beta 2

Beta 2 integrins are involved in the adhesion of leukocytes to other cells and surfaces. Although adhesion is required for cell locomotion, little is known regarding the way beta 2 integrin-receptors affect the actin network in leukocytes. In the present study filamentous actin (F-actin) levels in non-adherent human neutrophils have been measured by phalloidin staining after antibody cross-linking of beta 2 integrins. Antibody engagement of beta 2 integrins resulted in a rapid and sustained (146 and 131% after 30 and 300 s, respectively) increase in the neutrophil F-actin content. This is in contrast to stimulation with N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP), which causes a prompt and pronounced but rapidly declining rise in F-actin (214 and 127% after 15 and 300 s, respectively). Priming neutrophils with 1 nM PMA, a low concentration that did not influence the F-actin content per se, increased the magnitude of the beta 2 integrin-induced response but had no effect on the kinetics (199% after 30 s and 169% after 300 s). Removal of extracellular Ca2+ only marginally affected the beta 2 integrin-induced F-actin response for cells that were pretreated with PMA whereas the response for nonprimed cells was reduced by half. This suggests that even though extracellular Ca2+ has a modulatory effect it is not an absolute requirement for beta 2 integrin-induced actin polymerization. beta 2 integrin engagement did not affect the resting cellular level of cAMP arguing against a role of cAMP in beta 2 integrin-induced actin assembly.(ABSTRACT TRUNCATED AT 250 WORDS)

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Protein phosphatase inhibitors okadaic acid and calyculin A alter cell shape and F-actin distribution and inhibit stimulus-dependent increases in cytoskeletal actin of human neutrophils

Blood ◽

10.1182/blood.v80.11.2911.2911 ◽

1992 ◽

Vol 80 (11) ◽

pp. 2911-2919 ◽

Cited By ~ 46

Author(s):

P Kreienbuhl ◽

H Keller ◽

V Niggli

Keyword(s):

Okadaic Acid ◽

Human Neutrophils ◽

Resting Cells ◽

Calyculin A ◽

Chemotactic Peptide ◽

Phosphatase Inhibitors ◽

Protein Phosphatase Inhibitors ◽

Actin Localization ◽

Shape Changes

Abstract The phosphatase inhibitors okadaic acid and calyculin A were found to elicit or to modify several neutrophil responses, suggesting that dephosphorylation plays a regulatory role. The concentrations of okadaic acid (> or = 1 mumol/L) that were effective on neutrophil functions (shape changes and marginal stimulation of pinocytosis) were shown to stimulate the incorporation of 32PO4 into many neutrophil proteins several-fold. Calyculin A was effective at 50-fold lower concentrations. In the presence of the inhibitors, the cells exhibited a nonpolar shape and the polarization response induced by chemotactic peptide was inhibited. Both phosphatase inhibitors also induced the association of F-actin with the cell membrane. A steady-state phosphatase activity is thus involved in maintaining shape and F-actin localization of resting cells. Inhibitors alone had no significant effect on the amount of cytoskeleton-associated actin. The increase in cytoskeletal actin observed at 30 minutes of stimulation with phorbol ester or 5 to 30 minutes of stimulation with chemotactic peptide, however, was abolished by okadaic acid or calyculin A, suggesting an important role of a phosphatase. In contrast, the early increase in cytoskeleton-associated actin observed at 1 minute of stimulation with peptide was not affected. This finding indicates that the increased association of actin with the cytoskeleton in the early and the later stages of neutrophil activation may be mediated by different signalling pathways.

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Actin Polymerization, Calcium-Transients, and Phospholipid Metabolism in Human Neutrophils After Stimulation with Interleukin-8 and N-formyl Peptide

Journal of Investigative Dermatology ◽

10.1111/1523-1747.ep12371788 ◽

1994 ◽

Vol 102 (3) ◽

pp. 310-314 ◽

Cited By ~ 33

Author(s):

Johannes Norgauer ◽

Jean Krutmann ◽

Gustav J. Dobos ◽

Alexis E. Traynor-Kaplan ◽

Zenaida G. Oades ◽

...

Keyword(s):

Actin Polymerization ◽

Phospholipid Metabolism ◽

Interleukin 8 ◽

Calcium Transients ◽

Human Neutrophils ◽

Formyl Peptide

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Activation of human neutrophils by mastoparan. Reorganization of the cytoskeleton, formation of phosphatidylinositol 3,4,5-trisphosphate, secretion up-regulation of complement receptor type 3 and superoxide anion production are stimulated by mastoparan

Biochemical Journal ◽

10.1042/bj2820393 ◽

1992 ◽

Vol 282 (2) ◽

pp. 393-397 ◽

Cited By ~ 27

Author(s):

J Norgauer ◽

M Eberle ◽

H D Lemke ◽

K Aktories

Keyword(s):

Pertussis Toxin ◽

Actin Polymerization ◽

Cytochalasin B ◽

Superoxide Radical ◽

Human Neutrophils ◽

Superoxide Anion Production ◽

Radical Production ◽

Rapid Formation ◽

Primary Granules ◽

Type 3

In human neutrophils, mastoparan induced rapid F-actin polymerization which was followed by a slow and sustained depolymerization to below the initial F-actin content. Incubation of neutrophils with pertussis toxin inhibited mastoparan-stimulated actin polymerization; however it did not prevent sustained depolymerization of F-actin. Analyses of phospholipids performed in parallel revealed that mastoparan stimulated rapid formation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and consumption of phosphatidylinositol 4,5-bisphosphate (PIP2). Pertussis toxin treatment blocked mastoparan-induced formation of PIP3. Furthermore, mastoparan stimulated the release of N-acetylglucosaminidase from primary granules. Cytochalasin B enhanced mastoparan-stimulated secretion. Mastoparan triggered superoxide radical production in a cytochalasin B-sensitive manner and induced complement type 3 receptor (CR3) up-regulation.

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Primary granule exocytosis in human neutrophils is regulated by Rac-dependent actin remodeling

AJP Cell Physiology ◽

10.1152/ajpcell.00239.2008 ◽

2008 ◽

Vol 295 (5) ◽

pp. C1354-C1365 ◽

Cited By ~ 59

Author(s):

Troy Mitchell ◽

Andrea Lo ◽

Michael R. Logan ◽

Paige Lacy ◽

Gary Eitzen

Keyword(s):

Actin Polymerization ◽

Microscopic Analysis ◽

Formyl Peptide Receptor ◽

Human Neutrophils ◽

Cell Cortex ◽

Secretory Cells ◽

Actin Remodeling ◽

Granule Exocytosis ◽

Primary Granule

The actin cytoskeleton regulates exocytosis in all secretory cells. In neutrophils, Rac2 GTPase has been shown to control primary (azurophilic) granule exocytosis. In this report, we propose that Rac2 is required for actin cytoskeletal remodeling to promote primary granule exocytosis. Treatment of neutrophils with low doses (≤10 μM) of the actin-depolymerizing drugs latrunculin B (Lat B) or cytochalasin B (CB) enhanced both formyl peptide receptor- and Ca2+ionophore-stimulated exocytosis. Higher concentrations of CB or Lat B, or stabilization of F-actin with jasplakinolide (JP), inhibited primary granule exocytosis measured as myeloperoxidase release but did not affect secondary granule exocytosis determined by lactoferrin release. These results suggest an obligatory role for F-actin disassembly before primary granule exocytosis. However, lysates from secretagogue-stimulated neutrophils showed enhanced actin polymerization activity in vitro. Microscopic analysis showed that resting neutrophils contain significant cortical F-actin, which was redistributed to sites of primary granule translocation when stimulated. Exocytosis and actin remodeling was highly polarized when cells were primed with CB; however, polarization was reduced by Lat B preincubation, and both polarization and exocytosis were blocked when F-actin was stabilized with JP. Treatment of cells with the small molecule Rac inhibitor NSC23766 also inhibited actin remodeling and primary granule exocytosis induced by Lat B/fMLF or CB/fMLF, but not by Ca2+ionophore. Therefore, we propose a role for F-actin depolymerization at the cell cortex coupled with Rac-dependent F-actin polymerization in the cell cytoplasm to promote primary granule exocytosis.

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Small GTP-binding Protein TC10 Differentially Regulates Two Distinct Populations of Filamentous Actin in 3T3L1 Adipocytes

Molecular Biology of the Cell ◽

10.1091/mbc.01-10-0490 ◽

2002 ◽

Vol 13 (7) ◽

pp. 2334-2346 ◽

Cited By ~ 70

Author(s):

Makoto Kanzaki ◽

Robert T. Watson ◽

June Chunqiu Hou ◽

Mark Stamnes ◽

Alan R. Saltiel ◽

...

Keyword(s):

Protein Trafficking ◽

Actin Polymerization ◽

Coat Proteins ◽

Filamentous Actin ◽

Cortical Actin ◽

Amino Terminal ◽

Gtp Binding ◽

Subcellular Compartmentalization ◽

Constitutively Active

TC10 is a member of the Rho family of small GTP-binding proteins that has previously been implicated in the regulation of insulin-stimulated GLUT4 translocation in adipocytes. In a manner similar to Cdc42-stimulated actin-based motility, we have observed that constitutively active TC10 (TC10/Q75L) can induce actin comet tails in Xenopus oocyte extracts in vitro and extensive actin polymerization in the perinuclear region when expressed in 3T3L1 adipocytes. In contrast, expression of TC10/Q75L completely disrupted adipocyte cortical actin, which was specific for TC10, because expression of constitutively active Cdc42 was without effect. The effect of TC10/Q75L to disrupt cortical actin was abrogated after deletion of the amino terminal extension (ΔN-TC10/Q75L), whereas this deletion retained the ability to induce perinuclear actin polymerization. In addition, alteration of perinuclear actin by expression of TC10/Q75L, a dominant-interfering TC10/T31N mutant or a mutant N-WASP protein (N-WASP/ΔVCA) reduced the rate of VSV G protein trafficking to the plasma membrane. Furthermore, TC10 directly bound to Golgi COPI coat proteins through a dilysine motif in the carboxyl terminal domain consistent with a role for TC10 regulating actin polymerization on membrane transport vesicles. Together, these data demonstrate that TC10 can differentially regulate two types of filamentous actin in adipocytes dependent on distinct functional domains and its subcellular compartmentalization.

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Regulation of CD18 expression in human neutrophils as related to shape changes

Journal of Cell Science ◽

10.1242/jcs.106.2.493 ◽

1993 ◽

Vol 106 (2) ◽

pp. 493-501

Author(s):

A. Volz

Keyword(s):

Phorbol Myristate Acetate ◽

Surface Expression ◽

Cytochalasin D ◽

Human Neutrophils ◽

Chemotactic Peptide ◽

Myristate Acetate ◽

Surface Distribution ◽

Quantitative Expression ◽

High Concentrations ◽

Shape Changes

The study analyses the distribution and quantitative expression of surface CD18 of neutrophils exposed to distinct stimuli that produce different types of continuous shape changes, including types that are associated with locomotion and others that are not. The chemotactic peptide N-formyl-L-norleucyl-L-leucyl-L-phenylalanine, colchicine and nocodazole were used to induce a polarized locomotor morphology, phorbol myristate acetate, 1,2-dioctanoylglycerol and 1-oleoyl-2-acetyl-glycerol to induce non-polar motile cells ruffling all over the surface and 2H2O to induce non-polar cells performing circus movements as have been previously described. Except for colchicine and nocodazole, these stimuli increased surface expression of CD18. Thus, stimulated shape changes are frequently, though not always, associated with increased surface expression of CD18. High concentrations (10(−7) to 10(−5) M) of phorbol myristate acetate but not of chemotactic peptide induced down-regulation of surface CD18. Cytochalasin D (10(−4) M) stimulated CD18 expression even though it inhibited shape changes. The surface distribution of CD18 determined by light microscopy was uniform in unstimulated cells or in various forms of stimulation except for cells treated with 10(−5) M cytochalasin D. Cytochalasin D (10(−5) M) produced CD18 accumulation at the pole opposite the F-actin cap. Experiments with colchicine, nocodazole, 2H2O and cytochalasin D suggest that microtubules as well as microfilaments modulate surface expression of CD18. The results suggest that protein kinase C and phosphatases play a role in regulating surface expression of CD18 in neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)

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Coxiella burnetii Induces Reorganization of the Actin Cytoskeleton in Human Monocytes

Infection and Immunity ◽

10.1128/iai.66.11.5527-5533.1998 ◽

1998 ◽

Vol 66 (11) ◽

pp. 5527-5533 ◽

Cited By ~ 40

Author(s):

Sonia Meconi ◽

Véronique Jacomo ◽

Patrice Boquet ◽

Didier Raoult ◽

Jean-Louis Mege ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Coxiella Burnetii ◽

Actin Polymerization ◽

Q Fever ◽

Morphological Changes ◽

Critical Role ◽

Filamentous Actin ◽

Cytoskeleton Reorganization ◽

Membrane Protrusions ◽

Actin Protrusions

ABSTRACT Coxiella burnetii, an obligate intracellular bacterium which survives in myeloid cells, causes Q fever in humans. We previously demonstrated that virulent C. burnetiiorganisms are poorly internalized by monocytes compared to avirulent variants. We hypothesized that a differential mobilization of the actin cytoskeleton may account for this distinct phagocytic behavior. Scanning electron microscopy demonstrated that virulent C. burnetii stimulated profound and polymorphic changes in the morphology of THP-1 monocytes, consisting of membrane protrusions and polarized projections. These changes were transient, requiring 5 min to reach their maximum extent and vanishing after 60 min of incubation. In contrast, avirulent variants of C. burnetii did not induce any significant changes in cell morphology. The distribution of filamentous actin (F-actin) was then studied with a specific probe, bodipy phallacidin. Virulent C. burnetii induced a profound and transient reorganization of F-actin, accompanied by an increase in the F-actin content of THP-1 cells. F-actin was colocalized with myosin in cell protrusions, suggesting that actin polymerization and the tension of actin-myosin filaments play a role in C. burnetii-induced morphological changes. In addition, contact between the cell and the bacterium seems to be necessary to induce cytoskeleton reorganization. Bacterial supernatants did not stimulate actin remodeling, and virulent C. burnetii organisms were found in close apposition with F-actin protrusions. The manipulation of the actin cytoskeleton by C. burnetiimay therefore play a critical role in the internalization strategy of this bacterium.

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