Phorbol ester induces rapid actin assembly in neutrophil leucocytes independently of changes in [Ca2+]i and pHi

Sheterline, Peter; Je, Rickard; Boothroyd, B.; Rc, Richards

doi:10.1007/bf01753583

Cited by 50 publications

(18 citation statements)

References 37 publications

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“…The actin assembly induced by PMA is slower and more sustained that the actin polymerization response to chemoattractants and growth factors, and PMA, unlike these other stimuli, does not induce cell translocation (26,28,29). In platelets, PMA generates an irreversible shape change (18,30), centripetal contractions (31, 32) and cell-cell aggregation (30).…”

Section: Discussionmentioning

confidence: 99%

D3 Phosphoinositides and Outside-in integrin Signaling by Glycoprotein IIb-IIIa Mediate Platelet Actin Assembly and Filopodial Extension Induced by Phorbol 12-Myristate 13-Acetate

Hartwig

Kung

Kovacsovics

et al. 1996

Journal of Biological Chemistry

125

109

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Phorbol 12-myristate 13-acetate (PMA) uncaps a small number of the fast-growing (barbed) ends of actin filaments, thereby eliciting slow actin assembly and extension of filopodia in human blood platelets. These reactions, which also occur in response to immunologic perturbation of the integrin glycoprotein (GP) IIb-IIIa, are sensitive to the phosphoinositide 3-kinase inhibitor wortmannin. Platelets deficient in GPIIb-IIIa integrins or with GPIIb-IIIa function inhibited by calcium chelation or the peptide RGDS have diminished PMA responsiveness. The effects of PMA contrast with thrombin receptor stimulation by 5 M thrombin receptor-activating peptide (TRAP), which causes rapid and massive wortmannin-insensitive actin assembly and lamellar and filopodial extension. However, we show here that wortmannin can inhibit filopod formation if the thrombin receptor is ligated using suboptimal doses (<1 M) of TRAP. Phosphatidylinositol 3,4-bisphosphate inhibits actin filament severing and capping by human gelsolin in vitro. The findings implicate D3 polyphosphoinositides and integrin signaling in PMA-mediated platelet stimulation and implicate D3 containing phosphoinositides generated in response to protein kinase C activation and GPIIb-IIIa signaling as late-acting intermediates leading to filopodial actin assembly.Extracellular perturbations cause cells to change cell shape and translocate by remodeling diverse intracellular actin-based structures. The relative functional simplicity of these changes in the anucleate human blood platelet makes it a useful subject for working out the pathways leading to this remodeling. Of particular utility is the fact that actin remodeling events in the platelet predominantly take place sequentially in time rather than the actin rearrangements that occur simultaneously in space during cell locomotion.The resting platelet is a rigid disc stabilized by an actin filament gel that links to a submembrane skeleton and to transmembrane proteins (1, 2). Over 98% of the actin filaments in resting platelets have their fast growing ("barbed" as defined by myosin head fragment binding) ends stabilized by specific barbed-end capping proteins. In response to thrombin, a calcium transient in the platelet activates gelsolin, which then severs actin filaments in the periphery of the actin filament gel and caps the newly formed barbed ends of the severed filaments (3). Subsequently, phosphoinositides accumulate and inactivate gelsolin as well as another abundant barbed end binding factor, capping protein. Phosphoinositide-mediated uncapping of at least 25% of the filament barbed ends eventuates in massive actin assembly as micromolar quantities of monomeric subunits, previously prevented from spontaneous nucleation by sequestering proteins, add onto the uncapped filament barbed ends (4 -6).The actin assembly following thrombin-induced platelet activation mediates the appearance of two types of surface protrusions, ruffling lamellae that spread circumferentially and threadlike filopodial protrusions. The fi...

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Section: Discussionmentioning

confidence: 99%

D3 Phosphoinositides and Outside-in integrin Signaling by Glycoprotein IIb-IIIa Mediate Platelet Actin Assembly and Filopodial Extension Induced by Phorbol 12-Myristate 13-Acetate

Hartwig

Kung

Kovacsovics

et al. 1996

Journal of Biological Chemistry

125

109

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“…In neutrophils activated by stimuli such as the chemotactic peptide fMLP or the phorbol ester PMA, increases in F-actin are accompanied by spatial reorganization of the actin cytoskeleton (Downey et al, 1992;Howard and Wang, 1987;Roos et al, 1987Roos et al, , 1989Sheterline et al, 1986). The aim of the current section was to determine if the increase in amount of F-actin induced by okadaic acid was similarly accompanied by morphological alterations in the spatial arrangement of the actin cytoskeleton.…”

Section: Okadaic Acid Alters Cell Morphology and Spatial Arrangement mentioning

confidence: 93%

Okadaic acid‐induced actin assembly in neutrophils: Role of protein phosphatases

Downey

Takai

Zamel

et al. 1993

Journal Cellular Physiology

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Activation of neutrophils results in morphological and functional alterations including changes in cell shape and initiation of motile bchavior that depend on assembly and reorganization ot the actin cytoskeleton. Phosphoproteins are thought to be key intermediates in the regulation of cytoskeletal alterations and whereas much attention has been directed at the role of protein kinases, relatively little information is available on the importance of phosphatases. To elucidate the role of protein phosphatases, we studied thc effects of the phosphatase inhibitors okadaic acid and calyculin A on the actin cytoskeleton of human neutrophils. Exposure of cells to okadaic acid resulted in assembly and spatial redistribution of actin, which peaked at 25 min and returned to baseline levels by 45 min, as assessed by flow cytornetric analysis of NBD-phallacidin stained cells and confocal fluorescence microscopy, rcspec-tively. These effects correlated with an increase in protein phosphorylation, determined by incorporation of "P into cellular proteins using SDS-PAGE arid autoradiography. Similar but more rapid responses were observed in electropermeabilized cells treated with okadaic acid or calyculin A. The dose dependence of these effects was compatible with a role for phosphatase type 1 as the target enzyme. These findings also suggested the presence of constitutively active protein kinases capable of effecting actin polymerization. Phosphorylation of myosin light chain IMLC) has been postulated to promote actin assembly, but myosin light chain kinase (MLCK) appeared not to be involved because: (1) the effect of okadaic acid was not inhibited by the MLCK inhibitor KT5926 and ( 2 ) in permeabilized cells suspended in medium with free calcium [Ca"l < 10 n M (conditions under which MLCK is inactive), the effect of okadaic acid pcrsisted. The role of phosphatases in stimulus-induced actin assembly was assessed in cells preincubated with okadaic acid for 45 rnin, after F-actin levels had returned to baseline. Under these conditions, okadaic acid completely abrogated actin assembly induced by phorbol niyristate acetate, platelet activating factor, and leukotriene B,, whereas the effects of the chemotactic peptide fMLP and opsonized zymosan (OpZ) were unaffected. We conclude that swine and threonine phosphatases exert a tonic negative influence on actin assembly and organization. Furthermore, divergent pathways seem to mediate the response to lipidic stimuli, on one hand, and fMLP and OpZ, on the other, as evidenced by the difterential susceptibility to inhibition by okadaic acid. N e u t r o p h i l leukocytes are highly m o t i l e cells, a prope r t y w e l l suited t o t h e i r role as i t i n e r a n t phagocytes in i n f l a m m a t i o n . Locomotion, as w e l l as other functions crucial t o t h e process o f cell activation in response t o i n f l a m m a t o r y stimuli, i n c l u d i n g r e g u l a t i o n of cell shape, phagocytosis, and projection o f membrane ruffles and pseudopodia, are dependent o n t h e highl...

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“…One pathway that promotes migration is mediated by protein kinase C (PKC). PKC is a serine-threonine kinase involved in many cytoskeletal-dependent processes of neutrophils, including actin assembly (8)(9)(10)(11), degranulation (12), oxidative burst (13)(14)(15), adhesion (16,17), and migration (17)(18)(19)(20). Neutrophils contain a range of PKC isoforms, including the classical Ca 21 sensitive PKCa and PKCb, the novel Ca 21 -independent PKCd, and the atypical Ca 21 -and diacylglycerol-independent PKCz (21).…”

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confidence: 99%

Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) Protein Regulation of Human Neutrophil Migration

Eckert

Neuder²,

Park³

et al. 2010

Am J Respir Cell Mol Biol

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Neutrophil migration into infected tissues is essential for host defense, but products of activated neutrophils can be quite damaging to host cells. Neutrophil influx into the lung and airways and resultant inflammation characterizes diseases such as chronic obstructive pulmonary disease, bronchiectasis, and cystic fibrosis. To migrate, neutrophils must reorganize the actin cytoskeleton to establish a leading edge pseudopod and a trailing edge uropod. The actin-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) has been shown to bind and cross-link actin in a variety of cell types and to co-localize with F-actin in the leading edge lamellipodium of migrating fibroblasts. The hypothesis that MARCKS has a role in the regulation of neutrophil migration was tested using a cell-permeant peptide derived from the MARCKS myristoylated aminoterminus (MANS peptide). Treatment of isolated human neutrophils with MANS significantly inhibited both their migration and b2 integrin-dependent adhesion in response to N-formyl-methionyl-leucyl-phenylalanine (fMLF), IL-8, or leukotriene (LT)B 4 . The IC 50 for fMLF-induced migration and adhesion was 17.1 mM and 12.5 mM, respectively. MANS significantly reduced the F-actin content in neutrophils 30 seconds after fMLF stimulation, although the peptide did not alter the ability of cells to polarize or spread. MANS did not alter fMLF-induced increases in surface b2 integrin expression. These results suggest that MARCKS, via its myristoylated aminoterminus, is a key regulator of neutrophil migration and adhesion.

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Phorbol ester induces rapid actin assembly in neutrophil leucocytes independently of changes in [Ca2+]i and pHi

Cited by 50 publications

References 37 publications

D3 Phosphoinositides and Outside-in integrin Signaling by Glycoprotein IIb-IIIa Mediate Platelet Actin Assembly and Filopodial Extension Induced by Phorbol 12-Myristate 13-Acetate

D3 Phosphoinositides and Outside-in integrin Signaling by Glycoprotein IIb-IIIa Mediate Platelet Actin Assembly and Filopodial Extension Induced by Phorbol 12-Myristate 13-Acetate

Okadaic acid‐induced actin assembly in neutrophils: Role of protein phosphatases

Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) Protein Regulation of Human Neutrophil Migration

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