Experimental evidence for the limiting role of enzymatic reactions in chemoattractant-induced pseudopod extension in human neutrophils.
Chemoattractant-stimulated pseudopod growth in human neutrophils was used as a model system to study the rate-limiting mechanism of cytoskeleton rearrangement induced by activated G-protein-coupled receptors. Cells were activated with N-formyl-Met-Leu-Phe, and the temperature dependence of the rate of pseudopod extension was measured in the presence of pharmacological inhibitors with known mechanisms of action. Three groups of inhibitors were used: (i) inhibitors sequestering substrates involved in F-actin polymerization (latrunculin A for G-actin and cytochalasin D for actin filament-free barbed ends) or sequestering secondary messengers (PIP-binding peptide for phosphoinositide lipids); (ii) competitively binding inhibitors (Akt-inhibitor for Akt/protein kinase B); and (iii) inhibitors that reduce enzyme activity (wortmannin for phosphoinositide 3-kinase and chelerythrine for protein kinase C). The experimental data are consistent with a model in which the relative involvement of a given pathway of F-actin polymerization to the measured rate of pseudopod extension is limited by a slowest (bottleneck) reaction in the cascade of reactions involved in the overall signaling pathway. The approach we developed was used to demonstrate that chemoattractant-induced pseudopod growth and mechanically stimulated cytoskeleton rearrangement are controlled by distinct pathways of F-actin polymerization.
Chodniewicz
David
D
Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC 27708-0300, USA.
Alteraifi
Abdullatif M
AM
Zhelev
Doncho V
DV
D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000470:Alkaloids; D000730:Androstadienes / Q000494:pharmacology; D053119:Benzophenanthridines; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000737:chemistry; D015638:Cytochalasin D / Q000737:chemistry; D003599:Cytoskeleton / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004798:Enzymes / Q000737:chemistry* / Q000502:physiology; D006801:Humans; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D008956:Models, Chemical; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D010617:Phenanthridines / Q000737:chemistry; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011485:Protein Binding; D011493:Protein Kinase C / Q000737:chemistry; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D013696:Temperature; D013844:Thiazoles / Q000737:chemistry; D053778:Thiazolidines; D013997:Time Factors; D000077191:Wortmannin
D000199:Actins; D000470:Alkaloids; D000730:Androstadienes; D053119:Benzophenanthridines; D019086:Bridged Bicyclo Compounds, Heterocyclic; D004791:Enzyme Inhibitors; D004798:Enzymes; D008055:Lipids; D010455:Peptides; D010617:Phenanthridines; D043562:Receptors, G-Protein-Coupled; D013844:Thiazoles; D053778:Thiazolidines; D015638:Cytochalasin D; D009240:N-Formylmethionine Leucyl-Phenylalanine; C016299:chelerythrine; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; C037067:latrunculin A; D000077191:Wortmannin