title: Counterregulation of clathrin-mediated endocytosis by the actin and microtubular cytoskeleton in human neutrophils.
abstract: We have recently reported that disruption of the actin cytoskeleton enhanced N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated granule exocytosis in human neutrophils but decreased plasma membrane expression of complement receptor 1 (CR1), a marker of secretory vesicles. The present study was initiated to determine if reduced CR1 expression was due to fMLP-stimulated endocytosis, to determine the mechanism of this endocytosis, and to examine its impact on neutrophil functional responses. Stimulation of neutrophils with fMLP or ionomycin in the presence of latrunculin A resulted in the uptake of Alexa fluor 488-labeled albumin and transferrin and reduced plasma membrane expression of CR1. These effects were prevented by preincubation of the cells with sucrose, chlorpromazine, or monodansylcadaverine (MDC), inhibitors of clathrin-mediated endocytosis. Sucrose, chlorpromazine, and MDC also significantly inhibited fMLP- and ionomycin-stimulated specific and azurophil granule exocytosis. Disruption of microtubules with nocodazole inhibited endocytosis and azurophil granule exocytosis stimulated by fMLP in the presence of latrunculin A. Pharmacological inhibition of phosphatidylinositol 3-kinase, ERK1/2, and PKC significantly reduced fMLP-stimulated transferrin uptake in the presence of latrunculin A. Blockade of clathrin-mediated endocytosis had no significant effect on fMLP-stimulated phosphorylation of ERK1/2 in neutrophils pretreated with latrunculin A. From these data, we conclude that the actin cytoskeleton functions to limit microtubule-dependent, clathrin-mediated endocytosis in stimulated human neutrophils. The limitation of clathrin-mediated endocytosis by actin regulates the extent of both specific and azurophilic granule exocytosis.
authors.affiliation: Department of Medicine, University of Louisville, Louisville, KY, USA. smuria01@louisville.edu
authors.forename: Silvia M
authors.initials: SM
authors.lastname: Uriarte
authors.forename: Neelakshi R
authors.initials: NR
authors.lastname: Jog
authors.forename: Gregory C
authors.initials: GC
authors.lastname: Luerman
authors.forename: Samrath
authors.initials: S
authors.lastname: Bhimani
authors.forename: Richard A
authors.initials: RA
authors.lastname: Ward
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: McLeish
mesh_terms: D000199:Actins / Q000378:metabolism*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002103:Cadaverine / Q000031:analogs & derivatives / Q000494:pharmacology; D002746:Chlorpromazine / Q000494:pharmacology; D002966:Clathrin / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects; D005089:Exocytosis; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D008870:Microtubules / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D018375:Neutrophil Activation / Q000187:drug effects; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017463:Receptors, Complement 3b / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013395:Sucrose / Q000494:pharmacology; D053778:Thiazolidines / Q000494:pharmacology; D014168:Transferrin / Q000378:metabolism; D050257:Tubulin Modulators / Q000494:pharmacology
chemical_list: D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002103:Cadaverine; D002746:Chlorpromazine; D002966:Clathrin; D015759:Ionomycin; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D017463:Receptors, Complement 3b; D012710:Serum Albumin, Bovine; D013395:Sucrose; D053778:Thiazolidines; D014168:Transferrin; D050257:Tubulin Modulators; C509991:CR1 protein, human; C008542:monodansylcadaverine; C037067:latrunculin A