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Am J Physiol Cell Physiol 296: C857-C867, 2009. First published January 28, 2009; doi:10.1152/ajpcell.00454.2008
0363-6143/09 $8.00
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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON

Counterregulation of clathrin-mediated endocytosis by the actin and microtubular cytoskeleton in human neutrophils

Silvia M. Uriarte,1,* Neelakshi R. Jog,2,* Gregory C. Luerman,3 Samrath Bhimani,1 Richard A. Ward,1 and Kenneth R. McLeish1,3,4

Departments of 1Medicine, 2Microbiology and Immunology, and 3Biochemistry and Molecular Biology, University of Louisville, and 4Veterans Affairs Medical Center, Louisville, Kentucky

Submitted 2 September 2008 ; accepted in final form 24 January 2009

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.

exocytosis; signal transduction; intracellular granules


Address for reprint requests and other correspondence: S. M. Uriarte, Kidney Disease Program, Baxter I Research Bldg., 570 S. Preston St., Louisville, KY 40202 (e-mail: smuria01{at}louisville.edu)






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