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Am J Physiol Cell Physiol (January 3, 2007). doi:10.1152/ajpcell.00384.2006
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Submitted on July 12, 2006
Accepted on January 2, 2007

THE ACTIN CYTOSKELETON REGULATES EXOCYTOSIS OF ALL NEUTROPHIL GRANULE SUBSETS

Neelakshi R. Jog1, Madhavi J Rane, George Lominadze2, Gregory C. Luerman3, Richard A. Ward4, and Kenneth R. McLeish4*

1 Microbiology and Immunology, University of Louisville, Louisville, Kentucky, United States
2 Biochemistry and Molecular Biology, University of Louisville, Louisville, Kentucky, United States
3 Louisville, Kentucky, United States; Biochemistry and Molecular Biology, University of Louisville, Louisville, Kentucky, United States
4 Medicine, University of Louisville, Louisville, Kentucky, United States

* To whom correspondence should be addressed. E-mail: k.mcleish{at}louisville.edu.

A comprehensive analysis of the role of the actin cytoskeleton in exocytosis of the four different neutrophil granule subsets had not been performed previously. Immunoblot analysis showed that, compared to plasma membrane, there was less actin associated with secretory vesicles (SV) (75%), gelatinase granules (GG) (40%), specific granules (SG) (10%), and azurophil granules (AG) (5%). Exocytosis of SV, SG, and AG was measured as increased plasma membrane expression of CD35, CD66b, and CD63, respectively, using flow cytometry; and GG exocytosis was measured as gelatinase release using an ELISA. FMLP stimulated exocytosis of SV, GG, and SG with an ED50 of 15, 31, and 28 nM, respectively, with maximal response at 10-7 M FMLP by 5 min, while no exocytosis of AG was detected. Disruption of the actin cytoskeleton by latrunculin A and cytochalasin D induced a decrease in FMLP-stimulated CD35 expression after an initial increase. Both drugs enhanced the rate and extent of FMLP-stimulated GG, SG, and AG exocytosis, while the EC50 for FMLP was not altered. We conclude that the actin cytoskeleton controls access of neutrophil granules to the plasma membrane, thereby limiting the rate and extent of exocytosis of all granule subsets. Differential association of actin with the four granule subsets was not associated with graded exocytosis.




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