Degranulation is one of the most important response of neutrophils in inflammation and bacterial killing. Membrane fusion, which is required for degranulation, is a process which is virtually ubiquitous, as it must be employed by any cell engaged in endocytosis or exocytosis. Yet, fusion has been difficult to model in the laboratory and is consequently poorly understood. Several recent advances, such as isolation of endogenous fusogens and development of contents-mixing fusion assays have permitted an investigation of membrane fusion and secretion by human neutrophils. Working Hypothesis--Both membrane and cytosolic (annexin and non-annexin) components regulate fusion in neutrophils. We propose to study membrane fusion in neutrophils using a variety of established and novel techniques, emphasizing thoroughness and rigor, and minimizing assumptions. 1. Continued Isolation of Endogenous Neutrophil Fusogens Using a """"""""first principles"""""""" approach, we have isolated one endogenous protein fusogen from neutrophil cytosol. At least three more fusogenic activities have been found that require purification and identification. This will permit us to determine the potential of the cytosol to support degranulation. 2. DEVELOPMENT OF EXPERIMENTAL SYSTEMS FOR MEASURING GRANULE-LIPOSOME AND GRANULE-MEMBRANE FUSION We propose to develop contents-mixing assays suitable for studying the fusion of neutrophil specific granules (SG). This is necessary since lipid-mixing assays currently used to study fusion ar fraught with experimental limitations. These studies will allow us to explore the potential of neutrophil membranes to fuse. 3. USE OF """"""""MODEL"""""""", RECONSTITUTED GRANULES TO DISSECT FUSION We have found that isolated neutrophil granules, in contrast to plasma membrane (PM) vesicles, are extremely resistant to fusion. We therefore propose to isolate membrane-associated components from SG and reconstitute them into liposomes, to study the involvement of granule membrane components in fusion or regulating fusion. In particular, we will look at both the lipid and protein components of SG. These studies will allow us to separately dissect and reconstruct the elements of the fusion apparatus. 4. RECONSTITUTION OF SECRETION IN PERMEABILIZED NEUTROPHIL SYSTEMS As an intermediate step towards applying our findings from Specific Aims 1-3 to intact cells, we will attempt to reconstitute secretion in permeabilized neutrophils. The permeabilized cell system will be used as a tool for 1) identifying potential fusion modulators for use in vitro studies; and 2) testing fusion modulators identified in vitro in a more physiologically relevant system. The development of these in vitro model systems and the isolation of endogenous members of the fusion regulator system will also help clarify the mechanisms of secretion in intact and permeabilized cells.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
Project #
Application #
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Michigan Ann Arbor
Schools of Medicine
Ann Arbor
United States
Zip Code
Smolen, J E; Hessler, R J; Nauseef, W M et al. (2001) Identification and cloning of the SNARE proteins VAMP-2 and syntaxin-4 from HL-60 cells and human neutrophils. Inflammation 25:255-65
Smolen, J E; Petersen, T K; Koch, C et al. (2000) L-selectin signaling of neutrophil adhesion and degranulation involves p38 mitogen-activated protein kinase. J Biol Chem 275:15876-84
Hessler, R J; Blackwood, R A; Brock, T G et al. (1998) Identification of glyceraldehyde-3-phosphate dehydrogenase as a Ca2+-dependent fusogen in human neutrophil cytosol. J Leukoc Biol 63:331-6
Blackwood, R A; Transue, A T; Harsh, D M et al. (1996) PLA2 promotes fusion between PMN-specific granules and complex liposomes. J Leukoc Biol 59:663-70
Blackwood, R A; Smolen, J E; Hessler, R J et al. (1996) Development of an aqueous-space mixing assay for fusion of granules and plasma membranes from human neutrophils. Biochem J 314 ( Pt 2):469-75
Francis, J W; Balazovich, K J; Smolen, J E et al. (1992) Human neutrophil annexin I promotes granule aggregation and modulates Ca(2+)-dependent membrane fusion. J Clin Invest 90:537-44
Smolen, J E; Stoehr, S J; Kuczynski, B (1991) Cyclic AMP inhibits secretion from electroporated human neutrophils. J Leukoc Biol 49:172-9
Smolen, J E; Stoehr, S J; Kuczynski, B et al. (1991) Dual effects of guanosine 5'-[gamma-thio]triphosphate on secretion by electroporated human neutrophils. Biochem J 279 ( Pt 3):657-64
Stoehr, S J; Smolen, J E; Suchard, S J (1990) Lipocortins are major substrates for protein kinase C in extracts of human neutrophils. J Immunol 144:3936-45
Stoehr, S J; Smolen, J E (1990) Human neutrophils contain a protein kinase C-like enzyme that utilizes guanosine triphosphate as a phosphate donor. Cofactor requirements, kinetics, and endogenous acceptor proteins. Blood 75:479-87

Showing the most recent 10 out of 12 publications