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Increased membrane heterogeneity in stimulated human granulocytes
M Valentino1, M Governa, E Gratton
1Occupational Medicine Institute, Faculty of Medicine, University of Ancona, Italy.
Abstract:
TMA-DPH fluorescence decay in human PMN before and after stimulation with FMLP was studied using frequency domain fluorometry. Membrane heterogeneity was assessed by the width of the continuous distributions of lifetime values of Lorentzian shape used to describe the fluorescence decay. In non-stimulated granulocytes TMA-DPH fluorescence decay is characterized by two distributions of lifetime values centered at 6.5 and 1.0 ns and full width at half maximum of 0.3 and 1.2 ns, respectively. Within 15 min after stimulation, the center values of the two distribution components were 5.1 and 0.8 ns and the distribution width was 0.8 and 0.6 ns, respectively. These results indicate changes of membrane domain organization which can be ascribed to compositional changes and redistribution of membrane components.
Insights
Stimulating human neutrophils with FMLP alters their cell membrane organization. Frequency domain fluorometry reveals changes in membrane domain heterogeneity after FMLP stimulation, indicating component redistribution.
Area of Science:
- Cellular Biology
- Biophysics
- Membrane Biophysics
Background:
- Neutrophil activation involves dynamic changes in cell membrane organization.
- Understanding membrane heterogeneity is crucial for elucidating cellular signaling pathways.
Purpose of the Study:
- To investigate the effects of formyl-methionyl-leucyl-phenylalanine (FMLP) stimulation on the membrane heterogeneity of human neutrophils (PMN).
- To assess changes in membrane domain organization using fluorescence decay analysis.
Main Methods:
- Utilized frequency domain fluorometry to measure the fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene-3-trimethylammonium (TMA-DPH) in human PMN.
- Analyzed membrane heterogeneity by characterizing the width of continuous lifetime distributions of Lorentzian shape.
Main Results:
- Non-stimulated PMN exhibited two distinct TMA-DPH fluorescence lifetime distributions centered at 6.5 ns and 1.0 ns.
- Following FMLP stimulation, the lifetime distributions shifted to 5.1 ns and 0.8 ns, with altered distribution widths (0.8 ns and 0.6 ns).
- These changes indicate significant alterations in membrane domain organization and component distribution.
Conclusions:
- FMLP stimulation induces rapid and measurable changes in human neutrophil membrane organization.
- The observed alterations in membrane heterogeneity suggest compositional changes and redistribution of membrane components upon activation.
- Frequency domain fluorometry is a valuable tool for probing dynamic changes in cell membrane structure.