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Relation between membrane fluidity and signal transduction in the human megakaryoblastic cell line MEG-01
A Schootemeijer1, G Gorter, L G Tertoolen
1Department of Haematology, University Hospital Utrecht, The Netherlands.
Abstract:
The fluidity of the plasma membrane is thought to affect the responsiveness of blood platelets. We measured membrane fluidity in a single cell by Fluorescence Recovery after Photobleaching (FRAP) of the lipophilic probe DiIC14. Since platelets are too small for this technique, we used the human megakaryoblastic cell-line MEG-01, which shares many properties with platelets. MEG-01 cells were cultured for 44 h with simvastatin or mevalonate to change the cholesterol content, enabling analysis of signal processing at cholesterol/phospholipid ratios (C/P) between 0.20 and 0.31. The diffusion of DiIC14 correlated inversely with the C/P ratio with lateral diffusion coefficients (D) of 3.28 x 10(-9) cm2/s at a low C/P decreasing to 2.55 x 10(-9) cm2/s at a high C/P ratio. The mobile fraction was 65% and constant at the different C/P ratios. The relation between lipid diffusion and signal processing was measured following stimulation with 10 U/ml thrombin at 22 degrees C. There were only little differences in phosphatidylinositol metabolism, Ca2+ influx or mobilization and prostaglandin I2-induced formation of cyclic AMP. At 37 degrees C, cells with a high C/P ratio showed increased phosphatidylinositol metabolism, but these differences had no major effect on the Ca2+ responses. These data demonstrate that in megakaryoblasts the lateral diffusion of lipids is inversely correlated with the C/P ratio, but within the range of 0.20-0.31 the influence on signal processing is minor.
Insights
Plasma membrane fluidity, measured by Fluorescence Recovery after Photobleaching (FRAP), inversely correlates with cholesterol/phospholipid (C/P) ratio in megakaryoblasts. Changes in fluidity had minor effects on cellular signal processing.
Area of Science:
- Cell Biology
- Membrane Biophysics
- Biochemistry
Background:
- Plasma membrane fluidity influences cell signaling and responsiveness.
- Platelet function is critical in hemostasis and thrombosis.
- Cholesterol content significantly impacts membrane properties.
Purpose of the Study:
- To investigate the relationship between membrane fluidity and cholesterol/phospholipid ratio in megakaryoblasts.
- To determine how altered membrane fluidity affects signal processing in these cells.
- To understand the implications for platelet responsiveness.
Main Methods:
- Utilized Fluorescence Recovery after Photobleaching (FRAP) with DiIC14 probe on MEG-01 cells.
- Manipulated cellular cholesterol content using simvastatin and mevalonate.
- Analyzed lipid diffusion, mobile fraction, and signal transduction pathways (inositol phosphate metabolism, Ca2+ influx, cAMP formation).
Main Results:
- Lipid diffusion coefficient (D) decreased with increasing cholesterol/phospholipid (C/P) ratio (from 3.28 to 2.55 x 10(-9) cm2/s).
- Mobile fraction remained constant at 65% across varying C/P ratios.
- Signal processing showed minor alterations with changes in membrane fluidity, particularly at 37°C.
Conclusions:
- Lateral lipid diffusion in megakaryoblasts is inversely correlated with the C/P ratio.
- Within the studied C/P range (0.20-0.31), membrane fluidity has a limited impact on signal processing.
- Findings suggest that moderate changes in membrane cholesterol do not significantly impair megakaryoblast signal transduction.