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Small unilamellar vesicles are able to fuse with Mycoplasma capricolum cells
M Salman1, M Tarshis, S Rottem
1Department of Membrane and Ultrastructure Research, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Abstract:
We have investigated the fusion characteristics of intact Mycoplasma capricolum cells and small unilamellar vesicles (SUV). The rate and extent of fusion was monitored continuously by octadecylrhodamine B (R18) fluorescence dequenching assay, as well as by intracellular contents mixing, and by sucrose density gradient analysis. The fusion of SUV with M. capricolum cells was found to be dependent on poly(ethylene glycol) (PEG 8000), divalent cations in the medium, and on the cholesterol content of the lipid vesicles. Maximal levels of fusion were obtained with SUV containing 40 mol% cholesterol in the presence of 5% PEG. The rate and extent of fusion were affected by temperature, pH, osmotic pressure, and SUV/mycoplasma ratio. Under optimal fusion conditions, PEG did not increase the rate of exchange of either cholesterol or phospholipids between M. capricolum cells and SUV. Throughout the fusion process, M. capricolum cells remained intact as measured by the retention of [3H]thymidine-labeled components, and viable. M. capricolum cells were rendered nonfusogenic by treatment with glutaraldehyde (greater than 0.01%) or chlorpromazine (greater than 10 microM). Fusion was partially inhibited by treating the cells with the uncoupler CCCP (5 microM) or proteolytic enzymes, suggesting that a proton gradient across the cell membrane is required for the fusion, and that the cells possess proteinase-sensitive receptors that are responsible for a tighter contact with the lipid vesicles.
Insights
We studied how Mycoplasma capricolum cells fuse with lipid vesicles. Fusion efficiency depends on cholesterol content, divalent cations, and poly(ethylene glycol), with cells remaining intact and viable throughout the process.
Area of Science:
- Membrane biophysics
- Cell biology
- Mycoplasma research
Background:
- Understanding cell fusion is crucial for drug delivery and studying cellular processes.
- Mycoplasma capricolum offers a model system for investigating membrane fusion mechanisms.
- Small unilamellar vesicles (SUV) are widely used to mimic cellular membranes.
Purpose of the Study:
- To investigate the fusion characteristics of Mycoplasma capricolum cells and SUV.
- To identify factors influencing the rate and extent of cell-vesicle fusion.
- To determine the role of cell integrity and specific cellular components in the fusion process.
Main Methods:
- Octadecylrhodamine B (R18) fluorescence dequenching assay for monitoring fusion.
- Intracellular contents mixing assays to confirm fusion events.
- Sucrose density gradient analysis for separating fused and unfused components.
- Treatment of cells with chemical agents (glutaraldehyde, chlorpromazine, CCCP) and enzymes to assess fusogenicity and identify key factors.
Main Results:
- Fusion is dependent on poly(ethylene glycol) (PEG 8000), divalent cations, and SUV cholesterol content (optimal at 40 mol%).
- Fusion rate and extent are influenced by temperature, pH, osmotic pressure, and SUV/mycoplasma ratio.
- Mycoplasma capricolum cells maintain integrity and viability during fusion; PEG does not enhance lipid exchange.
- Cell treatments with glutaraldehyde or chlorpromazine render cells nonfusogenic; CCCP and proteolytic enzymes partially inhibit fusion.
Conclusions:
- Mycoplasma capricolum cell fusion with SUV is a complex process modulated by lipid composition and environmental factors.
- A proton gradient and proteinase-sensitive receptors on the cell surface are likely involved in mediating efficient fusion.
- The study provides insights into the mechanisms of microbial cell fusion and potential targets for modulating this process.