Cell Membrane Perturbation and Micro-Confinement Induced Variation in Giant Plasma Membrane Vesicles

Giant Plasma Membrane Vesicles(GPMVs)are cell-membrane model systems;characteristically they are fluid filled irreversible membrane blebs.Thus they are used to decipher the membrane dynamics.In this study we generated GPMVs with the help of chemical vesiculants that induce irreversible blebs from four different cell lines namely;HaCaT (immortalized human keratinocytes),Hela(cervical adenocarcinoma),HT-29(colon cancer)and MG-63 (osteosarcoma).The experiments were carried out both under macro conditions and micro conditions.Different cell lines showed distinct patterns in vesicle generation in both macro and micro conditions.MG-63 cells were taken up for further studies to identify the role of cytoskeletal reorganization in vesicle generation.In order to analyze this;Cytochalasin-D,an actin polymerization blocker and Methyl-ß-cyclodextrin(MßCD),a cholesterol depleting agent were used to disrupt the lipid raft arrangement.Using fluorescence microscopy,the growth kinetics of the vesicles was studied with the help of lipophilic dyes such as DiI and DiO.It was observed that highest number of vesicles and vesicles with largest diameter were obtained when MG-63 cells were treated with MßCD.DPH-Anisotropy was calculated to identify the membrane fluidity and it was found that MßCD treated cells were exhibiting higher membrane rigidity due to the absence of cholesterol.In order to assess the phase partitioning of the plasma membrane,confocal microscopy was done to observe the lipid ordered state(Lo)and the lipid disordered state (Ld).Lipid profiling was done after lipid extraction and extracts were used further for FTIR spectroscopy,phosphorous assay,and cholesterol estimation.Upon optimizing the culture conditions for GPMV synthesis the cells were grown in microfluidic cell culture platforms...