Understanding the Role of Cytoskeleton and Membrane Tension in Membrane Rupture and Repair

Sengupta, Titas (2014) Understanding the Role of Cytoskeleton and Membrane Tension in Membrane Rupture and Repair. Masters thesis, Indian Institute of Science Education and Research Kolkata.

PDF (MS dissertation of Titas Sengupta) Titas_Sengupta_09MS023.pdf - Submitted Version Restricted to Repository staff only Download (1MB)

Abstract

Membranes of animal cells (some of which inhabit mechanically stressful environments) are highly susceptible to membrane rupture and also undergo membrane repair. Through separate events the mechanism of repair is closely related to the diameter of the lesion formed by rupture. In order to understand the factors determining rupture size, we have in this thesis explored assays to impart mechanical stress on cells – by hypo-osmotic shocks and studied the role of cytoskeleton integrity and active processes – in both re-enforcing the cell membrane to prevent rupture as well as their role in determining the size of membrane rupture. Certain cells of the kidney are exposed to sharp osmolarity changes and as they start swelling on hypo-osmotic shock, volume and membrane tension regulatory mechanisms come into play. As a first step towards understanding the crosstalk between these two regulatory mechanisms and their role in maintaining membrane integrity, a standard laboratory cell line (CHO) is subjected to treatments which compromise the cell of its volume and membrane tension regulatory capacity, causing a certain proportion of the cells to rupture. Our experiments aim to characterize the rupture process, specifically to quantify the diameter of the lesion formed, and to draw possible connections between a cell’s ability to regulate its volume and maintain the integrity of its membrane. Cells under normal conditions as well as those treated with cytoskeleton disrupting agents, ATP depleting agents, or both, are exposed to hypo-osmotic shock at physiological (370C) and cold temperatures (40C). Rupture properties (size, propensity, etc.) and fold changes in volume are studied under all conditions. Rupture propensities are found to increase with the number and variety of treatments compared to controls. Average rupture size (diameter of lesion) is not significantly different across treatments as it presumably depends on bilayer properties. Volume regulation seems to be similar across treatments and between cells subjected to the same treatment. Our results seem to suggest that the treatments render different parts of the cell membrane differently susceptible to rupture and although the stress imposed by hypo-osmotic shock acts uniformly on all cells, certain cells rupture while others do not under the same conditions. Future investigations using reflection interference contrast microscopy will aim to characterize local heterogeneities in the membrane that result in varied rupture propensities under a global stress mechanism.

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