Cell Cycle Phase Determines Critical Temperature in Plasma Membrane Vesicles

Abstract

Giant plasma membrane vesicles (GPMVs) isolated from RBL-2H3 cells appear uniform at physiological temperatures, contain coexisting liquid-ordered and liquid-disordered phases at low temperatures, and experience micron-sized critical fluctuations close to their critical temperature. We observe a broad distribution of critical temperatures in GPMVs isolated from a dish of cells even though individual vesicles have a well-defined critical temperature. Interestingly, we find that densely plated cells yield GPMVs with lower average critical temperatures than GPMVs from less densely plated cells. Since it is known that cellular doubling times are reduced in cells plated at high density due to contact inhibition, we hypothesized that critical temperatures are linked to the cell cycle. To test this hypothesis, we isolated GPMVs from cells at various stages in their cell cycle. We plated cells in low serum media to produce a population of cells with arrested growth, and found low critical temperatures in GPMVs isolated from these cells (∼10°C). Critical temperatures recovered to typical values (∼20°C) after incubating the growth-arrested cells in serum rich media for 24h. Populations of cells are synchronized at S, G2, M, and G1 stages using a double Thymidine block that arrests cells at the border between G1 and S phases. Critical temperatures are elevated in GPMVs from populations of cells in cell cycle phases that immediately precede cell division (G2 and M) compared to the other stages (G1 and S). These results suggest that the magnitude of plasma membrane heterogeneity may be dependent on the cell cycle. We are currently investigating how position in the cell cycle influences the organization of plasma membrane proteins using quantitative super-resolution microscopy with multiple colors. We are also investigating cell cycle position's impact on outcomes of functional processes known to depend on lipid heterogeneity.