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Many cell types undergo a hypoxic response in the presence of low oxygen, which can lead to transcriptional, metabolic, and structural changes within the cell. Many biophysical studies to probe the localization and dynamics of single fluorescently labeled molecules in live cells either require or benefit from low-oxygen conditions. In this study, we examine how low-oxygen conditions alter the mobility of a series of plasma membrane proteins with a range of anchoring motifs in HeLa cells at 37°C. Under high-oxygen conditions, diffusion of all proteins is heterogeneous and confined. When oxygen is reduced with an enzymatic oxygen-scavenging system for ≥15 min, diffusion rates increase by >2-fold, motion becomes unconfined on the timescales and distance scales investigated, and distributions of diffusion coefficients are remarkably consistent with those expected from Brownian motion. More subtle changes in protein mobility are observed in several other laboratory cell lines examined under both high- and low-oxygen conditions. Morphological changes and actin remodeling are observed in HeLa cells placed in a low-oxygen environment for 30 min, but changes are less apparent in the other cell types investigated. This suggests that changes in actin structure are responsible for increased diffusion in hypoxic HeLa cells, although superresolution localization measurements in chemically fixed cells indicate that membrane proteins do not colocalize with F-actin under either experimental condition. These studies emphasize the importance of controls in single-molecule imaging measurements, and indicate that acute response to low oxygen in HeLa cells leads to dramatic changes in plasma membrane structure. It is possible that these changes are either a cause or consequence of phenotypic changes in solid tumor cells associated with increased drug resistance and malignancy.

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.

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. Individual vesicles have well-defined critical temperatures yet there is significant vesicle to vesicle variation even when GPMVs are isolated from a plate of seemingly identical cells. In this study, we explore if heterogeneity in critical temperatures arise, at least in part, from cells being at different stages of the cell cycle. Populations of cells were 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 were elevated in GPMVs isolated from cells in cell cycle phases that precede cell division (G2 and M) compared to other stages (G1 and S). In unsynchronized cells, critical temperatures were found to be inversely proportional to cell density, suggesting that contact inhibition and associated arrest of cell growth results in lower plasma membrane critical temperatures. Lower critical temperatures were also observed when growth was arrested through overnight serum starvation, and elevated critical temperatures were restored 24h after the addition of serum containing medium. Transition temperatures are also lowered in GPMVs prepared from cells undergoing apoptosis through the application of TRAIL, and vesicles contain a more rigid liquid-ordered or gel phase at low temperature. These results are in agreement with past studies that have indicated that plasma membrane composition varies within the cell cycle. Since GPMV critical temperatures are hypothesized to reflect on the magnitude of lipid-mediated heterogeneity in intact cells, these results suggest that membrane heterogeneity is greatest in cells undergoing rapid growth and cell division and is suppressed in cells under low growth conditions.

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 seemingly identical cells even though each individual vesicle has a well defined critical temperature. In addition, we observe that the average transition temperature of GPMVs isolated from a population of cells is inversely proportional to the surface density of cells growing in the culture dish. Since it is known that cellular doubling times are reduced in more densely plated cells due to contact inhibition, we hypothesized that critical temperatures are linked to the cell cycle. To test this hypothesis, we isolated GPMVs from populations of cells synchronized using a double Thymidine block that arrests cells at the border between G1 and S phases. After Thymidine is removed from the culture media, cells proceed synchronously through one cell cycle and GPMVs are isolated at time-points corresponding to S, G2, M, and G1 phases. We find that critical temperatures are significantly elevated in the cell cycle phases that immediately preceding cell division (G2 and M) compared to the remainder of the cell cycle phases (G1 and S), consistent with our observations of higher overall transition temperatures in more rapidly dividing cells. We speculate that membrane heterogeneity arising from this critical point may play a role in cytokinesis or other processes vital for cell division. If this hypothesis is correct, it may suggest that biochemical perturbations that lower critical temperatures in GPMVs can be used to inhibit cellular proliferation, possibly providing a novel treatment strategy for some cancers.

Diverse molecules induce general anesthesia with potency strongly correlated with both their hydrophobicity and their effects on certain ion channels. We recently observed that several n-alcohol anesthetics inhibit heterogeneity in plasma-membrane-derived vesicles by lowering the critical temperature (Tc) for phase separation. Here, we exploit conditions that stabilize membrane heterogeneity to further test the correlation between the anesthetic potency of n-alcohols and effects on Tc. First, we show that hexadecanol acts oppositely to n-alcohol anesthetics on membrane mixing and antagonizes ethanol-induced anesthesia in a tadpole behavioral assay. Second, we show that two previously described “intoxication reversers” raise Tc and counter ethanol’s effects in vesicles, mimicking the findings of previous electrophysiological and behavioral measurements. Third, we find that elevated hydrostatic pressure, long known to reverse anesthesia, also raises Tc in vesicles with a magnitude that counters the effect of butanol at relevant concentrations and pressures. Taken together, these results demonstrate that ΔTc predicts anesthetic potency for n-alcohols better than hydrophobicity in a range of contexts, supporting a mechanistic role for membrane heterogeneity in general anesthesia.