Fig 1.
GPMVs from RBL-2H3 exhibit a broad distribution of miscibility transition temperatures.
(A) A single field of GPMVs imaged at several distinct temperatures. Vesicles identified as containing coexisting liquid phases are marked with a yellow triangle. In this field, some vesicles contain coexisting phases and others are uniform at both 20° and 24°C. (B) Heterogeneity in the transition is quantified in a single GPMV preparation by measuring the fraction of vesicles containing two coexisting liquid phases as a function of temperature over many fields of vesicles like those shown in A. The average transition temperature is defined as the extrapolated temperature where 50% of vesicles are phase separated, which in this case is 21.0±0.2°C (dashed line). The width of the transition typically spans 10°C.
Fig 2.
Transition temperatures are reduced in GPMVs isolated from more densely plated cells.
(A) The percentage of GPMVs with coexisting liquid phases as a function of temperature varies significantly when GPMVs are prepared from cells plated at different densities. Images at right are representative fields of DiI-C12 labeled cells imaged prior to GPMV isolation for the indicated symbols. (B) Average GPMV transition temperature as a function of average cellular plating density. Color indicates the number of days between seeding and GPMV preparation, with an arrow pointing to a crowded sample that spent one day in culture. Average transition temperatures for representative samples shown in A are plotted with the same symbols in B. The solid line is a linear unweighted fit to the points and the dotted lines represent a standard deviation of the linear prediction. (C) GPMV transition temperatures are more heterogeneous when prepared from a dish of cells with large variations in local density. A dish of cells containing regions of high crowding and sparsely distributed cells was prepared as described in Methods. Images show representative regions of sparse, crowded, and border region cells. Points describing the percentage of phase separated vesicles as a function of temperature for GPMVs prepared from these cells are well described by a sum of two sigmoidal functions (green line). The inflection points correspond to the expected transition temperatures obtained using measured local cell densities and the trend line and confidence intervals shown in (B), as indicated by shaded boxes.
Fig 3.
Transition temperatures are reduced in cells with arrested growth due to serum starvation.
(A) Transition temperatures are suppressed in GPMVs isolated from cells incubated overnight in serum free medium when compared to GPMVs isolated from cells incubated in normal (20%) serum. (B) Elevated transition temperatures are restored in GPMVs isolated from serum starved cells in which serum has been restored for >15h. Points are a weighted average over three distinct measurements. Line is drawn to guide the eye and is not a fit to any theory.
Fig 4.
Transition temperatures vary systematically as RBL-2H3 cells synchronized to the G1/S boundary progress through the cell cycle.
(A) Representative fields of GPMVs prepared at the indicated time-points after release from a double thymidine block and imaged at 23°C. GPMVs isolated immediately after release from block (top) are mostly in a single phase whereas GPMVs isolated at later time-points often contain two phases in coexistence. (B) The fraction of phase separated GPMVs as a function of temperature for GPMVs isolated at the indicated times after release from block. These curves are used to quantify the average transition temperature (Tmisc). The dashed line is drawn at 23°C for comparison to A. (C) Tmisc as a function of time after release from the double thymidine block. Larger colored symbols represent values obtained in B and black symbols include data from 4 separate trials. The black line is smoothed from black points using a lowess filter.
Fig 5.
Tmisc is reduced in GPMVs from apoptotic cells.
(A) GPMVs isolated from RBL-2H3 cells pretreated with 100ng/ml TRAIL for 30min have lower transition temperatures than GPMVs isolated from untreated cells. At elevated temperatures, some TRAIL treated GPMVs contain non-circular domains. (B) GPMVs isolated from RBL-2H3 cells pretreated with purified sphingomyelinase (SMase) contain rigid and elongated gel domains at elevated temperature and more rigid liquid-like domains at lower temperature. GPMVs from sphingomyelinase treated cells have lower Tmisc than control GPMVs, where Tmisc is defined as the onset of the liquid appearing domains. (C) Elongated gel domains are also observed in GUVs containing purified DOPC, BSM, and Chol when 4–5 mol% of BSM is replaced by Brain ceramide. Images were acquired at 25°C.
Fig 6.
Predicted consequences of changing plasma membrane critical temperatures.
(A) One model of lipid-mediated membrane heterogeneity postulates that intact cell plasma membranes are tuned to be slightly above a critical point under growth conditions, as represented by the schematic phase diagram shown. In this model, conditions that alter Tmisc also alter ΔTC, or the difference between growth temperature and Tmisc. (B) Conditions that give rise to higher Tmisc are predicted to also give rise to larger and more long-lived composition fluctuations at growth temperature (left) when compared to conditions with lower Tmisc (right). (C) Membranes with higher Tmisc are also predicted to be more susceptible to subtle perturbations, making it easier to stabilize large and long-lived structures, as evident in the time-averaged simulated images shown. Images in B and C have the same scale with scale-bar of 50 pixels and simulations were conducted at T = 1.05TC (left images) and T = 1.2TC (right images), where TC is the critical temperature of the 2D Ising model. Methods used to generate the simulated images in B, C are described in Methods.