Growth Conditions and Cell Cycle Phase Modulate Phase Transition Temperatures in RBL-2H3 Derived Plasma Membrane Vesicles

<div><p>Giant plasma membrane vesicle (GPMV) isolated from a flask of RBL-2H3 cells appear uniform at physiological temperatures and contain coexisting liquid-ordered and liquid-disordered phases at low temperatures. While a single GPMV transitions between these two states at a well-defined temperature, there is significant vesicle-to-vesicle heterogeneity in a single preparation of cells, and average transition temperatures can vary significantly between preparations. In this study, we explore how GPMV transition temperatures depend on growth conditions, and find that average transition temperatures are negatively correlated with average cell density over 15°C in transition temperature and nearly three orders of magnitude in average surface density. In addition, average transition temperatures are reduced by close to 10°C when GPMVs are isolated from cells starved of serum overnight, and elevated transition temperatures are restored when serum-starved cells are incubated in serum-containing media for 12h. We also investigated variation in transition temperature of GPMVs isolated from cells synchronized at the G1/S border through a double Thymidine block and find that average transition temperatures are systematically higher in GPMVs produced from G1 or M phase cells than in GPMVs prepared from S or G1 phase cells. Reduced miscibility transition temperatures are also observed in GPMVs prepared from cells treated with TRAIL to induce apoptosis or sphingomyelinase, and in some cases a gel phase is observed at temperatures above the miscibility transition in these vesicles. We conclude that at least some variability in GPMV transition temperature arises from variation in the local density of cells and asynchrony of the cell cycle. It is hypothesized that GPMV transition temperatures are a proxy for the magnitude of lipid-mediated membrane heterogeneity in intact cell plasma membranes at growth temperatures. If so, these results suggest that cells tune their plasma membrane composition in order to control the magnitude of membrane heterogeneity in response to different growth conditions.</p></div

GPMVs from RBL-2H3 exhibit a broad distribution of miscibility transition temperatures.

<p>(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.</p

Transition temperatures are reduced in GPMVs isolated from more densely plated cells.

<p>(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.</p

T_misc is reduced in GPMVs from apoptotic cells.

<p>(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 T_misc than control GPMVs, where T_misc 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.</p

Transition temperatures vary systematically as RBL-2H3 cells synchronized to the G1/S boundary progress through the cell cycle.

<p>(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 (T_misc). The dashed line is drawn at 23°C for comparison to A. (C) T_misc 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.</p

Predicted consequences of changing plasma membrane critical temperatures.

<p>(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 T_misc also alter ΔT_C, or the difference between growth temperature and T_misc. (B) Conditions that give rise to higher T_misc are predicted to also give rise to larger and more long-lived composition fluctuations at growth temperature (left) when compared to conditions with lower T_misc (right). (C) Membranes with higher T_misc 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 = 1.05T_C (left images) and T = 1.2T_C (right images), where T_C is the critical temperature of the 2D Ising model. Methods used to generate the simulated images in B, C are described in Methods.</p

Modulating transition temperature affects cisplatin mediated cellular response in UMSCC1 cells.

<p>(A) Transition temperature shifts measured for GPMVs isolated from UMSCC1 in the presence of 50mM isopropanol or 100 μM menthol or each of these treatments in combination with 10μM cisplatin. (B) Efficacy of 50mM isopropanol and 100μM menthol action on UMSCC1 cells calculated as the number of cells present after 24h of treatment divided by the number of cells present in an untreated control. (C) Efficacy of cisplatin action as a function of the transition temperature shift effected by the treatment in isolated GPMVs shown in A. Efficacy of cisplatin action on UMSCC1 cells as above was computed as above in (B) by dividing the number of cells present after 24h of treatment compared to the number of cells present in an untreated control. (D) Plots show relative cell counts as a function of cisplatin concentration either in the presence or absence of 50mM isopropanol or 100μM menthol. Each point represents the average and SEM of at least 4 independent measurements, and lines are fit to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140925#pone.0140925.e001" target="_blank">Eq 1</a>. (E) Average IC₅₀ values as determined by fitting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140925#pone.0140925.e001" target="_blank">Eq 1</a> to individual dose response curves. Values represent an average and SEM over at least 4 independent measurements.</p

Changes in transition temperature in GPMVs correlate with the cell lines resistance to cisplatin.

<p>(A) GPMVs were isolated from four cell-lines as described in the Methods section. The transition temperature shifts are reported by comparing the transition temperatures of GPMVs probed in the presence of 10 μM cisplatin to untreated GPMVs. (B) Data points in panel A were plotted against a previously reported measure of surviving fraction to cisplatin obtained using clonogenic assays for the same four cell lines [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140925#pone.0140925.ref008" target="_blank">8</a>]. Surviving fraction was measured using a clonogenic survival assay. Surviving fractions were measured 72 hours after treatment with 10uM cisplatin. The straight line is drawn to visually distinguish sensitive and resistant celllines. Transition temperature shifts upon incubation with 10 μM cisplatin or exposure to 10 Gy irradiation for GPMVs isolated from ME-180 pt cells (C) and RBL cells (D). In all cases, points represent the average of at least 3 independent measurements and error bounds represent the standard error of the mean. Significance between transition temperature shift measurements were evaluated using t-tests.</p

Co-incubation of cisplatin with isopropanol leads to enhanced apoptosis without an increase in intercellular cisplatin concentration.

<p>(A) Expression levels of cleaved PARP, an apoptotic marker, as measured by western blot for cells incubated with 50mM isopropanol plus 10μM cisplatin, with 100μM menthol plus 10μM cisplatin or with 10μM cisplatin alone along with cisplatin free controls (B) Levels of intracellular cisplatin were measured using optical emission spectrometry for the three treatments, 50mM isopropanol plus 10μM cisplatin, with 100μM menthol plus 10μM cisplatin or with 10μM cisplatin alone. The differences between the three treatments are not statistically significant (n = 8 trials). Also shown are predicted levels of cisplatin obtained by assuming that intracellular cisplatin that is directly proportional to the external concentration determines the extent of cell death (as described in Methods). The solid line denotes the predicted mean theoretical value and corresponding dashed lines denote error bounds. (C) Levels of intracellular cisplatin for UMSCC1 and the more resistant cell line Me-180pt treated with 10μM cisplatin. The solid line as described previously denotes predicted levels given the assumptions stated in 4B and dashed lines indicate error bounds.</p

Measuring effective resolution of reconstructed super-resolution images with explicit over-counting.

<p>(A,B) Reconstructed super-resolution fluorescence localization images of labeled IgE on the bottom surface of RBL-2H3 mast cells. The region enclosed in the red box is magnified in the right panel. The image shown in A is reconstructed from raw data where each localized signal is counted independently. In B, intentional over-counting arising from probes remaining activated for multiple sequential frames is removed by grouping localized signals found at the same location within a small radius in sequential raw images. Grouping methods are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031457#s3" target="_blank">Materials and Methods</a>, and several locations which differ between the grouped and raw images are highlighted with green squares in the zoomed images. (C) Correlation functions are calculated from both the raw image to obtain and from the grouped image to obtain . The correlation function of the raw image contains more apparent clustering at short radii than the measured correlation function of the grouped image because there are additional contributions in the raw image from intentional over-counting. Subtracting from results in a curve that is proportional to the correlation function of the effective point spread function, . This is a measure of the effective resolution of the measurement. In this example, the black points are fit assuming a Gaussian PSF, , where σ is determined to be 9.6 nm and A = 4.9 is an constant related to the average number of times each probe was deliberately over-counted. In A and B, images on the left are filtered with a Gaussian PSF with standard deviation of 75 nm and zoomed images on the right are filtered with a Gaussian PSF with standard deviation of 10 nm for display purposes.</p