Report

Membrane potential modulates plasma membrane phospholipid dynamics and K-Ras signaling

See allHide authors and affiliations

Science  21 Aug 2015:
Vol. 349, Issue 6250, pp. 873-876
DOI: 10.1126/science.aaa5619
  • Fig. 1 PM depolarization enhances nanoclustering of lipids and K-Ras.

    (A) Whole-cell patch clamping of BHK cells to measure Vm in isotonic buffers containing different [K+]. (B) Weighted mean K functions shown as L(r) – r for a PS lipid probe (n ≥ 8) in control (CON) and depolarized BHK cells. L(r) – r values >99% confidence interval (C.I.) for a random pattern indicate clustering. Depolarization (100mM [K+]) significantly increased PS clustering (P < 0.001, bootstrap test). (C) Peak L(r) – r values, Lmax, derived from curves as in (B), quantify the extent of nanoclustering of lipid probes for PS, PIP2, PA, or PIP3 in control and depolarized (100 mM [K+]) BHK cells. (D) Short time course (<5 min) of changes in Lmax values for PS, PIP2, and GFP–K-RasG12V (KG12V) in BHK cells depolarized with 100 mM [K+] at t = 0 min. (E) Time course of depolarization (5 to 100 mM [K+] at t = 0 min) and repolarization (100 to 5 mM [K+] at t = 60 min), changes in Lmax for PS, PIP2, and K-RasG12V. (F) Dependence of GFP–K-RasG12V or GFP-tK clustering, quantified as Lmax values, on Vm varied as in (A). (G) FLIM images (GFP) of cells expressing GFP/RFP–K-RasG12V and GFP/RFP-tK FRET pairs. (H) Fluorescence lifetime of GFP–K-RasG12V or GFP-tK in cells expressing the cognate RFP-FRET pair plotted against Vm. Each point is the mean (±SEM) GFP lifetime measured in >60 individual cells. Significant differences (*P < 0.001) were evaluated by using one-way analysis of variance (ANOVA). Error bars in all panels represent SEM.

  • Fig. 2 Plasma membrane depolarization enhances K-Ras nanoclustering in mouse neuroblastoma cells.

    (A) FLIM images of differentiated N2A cells expressing the FRET pairs: RFP/GFP-tK or RFP/GFP–K-RasG12V (GFP-KG12V and depolarized with 100 mM [K+] or 50 to 100 μM glutamate (Glu). (B and C) Quantification of fluorescence lifetime of GFP–K-RasG12V or GFP-tK in N2A cells expressing the cognate RFP-FRET pair treated as in (A). Each data point is the mean (±SEM) GFP lifetime measured in >60 individual cells. Significant differences (*P < 0.01) were evaluated by using one-way ANOVA.

  • Fig. 3 PS mediates Vm-induced changes in K-Ras nanoclustering and signaling.

    (A) PM sheets from BHK cells expressing RFP–K-RasG12V, and a GFP-tagged lipid-binding probe for PS or PIP2, were labeled with anti-GFP-6nm gold and anti-RFP-2nm gold and visualized by EM. Bivariate K functions (summarized as LBI values) were used to quantify the colocalization of PS or PIP2 with GFP–K-RasG12V and GFP-tH nanoclusters. Statistical significance was evaluated in Mann-Whitney tests (*P < 0.05). Additional lipid reorganization results are shown in fig. S10E. (B) Univariate EM-spatial mapping of PM sheets prepared from PSA-3 cells expressing GFP–K-RasG12V and grown with or without ethanolamine (±Etn). (C) FLIM imaging of PSA-3 cells expressing GFP–K-RasG12V or coexpressing RFP–K-RasG12V and grown ±Etn. (D) MAPK activation in K-RasG12V–transformed BHK cells (and wild-type cells fig. S11) measured by quantitative immunoblotting for phosphorylated ERK (pERK) after PM depolarization as in Fig. 1A.

  • Fig. 4 Enhanced K-Ras clustering and MAPK signaling in intact fly embryo after PM depolarization.

    (A) Confocal images of flies expressing GFP-tK, or coexpressing GFP-tK and RFP-tK, from a neuronal-specific promoter. (B) FLIM imaging of brains from flies in (A) in control buffer and after depolarization with high [K+]. (C) Quantification of fluorescence lifetime of GFP-tK in fly brains expressing the cognate RFP-FRET pair treated as in (B); results are mean (±SEM, n = 9). High [K+] had no effect on the fluorescence lifetime of GFP-tH in fly brains expressing GFP-tH and RFP-tH. (D) Fly embryos from wild-type (WT) and mutant atp8b flies incubated in 5 or 90 mM K+ for 15 min and then immunoblotted for pERK. Results are quantified as mean ± SEM (n = 3).

Supplementary Materials

  • Membrane potential modulates plasma membrane phospholipid dynamics and K-Ras signaling

    Yong Zhou, Ching-On Wong, Kwang-jin Cho, Dharini van der Hoeven, Hong Liang, Dhananiay P. Thakur, Jialie Luo, Milos Babic, Konrad E. Zinsmaier, Michael X. Zhu, Hongzhen Hu, Kartik Venkatachalam, John F. Hancock

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

    Download Supplement
    • Materials and Methods
    • Figs. S1 to S16

Navigate This Article