Fig. 1: pH-dependent translocation of PIP2 to the outer leaflet of the plasma membrane.
From: Cells adapt to extracellular acidic pH through TM9SF3-mediated PI(4,5)P2 flop
a Confocal single-scanned cell images of isolated blastoderm cells at 4.5 hours post-fertilization (hpf), showing plasma membrane (PM) localization of various lipid-binding probes: TagRFP-PLCδ1-PH (PIP2; hereafter referred to as PIP2 probe), TagRFP-P4M-SidM (PIP), TagRFP-Akt-PH (PIP3), Annexin-V (PS), and FITC-SA-Ro (PE). Cells were incubated in Danieau’s buffer adjusted to pH 8.1 or pH 7.2, except for Annexin-V, which was used in a binding medium adjusted to pH 8.2 or pH 7.0. b Confocal single-scanned cell images of isolated blastoderm cells from Flag-Bax/H2A-BFP encoding mRNA-injected embryos at 4.5 hpf, incubated with Annexin-V in the binding medium adjusted to pH 8.2 or pH 7.0. c Quantification of fluorescence intensity at PM for each probe shown in (a). Individual cell values were normalized to the average intensity at pH 7.2 (set as 1). An identical methodology was employed for the quantitative analysis of lipid probes in this study. (PIP2, n = 288 (pH 8.1), n = 289 (pH 7.2); PE, n = 209 (pH 8.1), n = 168 (pH 7.2)). PM localization was not detected for PIP, PIP3, and PS. P-values were calculated using a two-tailed unpaired t-test for each lipid probe. The P values for PIP2 is <0.0001, and for PE is 0.0811. d Confocal single-scanned cell images of isolated blastoderm cells at 4.5 hpf embryos having TagRFP-P4M-SidM probe, PIP2 probe, and TagRFP-Akt-PH probe, injected at one-cell stage and imaged in Danieau’s buffer adjusted to pH 7.2. e Confocal single-scanned cell images of PIP2 probe on the PM of isolated blastoderm cells at 4.5 hpf, treated with DMSO or Ionomycin in L-15 medium. f Quantitative analysis of e. PM fluorescence intensity of TagRFP was measured in DMSO-treated (n = 121) and Ionomycin-treated (n = 160) cells. Individual cell values were normalized to the DMSO control (set as 1). P-values were calculated using a two-tailed unpaired t-test; **** indicates P < 0.0001. g Confocal single-scanned cell images of PIP2 probe of isolated blastoderm cells treated with DMSO or Nigericin/Valinomycin in Danieau’s buffer adjusted to pH 7.2. h Quantitative analysis of g. PM fluorescence intensity of TagRFP was measured for Nigericin/Valinomycin-treated (n = 141), FCCP-treated (n = 197), and control cells treated with DMSO (n = 140) or EtOH (n = 263). Values were normalized to their respective controls (set as 1). P-values were calculated using a two-tailed unpaired t-test; **** indicates P < 0.0001. All source data supporting the findings of this study are provided as a Source Data file. BF bright field, PS phosphatidylserine, PE phosphatidylethanolamine; PI, phosphatidylinositol, ND not detected, ns not significant, hpf hours post-fertilization. Scale bars, 20 µm.
