Supplementary Figure 6: PES formation does not require de novo PtdIns(4,5)P2 synthesis.
From: Decrease in plasma membrane tension triggers PtdIns(4,5)P2 phase separation to inactivate TORC2
(a) Quantification of the relative PtdIns(4,5)P2 levels at the cortex of the indicated cell upon PalmC treatment. A 2-px-wide line was drawn around the cell perimeter and the GFP-2xPHPLCδ fluorescence intensity was measured. Peaks representing PtdIns(4,5)P2 clusters are marked with asterisks. Presented results are representative of n=20 cells from two independent experiments. (b) Total GFP-2xPHPLCδ fluorescence intensities, expressed as afu/pixel and normalized to the control condition, after 5min PalmC treatment or 1M hyper-osmotic shock. Results are presented as mean +/-SD (n=40 cells pooled from two independent experiments, n.s: not significant change, p>0.05 in two-tailed unpaired t-test, see Supplementary Table 3 for exact p values). Source data are included in Supplementary Table 3. (c) Mss4 activity is necessary for the maintenance of PES. Timelapse of WT or inp51Δinp52Δ cells expressing the GFP-2xPHPLCδ biosensor and treated with 10µM PalmC for 5 min prior to ATP depletion. Images are maximum projections of 0.5μm-spaced Z-planes of the cells, and representative of results observed in three independent experiments. (d) Endocytic BAR protein do not play a major role in PES formation. Representative equatorial sections of cells expressing Rvs167-mCherry and the GFP-2xPHPLCδ biosensor, treated or not for 5min with 10µM PalmC. This experiment was repeated three times with similar results. All scale bars, 5µM.
