Supplementary Figure 4: PES are membrane invaginations, unrelated to endocytic structures.

(a, c) Distributions of the peak intensities of Avo3-GFP (a) or Slm1-mCherry (c) foci (n=100 foci, pooled from three independent experiments) and the thresholds used to define the clusters. (b) A hyper-osmotic shock induces TORC2 (Avo3-GFP) aggregation, but direct inhibition of TORC2 by NVP-BHS345 in TORC1^M2282T cells²⁸ does not. Images are maximum projections for 0.5-μm-spaced Z-planes, and representative of three independent experiments; scale bar, 5μm. (d) Illustration of the Rapamycin-mediated anchor-away system. Slm1–FRB dynamically relocalizes to and from eisosomes (left), but irreversible binding of Rapamycin locks it to Sur7–FKBP12 at eisosomes (right). (e) Anchoring of Slm1 to eisosomes impairs TORC2 reactivation following PalmC treatment. WT or TOR1-1 fpr1Δ slm2Δ SLM1–FRB SUR7–2×FKBP12 cells were treated with Rapamycin (5 min), then PalmC, and Ypk1 T⁶⁶² phosphorylation was monitored. This experiment was repeated twice with similar results, and unprocessed scans of the blots are shown in Supplementary Figure 7. (f) PalmC-treated cells expressing the PtdIns(4,5)P₂ biosensor display large PM invaginations enriched in PtdIns(4,5)P₂ when visualized by Electron Microscopy after anti-GFP immunogold labelling. For each condition, the middle and right images are a magnification of the indicated region from the left picture and the right image highlights the gold particules in red. Scale bar, 2μm. This experiment was repeated twice with similar results. (g) Filamentous actin is not required for PES formation. Equatorial sections of cells expressing Abp1-mCherry and the GFP-2xPH^PLCδ biosensor, treated with 10µM PalmC +/- 2µM LatA. Images are representative from three independent experiments. Scale bar, 5μm. (h) Filamentous actin is not required for PES resolution. Evolution of the percentage of cells displaying PES after PalmC treatment, +/- pre-treatment with LatA. Error bars represent the SD of mean values of three independent experiments (n=50 cells). Source data are included in Supplementary Table 3.