Figure 6.

cER Peaks and PM Integrity in Heat-Shocked Tcb3 Truncation Mutants

(A–C) 1.4-nm-thick tomographic slices of cER in the indicated strains (left) and 3D renderings of cER curvature (right). Agg: aggregate; cER: cortical ER; Mito: mitochondrion; Nuc: nucleus; PM: plasma membrane. (A) tcb3Δ + Tcb3-GFP HS, (B) tcb3Δ + Tcb3 SMPΔ- GFP HS, (C) tcb3Δ + Tcb3 C2Δ-GFP HS. Insets show cER peaks (blue arrowhead in the tomographic slice inset). Scale bars for tomographic slices: 300 nm (main panels); 25 nm (insets). The contrast of the tomographic slices in (A), (B), and (C) was enhanced using a deconvolution filter. (D) cER peak density per μm² of cER membrane area showing average values (gray bars) ± SE (error bars). N = 7 (WT HS), 3 (tcb3Δ + Tcb3-GFP HS), 3 (tcb3Δ + Tcb3 SMPΔ-GFP HS), and 3 (tcb3Δ + Tcb3 C2Δ-GFP HS) ER-PM MCS. n.s. indicates p > 0.05 by Mann-Whitney U test. (E) PM integrity assay of tcb3Δ cells complemented with Tcb3 truncation mutants upon 10-min incubation at 42°C. The plot shows average values (white/gray bars) for each condition ± SE (error bars). n.s., ^∗, ^∗∗, and ^∗∗∗, respectively, indicate p > 0.05, p < 0.05, p < 0.01, and p < 0.001 by unpaired t test. Four independent biological repeats were performed for all conditions. (F) Model for the function of cER peaks in maintaining PM integrity. In WT cells, Tcbs generate membrane peaks of extreme curvature on the cER membrane. This may facilitate the extraction of cER lipids and their delivery to the PM (top left). The generation of cER peaks is the main structural role of Tcbs at ER-PM MCS, as overall ER-PM tethering is not substantially affected by Tcb1/2/3 deletion. However, tcb1/2/3Δ cells lack cER peaks (bottom left). Under heat stress, influx of extracellular Ca²⁺ through a damaged PM drives the localized formation of additional Tcb-mediated cER peaks, which in turn facilitate sufficient delivery of cER lipids to the PM to maintain PM integrity (top right). Absence of cER peaks in heat stressed tcb1/2/3Δ cells leads to PM integrity defects allowing influx of propidium iodide (bottom right). See also Figure S4; Table S1.