A subset of ER-PM tethering proteins marks cortically retained ER islands. (A) Time-lapse microscopy of cells expressing the indicated ER-PM tether tagged with GFP (tether) and Htb1-mCherry (Htb1) imaged during meiosis. A representative cell is shown before meiosis (top) and during anaphase II (ana II; bottom). Tethers are categorized as retained or collapsed based on anaphase II localization. Arrowheads highlight large stretches of cortical signal for each of the retained tethers. (B) Cells expressing GFP-HDEL (ER) and Tcb3-mKate (Tcb3) imaged at 0 h (top) and 6 h (bottom) after introduction to SPO. Arrowheads mark islands of colocalized ER lumen and Tcb3 signal. (C) EM of a WT cell following spore closure (top). Yellow box outlines the area shown zoomed in on the bottom. White arrows indicate cortically retained ER fragments. S marks the four spores. V marks the vacuole. (D) Time-lapse microscopy of cells expressing GFP-Ist2 and Vph1-mCherry (vacuole) imaged every 30 min in meiosis. White arrowheads mark Vph1 signal at the mother cell vacuole membrane, which becomes diffuse upon vacuolar lysis at time 0. Blue arrowheads mark diffuse Vph1 signal following vacuole lysis. Yellow asterisks mark spore vacuoles, which do not lyse. Yellow arrowheads mark bright clusters of Ist2 that are degraded upon vacuole lysis. (E) Images of cells expressing GFP-HDEL (ER) and Htb1-mCherry (Htb1) taken at anaphase II. A representative cell is shown for each cortical ER classification. Yellow arrowheads highlight cortically retained ER. (F) Quantification of at least n = 100 cells for the indicated genotypes following the classification system in E. P values determined by χ² test comparing to WT. ns, P > 0.05; **, P < 0.01. (G) Time-lapse microscopy of cells expressing GFP-Ist2 and mCherry-HDEL (ER) imaged every 30 min in meiosis. The Gini score based on quantification of Ist2 signal is shown below each time point. Min 0 is defined as the time of ER collapse. (H) Gini quantification based on cortical Ist2 signal over time for the indicated genotypes. Values are the average of n = 10 WT or n = 3 ndt80Δ cells scored across each time point. Error bars represent SD. Gini score is significantly higher for WT than ndt80Δ for all time points starting at −90 min onward (P < 0.05 by Student’s t test). (I) Schematic showing ER morphology and tether localization in premeiotic and anaphase II cells. Scale bar = 2 µm for all panels except C, for which scale bar = 1 µm.