Artificial cortical ER tethering does not prevent ER collapse. (A) Schematic of artificial cortical ER tethering using Pil1-antiGFP. (B) Time-lapse microscopy of cells expressing GFP-Scs2 and mCherry-HDEL (ER) imaged every 10 min during meiosis. 0 min is defined as the time of ER collapse. (C) As in B but with cells expressing Pil1-antiGFP nanobody. (D) As in B but with cells expressing Rtn1-GFP instead of GFP-Scs2. Yellow arrowheads indicate cell cortex devoid of ER. White arrow indicates collapsed ER. (E) As in D but with cells expressing Pil1-antiGFP nanobody. Yellow arrowheads indicate small patches of cell cortex devoid of ER. White arrow indicates collapsed ER. (F) Cells expressing GFP-HDEL (ER) and Htb1-mCherry treated with DMSO (vehicle) at 4.5 h in meiosis and imaged every 10 min. 0 min is defined as the onset of anaphase II. Dashed white line denotes cell boundary. (G) As in F but cells were treated with 200 µM LatA instead of vehicle. Note the reduced ER mass in the cell center in G compared with F. (H) Quantification of cortical ER appearance for LatA-treated (n = 34) and untreated (n = 43) cells from the experiment shown in F and G. P values determined by χ2 test. ****, P < 0.0001. Scale bar = 2 µm for all panels. ana, anaphase.
