FIGURE 1:

Tethering of ER membranes to mitotic chromatin impairs chromosome segregation and cell division. (A) Scheme of the chromatin-ER membrane tethering system. FRB-WALP17-EGFP (membrane–chromatin tether, MCT); H2B-mPlum-FKBP (chromatin anchor, H2B*). (B) Flowchart of cell synchronization and drug treatment. Confocal images of interphase and mitotic MCT/H2B* cells. The ER was stained for calreticulin (α-CRT). (C) Flowchart of the experimental setup for D. (D) Time-lapse confocal microscopy of synchronized MCT/H2B* cells progressing through mitosis in the presence of DMSO or 200 nM rapamycin at the indicated times relative to anaphase onset. Quantification of anaphase DNA bridges (E), cytokinesis defects (F), and the time span between NEBD and anaphase onset (G) from time-lapse wide-field microscopy of synchronized MCT/H2B* cells. Note that only 70–80% of all cells express MCT in the presence of tetracycline. Only GFP-positive cells were analyzed. N = 3; mean ± SEM; n, number of cells. Dashed lines represent spindle axes. ****p < 0.0001; ns, not significant. Bars, 10 μm.