Abstract 2258 Yeast ER-PM contact sites coordinate mitotic regulation of nuclear segregation

The endoplasmic reticulum (ER) radiates out from the nuclear membrane into the cytoplasm and makes directed contacts with the plasma membrane (PM) at the cell cortex. Membrane contact sites (MCSs) between the ER and PM not only affix ER membrane to the cell cortex but, as a contiguous membrane extension of nuclear ER, we propose that cortical ER attachment serves to properly position nuclei within the cell. In budding yeast, there is no nuclear breakdown during mitosis and "closed" intact nuclei are divided and segregated between mother and daughter cells. We hypothesized that in yeast lacking ER-PM MCSs, nuclear ER positioning defects might disrupt the fidelity of mitotic nuclear segregation. In Δ-super-tether (Δ-s-tether) cells, in which 7 ER-PM tethering factors have been deleted and cortical ER-PM association is all but gone, nuclear division and segregation is in fact impaired. As a result, Δ-s-tether cells accumulate as large-budded cells each of which often contains a single mispositioned multiploid nucleus. A significant proportion of Δ-s-tether cells also contain ruptured nuclear ER membranes, and Δ-s-tether cells are sensitive to membrane fluidizing agents that affect nuclear integrity. We find that these defects trigger an anaphase cell-cycle delay as controlled by the spindle position checkpoint (SPOC), which is a mechanism that otherwise monitors anaphase spindle alignment and controls the spatiotemporal regulation of mitotic exit. Growth is severely inhibited if the SPOC regulators are deleted in Δ-s-tether cells and the polo-like kinase Cdc5p, which also controls anaphase checkpoints, is upregulated and mis-localized in cells lacking ER-PM tethers. Furthermore, suppressors that rescue Δ-s-tether growth defects demonstrate that changes in membrane phospholipid composition or non-specific ER-PM re-attachment can restore proper nuclear segregation. We present a model in which ER-PM MCSs promote nuclear positioning between mother and daughter cells for the Cdc5p-dependent coordination of nuclear ER membrane division and cytokinesis prior to mitotic exit. This work was supported by a Discovery Grant and an Accelerator Supplement from the Natural Sciences and Engineering Research Council (NSERC) of Canada (https://www.nserc-crsng.gc.ca/).