Abstract 1725 ER-PM membrane contact sites promote nuclear segregation in budding yeast

The direct physical attachment of cortical endoplasmic reticulum (cER) to the plasma membrane (PM) plays a pivotal role in maintaining cell membrane integrity by facilitating lipid exchange at membrane contact sites (MSCs). In budding yeast, we generated the Δ-super-tether (Δ-s-tether) strain by eliminating 7 tethering proteins that otherwise generate ER-PM MCSs. Under normal growth conditions, almost all cER association along the PM is gone in Δ-s-tether cells, and Δ-s-tether cells exhibit drastic ER morphological and phospholipid defects that induces ER membrane stress. To our surprise, Δ-s-tether cells are viable and grow well despite these significant membrane defects. However, when viewed under the microscope, many Δ-s-tether cells have a cell-cycle delay during late mitosis and many are polyploid. Moreover, a significant proportion of the Δ-s-tether cells exhibit abnormal nuclear positioning during anaphase, and nuclear ER membrane rupturing. Based on these results, we propose that ER-PM MCSs affect nuclear segregation and membrane fission, which is primarily monitored by the Spindle Positioning Checkpoint (SPOC) during late anaphase. The deletion of BUB2, a key regulator of SPOC, in Δ-s-tether cells results in severe growth inhibition confirming that defects in ER-PM MCSs triggers the late anaphase checkpoint. Unlike mammalian cells, during budding yeast mitosis there is no nuclear ER membrane breakdown and intact nuclei are divided and then segregated between dividing cells prior to cell division. During this yeast "closed" mitosis, the division of the nuclear ER membrane is spatially and temporally coupled with cell division and nuclear ER membrane fission. Because the cER is contiguous with the nuclear ER, we hypothesize that without ER-PM MCSs the extended anaphase nucleus is improperly positioned between the dividing mother and daughter cells. The defect in nuclear positioning is predicted to cause dysregulation in the coordination of cell and nuclear division. As a master regulator of anaphase checkpoints and mitotic exit, the Polo-like kinase Cdc5p coordinates the events leading to cell division. Consistent with the cell-cycle defects in Δ-s-tether cells, we find that Cdc5p levels are increased in cells lacking ER-PM MCSs where during early anaphase Cdc5p prematurely localizes to sites of cell division. We propose that the dysregulation of Cdc5p expression and distribution in Δ-s-tether cells disrupts the coordination of nuclear segregation with cell division resulting in both unseparated multiploid nuclei and ruptured nuclear ER membranes. These defects in Δ-s-tether cells can be suppressed by two distinct mechanisms: (1) re-establishing ER-PM contact via an "artificial tether," constructed from several different non-yeast proteins; (2) increasing phospholipid flux by overexpressing yeast phospholipid biosynthetic enzymes. Because the artificial tether has no effect on cellular lipid levels, it appears to restore the necessary positioning of the dividing nucleus during late anaphase. We predict that changes in nuclear phospholipid composition reduce shearing forces on the nuclear ER membrane and thereby reduce membrane rupturing. Our results implicate ER-PM MCSs in the spatial and temporal regulation of nuclear segregation in yeast, which contributes to Cdc5p regulation of anaphase progression and mitotic exit. Funding for this research comes from an operating grant awarded from the Natural Sciences and Engineering Council (NSERC) of Canada.