Project Summary To ensure that each dividing cell receives a complete set of the correct genome, cell cycle checkpoints are in place throughout the cell cycle. Yet, less is known about whether similar checkpoints exist for division of the cytoplasmic components or organelles of the cell. The endoplasmic reticulum (ER) is a gateway for the secretory pathway, generating almost all of the secreted and cell surface membrane proteins as well as synthesizing cellular lipids. Previously, we identified a cell cycle checkpoint, termed the ER stress surveillance (ERSU) pathway, that ensures the inheritance of sufficient levels of functional ER in the model organism S. cerevisiae. In response to ER stress, the ERSU pathway (1) blocks the inheritance of the stressed ER into the daughter cell, (2) mislocalizes the septin ring from the bud neck, the site of cytokinesis, and ultimately, (3) leads to temporary cell cycle arrest at cytokinesis until ER functional homeostasis is re-established. Cells that lack components of the ERSU, and thus cannot mount the ERSU pathway, die upon ER stress, underscoring the importance of this checkpoint. The ERSU pathway is distinct from the well-studied unfolded protein response. We have found that levels of phytosphingosine (PHS), an early intermediate of sphingolipid biosynthesis, increase upon ER stress, setting in motion the ERSU hallmark events. Moreover, we defined a PHS binding motif that is found within two different transmembrane domains of reticulon family proteins (i.e., Rtn1 and Yop1), leading to the activation of the ERSU events. In the current proposal, in AIM 1, we will apply molecular and cell biological approaches to dissect how PHS binding to Rtn1 or Yop1 results in ERSU activation. In AIM 2, we will extend the scope of the ERSU by dissecting the impact of ER lipotoxic stress induced by ER morphological changes, on the ERSU molecular events. In AIM 3, we will investigate how ER stress impacts the mammalian cell cycle. As the nuclear membrane breaks down during mitosis in mammalian cells, the division of functional ER may be even more tightly choreographed with the nuclear mitotic mechanisms. Our preliminary result of a mammalian septin subunit holds great promise for the presence of regulatory events that may share similarity to the yeast ERSU. Thus, we will fully investigate the impact of ER stress on (A) the mammalian septin subunits and cytokinetic components, and (B) major mitotic cell cycle structural changes that involve the ER, such as ER clearing from the “mitotic exclusion zone” and nuclear disassembly and reassembly. Understanding the molecular mechanisms that integrate ER homeostasis with cell cycle events will provide unprecedented insights into human diseases caused by the failure of ER regulation.