Roles of the ER Stress Surveillance Pathway During the Cell Cycle

项目总结 为了确保每个分裂细胞获得一套完整的正确基因组，细胞周期检查点在 在整个细胞周期中放置。然而，关于是否存在类似的检查站来划分 细胞质的成分或细胞器。内质网(ER)是血管内皮细胞 分泌途径，产生几乎所有的分泌和细胞表膜蛋白以及 合成细胞脂质。之前，我们确定了一个细胞周期检查点，称为内质网应激监视 (ERSU)途径，确保在模式生物S. 酿酒。作为对内质网应激的响应，ERSU途径(1)阻止了应激后内质网进入 子细胞，(2)错位花蕾颈的隔质环，细胞质分裂的部位，最终，(3) 导致细胞质分裂时细胞周期暂时停滞，直到内质网功能稳态重新建立。细胞 缺乏ERSU的组成部分，因此无法启动ERSU途径，死于内质网应激，强调 这个检查站的重要性。ERSU途径不同于研究得很好的未折叠蛋白 回应。我们发现，鞘磷脂的早期中间体--植酸鞘氨醇(PHS)的水平 生物合成，增加内质网压力，启动ERSU标志事件。此外，我们还定义了一个 在网状蛋白家族蛋白的两个不同跨膜域中发现的PHS结合基序(即， Rtn1和Yop1)，导致ERSU事件的激活。在目前的提案中，在目标1中，我们将适用 分子和细胞生物学方法研究PHS与Rtn1或Yop1结合如何导致ERSU 激活。在目标2中，我们将通过剖析内质网脂毒应激的影响来扩大ERSU的范围 诱导的内质网形态变化，对ERSU分子事件的影响。在目标3中，我们将研究ER如何 压力会影响哺乳动物的细胞周期。随着哺乳动物有丝分裂过程中核膜的破裂 细胞，功能性内质网的分裂可能与核有丝分裂更加紧密地编排在一起 机械装置。我们对哺乳动物隔膜蛋白亚基的初步研究结果显示， 可能与酵母ERSU相似的调控事件。因此，我们将全面调查ER的影响 强调(A)哺乳动物的隔膜蛋白亚基和细胞动力学成分，以及(B)主要有丝分裂细胞周期 涉及内质网的结构性变化，例如内质网从有丝分裂隔离区和核中清除 拆卸和重新组装。了解内质网内稳与内质网整合的分子机制 细胞周期事件将为人类因ER失败而导致的疾病提供前所未有的洞察力 监管。。

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. .