Roles of the ER Stress Surveillance Pathway During the Cell Cycle

项目摘要 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 检查点在细胞周期的整个过程中都存在。然而，关于是否相似，人们知之甚少。 细胞质成分或细胞器的分裂存在检查点。这个 内质网(ER)是分泌途径的门户，产生几乎所有的 分泌和细胞表面膜蛋白以及合成细胞脂。此前， 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， 确保在模式生物酿酒酵母中遗传足够水平的功能性内质网。 作为对内质网应激的响应，ERSU途径(1)阻断了应激的内质网遗传进入 子细胞，(2)错误定位花蕾颈部的隔素环，细胞质分裂的位置，以及 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 是重新建立的。缺少ERSU组件的电池，因此无法安装ERSU 途径，死于内质网应激，强调了这个检查点的重要性。ERSU途径 不同于研究得很好的未折叠蛋白反应。我们已经发现， 鞘磷脂生物合成的早期中间体--植酸鞘磷脂(PHS)对内质网的促进作用 压力，启动了ERSU的标志性事件。此外，我们还定义了PHS结合基序 在网状蛋白家族的两个不同的跨膜区(即Rtn1)中发现 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 应用分子和细胞生物学方法分析PHS如何与Rtn1或Yop1结合 导致ERSU激活。在目标2中，我们将通过剖析 内质网形态变化诱导的内质网脂毒性应激对ERSU分子的影响 事件。在目标3中，我们将研究内质网应激对哺乳动物细胞周期的影响。作为 哺乳动物细胞有丝分裂过程中核膜破裂，即功能内质网的分裂 可能与核有丝分裂机制更紧密地结合在一起。我们的预赛 哺乳动物Septin亚基的结果为调节事件的存在带来了巨大的希望 这可能与酵母ERSU有相似之处。因此，我们将全面调查内质网应激的影响 关于(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 涉及内质网的循环结构变化，例如内质网从有丝分裂排斥中清除 《无核武器区》以及核拆解和重新组装。了解分子机制， 将ER动态平衡与细胞周期事件相结合将为人类提供前所未有的洞察 内质网调控失灵引起的疾病。

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.