Defining the role of bilayer lipid composition in nuclear envelope dynamics and genome protection

项目说明 核膜是内质网(ER)的一个亚域，它包裹和保护 基因组。与先前的教条相反，核包膜不是一个稳定的结构，但在两者中都是高度动态的 间期和有丝分裂。核膜动力学缺陷在癌细胞和遗传性疾病中是常见的 由核膜蛋白突变引起的疾病，包括肌肉营养不良和早衰症。 因此，阐明核包膜动力学背后的机制将有助于我们对 疾病机制从癌症到加速衰老。驱动膜的基本原理 几乎所有膜结合细胞器的动力学都是由双层磷脂组成的，但很少是 已知磷脂在核膜动力学中的作用。这项建议的重点是一种独特的 磷脂合成的调节剂，CTDNEP1(以前称为杜拉德)，在细胞核中浓缩 包膜是核包膜动力学所必需的。CTDNEP1及其专性结合伴侣NEP1R1 是一种无处不在的保守的蛋白磷酸酶复合体，激活磷脂酸磷酸酶 脂类；脂类的活性是磷脂从头合成的中心。这里提出的工作主要有两个方面 目的：1)建立CTDNEP1-NEP1R1核膜定位的功能意义 磷脂合成的局部调节和核膜动力学以及2)阐明核的作用 被膜脂通过调节有丝分裂核被膜重塑来保护基因组完整性。完成 这些目标将阐明一个潜在的重要作用，但尚未得到充分研究的脂质介导的调节 核膜动力学以确保基因组保护。目标1将重点放在 在NEP1R1中我们发现了靶/在核膜/内质网中保留CTDNEP1的保守区 人类细胞。我们将在NEP1R1中找出特异性破坏CTDNEP1-NEP1R1富集率的突变体 核膜，使其成为一种更通用的内质网蛋白，并评估其对脂质成分和 核包膜动力学。目标2建立在我们的初步数据基础上，即CTDNEP1调节核膜 改造和防止癌细胞分裂过程中微核的形成。这些数据表明，核心核 内质网来源的核膜蛋白可能受特定的磷脂调节而形成核膜 膜。我们将使用内源性标记结合常规和超分辨率活细胞成像 系统定义核心核膜蛋白相对于新生核膜蛋白的定位和动态 野生型和ctdnep1缺陷癌细胞核膜重塑过程中的核膜。在……里面 体外脂结合分析将确定核膜蛋白中直接与主要 磷脂。这样的研究将允许设计具有改变的脂质结合特性的突变蛋白质来测试 对于在ER膜募集和重塑中产生核膜的功能意义。

PROJECT DESCRIPTION The nuclear envelope is a subdomain of the endoplasmic reticulum (ER) that surrounds and protects the genome. Contrary to prior dogma, the nuclear envelope is not a stable structure, but is highly dynamic in both interphase and mitosis. Defects in nuclear envelope dynamics are common to cancer cells and to genetic disorders caused by mutations in nuclear envelope proteins that include muscular dystrophy and progeria. Thus, elucidating mechanisms that underlie nuclear envelope dynamics will inform on our understanding of disease mechanisms ranging from cancer to accelerated aging. An underlying principle that drives membrane dynamics at nearly all membrane-bound organelles is the composition of bilayer phospholipids, yet little is known about the role of phospholipids in nuclear envelope dynamics. This proposal focuses on a distinct regulator of phospholipid synthesis, CTDNEP1 (formerly known as Dullard), that is enriched at the nuclear envelope and is required for nuclear envelope dynamics. CTDNEP1 and its obligate binding partner NEP1R1 are a ubiquitously conserved protein phosphatase complex that activate the phosphatidic acid phosphatase lipin; lipin's activity is central to the de novo phospholipid synthesis. The work proposed here has two main aims: 1) to establish the functional significance of CTDNEP1-NEP1R1 localization at the nuclear envelope in local regulation of phospholipid synthesis and nuclear envelope dynamics and 2) to elucidate a role for nuclear envelope lipids in protecting genome integrity by regulating mitotic nuclear envelope reformation. Completion of these goals will elucidate a potentially significant, yet understudied role for lipid-mediated regulation of membrane dynamics at the nuclear envelope to ensure genome protection. Aim 1 will focus on the role of a conserved region in NEP1R1 that we discovered targets/retains CTDNEP1 in the nuclear envelope/ER in human cells. We will identify mutants in NEP1R1 that specifically disrupt enrichment of CTDNEP1-NEP1R1 at the nuclear envelope so that it is a more generic ER protein and assess the effects on lipid composition and nuclear envelope dynamics. Aim 2 builds on our preliminary data that CTDNEP1 regulates nuclear envelope reformation and prevents micronucleus formation in dividing cancer cells. These data suggest that core nuclear envelope proteins may be regulated by specific phospholipids to form the nuclear envelope from ER-derived membranes. We will use endogenous tags combined with conventional and super-resolution live-cell imaging to systematically define the localization and dynamics of core nuclear envelope proteins relative to nascent nuclear membranes during nuclear envelope reformation in wild type and ctdnep1-deficient cancer cells. In vitro lipid binding assays will identify domains in nuclear envelope proteins that directly bind to major phospholipids. Such studies will allow the design of mutant proteins with altered lipid binding properties to test for functional significance in ER membrane recruitment and remodeling to generate the nuclear envelope.