Cellular reprogramming during cell cycle progression

问题概述 真核细胞分裂前的准备工作需要大量的细胞重组，影响细胞骨架、染色质和细胞器。细胞结构的这些变化确保了染色体的正确分离和细胞器的遗传。有丝分裂重组缺陷可影响有丝分裂进入、染色体分离、细胞器遗传和细胞命运。在有丝分裂进入时，细胞聚集以产生用于有丝分裂纺锤体组装和胞质分裂的最佳细胞几何形状。这些变化在很大程度上是由中央细胞周期调节因子Cdk1调节的，Cdk1控制着许多影响细胞形状和细胞粘附的事件。虽然Cdk1几乎调节有丝分裂期间发生的所有细胞内事件，但对其在细胞表面的潜在影响知之甚少。与其他细胞区室一样，质膜在有丝分裂开始时经历戏剧性的重塑，但这些变化的整体功能知之甚少。虽然有丝分裂细胞变圆尚未完全了解，但它似乎是由肌动蛋白细胞骨架的变化和粘附系统的下调驱动的。一些受体被证明是从有丝分裂细胞的表面耗尽，但需要更全面的分析，以充分了解细胞表面蛋白在有丝分裂过程中的作用和调节。 目标 由于技术上的限制，质膜蛋白历来研究不足。靶向细胞表面蛋白质组的化学蛋白质组学技术（即，Surfaceome）已经开始通过提供表面可及蛋白质结构域的综合分析来克服这些局限性。我们已经采用这种技术来研究细胞表面蛋白在有丝分裂中的作用，并且该项目有两个主要目标： 1)我们希望在细胞周期的不同阶段生成细胞的高分辨率表面组图。在这个目标中，我们将使用蛋白质组学方法，生物化学和细胞测定，以监测细胞周期中的细胞表面蛋白。 2)我们想确定细胞表面变化的生物学意义，因为它们与有丝分裂有关。在这个目标中，我们将使用细胞生物学方法和显微镜来研究细胞表面蛋白在有丝分裂中的作用。 工作的新奇和预期意义 利用蛋白质组学的创新方法，我们希望揭示细胞周期中细胞表面发生的精确变化。这些数据将有助于确定有丝分裂细胞如何重组细胞表面，并可能揭示有关这一基本生物学过程的重要信息。 我们的研究结果将成为我们实验室和科学界的资源，并可能在未来几年内激发新的研究途径。该项目将为培训下一代科学家提供一个出色的平台，以解决与细胞周期有关的高度复杂的生物学问题。

OVERVIEW OF THE PROBLEMATIC The preparation of eukaryotic cells for division requires an extensive cellular reorganization, affecting cytoskeletal elements, chromatin, and organelles. These changes in cellular architecture ensure the proper segregation of chromosomes and inheritance of organelles. Defects in mitotic reorganization can affect mitotic entry, chromosome segregation, organelle inheritance, and cell fate. At mitosis entry, cells round up to generate optimal cell geometry for mitotic spindle assembly and cytokinesis. These changes are, for the most part, regulated by the central cell cycle regulator Cdk1, which controls many events affecting cell shape and cell adherence. While Cdk1 regulates nearly all intracellular events occurring during mitosis, much less is known about its potential impact at the cell surface. As with other cellular compartments, the plasma membrane undergoes dramatic reshaping at the onset of mitosis, but the global function of these changes is poorly known. While mitotic cell rounding is not fully understood, it appears to be driven by changes in the actin cytoskeleton and by the downregulation of adhesive systems. Some receptors were shown to be depleted from the surface of mitotic cells, but a more global analysis would be required to fully understand the role and regulation of cell surface proteins during mitosis. OBJECTIVES Plasma membrane proteins have been historically understudied because of technical limitations. Chemoproteomic techniques that target the cell surface proteome (i.e., surfaceome) have begun to overcome these limitations by providing comprehensive analyses of surface-accessible protein domains. We have adapted this technology to study the role of cell surface proteins in mitosis, and have two main goals for this project: 1) We want to generate high-resolution surfaceome maps of cells at different phases of the cell cycle. In this aim, we will use proteomic approaches, biochemical and cellular assays, to monitor cell-surface proteins during the cell cycle. 2) We want to determine the biological significance of cell-surface changes as they relate to mitosis. In this aim, we will use cell-biological methods and microscopy to study the role of cell-surface proteins in mitosis. NOVELTY and EXPECTED SIGNIFICANCE OF THE WORK Using innovative approaches in proteomics, we expect to uncover the precise changes that occur at the cell surface during the cell cycle. These data will help define how mitotic cells reorganize the cell surface, and will likely reveal important information about this basic biological process. Our results will be a resource for our lab and the scientific community, and will likely stimulate new investigation paths for years to come. This project will provide an outstanding platform for training the next generation of scientists on highly complex biological questions relating to the cell cycle.