Cellular reprogramming during cell cycle progression

问题概述真核细胞分裂的制备需要广泛的细胞重组，影响细胞骨架元件、染色质和细胞器。细胞结构的这些变化保证了染色体的适当分离和细胞器的遗传。有丝分裂重组中的缺陷会影响有丝分裂的进入、染色体分离、细胞器遗传和细胞命运。在有丝分裂进入时，细胞聚集为有丝分裂纺锤体组装和细胞分裂生成最佳的细胞几何形状。这些变化在很大程度上是由中央细胞周期调节因子Cdk1调控的，它控制着许多影响细胞形状和细胞粘附的事件。虽然Cdk1调节有丝分裂过程中发生的几乎所有细胞内事件，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了戏剧性的重塑，但这些变化的整体功能尚不清楚。虽然有丝分裂细胞的圆缩尚不完全清楚，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所驱动的。一些受体从有丝分裂细胞表面消失，但需要更全面的分析来充分了解细胞表面蛋白在有丝分裂过程中的作用和调节。目的由于技术上的限制，质膜蛋白的研究一直不足。针对细胞表面蛋白质组（即表面组）的化学蛋白质组学技术已经开始通过提供对表面可接近的蛋白质结构域的全面分析来克服这些限制。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，这个项目有两个主要目标：1)我们想要生成细胞周期不同阶段的高分辨率表面体图。在这个目标中，我们将使用蛋白质组学方法，生化和细胞分析，监测细胞周期中的细胞表面蛋白质。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。为此，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。研究的新颖性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。这些数据将有助于定义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，并可能在未来几年激发新的研究路径。该项目将为培养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 PROBLEMATICThe 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.OBJECTIVESPlasma 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 WORKUsing 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.