Mechanisms of mitosis and size control in Xenopus

非洲爪蟾有丝分裂和大小控制的机制

• 批准号: 9896841
• 负责人: Rebecca W Heald
• 金额: $ 81.63万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-12 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9896841
• 关键词: Address; Affect; Architecture; Area; Biochemical; Biological; Biological Assay; Biophysical Process; Biophysics; Cell Size; Cell division; Cell membrane; Cell physiology; Cell surface; Cells; Cellular Structures; Chromatin; Chromosome Segregation; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Computer Models; Cytoplasm; Cytoskeleton; Development; Embryo; Embryonic Development; Ensure; Event; Genetic Transcription; Genome; Hybrids; Imaging Techniques; In Vitro; Individual; Kinetochores; Label; Laboratories; Lead; Life; Malignant Neoplasms; Mediating; Methods; Microfluidics; Microscopy; Microtubules; Mitosis; Mitotic; Mitotic Chromosome; Mitotic spindle; Molecular; Morphology; Nuclear; Organelles; Organism; Phylogenetic Analysis; Process; Proteomics; RNA; RNA Processing; Rana; Research; Resolution; Role; Shapes; Structure; Subcellular structure; Surface; System; Techniques; Testing; Transcript; Variant; Xenopus; alpha Karyopherins; base; cell type; daughter cell; human disease; in vitro Assay; in vivo; innovation; insight; novel; public health relevance; reconstitution; segregation; self organization; sensor; transcriptome sequencing; virtual

 DESCRIPTION (provided by applicant): Research in my laboratory is supported by two highly productive R01s and has focused on two major areas: Cell division is arguably the most dramatic event in the life of a cell. Chromosomes condense, organelles vesiculate, and the microtubule cytoskeleton rearranges into a bipolar spindle that attaches to chromosomes at their kinetochores and segregates a complete set to each daughter cell. Although the morphological changes that occur during mitosis were first observed over a century ago, we still do not understand how these dynamic events are orchestrated. Many factors have been identified that contribute to spindle assembly and function, but the molecular and biophysical mechanisms and interactions that ensure mitotic fidelity remain unclear. Our current projects address outstanding questions including 1) What are the molecular underpinnings and functional consequences of different spindle architectures? Spindle size and organization varies dramatically across cell types and organisms, and factors known to affect these parameters are altered in many cancers, but how specific spindle features are established and their effects on chromosome segregation and cell division are poorly understood. We will leverage morphometric and phylogenetic comparisons together with biochemical and functional assays to investigate the basis and significance of variation in astral microtubule morphology at spindle poles. 2) What activities are sufficient to establish the mechanochemical core of the spindle? Whereas the functions of many individual spindle factors have been studied extensively, reconstituting the spindle from purified components remains a holy grail as the key to a complete understanding of the process. We will extend our bead-based spindle assembly system to define the chromatin-associated activities sufficient for spindle self-organization. 3) What is the role of RNA in kinetochore assembly? Transcription of centromeric sequences appears to be a conserved mechanism required for kinetochore formation, but the fate and mitotic function of nascent transcripts is unclear. We will examine centromeric transcription and RNA processing during mitotic progression using a novel in vitro assay and elucidate its role in spindle assembly. Together, these projects elucidate mitotic mechanisms and advance the field toward a systems-level understanding of the spindle. Absolute and relative size of biological entities varies widely, both within and among species at all levels of organization above the atomic/molecular: the organism, the cells that make up the organism, and the components of the cells. How does scaling occur so that everything fits and functions properly? Correct scaling inside cells is crucial for cell function, architecture, and division, but until recently the contrl systems that a cell uses to regulate the size of its internal structures were virtually unknown. We have established assays to elucidate mechanisms of intracellular scaling between different-sized frog species and during the rapid, reductive cell divisions of early embryogenesis. We are further developing these systems to ask: 1) What scales mitotic chromosome size to cell size? The determinants of mitotic chromosome architecture are poorly understood, and a major challenge in addressing this question is to establish live chromosome labeling methods. We will utilize our new CRISPR-based imaging technique to test the role of candidate factors in chromosome scaling during development. 2) Is there a scaling mechanism that senses the cell surface area-to-volume ratio? Accumulating evidence suggests that cells sense surface area-to-volume as a direct readout for size, and that this information is used to scale subcellular structures. We hypothesize that importin α, an abundant regulator of spindle and nuclear size that also associates with the plasma membrane and is depleted from the cytoplasm of small cells relative to large cells, acts as a cell size sensor. We will use our size-tunable microfluidi droplet system to test this hypothesis. 3) How is size regulated at the cellular and organism levels? The relative contribution of maternal cytoplasmic factors versus genome content and expression to cell and organism size is unclear. The close phylogenetic relationship between the two Xenopus species used in our lab enables us to generate hybrid frogs of intermediate size and evaluate the role of genome size and content on size relationships. Together, our projects utilizing in vitro and in vivo approaches are identifying cellular and molecular mechanisms underlying biological size control and scaling. The means to address these fundamental cell biological questions is enabled by powerful experimental systems based on cytoplasmic extracts and functional, in vivo assays in vertebrate (Xenopus) embryos. We have established productive collaborations and apply diverse techniques including high-resolution microscopy, biophysical assays, proteomics, RNA sequencing, microfluidics and computational modeling to create new and innovative approaches. Our research will continue to provide novel insight into cell division and size control, processes essential for viability and development, and defective in human diseases including cancer.

 描述（由适用提供）：我的实验室的研究得到了两个高生产力R01的支持，并重点介绍了两个主要领域：细胞分裂可以说是细胞生命中最戏剧性的事件。染色体凝结，细胞器囊泡和微管细胞骨架重排成双极纺锤体，该双极纺锤体连接到其动物学上的染色体上，并将完整的套件分离到每个子细胞。尽管一个世纪前首次观察到有丝分裂过程中发生的形态变化，但我们仍然不了解这些动态事件是如何精心策划的。已经确定了许多有助于主轴组装和功能的因素，但是确保有丝分裂保真度的分子和生物物理机制和相互作用尚不清楚。我们当前的项目解决了未来的问题，包括1）不同纺锤体体系结构的分子基础和功能后果是什么？主轴大小和组织在细胞类型和组织之间发生了巨大变化，许多癌症中已知影响这些参数的因素发生了变化，但是如何确定特定的纺锤体特征以及它们对染色体分离和细胞分裂的影响知之甚少。我们将利用形态计量和系统发育比较以及生化和功能评估来研究脊柱螺旋杆上星体微管形态变异的基础和重要性。 2）哪些活动足以建立主轴的机械化学核心？尽管已经对许多单个主轴因素的功能进行了广泛的研究，但从纯化的组件中重新建立了主轴仍然是圣杯，这是对过程完全理解的关键。我们将扩展基于珠的主轴组装系统，以定义足够用于主轴自组织的染色质相关活性。 3）RNA在动力学组装中的作用是什么？丝粒序列的转录似乎是动力学形成所需的组成机制，但是新生转录本的命运和有丝分裂功能尚不清楚。我们将使用新型的体外测定法检查丝粒转录和RNA处理，并阐明其在纺锤体组装中的作用。这些项目共同阐明了有丝分裂机制，并将该领域推进了对纺锤体的系统水平的理解。生物实体的绝对和相对大小在原子/分子以上的各个组织的物种内部和物种之间都差异很大：组织，组成组织的细胞以及细胞的组成部分。如何进行缩放以使一切都适合和正常运行？正确缩放细胞内部对于细胞功能，体系结构和除法至关重要，但是直到最近，单元用于调节其内部结构大小的控制系统实际上是未知的。我们 已经建立了刺客，以阐明不同大小的青蛙物种之间的细胞内尺度机制以及在早期胚胎发生的快速，减少的细胞分裂中。我们正在进一步开发这些系统以询问：1）哪些尺度有丝分裂染色体大小到细胞大小？有丝分裂染色体架构的决定剂知之甚少，解决此问题的主要挑战是建立活染色体标记方法。我们将利用我们的新基于CRISPR的成像技术来测试候选因素在开发过程中染色体缩放中的作用。 2）是否有一种缩放机制可以感觉到细胞表面积与体积比率？积累的证据表明，细胞将表面积之间的体积视为大小的直接读数，并且该信息用于扩展亚细胞结构。我们假设Imputinα是大量的纺锤体和核大小调节剂，也与质膜相关联，并且从小细胞相对于大细胞的细胞质耗尽，充当细胞大小传感器。我们将使用尺寸可调的微流体液滴系统来检验此假设。 3）在细胞和生物水平上如何调节大小？母体细胞质因子与基因组含量和表达对细胞和生物体大小的相对贡献尚不清楚。我们实验室中使用的两种爪蟾物种之间的紧密系统发育关系使我们能够生成中间大小的混合青蛙，并评估基因组大小和含量在尺寸关系中的作用。共同使用体外和体内方法的我们的项目正在识别生物学大小控制和缩放的细胞和分子机制。解决这些基本细胞生物学问题的手段是通过基于细胞质提取物和功能性的强大实验系统在脊椎动物（Xenopus）胚胎中的体内分析的。我们已经建立了产品合作，并应用了潜水技术，包括高分辨率显微镜，生物物理测定，蛋白质组学，RNA测序，微流体和计算建模来创建新的创新方法。我们的研究将继续提供有关细胞分裂和大小控制的新颖洞察力，对于生存能力和开发至关重要的过程以及 包括癌症在内的人类疾病缺陷。