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（由申请人提供）：我的实验室的研究得到了两个高产 R01 的支持，并集中在两个主要领域：细胞分裂可以说是细胞生命中最引人注目的事件，染色体凝缩、细胞器囊泡和微管。细胞骨架重新排列成双极纺锤体，在其动粒处附着在染色体上，并将完整的一组染色体分离到每个子细胞中，尽管形态发生了变化。有丝分裂过程中发生的现象在一个多世纪前首次被观察到，我们仍然不明白这些动态事件是如何精心策划的，许多因素已被确定有助于纺锤体的组装和功能，但确保有丝分裂保真度的分子和生物物理机制和相互作用仍然存在。我们目前的项目解决了一些突出的问题，包括 1) 不同纺锤体结构的分子基础和功能后果是什么？纺锤体大小和组织在细胞类型和生物体中存在显着差异，并且已知影响这些参数的因素存在于许多癌症中，但是如何影响这些参数？具体的纺锤体特征已经建立，但它们对染色体分离和细胞分裂的影响知之甚少。我们将利用形态学和系统发育比较以及生化和功能测定来研究纺锤体极星体微管形态变化的基础和意义。 2) 哪些活动。足以建立纺锤体的机械化学核心吗？尽管许多单独的纺锤体因素的功能已被广泛研究，但从纯化的成分中重建纺锤体仍然是全面理解纺锤体的关键。我们将扩展基于珠子的纺锤体组装系统，以定义足以实现纺锤体自组织的染色质相关活性。 3) RNA 在着丝粒组装中的作用是什么？着丝粒形成所需的，但新生转录本的命运和有丝分裂功能尚不清楚，我们将使用一种新的体外测定来检查有丝分裂进展过程中的着丝粒转录和RNA加工并阐明其。这些项目共同阐明了有丝分裂机制，并推动了该领域对纺锤体的系统级理解，生物实体的绝对和相对大小在原子/组织之上的物种内部和物种之间存在很大差异。分子：有机体、构成有机体的细胞以及细胞的组成部分是如何发生的，以便细胞内的一切正常运转并发挥作用？控制我们几乎不知道细胞用来调节其内部结构大小的系统。 已经建立了分析方法来阐明不同大小的青蛙物种之间以及早期胚胎发生的快速、还原性细胞分裂过程中的细胞内缩放机制，我们正在进一步开发这些系统来询问：1）有丝分裂染色体大小与细胞大小的决定因素是什么？我们对有丝分裂染色体结构知之甚少，解决这个问题的一个主要挑战是建立活染色体标记方法，我们将利用我们新的基于 CRISPR 的成像技术来测试候选因子在发育过程中染色体缩放的作用 2)。是否存在感知细胞表面积与体积之比的缩放机制？越来越多的证据表明，细胞将表面积与体积的比值作为尺寸的直接读数，并且该信息用于缩放亚细胞结构。纺锤体和核大小的丰富调节剂也与质膜相关，并且相对于大细胞而言，小细胞的细胞质被耗尽，我们将使用我们的尺寸可调微流体液滴系统来检测细胞大小。 3）在细胞和生物体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。我们实验室使用的两种非洲爪蟾物种之间的密切系统发育关系尚不清楚。使我们能够产生中等大小的杂交青蛙，并评估基因组大小和内容对大小关系的作用，我们的项目利用体外和体内方法来确定生物大小控制和缩放的细胞和分子机制。这些基本的细胞生物学问题是通过基于细胞质提取物和脊椎动物（非洲爪蟾）胚胎的功能性体内测定的强大实验系统，我们建立了富有成效的合作，并应用多种技术，包括高分辨率显微镜、生物物理测定、蛋白质组学、RNA 测序、微流体和计算模型来创建新的。我们的研究将继续为细胞分裂和大小控制、生存和发育所必需的过程提供新的见解。 包括癌症在内的人类疾病有缺陷。