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(申请人提供)：我的实验室的研究由两个高生产率的R01支持，并专注于两个主要领域：细胞分裂可以说是细胞生命中最戏剧性的事件。染色体浓缩，细胞器泡化，微管细胞骨架重新排列成两极纺锤体，在着丝点附着在染色体上，并将一整套分离到每个子细胞。虽然在有丝分裂过程中发生的形态变化在一个多世纪前就被首次观察到，但我们仍然不知道这些动态事件是如何安排的。许多影响纺锤体组装和功能的因素已被确定，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不清楚。我们目前的项目致力于解决悬而未决的问题，包括1)不同主轴结构的分子基础和功能后果是什么？纺锤体的大小和组织在不同的细胞类型和生物体中差异很大，已知的影响这些参数的因素在许多癌症中都会发生变化，但具体的纺锤体特征是如何建立的，以及它们对染色体分离和细胞分裂的影响尚不清楚。我们将利用形态计量学和系统发育比较以及生化和功能分析来研究纺锤体两极星形微管形态变异的基础和意义。2)什么活动足以建立纺锤体的机械力化学核心？尽管许多单独的纺锤体因子的功能已经被广泛研究，但从纯化的组件重组纺锤体仍然是一个圣杯，因为它是完全理解这一过程的关键。我们将扩展我们的基于珠子的纺锤体组装系统，以定义足以进行纺锤体自组织的染色质相关活动。3)RNA在动粒组装中的作用是什么？着丝粒序列的转录似乎是动粒形成所需的一种保守机制，但新生转录产物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方法检测着丝粒转录和有丝分裂过程中的RNA处理，并阐明其在纺锤体组装中的作用。总之，这些项目阐明了有丝分裂的机制，并将该领域推向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差异很大，无论是在物种内部还是在物种之间，在原子/分子以上的各级组织：有机体、组成有机体的细胞和细胞的组成部分。如何进行伸缩，以使一切都适合并正常运行？细胞内的正确伸缩对细胞的功能、结构和分裂至关重要，但直到最近，细胞用来调节其内部结构大小的控制系统几乎是未知的。我们 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在早期胚胎发育的快速、还原细胞分裂期间细胞内结垢的机制。我们正在进一步开发这些系统，以问：1)有丝分裂染色体大小与细胞大小的比例是什么？有丝分裂染色体结构的决定因素知之甚少，解决这个问题的一个主要挑战是建立活的染色体标记方法。我们将利用我们新的基于CRISPR的成像技术来测试候选因素在发育过程中对染色体比例的作用。2)有没有一种能感知细胞表面积体积比的伸缩机制？越来越多的证据表明，细胞感觉表面积与体积之比是大小的直接读数，这一信息被用来衡量亚细胞结构。我们推测，Importinα是一种丰富的纺锤体和核大小调节因子，也与质膜相关，相对于大细胞，它从小细胞的细胞质中被耗尽，起到细胞大小传感器的作用。我们将使用我们的尺寸可调的微流控液滴系统来验证这一假设。3)在细胞和生物体水平上，大小是如何调节的？母体细胞质因子相对于基因组含量和表达对细胞和有机体大小的相对贡献尚不清楚。我们实验室中使用的两个非洲爪哇物种之间密切的系统发育关系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含量对大小关系的作用。总之，我们的项目利用体外和体内的方法，正在识别潜在的生物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由基于细胞质提取和脊椎动物(非洲爪哇)胚胎体内功能分析的强大实验系统实现的。我们已经建立了富有成效的合作，并应用了各种技术，包括高分辨率显微镜、生物物理分析、蛋白质组学、RNA测序、微流体和计算建模，以创造新的和创新的方法。我们的研究将继续为细胞分裂和大小控制、对生存和发育至关重要的过程提供新的见解，以及 在包括癌症在内的人类疾病方面有缺陷。