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: 9071807
• 负责人: Rebecca W Heald
• 金额: $ 62.35万
• 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/9071807
• 关键词: Address; Affect; Architecture; Area; Biochemical; Biological; Biological Assay; Biophysical Process; 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 Simulation; 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; Transcription Process; 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

 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.