Mechanisms of Intracellular Scaling

描述(申请人提供)：研究细胞内伸缩的机制细胞大小在不同生物体以及同一生物体内不同组织类型和发育过程中差异很大，对细胞器和内部结构提出不同的代谢和功能需求。一个基本的问题是，如何调节基本的亚细胞成分，如核、有丝分裂纺锤体和染色体，以适应细胞大小的差异。非洲爪蛙提供了两种生理环境，我们可以在其中研究这个问题。首先，我们可以将非洲爪哇与体型较小的近缘物种非洲爪哇进行比较，热带非洲爪哇产卵较小，在整个发育过程中细胞比例较小。其次，我们可以比较非洲爪哇胚胎发育的不同阶段，直径约1毫米的卵子迅速分裂形成更小的卵裂球，到第15次分裂时，卵裂球的直径减少到40微米。我们方法的一个独特方面是从卵子和胚胎中制备细胞质提取液，这些提取液可以概括细胞器在体外的缩放情况，我们可以使用这些提取液来识别导致大小变化的分子差异。我们的第一个具体目标是有丝分裂纺锤体，我们利用计算机模拟来确定微管动力学和组织参数，这些参数可能有助于纺锤体大小和形态在物种之间和发育过程中的变化。这一目标还开发了通过物理限制不同大小液滴内的主轴组装反应来研究外部缩放机制的新方法，这将揭示是否存在从外部缩放主轴或改变组装路径的尺寸阈值。目的2研究有丝分裂染色体的大小在发育过程中如何改变以与纺锤体长度相协调，从而实现完全分离。在目标3中，我们通过研究非洲爪哇在发育过程中改变核大小的后果，开始讨论细胞器缩放的重要性。这些实验将深入了解结垢是如何发生的，并有助于细胞内的形态发生和细胞分裂，对生存和发育至关重要的过程，以及包括癌症在内的人类疾病的缺陷。 与公共健康相关：在生物学的许多层面上，大小差异很大：动物，组成动物的细胞，以及细胞的内容物，但我们不知道适当的比例是如何发生的，从而使一切都适合并正常发挥作用。这项建议研究了细胞如何调节其内部结构的大小，包括包含基因组的染色体和将染色体分离到子细胞的有丝分裂纺锤体，并阐述了内部大小调节在发育中的青蛙胚胎中的重要性。由于细胞分裂和大小调节缺陷在癌症中经常被观察到，我们的研究可能会揭示其潜在的机制，并导致新的治疗方法。

DESCRIPTION (provided by applicant): Investigating Mechanisms of Intracellular Scaling Cell size varies widely among different organisms as well as within the same organism in different tissue types and during development, placing variable metabolic and functional demands on organelles and internal structures. A fundamental question is how essential subcellular components such as the nucleus, mitotic spindle and chromosomes are regulated to accommodate cell size differences. Xenopus frogs offer two physiological contexts in which we can investigate this question. First, we can compare Xenopus laevis to the smaller, related species Xenopus tropicalis, which lays smaller eggs and has proportionally smaller cells throughout development. Second, we can compare different stages of Xenopus laevis embryogenesis, as the ~1 millimeter diameter egg rapidly cleaves to form smaller blastomeres, which by the 15th division are reduced to 40 microns across. A unique aspect of our approach is to prepare cytoplasmic extracts from eggs and embryos that recapitulate organelle scaling in vitro, which we can use to identify molecular differences that underlie size changes. Our first specific aim focuses on the mitotic spindle, and we take advantage of computer simulations to identify parameters of microtubule dynamics and organization that could contribute to spindle size and morphology changes between species and during development. This aim also develops novel methods to examine extrinsic scaling mechanisms by physically confining spindle assembly reactions inside different sized droplets, which will reveal whether there are size thresholds that scale the spindle externally or alter assembly pathways. Aim 2 investigates how the size of mitotic chromosomes is altered during development to coordinate with spindle length so that complete segregation occurs. In Aim 3, we begin addressing the importance of organelle scaling by examining the consequences of altering nuclear size during development in Xenopus laevis. These experiments will provide insight into how scaling occurs and contributes to intracellular morphogenesis and cell division, processes essential for viability and development, and defective in human diseases including cancer. PUBLIC HEALTH RELEVANCE: Size varies widely in biology at many levels: the animal, the cells that make up the animal, and the contents of the cells, but we don't understand how proper scaling occurs so that everything fits and functions properly. This proposal investigates how cells regulate the size of their internal structures, including the chromosomes that contain the genome and the mitotic spindle that separates the chromosomes to daughter cells, and addresses the importance of internal size regulation in the developing frog embryo. Since defects in cell division and size regulation are often observed in cancer, our research may shed light on the underlying mechanisms and lead to new therapies.