Mechanisms of Intracellular Scaling

描述（由申请人提供）：研究细胞内缩放的机制细胞大小在不同生物体之间以及在不同组织类型的同一生物体内和发育期间变化很大，对细胞器和内部结构提出了可变的代谢和功能要求。一个基本问题是如何调节细胞核、有丝分裂纺锤体和染色体等重要亚细胞成分以适应细胞大小差异。非洲爪蟾蛙提供了两个我们可以研究这个问题的生理背景。首先，我们可以将非洲爪蟾与较小的相关物种热带爪蟾进行比较，热带爪蟾产卵较小，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蟾胚胎发生的不同阶段，因为直径约1毫米的卵迅速分裂形成较小的卵裂球，到第15次分裂时，卵裂球缩小到40微米。我们的方法的一个独特的方面是从卵和胚胎中制备细胞质提取物，这些细胞质提取物在体外重演细胞器缩放，我们可以用它来识别大小变化背后的分子差异。我们的第一个具体目标集中在有丝分裂纺锤体，我们利用计算机模拟，以确定参数的微管动力学和组织，可能有助于纺锤体的大小和形态的物种之间的变化和发展过程中。这一目标还开发了新的方法来检查外部缩放机制，通过物理限制不同大小的液滴内的纺锤体组装反应，这将揭示是否存在外部缩放纺锤体或改变组装途径的尺寸阈值。目的2研究有丝分裂染色体的大小在发育过程中是如何改变的，以协调纺锤体的长度，使完全分离发生。在目标3中，我们开始通过研究非洲爪蟾发育过程中改变细胞核大小的后果来解决细胞器缩放的重要性。这些实验将深入了解缩放如何发生，并有助于细胞内形态发生和细胞分裂，对生存力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。

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.