Mechanics of Microtubule Aster Growth and Positioning: Instrumentation Supplement

项目摘要/摘要(来自母公司赠款GM135568) 微管细胞骨架对组织细胞内部起着至关重要的作用。有时，在 细胞周期，这个网络采用星形阵列的形式，微管纤维从 一个中心焦点，并在整个细胞内向外分支。重要的是，该数组的中心是 通常在单元格的几何中心附近找到，在那里它锚定到 原子核。由微管本身产生的力移动细胞的内部器官。 在间期，因为网络也是一个脚手架，这些器官 附加时，其位置决定了它们在单元格内的空间排列。在有丝分裂过程中，相似的力量 定位细胞分裂机械并确定细胞分裂的最终位置 飞机。 尽管在所有单元格中居中可能都很关键，但在大型单元格中尤其重要 在受精后立即进行。在这里，男性原核(来自精子)必须横切大 到达细胞中心并确定有丝分裂纺锤体形成位置的距离和 分裂平面在随后的有丝分裂过程中的定位。因此，此过程中的错误可能会导致 错误的细胞分裂，对发育有有害影响。准确地说， 微管蛋白产生并响应向细胞中心移动的力残留物 无人接听。在这项工作中，我们描述了一个新的应用和继续开发的 旨在克服用于研究的其他方法固有的现有限制的方法 Aster定位。这种方法依赖于光不稳定水凝胶的使用，这可以是 由于暴露在特定波长的光下而聚合和降解。什么时候 与无细胞的细胞质提取物相结合，它提供了对细胞质形状的精细控制 和体积，该平台可使用标准广域和共焦进行可视化 光学显微镜。

PROJECT SUMMARY/ABSTRACT (from parent grant GM135568) The microtubule cytoskeleton is critical for organization the insides of cells. At times during the cell cycle, this network adopts the form of an astral array with microtubule fibers emanating from a central focus and branching outward throughout the cell. Importantly, the center of this array is typically found near the geometric center of the cell where it is anchored to the surface of the nucleus. Forces generated by the microtubules themselves move the internal organs of the cell during interphase, and because the network also serves as a scaffold upon which these organs attach, its position dictates their spatial arrangement within the cell. During mitosis similar forces positioning the cell division machinery and establish the eventual location of the cell division plane. Though aster centering is likely critical in all cells, it is particularly relevant in large cells immediately after fertilization. Here, the male pronucleus (from the sperm) must transverse large distances to reach the cell center and establish the location of mitotic spindle formation and division plane positioning during the subsequent mitosis. Thus, errors in this process can lead to erroneous cell division and have deleterious effects on development. Precisely how the microtubule aster generates and responds to forces to move to the cell center remains unanswered. In this work, we describe the application and continued development of a new approach engineered to overcome existing limitations inherent to other approaches used to study aster positioning. This approach relies on the use of photo-labile hydrogels, which can be polymerized and degraded in response to exposure to specific wavelengths of light. When combined with cell-free cytoplasmic extracts, it provides exquisite control of cytoplasmic shape and volume in a platform that is amenable to visualization using standard wide-field and confocal light microscopy.