Mechanics of Microtubule Aster Growth and Positioning: Instrumentation Supplement

项目概要/摘要（来自母基金GM 135568） 微管细胞骨架对于组织细胞内部是至关重要的。有时， 在细胞周期中，这个网络采用星形阵列的形式，微管纤维从细胞中发出， 一个中心焦点和整个细胞的向外分支。重要的是，这个阵列的中心是 通常在细胞的几何中心附近发现，在那里它被锚定到细胞的表面。 原子核微管自身产生的力移动细胞的内部器官 在间期，因为网络也作为这些器官的支架， 附着，其位置决定了它们在细胞内的空间排列。在有丝分裂过程中， 定位细胞分裂机制并确定细胞分裂的最终位置 飞机 尽管星形细胞居中在所有细胞中可能都是至关重要的，但它在大细胞中尤其重要 受精后立即在这里，雄性原核（来自精子）必须横大 到达细胞中心并确定有丝分裂纺锤体形成位置的距离， 在随后的有丝分裂过程中分裂平面的定位。因此，此过程中的错误可能导致 错误的细胞分裂并对发育产生有害影响。准确地说， 微管aster产生并响应力移动到细胞中心残留物 无人回应在这项工作中，我们描述了一种新的 设计方法以克服用于研究的其他方法固有的现有限制 aster定位。这种方法依赖于使用光不稳定的水凝胶，其可以是透明的。 聚合并降解以响应暴露于特定波长的光。当 结合无细胞细胞质提取物， 和体积，该平台适合于使用标准的宽视场和共聚焦成像技术进行可视化。 光镜

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.