Forces in Asymmetric Spindle Positioning in Mouse Oocytes

描述(由申请人提供)：本提案的总体目标是了解定位主轴的机构。在许多细胞中，纺锤体的位置决定了分裂平面的位置，因此对发育至关重要。主轴的定位 是一种力学现象，因此必须从力和分子活性两个方面来解释，但还没有对所涉及的力进行测量。我们最近开发了一个实验系统，它将允许我们测量小鼠卵母细胞中的相关力量。小鼠卵母细胞中纺锤体的定位过程尚不清楚，但已知它们依赖于肌动蛋白。我们将使用磁钳对小鼠卵母细胞减数分裂二中期组装的纺锤体施加作用力。我们将彻底确定定位纺锤体的力的特征，并使用分子扰动、激光消融和流变学测量来确定与纺锤体定位有关的过程的贡献：纺锤体与皮质的直接拴系、细胞质肌动蛋白网络产生的主动力以及肌动蛋白驱动的细胞质流动产生的力。这项工作将建立负责维持小鼠卵母细胞第二减数分裂纺锤体的生物物理机制，并可能为深入了解其他系统中纺锤体的定位提供依据。了解第二减数分裂纺锤体的定位机制可能具有医学意义，因为第二减数分裂纺锤体定位不当是导致受精率下降的已知原因，甚至在体外受精临床中被用作衡量卵母细胞质量的指标。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to understand the mechanisms which position the spindle. In many cells, the position of the spindle determines the position of the division plane, and is thus crucial for development. The positioning of the spindle is a mechanical phenomenon and thus must be explained in terms of both forces and molecular activities, but there have been no measurements of the forces involved. We have recently developed an experimental system which will allow us to measure the relevant forces in mouse oocytes. The processes which position the spindle in mouse oocytes are unclear, but it is known that they are actin dependent. We will use magnetic tweezers to exert forces on spindles assembled in metaphase of meiosis two in mouse oocytes. We will thoroughly characterize the forces that position the spindle and use molecular perturbations, laser ablation, and rheological measurements to determine the contribution of processes proposed to contribute to spindle positioning: direct tethering of the spindle to the cortex, active force production from the cytoplasmic actin meshwork, and forces generated by actin driven cytoplasmic streaming. This work will establish the biophysical mechanisms responsible for the maintenance of the second meiotic spindle in mouse oocytes and may provide insight into spindle positioning in other systems. Understanding the mechanisms of the positioning of the second meiotic spindle may have medical relevance because improper positioning of the second meiotic spindle is a known cause of reduced fertility and is even used as a measure of oocyte quality in in-vitro fertilizatio clinics.