Equipment Supplement for Centromere Interactions and Meiotic Chromosome Segregation in Yeast

母项目总结（R 01 GM 138889） 在减数分裂前期I，同源染色体配对并通过 跨界车跨界车提供的连接有助于合作伙伴连接到 从纺锤体两侧放射状分布的微管。这种两极依恋 当伴侣动粒被拉向相反的两极时， 连接的微管这使得染色体对在纺锤体上保持平衡 而其他对则正确地附着在微管上。我们的家长格兰特 专注于着丝粒配对。当伴侣的着丝粒 染色体聚集在一起，然后以一种鲜为人知的方式附着在一起， 像交叉一样，允许同源配偶体正确地形成双极连接， 即使他们未能通过交叉连接。我们提出 在父项目中进行实验，以监测着丝粒的行为， 在减数分裂I中，染色体伴侣附着在纺锤体上。近一半的 该提案中的实验涉及活的减数分裂酵母细胞的持续成像。我们 已经提出了在细胞中对荧光标记的着丝粒进行成像， 安装在显微镜载物台上的微流体室。在这些实验中， 可以通过在所述双取向过程中流过所述感兴趣区域来询问它们在所述双取向过程中的作用。 细胞化合物，触发特定基因的表达或降解， 特定蛋白质此外，我们还建议衡量跨界车的能力， 同源配对染色体之间的着丝粒连接传递张力 同源动粒之间的联系这将使我们能够询问有关 连接的生物物理性质，做，或不，稳定双向微管 附件的权限.这些实验测量动粒的布朗振动， 确定它们之间连接的弹簧刚度的装置（由 交叉或着丝粒连接）（图1）。这些实验将共同定义为0.5 s 软弹簧 刚性弹簧 图1. 记波器 关于GFP- 标记双 定向 着丝粒 \图像 分析跟踪了 的质心 每个GFP 重点和 变化 距离 之间 质心 图片来自M。 迦纳 通过着丝粒配对过程在伴侣着丝粒之间形成的桥的分子基础， 着丝粒配对和交换形成的连接的生物物理特征。

Summary of parent project (R01GM138889) In prophase of meiosis I, homologous chromosomes pair and become connected by crossovers. The connection provided by crossovers helps the partners attach to microtubules that radiate from opposite sides of the spindle. This bipolar attachment is stabilized by tension as the partner kinetochores are tugged towards opposite poles by the connected microtubules. This allows the chromosome pair to remain poised at the spindle mid-zone while other pairs become correctly attached to microtubules. Our parent grant is focused on centromere-pairing. This occurs when the centromeres of the partner chromosomes come together and then become attached in a poorly understood way that, like crossovers, allows the homologous partners to correctly form bipolar attachments, even if they have failed to become attached by a crossover. We have proposed experiments in the parent project to monitor the behavior of centromeres as the chromosome partners become attached to the spindle in meiosis I. Nearly half of the experiments in the proposal involve sustained imaging of living meiotic yeast cells. We have proposed to image fluorescently-tagged centromeres, in cells that are sustained in microfluidics chambers mounted on the microscope stage. In these experiments genes of interest can be interrogated for their roles in the biorientation process by flowing over the cells compounds that trigger the expression of specific genes or the degradation of specific proteins. In addition, we propose to measure the ability of crossovers and centromere connections between homologous partner chromosomes to transmit tension between the homologous kinetochores. This will allow us to ask questions about the biophysical properties of connections that do, or do not, stabilize bioriented microtubule attachments. These experiments measure the Brownian vibration of the kinetochores as a means to determine the spring-stiffness of the connection between them (provided by a crossover or centromere connection) (Fig. 1). Together the experiments will define to0.5 s Soft Spring Stiff Spring Figure 1. Kymograph of GFP- tagged bi- oriented centromeres. \Image analysis tracks the centroid of each GFP focus and changes in distance between centroids. Image from M. Gardner. molecular basis of the bridge formed between partner centromeres by the centromere pairing process and the biophysical characteristics of connections formed by centromere pairing and crossing-over.