Equipment Supplement for Centromere Interactions and Meiotic Chromosome Segregation in Yeast

酵母着丝粒相互作用和减数分裂染色体分离的设备补充

基本信息

批准号：
10580231
负责人：
DEAN S DAWSON
金额：
$ 4.9万
依托单位：
OKLAHOMA MEDICAL RESEARCH FOUNDATION
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-01 至 2022-12-31
项目状态：
已结题

项目摘要

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 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. These experiments will allow us to measure the ability of crossovers and centromere connections between homologous partner chromosomes to transmit tension between the homologous kinetochores when they are attached to microtubules. Further, we will be able to ask questions about the biophysical properties of connections (e.g. their stiffness) that do, or do not, stabilize bioriented microtubule attachments. The method measures the Brownian vibration of the kinetochores. Kinetochore pairs with stiff connections vibrate less then pairs with soft connections. Thus, vibration rates can be converted into measurements pulling forces on the kinetochores (Fig. 1). The experiments will define the molecular basis of0.5 s Soft Spring Stiff Spring Figure 1. Kymograph of GFP-tagged bi- oriented centromeres. Images are acquired at 50 frames per second. Image analysis tracks the centroid of each GFP focus distance between centroids. Image from M. Gardner and colleagues). and changes in the bridge that is formed between partner centromeres during the centromere pairing process and the biophysical characteristics of the connections between centromeres provided by both centromere pairing and crossing-over.

父项目摘要 (R01GM138889) 在减数分裂 I 前期，同源染色体配对并连接通过交叉。交叉提供的连接有助于合作伙伴依附于从纺锤体相对两侧辐射的微管。该双极附件是当伴侣动粒被拉向相反的两极时，通过张力稳定通过连接的微管。这使得染色体对保持平衡纺锤体中间区域，而其他对则正确附着到微管上。我们的父母资助的重点是着丝粒配对。当着丝粒伴侣染色体聚集在一起，然后以不良的方式附着理解的方式，就像交叉一样，允许同源伙伴正确地形成双极依恋，即使他们未能通过交叉而建立依恋。我们在父项目中提出了实验来监控着丝粒作为染色体伴侣在减数分裂中附着在纺锤体上 I. 该提案中近一半的实验涉及活体减数分裂酵母的成像细胞。我们建议对细胞中荧光标记的着丝粒进行成像维持在安装在显微镜载物台上的微流体室中。在这些实验可以询问感兴趣的基因在生物取向中的作用通过流过细胞的化合物触发特定表达的过程基因或特定蛋白质的降解。这些实验将使我们能够测量同源体之间的交叉和着丝粒连接的能力伴侣染色体在同源着丝粒之间传递张力它们附着在微管上。此外，我们还可以询问有关稳定或不稳定的连接的生物物理特性（例如其刚度）双取向微管附件。该方法测量布朗振动动粒。具有刚性连接的动粒对振动小于具有刚性连接的动粒对软连接。因此，振动速率可以转换为拉力测量值着丝粒上的力（图1）。实验将定义0.5 s的分子基础软弹簧僵硬的弹簧图 1. 的Kymograph GFP 标记的双导向的着丝粒。图像是 50岁时获得每帧数第二。图像分析轨迹的质心每个 GFP 焦点之间的距离质心。图像来自M.加德纳和同事）。和变化着丝粒配对过程中伙伴着丝粒之间形成的桥梁着丝粒配对和着丝粒之间连接的生物物理特征交叉。