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

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/10580231
• 关键词: Behavior monitoring; Cells; Centromere; Chromosome Pairing; Chromosome Segregation; Chromosomes; Congenital Abnormality; Equipment; Genes; Genetic Crossing Over; Germ Cells; Image; Image Analysis; Infertility; Kinetochores; Lead; Measurement; Measures; Meiosis; Mental Retardation; Methods; Microfluidics; Microscope; Microtubules; Molecular; Process; Prophase; Proteins; Role; Saccharomycetales; Side; Yeasts; biophysical properties; chromosome movement; experimental study; fluorescence imaging; interest; parent grant; parent project; prevent; vibration

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.

母项目总结（R01GM138889） 在减数分裂Ⅰ前期，同源染色体配对并连接 crossover的。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。这使得染色体对保持平衡， 纺锤体中间区，而其他对则正确地附着在微管上。我们 ParentGrant专注于着丝粒配对。这发生在当着丝粒的 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，像交叉，允许同源合作伙伴正确地 形成双极附件，即使它们未能通过交叉连接。 我们已经在父项目中提出了实验，以监控 在减数分裂中，作为染色体伴侣的着丝粒附着在纺锤体上 I.该提案中近一半的实验涉及活的减数分裂酵母的成像 细胞我们已经提出在细胞中对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 实验中，可以询问感兴趣的基因在双向取向中的作用， 这一过程是通过流过细胞的化合物来触发特定的 基因或特定蛋白质的降解。这些实验将使我们能够 测量同源染色体之间的交叉和着丝粒连接的能力 伴侣染色体传递张力之间的同源动粒时， 它们附着在微管上。此外，我们将能够提出问题， 连接的生物物理特性（例如，它们的刚度）是否稳定 双向微管附着该方法测量的布朗振动 动粒具有刚性连接的动粒对比具有刚性连接的动粒对振动少。 软连接。因此，振动率可以转换成测量拉 动粒上的力（图1）。实验将确定0.5 s的分子基础 软弹簧 刚性弹簧 图1. 记波器 GFP标记的双 定向 着丝粒 图像 在50岁时获得 帧 一下图像 分析跟踪了 的质心 每个GFP焦点 距离 质心图像 从麻省加德纳 同事们）。 和变化 在着丝粒配对过程中，在伴侣着丝粒之间形成的桥， 生物物理特性的连接着丝粒提供的两个着丝粒配对和 交换