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.

家长项目摘要（R01GM138889） 在减数分裂的预言中，同源染色体对并连接 通过跨界。交叉提供的连接可帮助合作伙伴附加 从主轴的相对侧辐射的微管。这个双极的附件是 由于伴侣的动物学将稳定稳定在张力上 通过连接的微管。这允许染色体对保持 纺锤体中区，而其他对正确连接到微管上。我们的 父母赠款的重点是丝粒对。当centromeres的中心 伴侣染色体聚集在一起，然后依附在一个很差的 理解的方式，就像跨界一样，允许同源伙伴正确 即使未能通过交叉附着，也形成双极附件。 我们已经在父项目中提出了实验，以监视 染色体伴侣随着染色体伴侣的固定在减数分裂的纺锤体上 I.提案中近一半的实验涉及活物质酵母的成像 细胞。我们提议在荧光标记的centromeres中成像， 在安装在显微镜阶段的微流体室中维持。在这些 可以质疑感兴趣的实验基因在生物学中的作用 通过流过细胞化合物来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 测量同源性之间的跨界和丝粒连接的能力 伴侣染色体以在同源动力学之间传递张力时 它们附着在微管上。此外，我们将能够询问有关 连接的生物物理特性（例如它们的刚度）具有或不稳定 生物导向的微管附件。该方法测量了布朗的振动 动力学。动力学对与紧密连接的振动较小，而不是对 软连接。因此，振动速率可以转换为测量值 动力学上的力（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。 Kymograph of GFP标记的双 定向 中心。 图像是 在50岁时获得 帧 /帧 第二。图像 分析轨道 质心 每个GFP焦点 之间的距离 质心。图像 来自M. Gardner 和同事）。 和变化 在中心粒配对过程中，伴侣中心粒之间形成的桥 丝粒配对和 越过。