Equipment Supplement for Centromere Interactions and Meiotic Chromosome Segregation in Yeast

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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.