Tension and Force Generation in the Yeast Kinetochore (2015)

Undergraduate: Benjamin Badger

Faculty Advisor: Kerry Bloom
Department: Biology

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Effective chromosome segregation is not possible without forces from microtubules and chromatin acting on kinetochores. We introduce a novel tension sensor in the yeast: a F¿¿rster/Fluorescence Resonance Energy Transfer (FRET) probe consisting of two fluorophores connected by a flexible linker inserted into Ndc80, an evolutionarily conserved subunit of the Ndc80 complex that directly links microtubules to the rest of the kinetochore. By measuring the FRET between excitation and emission fluorophores the relative distance between the donor and emitter may be extrapolated and thus tension in the Ndc80 molecule (which is necessary to pull the fluorophores apart) may be inferred. FRET varies significantly during the cell cycle: we find that Ndc80 experiences the most tension during metaphase and the least in late anaphase. We also inserted the FRET probe at the C-terminal end of the Nuf2 subunit of the Ndc80 complex as a control. By measuring the energy transfer we find that there is much less separation compared to the intramolecular Ndc80 probe and moreover that the force does not appreciably differ throughout the cell cycle. To find how the high tension in metaphase created, we correlating the intensity of a tdTomato-tagged Stu2 protein (a component of the spindle that associates with polymerizing microtubules) to the FRET during metaphase, we find that polymerization of kinetochore microtubules contributes to the higher average tension found at Ndc80 during metaphase