PROJECT SUMMARY An exquisite molecular machine, the mitotic spindle, separates duplicated chromosomes during cell division. To uncover how it operates, we are reconstituting spindle activities using purified components and developing biophysical tools to directly manipulate and track their dynamics at the single molecule level. The accuracy of eukaryotic chromosome segregation is incredible and depends critically on kinetochores, which are multiprotein complexes that maintain strong yet dynamic attachments between chromosomes and spindle microtubules in order to produce force and move the chromosomes. Kinetochores also carry out vital regulatory activities such as distinguishing and selectively stabilizing proper attachments, releasing erroneous attachments, and generating diffusible ‘wait’ signals to delay mitosis until proper attachments are achieved. Our reconstitution- based approach has enabled the first direct measurements of many fundamental kinetochore activities and direct tests of long-standing hypotheses from cytological studies, proving for example that tension stabilizes kinetochore attachments in at least two different ways. Our work has also revealed previously unrecognized kinetochore behaviors and motivated new genetic, cytological, and structural studies. Our preliminary data now reveal another striking and previously unrecognized behavior: yeast kinetochores grip the sides of microtubules with directionally asymmetric strength – much more strongly when pulled toward plus ends than when pulled toward minus ends. This asymmetric grip strength is likely to promote proper attachments in early mitosis when kinetochores first bind the sides of microtubules. We will uncover its molecular basis, test its conservation in human kinetochores, determine how it relates to the catch bond-like activity we previously uncovered and to the widely studied kinase-based mechanism for correcting erroneous attachments, and ultimately develop a new integrated explanation