PROJECT SUMMARY The alignment of chromosomes during metaphase is one of the most well conserved events in cell division, yet the molecular mechanisms that underlie this process are not understood. As cells progress from prometaphase into metaphase, chromosomes are aligned at the equator of the mitotic spindle through (A) the spatial restriction of their movements and (B) an increase in attachments between kinetochores and microtubules. The mechanisms that temporally coordinate the confinement of chromosome movements with increased attachments have not been identified. The kinesin-like motor Kif18A (kinesin-8) is required for the alignment of all chromosomes in mitotic cells, however, the motor's role in this process is not fully understood. During mitosis, Kif18A concentrates at the plus-ends of kinetochore-microtubules, which generate the primary forces that move chromosomes. The proposed studies will address key questions related to understanding how Kif18A functions mechanistically during chromosome alignment including: (A) how does Kif18A access the plus-ends of kinetochore-microtubules, which are embedded in a dense protein matrix, (B) how do Kif18A ensembles affect kinetochore-microtubule-generated forces, and (C) how does Kif18A increase attachments between kinetochores and microtubules. The primary goal of the proposed work is to answer these questions by determining how Kif18A's biochemical and biophysical properties translate to its complex effects on the movements and attachments of mitotic chromosomes. We hypothesize that the accumulation of Kif18A at kinetochore-microtubule plus-ends temporally coordinates the spatial confinement of chromosome movements with increased kinetochore-microtubule attachments through two independent mechanisms. We will use an approach combining single molecule biophysics, quantitative live cell imaging, and structural mutagenesis to test these predictions.