Deducing Kinetochore Protein Distributions along and Around a Microtubule Plus-End from FRET Measurements

Abstract

The eukaryotic kinetochore is a sophisticated motor that moves and segregates chromosomes during cell division. As a microtubule-based motor, the kinetochore is distinct from all other motor proteins and microtubule-associated proteins: it attaches end-on to ∼ 40 nm zone behind the plus-end of one or more microtubules, and it acts as a force coupler rather than an active force generator. These characteristics ensure that chromosome movement is tightly coupled with polymer growth and shortening at the plus-end. They also strongly suggest that kinetochore protein architecture, the distributions of the microtubule-binding kinetochore proteins along and around the microtubule plus-end and their dynamics, dictate the molecular mechanisms that generate movement. We have developed a FRET-based approach to determine the nanometer-scale distributions of multiple copies of the three principle microtubule-binding kinetochore complexes: Dam1, Ndc80, and Spc105. Sensitized emission measurements are carried out in metaphase and anaphase budding yeast (Saccharomyces cerevisiae) cells expressing kinetochore proteins labeled with Cerulean (donor) and Venus (acceptor) at desired locations. Preliminary measurements probing the distribution of the Ndc80 complex molecules show that the microtubule-binding head domains of adjacent Ndc80 molecules are ∼ five nm apart, but this distance increases significantly at the other end of the molecule. The Dam1 complex is located more than 10 nm away from these head domains of Ndc80. We are developing Monte Carlo approaches to simulate the energy transfer processes among a known number of donor and acceptor molecules using known protein structures of some of the kinetochore proteins and the cylindrical microtubule lattice. These simulations will identify molecular distributions consistent with the FRET data, and illuminate the structural basis for microtubule end-coupled movement of the kinetochore.