The Role of Microtubule-Associated Proteins During Microtubule Nucleation and Organization

Abstract

Successful mitotic division is fundamental to life. During mitosis, the cytoskeletal network is reorganized to form the bipolar spindle: microtubules extend from microtubule-organizing centers to attach to and separate chromosomes. The mitotic spindle is formed by 1) transport of existing microtubules and 2) targeted nucleation of new microtubules. These processes are governed by the microtubule-organizing center and proteins associated with the microtubule. Here I investigate how the MTOCs and the microtubule associated proteins work together to nucleate microtubules. Using a turbidity assay, I find that XMAP215 is a classically defined microtubule nucleator, as it reduces the nucleation lag seen in bulk tubulin assembly. I further characterize the role of XMAP215 in nucleation, finding its ability to nucleate correlates with its ability to elongate existing microtubules and this is true in both the presence and absence of gamma-tubulin. Then using a novel tool, I define the role of three microtubule associated proteins (Stu2, Bim1 and Bik1) and two motors (Kip3 and Vik1) during assembly of microtubule arrays from an ectopic microtubule nucleation site expressed in vivo. I found that Stu2 and Bim1 are required for microtubule nucleation from the ectopic site, whereas Bik1, Kip3 and Vik1 are required for organization of the microtubule arrays.