Perturbing the cryptococcus neoformans kinetochore : insights into kinetochore architecture, chromosome segregation and the spindle assembly checkpoint

Abstract

High fidelity chromosome segregation ensures proper inheritance of the genetic material, one of the most fundamental cellular processes. Chromosome segregation requires the linking of the chromosomes to a dynamic pulling and pushing force that enables equal division of chromosomes between the mother and daughter cells. The kinetochore is the chromosomal attachment site of this segregation machinery, the spindle, comprised of microtubules. Any errors occurring in this critical attachment are monitored with by the spindle assembly checkpoint (SAC). Several proteins, more than 80 known in yeast, assemble in a regulated manner to form the kinetochore on centromeric chromatin linking a chromosome to the dynamic plus ends of microtubules. Intriguingly, although the centromere DNA sequence in various eukaryotes is highly divergent, the basic architecture and function of the kinetochore remains conserved. However, the process of kinetochore assembly including localization patterns and localization dependency of various proteins at the kinetochore varies significantly. To understand the underlying principles involving the generation of this diversity, we studied the kinetochore assembly dynamics of the human fungal pathogen and an evolutionarily distinct basidiomycetous yeast Cryptococcus neoformans var. grubii. Utilizing a stringent controllable promoter of the GAL7 gene, we expressed fluorescently tagged kinetochore proteins and sought to address the essentiality, localization dependency and regulation of assembly among proteins that belong to different layers of the proposed tri-laminar kinetochore network. Our results suggest a putative kinetochore architecture wherein depleting the protein pools of an outer layer (farther from centromere DNA but nearer to microtubule ends) does not appreciably effect the localization of underlying layers, while disrupting the inner layers (nearer to centromere DNA) perturbs the whole multi-subunit assembly of the kinetochore. Yet, to our surprise we observed that centromeric localization of a middle kinetochore protein complex (Ndc80) is Cse4 (the inner kinetochore) independent, hinting at the existence of a parallel /bi-partite assembly via an unidentified protein. In addition to this finding it was observed that depletion of kinetochore proteins other than the fungal specific Dam1 complex did not result in the cell cycle arrest mediated by SAC.