Identification of a Novel Chromosomal Passenger Complex and Its Unique Localization during Cytokinesis in Trypanosoma brucei

Ziyin Li,Arthur Günzl,Feixia Chu,Ching C Wang,Ju Huck Lee,Alma L Burlingame,Clotilde K S Carlow

doi:10.1371/journal.pone.0002354

Abstract

Aurora B kinase is a key component of the chromosomal passenger complex (CPC), which regulates chromosome segregation and cytokinesis. An ortholog of Aurora B was characterized in Trypanosoma brucei (TbAUK1), but other conserved components of the complex have not been found. Here we identified four novel TbAUK1 associated proteins by tandem affinity purification and mass spectrometry. Among these four proteins, TbKIN-A and TbKIN-B are novel kinesin homologs, whereas TbCPC1 and TbCPC2 are hypothetical proteins without any sequence similarity to those known CPC components from yeasts and metazoans. RNAi-mediated silencing of each of the four genes led to loss of spindle assembly, chromosome segregation and cytokinesis. TbKIN-A localizes to the mitotic spindle and TbKIN-B to the spindle midzone during mitosis, whereas TbCPC1, TbCPC2 and TbAUK1 display the dynamic localization pattern of a CPC. After mitosis, the CPC disappears from the central spindle and re-localizes at a dorsal mid-point of the mother cell, where the anterior tip of the daughter cell is tethered, to start cell division toward the posterior end, indicating a most unusual CPC-initiated cytokinesis in a eukaryote.

Highlights

Trypanosoma brucei is an ancient protozoan parasite and the causative pathogen of sleeping sickness in human and nagana in various livestock in sub-Saharan Africa
Identification of TbAUK1-associated proteins Since no close homologs of chromosomal passenger proteins other than Aurora B were identified in the T. brucei genome, we employed tandem affinity purification to isolate proteins that form complexes with TbAUK1 in vivo using the PTP tagging and purification system, which was recently established in T. brucei [24]
Tryptic digest of each protein band was analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) which resulted in the identification of four novel proteins, designated TbKIN-A, TbKIN-B, TbCPC1 and TbCPC2 for reasons stated below (KIN stands for kinesin whereas CPC represents chromosomal passenger complex), and a fifth protein, which turned out to be the known membrane protein

Summary

Introduction

Inhibition of mitosis in bloodstream cells prevents cytokinesis, but allows continuous progression of the nuclear cycle and organelle replication producing giant polyploid cells with multiple kinetoplasts, basal bodies and flagella [9,10,11]. Exploration of these unique features in T. brucei may facilitate our understanding of the molecular mechanisms which coordinate the nuclear and kinetoplast cycles, initiate cytokinesis in two different ways between two developmental stages, and drive a novel mode of cell division in a deeply-branching eukaryote

Methods

Results

Conclusion