A Comparative Proteomic Analysis Reveals a New Bi-Lobe Protein Required for Bi-Lobe Duplication and Cell Division in Trypanosoma brucei

Qing Zhou,Teck Kwang Lim,Yixin Chen,Jun Zhang,Cynthia Y He,Shaowei Li,Qingsong Lin,Ningshao Xia,Ladan Gheiratmand,Binghai Liu,Michael Polymenis

doi:10.1371/journal.pone.0009660

Abstract

A Golgi-associated bi-lobed structure was previously found to be important for Golgi duplication and cell division in Trypanosoma brucei. To further understand its functions, comparative proteomics was performed on extracted flagellar complexes (including the flagellum and flagellum-associated structures such as the basal bodies and the bi-lobe) and purified flagella to identify new bi-lobe proteins. A leucine-rich repeats containing protein, TbLRRP1, was characterized as a new bi-lobe component. The anterior part of the TbLRRP1-labeled bi-lobe is adjacent to the single Golgi apparatus, and the posterior side is tightly associated with the flagellar pocket collar marked by TbBILBO1. Inducible depletion of TbLRRP1 by RNA interference inhibited duplication of the bi-lobe as well as the adjacent Golgi apparatus and flagellar pocket collar. Formation of a new flagellum attachment zone and subsequent cell division were also inhibited, suggesting a central role of bi-lobe in Golgi, flagellar pocket collar and flagellum attachment zone biogenesis.

Highlights

Trypanosoma brucei is a parasitic pathogen causing sleeping sickness in human and Nagana in cattle, both imposing major economic burdens in Sub-Saharan Africa [1]. This single-celled parasite contains a single nucleus, a single kinetoplast, a single Golgi apparatus and a single flagellum which exits the cell through an adhesion zone named flagellar pocket collar (FPC) and remains attached to the cell body via a cytoplasmic structure known as the flagellum attachment zone (FAZ)
While TbCentrin4 labeling was readily identifiable at the tip of each flagellum in the extracted flagellar complex, no TbCentrin4 staining was detected in the detached flagella fraction (Fig. 1A, B)
The observation that the bi-lobed structure was tightly associated with the flagellum, even after detergent extraction and high-salt wash, suggests that it is a core component of the flagellar complex, which includes the basal bodies that seed flagellum growth and the FPC that is required for flagellar pocket biogenesis

Summary

Introduction

Trypanosoma brucei is a parasitic pathogen causing sleeping sickness in human and Nagana in cattle, both imposing major economic burdens in Sub-Saharan Africa [1] This single-celled parasite contains a single nucleus, a single kinetoplast (aggregate of mitochondrial DNA), a single Golgi apparatus and a single flagellum which exits the cell through an adhesion zone named flagellar pocket collar (FPC) and remains attached to the cell body via a cytoplasmic structure known as the flagellum attachment zone (FAZ). The bi-lobed structure was first discovered using a pantopic antibody against centrins [6], which are highly conserved calciumbinding proteins frequently found associated with microtubule organizing centers and required for their duplication [7,8] Both TbCentrin and TbCentrin ( known as TbCen1 [9]) are found localized to both the bi-lobed structure and the basal bodies in T. brucei. While depletion of TbCentrin inhibits the duplication/segregation of basal bodies, Golgi, kinetoplast, FAZ and final cell division, depletion of TbCentrin did not affect the duplication of organelles, but did disrupt the timing between nuclear division and the subsequent cytokinesis through an unknown mechanism [10]

Methods

Results

Conclusion