A High-OrderTrans-Membrane Structural Linkage Is Responsible for Mitochondrial Genome Positioning and Segregation by Flagellar Basal Bodies in Trypanosomes

Abstract

In trypanosomes, the large mitochondrial genome within the kinetoplast is physically connected to the flagellar basal bodies and is segregated by them during cell growth. The structural linkage enabling these phenomena is unknown. We have developed novel extraction/fixation protocols to characterize the links involved in kinetoplast-flagellum attachment and segregation. We show that three specific components comprise a structure that we have termed the tripartite attachment complex (TAC). The TAC involves a set of filaments linking the basal bodies to a zone of differentiated outer and inner mitochondrial membranes and a further set of intramitochondrial filaments linking the inner face of the differentiated membrane zone to the kinetoplast. The TAC and flagellum-kinetoplast DNA connections are sustained throughout the cell cycle and are replicated and remodeled during the periodic kinetoplast DNA S phase. This understanding of the high-order trans-membrane linkage provides an explanation for the spatial position of the trypanosome mitochondrial genome and its mechanism of segregation. Moreover, the architecture of the TAC suggests that it may also function in providing a structural and vectorial role during replication of this catenated mass of mitochondrial DNA. We suggest that this complex may represent an extreme form of a more generally occurring mitochondrion/cytoskeleton interaction. INTRODUCTIONThe African trypanosome Trypanosoma brucei belongs to a large order of pathogenic and free-living flagellated protozoa designated the kinetoplastida. The majority of these organisms possess a single mitochondrion containing a structural mass of proteins and catenated circular DNA molecules, the kinetoplast. The kinetoplast is essential for survival and cyclical transmission of the parasite between mammalian host and tsetse fly. Early light microscope descriptions of trypanosomes recognized the precise location of the kinetoplast in proximity to the base of the flagellum (Robertson, 1912(Robertson, , 1913. The positions of the flagellum and kinetoplast have become the central features in classifying the different life cycle stages of kinetoplastids (Hoare and Wallace, 1966;Vickerman, 1976). Electron microscopy revealed the kinetoplast to be a highly complex disk-shaped structure located within a distended portion of the mitochondrial matrix adjacent to the flagellum basal bodies (Vickerman, 1973). The T. brucei kinetoplast consists of multiple copies of topologically interlocked circular DNA molecules termed maxicircles and minicircles, along with specific kinetoplast proteins. The maxicircle composition of the T. brucei network is 25-50 copies and represents 10% of the network mass (each maxicircle is 20 kb and all have identical DNA sequence). Maxicircles encode the mitochondrial proteins and some guide RNAs (gRNAs), whereas the minicircles are present in several thousand copies (1 kb in length), are heterogeneous in sequence, and encode gRNAs. The gRNAs are essential for the RNA editing process whereby the maxic...

The Trypanosoma brucei Cyclin, CYC2, Is Required for Cell Cycle Progression through G1 Phase and for Maintenance of Procyclic Form Cell Morphology

“…Interestingly, the cell cycle arrest seen with RNAi of CYC2 appears earlier in the cell cycle than that seen previously with either aphidicolin or acriflavine treatment of procyclic trypanosomes (6,25). Aphidicolin inhibits nuclear DNA synthesis but not kinetoplast DNA synthesis.…”

“…Treatment of procyclic trypanosomes with aphidicolin leads to an accumulation of cells in S phase, and at later time points 1N2K cells (with incompletely replicated nuclear DNA) divide, without undergoing mitosis, to give rise to a 1N1K daughter and a zoid (0N1K) (6). Acriflavine treatment of procyclic trypanosomes selectively inhibits kinetoplast replication (25), but basal body duplication and segregation are unaffected. The single kinetoplast associates with one of the two basal bodies (in the majority of cases, with the basal body subtending the old flagellum), and the cell cycle continues, leading to a cell division that results in asymmetric segregation of kinetoplast DNA, with the formation of a 1N1K daughter and a dyskinetoplastic cell (1N0K).…”

Centrin Gene Disruption Impairs Stage-specific Basal Body Duplication and Cell Cycle Progression in Leishmania

“…The kinetoplast is attached to the basal body through a tripartite attachment complex (30). Arresting the basal body segregation by treating Trypanosoma brucei cells with ansamitocin, an antimicrotubule drug, no segregation in the kinetoplasts was found (27).…”

The direct route: a simplified pathway for protein import into the mitochondrion of trypanosomes

“…Besides its clinical importance T. brucei has attracted interest because of its rich biology in general, and the many intriguing features of its mitochondrion in particular (Box 1). Trypanosoma brucei has only a single mitochondrion whose genome is physically connected to the flagellar basal bodies [5] by way of proteinaceous connections that span the two mitochondrial membranes (Figure 1). Whereas most organisms have a single type of mitochondrial chromosome, the mitochondrial DNA in T. brucei consists of two genetic elements, the maxicircles and the minicircles, which are topologically intertwined.…”