The mitochondrial DNA of Trypanosoma brucei is organized in a complex structure called the kinetoplast. In this study, we define the complete kinetoplast duplication cycle in T. brucei based on three-dimensional reconstructions from serial-section electron micrographs. This structural model was enhanced by analyses of the replication process of DNA maxi-and minicircles. Novel insights were obtained about the earliest and latest stages of kinetoplast duplication. We show that kinetoplast S phase occurs concurrently with the repositioning of the new basal body from the anterior to the posterior side of the old flagellum. This emphasizes the role of basal body segregation in kinetoplast division and suggests a possible mechanism for driving the rotational movement of the kinetoplast during minicircle replication. Fluorescence in situ hybridization with minicircleand maxicircle-specific probes showed that maxicircle DNA is stretched out between segregated minicircle networks, indicating that maxicircle segregation is a late event in the kinetoplast duplication cycle. This new view of the complexities of kinetoplast duplication emphasizes the dependencies between the dynamic remodelling of the cytoskeleton and the inheritance of the mitochondrial genome.Faithful inheritance of the genome through successive generations is of fundamental importance to every cell, and sophisticated mechanisms have evolved to perform this function. Trypanosoma brucei is a protozoan parasite of the order Kinetoplastida. During their life cycle, these parasites alternate between the tsetse fly vector and their mammalian host. Differentiation between these forms is accompanied by dramatic changes in metabolism and mitochondrial morphology. Procylic (insect form) T. brucei cells have a single mitochondrion in the form of an extended tubular network. Since there is only one mitochondrion per cell, the duplication cycles of the nucleus and mitochondrion must be coordinated. The mitochondrial genome of Kinetoplastida forms an intricately structured nucleoid, localized within a specialized region of the mitochondrial matrix, a structure termed the kinetoplast. The kinetoplast DNA (kDNA) is composed of two classes of DNA rings. A few dozen maxicircles (23 kb) encode genes for a few mitochondrial proteins (e.g., subunits of respiratory complexes) and rRNA. Several thousand minicircles (ϳ1 kb) encode a few hundred guide RNAs (usually three per minicircle) that direct the RNA editing (uridylate insertion and deletion) of cryptic maxicircle transcripts; thus, kDNA has many different minicircle species varying in copy number. The most remarkable feature of kDNA is that all of the mini-and maxicircles are topologically linked to form a single planar network. In situ, the T. brucei kDNA network is condensed into a compact disk, approximately 100 nm thick, with a diameter of ϳ650 nm, which is anchored to the base of the flagellum through cytoskeletal filaments (36).Once per cell cycle the kDNA network is replicated and positioned within the cell such tha...

The African sleeping sickness parasite Trypanosoma brucei evades the host immune system through antigenic variation of its variant surface glycoprotein (VSG) coat. Although the T. brucei genome contains ∼1500 VSGs, only one VSG is expressed at a time from one of about 15 subtelomeric VSG expression sites (ESs). For antigenic variation to work, not only must the vast VSG repertoire be kept silent in a genome that is mainly constitutively transcribed, but the frequency of VSG switching must be strictly controlled. Recently it has become clear that chromatin plays a key role in silencing inactive ESs, thereby ensuring monoallelic expression of VSG. We investigated the role of the linker histone H1 in chromatin organization and ES regulation in T. brucei. T. brucei histone H1 proteins have a different domain structure to H1 proteins in higher eukaryotes. However, we show that they play a key role in the maintenance of higher order chromatin structure in bloodstream form T. brucei as visualised by electron microscopy. In addition, depletion of histone H1 results in chromatin becoming generally more accessible to endonucleases in bloodstream but not in insect form T. brucei. The effect on chromatin following H1 knock-down in bloodstream form T. brucei is particularly evident at transcriptionally silent ES promoters, leading to 6–8 fold derepression of these promoters. T. brucei histone H1 therefore appears to be important for the maintenance of repressed chromatin in bloodstream form T. brucei. In particular H1 plays a role in downregulating silent ESs, arguing that H1-mediated chromatin functions in antigenic variation in T. brucei.

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Kinetoplastids are a group of parasites that includes several medically-important species. These human-infective species are transmitted by insect vectors in which the parasites undergo specific developmental transformations. For each species, this includes a stage in which parasites adhere to insect tissue via a hemidesmosome-like structure. Although this structure has been described morphologically, it has never been molecularly characterized. We are using Crithidia fasciculata , an insect parasite that produces large numbers of adherent parasites inside its mosquito host, as a model kinetoplastid to investigate both the mechanism of adherence and the signals required for differentiation to an adherent form. An advantage of C . fasciculata is that adherent parasites can be generated both in vitro , allowing a direct comparison to cultured swimming forms, as well as in vivo within the mosquito. Using RNAseq, we identify genes associated with adherence in C . fasciculata . As almost all of these genes have orthologs in other kinetoplastid species, our findings may reveal shared mechanisms of adherence, allowing investigation of a crucial step in parasite development and disease transmission. In addition, dual-RNAseq allowed us to explore the interaction between the parasites and the mosquito. Although the infection is well-tolerated, anti-microbial peptides and other components of the mosquito innate immune system are upregulated. Our findings indicate that C . fasciculata is a powerful model system for probing kinetoplastid-insect interactions.