DNA segregation in mitochondria and beyond: insights from the trypanosomal tripartite attachment complex.

The tripartite attachment complex (TAC) of the single mitochondrion of trypanosomes allows precise segregation of its single nucleoid mitochondrial genome during cytokinesis. It couples the segregation of the duplicated mitochondrial genome to the segregation of the basal bodies of the flagella. Here, we provide a model of the molecular architecture of the TAC that explains how its eight essential subunits connect the basal body, across the mitochondrial membranes, with the mitochondrial genome. We also discuss how the TAC subunits are imported into the mitochondrion and how they assemble to form a new TAC. Finally, we present a comparative analysis of the trypanosomal TAC with open and closed mitotic spindles, which reveals conserved concepts between these diverse DNA segregation systems.

Single p197 molecules of the mitochondrial genome segregation system of Trypanosoma brucei determine the distance between basal body and outer membrane.

The tripartite attachment complex (TAC) couples the segregation of the single unit mitochondrial DNA of trypanosomes with the basal body (BB) of the flagellum. Here, we studied the architecture of the exclusion zone filament (EZF) of the TAC, the only known component of which is p197, that connects the BB with the mitochondrial outer membrane (OM). We show that p197 has three domains that are all essential for mitochondrial DNA inheritance. The C terminus of p197 interacts with the mature and probasal body (pro-BB), whereas its N terminus binds to the peripheral OM protein TAC65. The large central region of p197 has a high α-helical content and likely acts as a flexible spacer. Ultrastructure expansion microscopy (U-ExM) of cell lines exclusively expressing p197 versions of different lengths that contain both N- and C-terminal epitope tags demonstrates that full-length p197 alone can bridge the ∼270-nm distance between the BB and the cytosolic face of the OM. Thus U-ExM allows the localization of distinct domains within the same molecules and suggests that p197 is the TAC subunit most proximal to the BB. In addition, U-ExM revealed that p197 acts as a spacer molecule, as two shorter versions of p197, with the repeat domain either removed or replaced by the central domain of the Trypanosoma cruzi p197 ortholog reduced the distance between the BB and the OM in proportion to their predicted molecular weight.

p166 links membrane and intramitochondrial modules of the trypanosomal tripartite attachment complex.

The protist parasite Trypanosoma brucei has a single mitochondrion with a single unit genome termed kinetoplast DNA (kDNA). Faithfull segregation of replicated kDNA is ensured by a complicated structure termed tripartite attachment complex (TAC). The TAC physically links the basal body of the flagellum with the kDNA spanning the two mitochondrial membranes. Here, we characterized p166 as the only known TAC subunit that is anchored in the inner membrane. Its C-terminal transmembrane domain separates the protein into a large N-terminal region that interacts with the kDNA-localized TAC102 and a 34 aa C-tail that binds to the intermembrane space-exposed loop of the integral outer membrane protein TAC60. Whereas the outer membrane region requires four essential subunits for proper TAC function, the inner membrane integral p166, via its interaction with TAC60 and TAC102, would theoretically suffice to bridge the distance between the OM and the kDNA. Surprisingly, non-functional p166 lacking the C-terminal 34 aa still localizes to the TAC region. This suggests the existence of additional TAC-associated proteins which loosely bind to non-functional p166 lacking the C-terminal 34 aa and keep it at the TAC. However, binding of full length p166 to these TAC-associated proteins alone would not be sufficient to withstand the mechanical load imposed by the segregating basal bodies.

Oxidative damage of SP-D abolishes control of eosinophil extracellular DNA trap formation.

The asthmatic airways are highly susceptible to inflammatory injury by air pollutants such as ozone (O3 ), characterized by enhanced activation of eosinophilic granulocytes and a failure of immune protective mechanisms. Eosinophil activation during asthma exacerbation contributes to the proinflammatory oxidative stress by high levels of nitric oxide (NO) production and extracellular DNA release. Surfactant protein-D (SP-D), an epithelial cell product of the airways, is a critical immune regulatory molecule with a multimeric structure susceptible to oxidative modifications. Using recombinant proteins and confocal imaging, we demonstrate here that SP-D directly bound to the membrane and inhibited extracellular DNA trap formation by human and murine eosinophils in a concentration and carbohydrate-dependent manner. Combined allergic airway sensitization and O3 exposure heightened eosinophilia and nos2 mRNA (iNOS) activation in the lung tissue and S-nitrosylation related de-oligomerisation of SP-D in the airways. In vitro reproduction of the iNOS action led to similar effects on SP-D. Importantly, S-nitrosylation abolished the ability of SP-D to block extracellular DNA trap formation. Thus, the homeostatic negative regulatory feedback between SP-D and eosinophils is destroyed by the NO-rich oxidative lung tissue environment in asthma exacerbations.

Photoconvertible pathogen labeling reveals nitric oxide control of Leishmania major infection in vivo via dampening of parasite metabolism.

The immune system can control infectious diseases through different modes of action, including direct killing or spatial confinement. Addressing how the immune system impacts pathogen biology in vivo has remained challenging. We expressed a photoconvertible fluorescent protein in pathogens in order to track their spatial dissemination in infected tissues. In addition, we developed the fluorescence recovery after photoconversion (FRAC) method in order to probe pathogen metabolic activity in vivo. Combining these two approaches in the context of Leishmania major infection of mice and pharmacologically inhibiting iNOS, we found that nitric oxide produced during the immune response to L. major reduces the metabolic activity of intracellular parasites without necessarily exerting direct killing. We propose that this chronic pressure on pathogen proliferation represents a sublethal mode of control required for ultimately resolving the infection. The ability to probe pathogen biology in response to immune defense mechanisms in vivo should create opportunities for better dissecting host-pathogen interactions.