Molecular model of the mitochondrial genome segregation machinery in Trypanosoma brucei.

In almost all eukaryotes, mitochondria maintain their own genome. Despite the discovery more than 50 y ago, still very little is known about how the genome is correctly segregated during cell division. The protozoan parasite Trypanosoma brucei contains a single mitochondrion with a singular genome, the kinetoplast DNA (kDNA). Electron microscopy studies revealed the tripartite attachment complex (TAC) to physically connect the kDNA to the basal body of the flagellum and to ensure correct segregation of the mitochondrial genome via the basal bodies movement, during the cell cycle. Using superresolution microscopy, we precisely localize each of the currently known TAC components. We demonstrate that the TAC is assembled in a hierarchical order from the base of the flagellum toward the mitochondrial genome and that the assembly is not dependent on the kDNA itself. Based on the biochemical analysis, the TAC consists of several nonoverlapping subcomplexes, suggesting an overall size of the TAC exceeding 2.8 mDa. We furthermore demonstrate that the TAC is required for correct mitochondrial organelle positioning but not for organelle biogenesis or segregation.

iMEM: Isolation of Plasma Membrane for Cryoelectron Microscopy.

The plasma membrane and the cell cortex are essential parts of the eukaryotic cell. The plasma membrane delimitates the cell and mediates communication with the outside. The cell cortex is the submembrane cytoskeleton shaping the cell and is able to reorganize for the passage of material. To study events at and near the plasma membrane, cryoelectron microscopy (cryo-EM) may be used. Most intact cells are too thick for direct cryo-EM imaging. Generating cell-free membrane patches could be a means to study features at the plasma membrane. Here we present an unroofing method, termed iMEM (isolation of membrane patches for cryo-EM) where the plasma membrane is isolated directly on an EM grid. The in situ isolation of membrane patches has several advantages: it is a one-step procedure providing a higher throughput than focused-ion beam cryomilling. It enables the time-precise control over biochemical events before cryofixation.

Mitochondrial growth during the cell cycle of Trypanosoma brucei bloodstream forms.

Mitochondrial organelles need to be replicated during cell division. Many aspects of this process have been studied in great detail, however the actual size increase and the position of organelle growth are less well understood. We use the protozoan parasite Trypanosoma brucei that contains a single mitochondrion to study organelle biogenesis by fluorescence microscopy. From the analysis of more than 1000 T. brucei bloodstream form cells of a nonsynchronous population we conclude that the mitochondrial network mostly grows from two areas along the main organelle axis, posterior and anterior of the nucleus. Loops and branches from these two areas eventually fuse to build a complex network. Together with the appearance of the division fold in the posterior part of the cell, pruning of the mitochondrial network and finally separation into the two daughter cells occurs. Overall organelle biogenesis is not continuous during cell growth and occurs mostly in the last part of the cell cycle. Furthermore, using 3D STED super resolution microscopy we reconstruct the volume of the organelle and characterize the region where the mitochondrial genome is positioned by serial block face scanning electron microscopy.