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To spatially organize chromosomes, ring-shaped protein complexes including condensin and cohesin have been hypothesized to extrude DNA loops. Condensin has been shown to exhibit a DNA-translocating motor function, but extrusion has not been observed directly. Using single-molecule imaging, Ganji et al. visualized in real time a condensin-mediated, adenosine triphosphate-dependent, fast DNA loop extrusion process. Loop extrusion occurred asymmetrically, with condensin reeling in only one end of the DNA. These data provide unambiguous evidence of a loop extrusion mechanism for chromosome organization. Science , this issue p. 102 Single-molecule imaging supports a loop extrusion mechanism for the spatial organization of chromosomes. It has been hypothesized that SMC protein complexes such as condensin and cohesin spatially organize chromosomes by extruding DNA into large loops. We directly visualized the formation and processive extension of DNA loops by yeast condensin in real time. Our findings constitute unambiguous evidence for loop extrusion. We observed that a single condensin complex is able to extrude tens of kilobase pairs of DNA at a force-dependent speed of up to 1500 base pairs per second, using the energy of adenosine triphosphate hydrolysis. Condensin-induced loop extrusion was strictly asymmetric, which demonstrates that condensin anchors onto DNA and reels it in from only one side. Active DNA loop extrusion by SMC complexes may provide the universal unifying principle for genome organization.

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.

The recently developed ultrastructure expansion microscopy (U-ExM) technique allows to increase the spatial resolution within a cell or tissue for microscopic imaging through the physical expansion of the sample. In this study we validate the use of U-ExM in Trypanosoma brucei by visualizing the nucleus and kDNA as well as proteins of the cytoskeleton, the basal body, the mitochondrion and the ER. T. brucei is a unicellular flagellated protozoan parasite and the causative agent of human African sleeping sickness and Nagana in cattle.The highly polarized parasite cell body is about 25 μm in length and is shaped by the subpellicular microtubule corset. Its single flagellum emanates from the posterior part of the cell and is attached along the entire cell body. T. brucei The cell contains all typical organelles of eukaryotic cells including ER, Golgi and mitochondrion. Interestingly, Golgi and mitochondrion are single unit organelles in this protozoan parasite. The signature feature of trypanosomes is the single unit mitochondrial genome, the kinetoplast DNA (kDNA) that is organized in a complex structure of interlocked mini- and maxicircles. The kDNA is segregated during cell division by the tripartite attachment complex (TAC) that connects it via the mitochondrial membranes to the base of the flagellum.

The tripartite attachment complex (TAC) couples the segregation of the single unit mitochondrial DNA of trypanosomes with the basal body of the flagellum. Here we studied the architecture of the exclusion zone filament of the TAC that connects the basal body with the mitochondrial outer membrane. The only known component of the exclusion zone filaments is p197. Using genetical, biochemical and microscopical methods we show that p197 has three domains all of which are essential for mitochondrial DNA inheritance. The C-terminus of p197 interacts with the mature and pro-basal body whereas its N-terminus binds to the peripheral outer membrane protein TAC65. The large central region of p197 has a high α-helical content and likely acts as a flexible spacer. Replacement of endogenous p197 with a functional version containing N- and C-terminal epitope tags together with expansion microscopy demonstrates that p197 alone can bridge the approximately 170 nm gap between the basal body and the periphery of the outer membrane. This demonstrates the power of expansion microscopy which allows to localize distinct regions within the same molecule and suggests that p197 is the TAC subunit most proximal to the basal body. Competing Interest Statement The authors have declared no competing interest.

Proper mitochondrial genome inheritance is key for eukaryotic cell survival, however little is known about the molecular mechanism controlling this process. Trypanosoma brucei, a protozoan parasite, contains a singular mitochondrial genome aka kinetoplast DNA (kDNA). kDNA segregation requires anchoring of the genome to the basal body via the tripartite attachment complex (TAC). Several components of the TAC as well as their assembly have been described, it however remains elusive how the TAC connects to the kDNA. Here, we characterize the TAC associated protein TAP110 and for the first time use ultrastructure expansion microscopy in trypanosomes to reveal that TAP110 is the currently most proximal kDNA segregation factor. The kDNA proximal positioning is also supported by RNAi depletion of TAC102, which leads to loss of TAP110 at the TAC. Overexpression of TAP110 leads to expression level changes of several mitochondrial proteins and a delay in the separation of the replicated kDNA networks. In contrast to other kDNA segregation factors TAP110 remains only partially attached to the flagellum after DNAse and detergent treatment and can only be solubilized in dyskinetoplastic cells, suggesting that interaction with the kDNA might be important for stability of the TAC association. Furthermore, we demonstrate that the TAC, but not the kDNA, is required for correct TAP110 localization in vivo and suggest that TAP110 might interact with other proteins to form a >669 kDa complex. Competing Interest Statement The authors have declared no competing interest.