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.

Ultrastructure expansion microscopy in Trypanosoma brucei.

The recently developed ultrastructure expansion microscopy (U-ExM) technique allows us 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 measuring the expansion factors of several different compartments/organelles and thus verify the isotropic expansion of the cell. We furthermore demonstrate the use of this sample preparation protocol for future studies by visualizing the nucleus and kDNA, as well as proteins of the cytoskeleton, the basal body, the mitochondrion and the endoplasmic reticulum. Lastly, we discuss the challenges and opportunities of U-ExM.

Characterization of the novel mitochondrial genome segregation factor TAP110 in Trypanosoma brucei.

Proper mitochondrial genome inheritance is important for eukaryotic cell survival. Trypanosoma brucei, a protozoan parasite, contains a singular mitochondrial genome, the kinetoplast (k)DNA. The kDNA is anchored to the basal body via the tripartite attachment complex (TAC) to ensure proper segregation. Several components of the TAC have been described; however, the connection of the TAC to the kDNA remains elusive. Here, we characterize the TAC-associated protein TAP110. We find that both depletion and overexpression of TAP110 leads to a delay in the separation of the replicated kDNA networks. Proteome analysis after TAP110 overexpression identified several kDNA-associated proteins that changed in abundance, including a TEX-like protein that dually localizes to the nucleus and the kDNA, potentially linking replication and segregation in the two compartments. The assembly of TAP110 into the TAC region seems to require the TAC but not the kDNA itself; however, once TAP110 has been assembled, it also interacts with the kDNA. Finally, we use ultrastructure expansion microscopy in trypanosomes for the first time, and reveal the precise position of TAP110 between TAC102 and the kDNA, showcasing the potential of this approach.This article has an associated First Person interview with the first author of the paper.

Real-time imaging of DNA loop extrusion by condensin.

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.

Molecular Architecture of Contactin-associated Protein-like 2 (CNTNAP2) and Its Interaction with Contactin 2 (CNTN2).

Contactin-associated protein-like 2 (CNTNAP2) is a large multidomain neuronal adhesion molecule implicated in a number of neurological disorders, including epilepsy, schizophrenia, autism spectrum disorder, intellectual disability, and language delay. We reveal here by electron microscopy that the architecture of CNTNAP2 is composed of a large, medium, and small lobe that flex with respect to each other. Using epitope labeling and fragments, we assign the F58C, L1, and L2 domains to the large lobe, the FBG and L3 domains to the middle lobe, and the L4 domain to the small lobe of the CNTNAP2 molecular envelope. Our data reveal that CNTNAP2 has a very different architecture compared with neurexin 1α, a fellow member of the neurexin superfamily and a prototype, suggesting that CNTNAP2 uses a different strategy to integrate into the synaptic protein network. We show that the ectodomains of CNTNAP2 and contactin 2 (CNTN2) bind directly and specifically, with low nanomolar affinity. We show further that mutations in CNTNAP2 implicated in autism spectrum disorder are not segregated but are distributed over the whole ectodomain. The molecular shape and dimensions of CNTNAP2 place constraints on how CNTNAP2 integrates in the cleft of axo-glial and neuronal contact sites and how it functions as an organizing and adhesive molecule.