Packaging DNA into condensed structures is integral to the transmission of genomes. The mammalian mitochondrial genome (mtDNA) is a high copy, maternally inherited genome in which mutations cause a variety of multisystem disorders. In all eukaryotic cells, multiple mtDNAs are packaged with protein into spheroid bodies called nucleoids, which are the fundamental units of mtDNA segregation. The mechanism of nucleoid formation, however, remains unknown. Here, we show that the mitochondrial transcription factor TFAM, an abundant and highly conserved High Mobility Group box protein, binds DNA cooperatively with nanomolar affinity as a homodimer and that it is capable of coordinating and fully compacting several DNA molecules together to form spheroid structures. We use noncontact atomic force microscopy, which achieves near cryo-electron microscope resolution, to reveal the structural details of protein-DNA compaction intermediates. The formation of these complexes involves the bending of the DNA backbone, and DNA loop formation, followed by the filling in of proximal available DNA sites until the DNA is compacted. These results indicate that TFAM alone is sufficient to organize mitochondrial chromatin and provide a mechanism for nucleoid formation. INTRODUCTIONMutations affecting mitochondrial oxidative phosphorylation cause a variety of multisystem disorders (DiMauro and Schon, 2003;Taylor and Turnbull, 2005) with an estimated incidence of 1:5000 live births (Thorburn, 2004). The majority of these mutations are found in the mitochondrial genome (mtDNA), which encodes the core hydrophobic proteins involved in oxidative phosphorylation and some of the molecules required for their expression, such as tRNAs and rRNAs. The mutant mtDNA genomes generally coexist alongside wild-type copies in affected patients, a situation referred to as heteroplasmy. The segregation pattern of heteroplasmic DNAs in different tissues is an important determinant of the severity of the clinical phenotype. To develop an animal model of heteroplasmy, our laboratory generated mice containing polymorphisms in otherwise normal mtDNA, and we identified three nuclear quantitative trait loci that affect mtDNA segregation in liver, spleen, and kidney (Jenuth et al., 1997;Battersby et al., 2003). The selection of one variant of the mtDNA over another could be explained by differences in their organization or packaging.Mammalian mtDNA is packaged in protein-DNA complexes termed nucleoids, which can be visualized as small submitochondrial bodies in the matrix (Nass, 1969). The nucleoid contains two to seven genomes, depending on cell type, and it is present as 450 -800 distinct foci in cultured cells (Nass, 1969;Iborra et al., 2004;Legros et al., 2004). Several mammalian mitochondrial nucleoid proteins have been identified after enrichment by sedimentation and immunoprecipitation (Wang and Bogenhagen, 2006;He et al., 2007). However, other than the components of the mitochondrial replisome and transcription apparatus, the only proteins that have been fo...
Photoswitchable fluorescent probes are central to localization-based super-resolution microscopy. Among these probes, fluorescent proteins are appealing because they are genetically encoded. Moreover, the ability to achieve a one to one labeling ratio between the fluorescent protein and the protein of interest makes them attractive for quantitative single molecule counting. The percentage of fluorescent protein that is photoactivated into a fluorescently detectable form (i.e. photoactivation efficiency) plays a critical role in properly interpreting the quantitative information. It is important to characterize the photoactivation efficiency at the single molecule level to replicate the conditions used in super-resolution imaging. Here, we used the human Glycine receptor expressed in Xenopus oocytes and stepwise photobleaching or single molecule counting-PALM to determine the percentage of photoactivated fluorescent protein for mEos2, mEos3.1, mEos3.2, Dendra2, mClavGR2, mMaple, PA-GFP and PA-mCherry. This analysis provides important information that must be considered when using these fluorescent proteins in quantitative super-resolution microscopy.
In Alzheimer disease and related disorders, the microtubule-associated protein tau aggregates and forms cytoplasmic lesions that impair neuronal physiology at many levels. In addition to affecting the host neuron, tau aggregates also spread to neighboring, recipient cells where the misfolded tau aggregates, in a manner similar to prions, actively corrupt the proper folding of soluble tau, and thereby impair cellular functions. One vehicle for the transmission of tau aggregates are secretory nanovesicles known as exosomes. Here, we established a simple model of a neuronal circuit using a microfluidics culture system in which hippocampal neurons A and B were seeded into chambers 1 and 2, respectively, extending axons via microgrooves in both directions and thereby interconnecting. This system served to establish two models to track exosome spreading. In the first model, we labeled the exosomal membrane by coupling tetraspanin CD9 with either a green or red fluorescent tag. This allowed us to reveal that interconnected neurons exchange exosomes only when their axons extend in close proximity. In the second model, we added exosomes isolated from the brains of tau transgenic rTg4510 mice (i.e. exogenous, neuron A-derived) to neurons in chamber 1 (neuron B) interconnected with neuron C in chamber 2. This allowed us to demonstrate that a substantial fraction of the exogenous exosomes were internalized by neuron B and passed then on to neuron C. This transportation from neuron B to C was achieved by a mechanism that is consistent with the hijacking of secretory endosomes by the exogenous exosomes, as revealed by confocal, super-resolution and electron microscopy. Together, these findings suggest that fusion events involving the endogenous endosomal secretory machinery increase the pathogenic potential and the radius of action of pathogenic cargoes carried by exogenous exosomes.Electronic supplementary materialThe online version of this article (10.1186/s40478-018-0514-4) contains supplementary material, which is available to authorized users.
The subunit stoichiometry of heteromeric glycine-gated channels (GlyRs) determines fundamental properties of these key inhibitory neurotransmitter receptors; however the ratio of α1 to β-subunits per receptor remains controversial. We used single molecule imaging and stepwise photobleaching in Xenopus oocytes to directly determine the subunit stoichiometry of a glycine receptor to be 3α1:2β. This approach allowed us to determine the receptor stoichiometry in mixed populations consisting of both heteromeric and homomeric channels, additionally revealing the quantitative proportions for the two populations.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.