To address fundamental questions within mitochondrial biology, super-resolution microscopy has proven to be a truly indispensable tool. This chapter details the automated process for achieving efficient mtDNA labeling and quantifying nucleoid diameters in fixed, cultured cells using STED microscopy.
Live cell DNA synthesis is selectively labeled using the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU) in metabolic labeling procedures. Newly synthesized DNA, incorporating EdU, can be post-extraction or in fixed cellular contexts modified through copper-catalyzed azide-alkyne cycloaddition click chemistry reactions. This permits bioconjugation to various substrates including fluorescent molecules, which is advantageous for imaging. EdU labeling, frequently employed to examine nuclear DNA replication, can additionally be harnessed for the detection of organellar DNA synthesis occurring within the cytoplasm of eukaryotic cells. Employing fluorescent EdU labeling and super-resolution light microscopy, this chapter details the methods for studying mitochondrial genome synthesis in fixed, cultured human cells.
Maintaining adequate mitochondrial DNA (mtDNA) levels is crucial for a wide array of cellular biological functions, and its correlation with aging and various mitochondrial disorders is well-established. Defects within the core constituents of the mtDNA replication apparatus contribute to a reduction in the abundance of mtDNA. Along with other indirect mitochondrial elements, ATP concentration, lipid profile, and nucleotide sequence all contribute to the sustained integrity of mtDNA. Furthermore, the mitochondrial network evenly distributes mtDNA molecules. This uniform distribution pattern is vital for oxidative phosphorylation and ATP synthesis, and its disruption has been implicated in numerous diseases. Therefore, for a comprehensive understanding of mtDNA, its cellular context must be considered. We detail, in these protocols, the visualization of mitochondrial DNA (mtDNA) within cells via fluorescence in situ hybridization (FISH). Salivary biomarkers Specificity and sensitivity are both achieved through the direct targeting of the mtDNA sequence by fluorescent signals. The dynamic visualization of mtDNA-protein interactions is enabled by combining this mtDNA FISH method with immunostaining.
Mitochondrial DNA, or mtDNA, dictates the production of multiple varieties of ribosomal RNA (rRNA), transfer RNA (tRNA), and proteins that play key roles in the cellular respiratory process. MtDNA's integrity underpins mitochondrial processes, impacting numerous physiological and pathological systems in significant ways. The occurrence of mutations in mtDNA frequently correlates with the appearance of metabolic diseases and the aging process. The mitochondrial matrix contains hundreds of nucleoids, each harboring segments of mtDNA within human cells. Insight into how mitochondrial nucleoids are arranged and dispersed is vital to grasping mtDNA structure and functions. Insights into the regulation of mtDNA replication and transcription can be effectively gained by visualizing the distribution and dynamics of mtDNA within the mitochondrial compartment. Within this chapter, we delineate the application of fluorescence microscopy to observe mtDNA and its replication processes in both fixed and living cells, utilizing a range of labeling methods.
For the majority of eukaryotic organisms, mitochondrial DNA (mtDNA) sequencing and assembly can be initiated from total cellular DNA; however, investigating plant mtDNA proves more difficult, owing to its reduced copy number, less conserved sequence, and intricate structural makeup. Plant mitochondrial genome analysis, sequencing, and assembly are further complicated by the large nuclear genome sizes and high ploidy levels frequently found in many plant species. Therefore, a substantial boost in mitochondrial DNA is required. To extract and purify mitochondrial DNA (mtDNA), plant mitochondria are first isolated and subsequently purified. Relative mtDNA enrichment can be determined through quantitative PCR (qPCR), whereas the absolute enrichment is deduced from the proportion of sequencing reads that map to each of the three plant genomes. We detail methods for mitochondrial isolation and mtDNA extraction, applicable across diverse plant species and tissues, subsequently analyzing the degree of mtDNA enrichment achieved using various protocols.
Crucial to the investigation of organellar proteomes and the determination of the precise cellular locations of newly identified proteins, as well as evaluating distinct organelle activities, is the isolation of organelles removed from other cellular structures. The isolation of crude and highly pure mitochondria from the yeast Saccharomyces cerevisiae, along with methods for evaluating their functional integrity, is detailed in this protocol.
Stringent mitochondrial isolations are insufficient to eliminate persistent nuclear contamination, thus limiting direct, PCR-free mtDNA analysis. This method, originating in our laboratory, merges commercially available mtDNA extraction protocols with exonuclease treatment and size exclusion chromatography (DIFSEC). Small-scale cell cultures yield highly enriched mtDNA extracts via this protocol, exhibiting virtually no detectable nuclear DNA contamination.
The double-membrane-bound eukaryotic organelles, mitochondria, are involved in diverse cellular activities, encompassing the conversion of energy, apoptosis mechanisms, cell signaling cascades, and the biosynthesis of enzyme cofactors. Within the mitochondria resides its own genetic material, mtDNA, which dictates the composition of oxidative phosphorylation components, and also the ribosomal RNA and transfer RNA vital for mitochondrial protein synthesis. Studies of mitochondrial function have been greatly advanced by the capability of isolating highly purified mitochondria from their cellular origins. Centrifugation, with its differential forces, has long been a reliable method for the isolation of mitochondria. Following osmotic swelling and disruption of the cells, centrifugation in isotonic sucrose solutions is employed to separate the mitochondria from the remaining cellular components. Hepatic portal venous gas This principle forms the basis of a method we propose for the isolation of mitochondria from cultured mammalian cell lines. Mitochondrial purification by this method allows for further fractionation to study protein location, or for initiating the procedure for isolating mtDNA.
A detailed evaluation of mitochondrial function is unattainable without the use of meticulously prepared samples of isolated mitochondria. In order to obtain a good outcome, the protocol for mitochondria isolation should be quick, ensuring a reasonably pure, intact, and coupled pool. Using isopycnic density gradient centrifugation, we outline a fast and straightforward procedure for the purification of mammalian mitochondria. When isolating mitochondria with functional integrity from differing tissues, adherence to specific steps is paramount. Analyzing various aspects of the organelle's structure and function is facilitated by this suitable protocol.
In cross-national studies of dementia, functional limitations are evaluated. Our study focused on evaluating the performance of survey items pertaining to functional limitations, encompassing diverse geographical areas and cultural backgrounds.
To determine the associations between items of functional limitations and cognitive impairment, we utilized data from the Harmonized Cognitive Assessment Protocol Surveys (HCAP) in five countries (N=11250).
South Africa, India, and Mexico's performance for many items was outdone by the United States and England. The Community Screening Instrument for Dementia (CSID) displayed the least amount of variation in its items across nations, a standard deviation of 0.73 being observed. 092 [Blessed] and 098 [Jorm IQCODE] were detected; however, their association with cognitive impairment was the least powerful, with a median odds ratio of 223. In a blessed state, 301, and 275, which represents the Jorm IQCODE.
Cultural diversity in the reporting of functional limitations is likely to affect the performance of functional limitation items, thus influencing the interpretation of data from major investigations.
A substantial disparity in item performance was observed between different parts of the nation. ML-236B Items from the Community Screening Instrument for Dementia (CSID) exhibited a lower level of variability across countries, but their performance scores were weaker. Instrumental activities of daily living (IADL) displayed more diverse performance levels in comparison to activities of daily living (ADL) items. The nuanced perspectives on aging, varying significantly across cultures, must be considered. Functional limitations necessitate novel assessment approaches, as evident in the results.
The national average item performance masked considerable differences across the geographical spectrum. Despite lower performance, the Community Screening Instrument for Dementia (CSID) items demonstrated reduced variability across different countries. Variability in instrumental activities of daily living (IADL) scores was more pronounced compared to the variability in activities of daily living (ADL) scores. The differing expectations surrounding aging across cultures deserve consideration. These results strongly suggest the importance of novel assessment methods for functional limitations.
Adult human brown adipose tissue (BAT), recently rediscovered, along with work done on preclinical models, demonstrates a potential to provide a diversity of positive metabolic outcomes. Lowered plasma glucose, improved insulin sensitivity, and reduced susceptibility to obesity and its accompanying diseases are encompassed by these outcomes. Consequently, further investigation into this area could potentially illuminate strategies for therapeutically altering this tissue, thereby enhancing metabolic well-being. Researchers have reported an enhancement of mitochondrial respiration and an improvement in whole-body glucose homeostasis following the targeted deletion of the protein kinase D1 (Prkd1) gene in the fat cells of mice.