Addressing fundamental questions within mitochondrial biology has been significantly advanced by the utility of super-resolution microscopy. An automated system for efficient mtDNA labeling and quantification of nucleoid diameter in fixed cultured cells, using STED microscopy, is described in this chapter.
The application of the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU) in metabolic labeling allows for selective labeling of DNA synthesis in live cells. Covalent modification of newly synthesized EdU-containing DNA is achievable after extraction or in fixed cells through the application of copper-catalyzed azide-alkyne cycloaddition click chemistry reactions. This allows bioconjugation with various substrates, such as fluorophores, for imaging studies. The EdU labeling procedure, routinely used to investigate nuclear DNA replication, is also capable of identifying the synthesis of organellar DNA within the cytoplasm of eukaryotic organisms. In fixed cultured human cells, this chapter elucidates the methods for applying fluorescent EdU labeling to investigate mitochondrial genome synthesis, employing super-resolution light microscopy.
Cellular biological processes necessitate proper mitochondrial DNA (mtDNA) levels, and its association with aging and numerous mitochondrial disorders is a well-known fact. Faults in the critical components of the mitochondrial DNA replication machinery cause a decline in the levels of mtDNA. The upkeep of mtDNA is not solely determined by direct mechanisms; various other indirect mitochondrial contexts, including ATP concentration, lipid composition, and nucleotide makeup, play a crucial role. Beyond that, there is an even distribution of mtDNA molecules within the mitochondrial network. This uniform distribution pattern, critical for oxidative phosphorylation and ATP production, is linked to numerous diseases when disrupted. Therefore, for a comprehensive understanding of mtDNA, its cellular context must be considered. This document elucidates the procedures for observing mtDNA in cells, employing fluorescence in situ hybridization (FISH). SF2312 With the fluorescent signals directly aimed at the mtDNA sequence, both high sensitivity and precision are achieved. Visualization of mtDNA-protein interactions and their dynamics can be achieved by combining this mtDNA FISH method with immunostaining procedures.
Ribosomal RNAs, transfer RNAs, and proteins of the respiratory chain are all specified by the mitochondrial genetic code, housed within mtDNA. Mitochondrial DNA integrity is essential for mitochondrial function and plays a critical role in a wide array of physiological and pathological processes. Genetic alterations in mitochondrial DNA can lead to the emergence of metabolic diseases and the progression of aging. Mitochondrial nucleoids, numbering in the hundreds, encapsulate the mtDNA present within the human mitochondrial matrix. Understanding the dynamic distribution and organization of nucleoids within mitochondria is crucial for comprehending mtDNA structure and function. Therefore, the visualization of mtDNA's distribution and dynamics inside mitochondria offers a valuable means of exploring the regulation of mtDNA replication and transcription. This chapter details fluorescence microscopy methods for observing mtDNA and its replication in both fixed and live cells, employing various labeling strategies.
Beginning with total cellular DNA, mitochondrial DNA (mtDNA) sequencing and assembly is usually feasible for most eukaryotic species. Nevertheless, the study of plant mtDNA is considerably more complex because of its low copy number, limited sequence conservation, and intricate structural layout. 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. As a result, the amplification of mitochondrial DNA is critical. To extract and purify mitochondrial DNA (mtDNA), plant mitochondria are first isolated and subsequently purified. By leveraging quantitative PCR (qPCR), the relative enrichment of mtDNA can be evaluated, while the absolute enrichment can be established by measuring the proportion of next-generation sequencing reads aligning with the respective genomes within the plant cell. Different plant species and tissues are addressed in this study concerning methods of mitochondrial purification and mtDNA extraction, which are further compared to evaluate mtDNA enrichment efficiency.
The isolation of organelles, excluding other cellular components, is essential for scrutinizing organellar protein profiles and the precise subcellular placement of newly identified proteins, and critically important for evaluating specific organelle functions. A procedure for obtaining both crude and highly pure mitochondrial fractions from Saccharomyces cerevisiae, coupled with techniques for evaluating the isolated organelles' functionality, is presented.
Persistent nuclear genome contaminants, even after meticulous mitochondrial isolation, restrict the direct PCR-free analysis of mtDNA. We present a laboratory-created method that merges established, commercially available mtDNA isolation procedures, exonuclease treatment, and size exclusion chromatography (DIFSEC). This protocol facilitates the isolation of mtDNA extracts from small-scale cell cultures, characterized by their high enrichment and near-absence of nuclear DNA contamination.
Eukaryotic mitochondria, double membrane-bound, participate in multifaceted cellular functions, encompassing the conversion of energy, apoptosis regulation, cellular communication, and the synthesis of enzyme cofactors. The genome of mitochondria, mtDNA, specifies the components of the oxidative phosphorylation system, and provides the ribosomal and transfer RNA required for their translation within the confines of the mitochondria. The process of isolating highly purified mitochondria from cells has proven instrumental in numerous studies pertaining to mitochondrial function. Centrifugation, with its differential forces, has long been a reliable method for the isolation of mitochondria. Centrifugation in isotonic sucrose solutions separates mitochondria from the rest of the cell's components after the cells are osmotically swollen and disrupted. Tooth biomarker Mitochondria isolation from cultured mammalian cell lines is achieved via a method that capitalizes on this principle. Using this purification method, mitochondria can be fractionated further to examine the cellular localization of proteins, or be employed as a preliminary stage in the purification of mtDNA.
A detailed evaluation of mitochondrial function is unattainable without the use of meticulously prepared samples of isolated mitochondria. To achieve optimal results, a quick mitochondria isolation protocol should produce a reasonably pure, intact, and coupled pool. This paper details a rapid and simple method for purifying mammalian mitochondria, employing the technique of isopycnic density gradient centrifugation. To ensure the isolation of functional mitochondria from various tissues, a specific set of procedures must be followed. This protocol proves suitable for the investigation of various facets of organelle structure and function.
Dementia measurement across countries is contingent upon assessing functional impairments. We investigated the effectiveness of survey items measuring functional limitations, focusing on the variation in cultures and geographic settings.
The Harmonized Cognitive Assessment Protocol Surveys (HCAP), encompassing data from five countries (total N=11250), were analyzed to determine quantitative associations between items representing functional limitations and cognitive impairment.
When evaluated against the performance in South Africa, India, and Mexico, numerous items in the United States and England performed better. 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. The presence of 092 [Blessed] and 098 [Jorm IQCODE] displayed a link to cognitive impairment, yet exhibited the weakest correlation strength; the median odds ratio [OR] was 223. 301, a blessed status, and 275, representing the Jorm IQCODE.
Functional limitations' varying cultural reporting norms probably impact the performance of functional limitation items, potentially altering the interpretation of findings from substantial studies.
The performance of items varied significantly from one region of the country to another. Postmortem biochemistry Despite exhibiting less cross-national variability, items from the Community Screening Instrument for Dementia (CSID) yielded lower performance. Activities of daily living (ADL) items displayed less variability in performance when compared to instrumental activities of daily living (IADL). Cultural expectations concerning older adults exhibit significant diversity, and this needs to be factored in. In light of the results, novel approaches to assessing functional limitations are indispensable.
A substantial discrepancy in item effectiveness was noted between different parts of the nation. Although the Community Screening Instrument for Dementia (CSID) items demonstrated less variability across countries, their performance scores were lower. The performance of instrumental activities of daily living (IADL) showed greater variance than that of activities of daily living (ADL). One should account for the diverse societal expectations surrounding the experiences of older adults across cultures. A significant implication of these results is the need for novel approaches in assessing 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. Lower plasma glucose, improved insulin sensitivity, and a reduced chance of obesity and its co-morbidities are integral components of the observed improvements. Given this, continued research on this topic could uncover ways to therapeutically modify this tissue, leading to improved metabolic health. Experiments have shown that eliminating the protein kinase D1 (Prkd1) gene within the mouse adipose tissue elevates mitochondrial activity and improves the body's handling of glucose.