Browsing by Subject "Ultrastructure"

Skeletal muscle is the most abundant tissue in the body, accounting for up to 40-50% of total bodyweight. Regeneration of this tissue is dependent on skeletal muscle stem cells, which are termed satellite cells (SCs) based on their anatomical position between the basal lamina and plasma membrane of muscle fibers. SCs exist under homeostatic conditions in a reversible G0 phase of the cell cycle. Quiescent SCs are recognized by the expression of the paired box 7 (Pax7) transcription factor, in the absence of other myogenic transcription factors such as myoblast determination protein 1 (MyoD) or myogenin (MyoG). Quiescent SCs are metabolically less active with a low oxygen consumption rate. They contain less ATP and have few mitochondria with a low membrane potential in comparison to activated SCs. Activated SCs enter the cell cycle and start to proliferate, undergoing metabolic rewiring to primarily utilize glycolysis for energy production. During early activation, there is an increase in mitochondrial content and ATP production, while the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) increase later during active proliferation. Although similar population dynamics, SCs are a heterogenous population of stem cells, with differences in the expression of notch receptors, stem cell markers, ATP and mitochondrial content, which in turn affect the myogenic potential of the cells. Mitochondria are semi-autonomous, double membrane organelles with various regulation within the cell, such as calcium homeostasis, apoptosis, production of metabolic intermediates, reactive oxygen species (ROS) metabolism, and ATP generation through oxidative phosphorylation (oxphos). Differentiation of various other stem cell types is accompanied by an increase in both mitochondrial content and oxidative phosphorylation, with ultrastructural changes that favour this shift in metabolism. The aim of this thesis was to quantify the ultrastructural changes that occur within SC mitochondria during the early proliferative phase, and to implement a method of Correlative Light and Electron Microscopy (CLEM) for identifying and studying subpopulations of SCs. After isolation and during early activation, SCs contain few mitochondria with a diffuse ultrastructure. Classification of the observed mitochondrial phenotypes revealed heterogeneity both within and between timepoints. During later phases of proliferation, there was an increase in the proportion of mature mitochondria, with an increase in cristae density and a decrease in cristae width. Utilizing genetically modified R26-Snaptag-Omp25 x PAX7CreErt2 mice in which recombination with tamoxifen initiates the expression of mitochondrial outer membrane protein 25 (omp25) bound with a SNAP-tag, allowed for specific and temporal labelling of SC mitochondria by fluorescent SNAP substrates. Performing CLEM on fluorescently labelled SC mitochondria enabled their identification during transmission electron microscopy (TEM). In addition to this, temporal labelling of pre-existing (old) and newly imported (young) omp25 revealed a few cells that contained more old mitochondria, with the cristae density being higher in these. While this indicates a correlation between mitochondrial content and ultrastructure within subpopulations of SCs, further studies are needed to validate these early observations.