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Subcellular Specialization of 3D Mitochondrial Morphology and Mitochondria‐Lipid Droplet Interactivity is Associated with Proximity to Oxygen Supply in Muscle Cells

Advancements in imaging technology have considerably expanded our capacity to investigate cell biology, and our laboratory has recently employed high‐resolution 3D microscopy to explore the subcellular structures and organelle‐organelle interactions that support muscle metabolism. While this initial work has demonstrated that mitochondrial structure and mitochondria‐organelle interactivity are regulated according to muscle cell type, it remains unclear how cellular metabolism is supported by the subcellular specialization of mitochondria. Certainly, several subcellular clusters of mitochondria have been identified within muscle cells, and these groups of mitochondria are often categorized according to their proximity to other cellular components (i.e. “intermyofibrillar” or “paravascular”). However, it is unknown how 3D mitochondrial morphology and organelle interactivity is altered to support the varying functional demands of different subcellular regions. Here, we combine 3D focused‐ion beam scanning electron microscopy (FIB‐SEM) with machine learning programs to quantify individual mitochondrial structure and organelle interactivity within large subcellular volumes (~8,000 µm3) (Fig. 1a‐c). Using this high‐throughput approach, we evaluate over 700 individual mitochondria per cell and leverage the statistical power of this large data set to identify 3D spatial relationships among multiple subcellular structures. We find that mitochondrial surface area to volume ratio (15.5±0.25 µm‐1) is significantly correlated (R2= 0.39) to the minimal distance to capillary, but not distance to sarcolemma (R2= 0.02) (Fig. 1d). Further analysis revealed that mitochondria‐organelle interactivity is also closely related to subcellular localization. Specifically, mitochondria within close proximity (<5 µm) to capillaries (paravascular) are ~3‐fold more likely to make contact with lipid droplets compared to mitochondria located in other subcellular regions (Fig. 1e). Moreover, the paravascular mitochondria in contact with lipid droplets have 75% greater volume (1.4±0.07 µm3) compared to those not connected to lipid droplets (0.8±0.03 µm3). These preliminary analyses suggest that mitochondria in close proximity to the muscle oxygen supply, but not cell boundary, exhibit distinct 3D morphology and organelle interactivity patterns that may function to support metabolism and other cellular processes.