Reorganization of mitochondria-organelle interactions during postnatal development in skeletal muscle.

Skeletal muscle cellular development requires the integrated assembly of mitochondria and other organelles adjacent to the sarcomere in support of muscle contractile performance. However, it remains unclear how interactions among organelles and with the sarcomere relates to the development of muscle cell function. Here, we combine 3D volume electron microscopy, proteomic analyses, and live cell functional imaging to investigate the postnatal reorganization of mitochondria-organelle interactions in skeletal muscle. We show that while mitochondrial networks are disorganized and loosely associated with the contractile apparatus at birth, contact sites among mitochondria, lipid droplets and the sarcoplasmic reticulum are highly abundant in neonatal muscles. The maturation process is characterized by a transition to highly organized mitochondrial networks wrapped tightly around the muscle sarcomere but also to less frequent interactions with both lipid droplets and the sarcoplasmic reticulum. Concomitantly, expression of proteins involved in mitochondria-organelle membrane contact sites decreases during postnatal development in tandem with a decrease in abundance of proteins associated with sarcomere assembly despite an overall increase in contractile protein abundance. Functionally, parallel measures of mitochondrial membrane potential, NADH redox status, and NADH flux within intact cells revealed that mitochondria in adult skeletal muscle fibres maintain a more activated electron transport chain compared with neonatal muscle mitochondria. These data demonstrate a developmental redesign reflecting a shift from muscle cell assembly and frequent inter-organelle communication toward a muscle fibre with mitochondrial structure, interactions, composition and function specialized to support contractile function. KEY POINTS: Mitochondrial network organization is remodelled during skeletal muscle postnatal development. The mitochondrial outer membrane is in frequent contact with other organelles at birth and transitions to more close associations with the contractile apparatus in mature muscles. Mitochondrial energy metabolism becomes more activated during postnatal development. Understanding the developmental redesign process within skeletal muscle cells may help pinpoint specific areas of deficit in muscles with developmental disorders.

High resolution spatial investigation of intracellular oxygen in muscle cells.

Molecular oxygen (O 2 ) is one of the most functionally relevant metabolites. O 2 is essential for mito-chondrial aerobic respiration. Changes in O 2 affect muscle metabolism and play a critical role in the maintenance of skeletal muscle mass, with lack of sufficient O 2 resulting in detrimental loss of muscle mass and function. How exactly O 2 is used by muscle cells is less known, mainly due to the lack of tools to address O 2 dynamics at the cellular level. Here we discuss a new imaging method for the real time quantification of intracellular O 2 in muscle cells based on a genetically encoded O 2 -responsive sensor, Myoglobin-mCherry. We show that we can spatially resolve and quantify intracellular O 2 concentration in single muscle cells and that the spatiotemporal O 2 gradient measured by the sensor is linked to, and reflects, functional metabolic changes occurring during the process of muscle differentiation. Highlights: Real time quantitation of intracellular oxygen with spatial resolutionIdentification of metabolically active sites in single cellsOxygen metabolism is linked to muscle differentiation.

Mitochondrial respiration reduces exposure of the nucleus to oxygen.

The endosymbiotic theory posits that ancient eukaryotic cells engulfed O2-consuming prokaryotes, which protected them against O2 toxicity. Previous studies have shown that cells lacking cytochrome c oxidase (COX), required for respiration, have increased DNA damage and reduced proliferation, which could be improved by reducing O2 exposure. With recently developed fluorescence lifetime microscopy-based probes demonstrating that the mitochondrion has lower [O2] than the cytosol, we hypothesized that the perinuclear distribution of mitochondria in cells may create a barrier for O2 to access the nuclear core, potentially affecting cellular physiology and maintaining genomic integrity. To test this hypothesis, we utilized myoglobin-mCherry fluorescence lifetime microscopy O2 sensors without subcellular targeting ("cytosol") or with targeting to the mitochondrion or nucleus for measuring their localized O2 homeostasis. Our results showed that, similar to the mitochondria, the nuclear [O2] was reduced by ∼20 to 40% compared with the cytosol under imposed O2 levels of ∼0.5 to 18.6%. Pharmacologically inhibiting respiration increased nuclear O2 levels, and reconstituting O2 consumption by COX reversed this increase. Similarly, genetic disruption of respiration by deleting SCO2, a gene essential for COX assembly, or restoring COX activity in SCO2-/- cells by transducing with SCO2 cDNA replicated these changes in nuclear O2 levels. The results were further supported by the expression of genes known to be affected by cellular O2 availability. Our study reveals the potential for dynamic regulation of nuclear O2 levels by mitochondrial respiratory activity, which in turn could affect oxidative stress and cellular processes such as neurodegeneration and aging.

Intracellular imaging of metmyoglobin and oxygen using new dual purpose probe EYFP-Myoglobin-mCherry.

The biological relevance of nitric oxide (NO) and reactive oxygen species (ROS) in signaling, metabolic regulation, and disease treatment has become abundantly clear. The dramatic change in NO/ROS processing that accompanies a changing oxygen landscape calls for new imaging tools that can provide cellular details about both [O2 ] and the production of reactive species. Myoglobin oxidation to the met state by NO/ROS is a known sensor with absorbance changes in the visible range. We previously employed Förster resonance energy transfer to read out the deoxygenation/oxygenation of myoglobin, creating the subcellular [O2 ] sensor Myoglobin-mCherry. We now add the fluorescent protein EYFP to this sensor to create a novel probe that senses both met formation, a proxy for ROS/NO exposure, and [O2 ]. Since both proteins are present in the construct, it can also relieve users from the need to measure fluorescence lifetime, making [O2 ] sensing available to a wider group of laboratories.

Developing Analysis Protocols for Monitoring Intracellular Oxygenation Using Fluorescence Lifetime Imaging of Myoglobin-mCherry.

Oxygen (O2) is a critical metabolite for cellular function as it fuels aerobic cellular metabolism; further, it is a known regulator of gene expression. Monitoring oxygenation within cells and organelles can provide valuable insights into how O2, or lack thereof, both influences and responds to cell processes. In recent years, fluorescence lifetime imaging microscopy (FLIM) has been used to track several probe concentration independent intracellular phenomena, such as pH, viscosity, and, in conjunction with Förster resonance energy transfer (FRET), protein-protein interactions. Here, we describe methods for synthesizing and expressing the novel FLIM-FRET intracellular O2 probe Myoglobin-mCherry (Myo-mCherry) in cultured cell lines, as well as acquiring FLIM images using a laser scanning confocal microscope configured for two-photon excitation and a time-correlated single photon counting (TCSPC) module. Finally, we provide step-by-step protocols for FLIM analysis of Myo-mCherry using the commercial software SPCImage and conversion of fluorescence lifetime values in each pixel to apparent intracellular oxygen partial pressures (pO2).

Single cell-based fluorescence lifetime imaging of intracellular oxygenation and metabolism.

Oxidation-reduction chemistry is fundamental to the metabolism of all living organisms, and hence quantifying the principal redox players is important for a comprehensive understanding of cell metabolism in normal and pathological states. In mammalian cells, this is accomplished by measuring oxygen partial pressure (pO2) in parallel with free and enzyme-bound reduced nicotinamide adenine dinucleotide (phosphate) [H] (NAD(P)H) and flavin adenine dinucleotide (FAD, a proxy for NAD+). Previous optical methods for these measurements had accompanying problems of cytotoxicity, slow speed, population averaging, and inability to measure all redox parameters simultaneously. Herein we present a Förster resonance energy transfer (FRET)-based oxygen sensor, Myoglobin-mCherry, compatible with fluorescence lifetime imaging (FLIM)-based measurement of nicotinamide coenzyme state. This offers a contemporaneous reading of metabolic activity through real-time, non-invasive, cell-by-cell intracellular pO2 and coenzyme status monitoring in living cells. Additionally, this method reveals intracellular spatial heterogeneity and cell-to-cell variation in oxygenation and coenzyme states.