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Mapping biological circuit connectivity has revolutionized our understanding of structure-function relationships. Although connectomic analyses have primarily focused on neural systems, electrical connectivity within muscle mitochondrial networks was recently demonstrated to provide a rapid mechanism for cellular energy distribution. However, tools to evaluate organelle connectivity with high spatial fidelity within single cells are currently lacking. Here, we developed a framework to quantitatively assess mitochondrial network connectivity and interactions with cellular sites of energy storage, utilization, and calcium cycling in cardiac, oxidative, and glycolytic muscle. We demonstrate that mitochondrial network configuration, individual mitochondrial size and shape, and the junctions connecting mitochondria within each network are consistent with the differing contraction demands of each muscle type. Moreover, mitochondria-lipid droplet interaction analyses suggest that individual mitochondria within networks may play specialized roles regarding energy distribution and calcium cycling within the cell and reveal the power of connectomic analyses of organelle interactions within single cells. Assessing biological circuit connections in single cells has been intractable due to lack of appropriate tools. Here, Bleck et al. develop a method to assess mitochondrial network connectivity in muscle cells and observe clear differences consistent with differing energy requirements.

Mitochondria are shown to form a conductive pathway throughout the cell in the form of a proton motive force, and throughout this network, mitochondrial protein localization seems to be varied, allowing optimized generation and utilization of the mitochondrial membrane potential; the rapid energy distribution network, which depends on conduction rather than diffusion, could explain how the muscle can rapidly respond to energy demands. A cellular power grid How is energy distributed within the cell? In the skeletal muscle, energy distribution has been proposed to occur through metabolite-facilitated diffusion, although genetic evidence has raised questions about the importance of this mode of distribution. Using various forms of high-resolution microscopy, Robert Balaban and colleagues explore whether the mitochondria themselves — as well as actually generating the energy — also have a role in its distribution. They find that they do, by forming a conductive pathway throughout the cell in the form of a proton-motive force. Throughout this network, the mitochondrial protein localization seems to be varied, allowing optimized generation and utilization of the mitochondrial membrane potential. This energy distribution network, which depends on conduction rather than diffusion, is potentially extremely rapid, thereby enabling muscle to respond almost instantaneously to new energy demands. Intracellular energy distribution has attracted much interest and has been proposed to occur in skeletal muscle via metabolite-facilitated diffusion 1 , 2 ; however, genetic evidence suggests that facilitated diffusion is not critical for normal function 3 , 4 , 5 , 6 , 7 . We hypothesized that mitochondrial structure minimizes metabolite diffusion distances in skeletal muscle. Here we demonstrate a mitochondrial reticulum providing a conductive pathway for energy distribution, in the form of the proton-motive force, throughout the mouse skeletal muscle cell. Within this reticulum, we find proteins associated with mitochondrial proton-motive force production preferentially in the cell periphery and proteins that use the proton-motive force for ATP production in the cell interior near contractile and transport ATPases. Furthermore, we show a rapid, coordinated depolarization of the membrane potential component of the proton-motive force throughout the cell in response to spatially controlled uncoupling of the cell interior. We propose that membrane potential conduction via the mitochondrial reticulum is the dominant pathway for skeletal muscle energy distribution.

Transmission electron microscopy (TEM) is widely used as an imaging modality to provide high-resolution details of subcellular components within cells and tissues. Mitochondria and endoplasmic reticulum (ER) are organelles of particular interest to those investigating metabolic disorders. A straightforward method for quantifying and characterizing particular aspects of these organelles would be a useful tool. In this protocol, we outline how to accurately assess the morphology of these important subcellular structures using open source software ImageJ, originally developed by the National Institutes of Health (NIH). Specifically, we detail how to obtain mitochondrial length, width, area, and circularity, in addition to assessing cristae morphology and measuring mito/endoplasmic reticulum (ER) interactions. These procedures provide useful tools for quantifying and characterizing key features of sub-cellular morphology, leading to accurate and reproducible measurements and visualizations of mitochondria and ER.

Human movement occurs through contraction of the basic unit of the muscle cell, the sarcomere. Sarcomeres have long been considered to be arranged end-to-end in series along the length of the muscle into tube-like myofibrils with many individual, parallel myofibrils comprising the bulk of the muscle cell volume. Here, we demonstrate that striated muscle cells form a continuous myofibrillar matrix linked together by frequently branching sarcomeres. We find that all muscle cells contain highly connected myofibrillar networks though the frequency of sarcomere branching goes down from early to late postnatal development and is higher in slow-twitch than fast-twitch mature muscles. Moreover, we show that the myofibrillar matrix is united across the entire width of the muscle cell both at birth and in mature muscle. We propose that striated muscle force is generated by a singular, mesh-like myofibrillar network rather than many individual, parallel myofibrils. Skeletal muscle cells have long been considered to be made primarily of many individual, parallel myofibrils. Here, the authors show that the striated muscle contractile machinery forms a highly branched, mesh-like myofibrillar matrix connected across the entire length and width of the muscle cell.

Across different cell types and within single cells, mitochondria are heterogeneous in form and function. In skeletal muscle cells, morphologically and functionally distinct subpopulations of mitochondria have been identified, but the mechanisms by which the subcellular specialization of mitochondria contributes to energy homeostasis in working muscles remains unclear. Here, we discuss the current data regarding mitochondrial heterogeneity in skeletal muscle cells and highlight potential new lines of inquiry that have emerged due to advancements in cellular imaging technologies.

Mitochondrial networks provide coordinated energy distribution throughout muscle cells. However, pathways specifying mitochondrial networks are incompletely understood and it is unclear how they might affect contractile fiber-type. Here, we show that natural energetic demands placed on Drosophila melanogaster muscles yield native cell-types among which contractile and mitochondrial network-types are regulated differentially. Proteomic analyses of indirect flight, jump, and leg muscles, together with muscles misexpressing known fiber-type specification factor salm , identified transcription factors H15 and cut as potential mitochondrial network regulators. We demonstrate H15 operates downstream of salm regulating flight muscle contractile and mitochondrial network-type. Conversely, H15 regulates mitochondrial network configuration but not contractile type in jump and leg muscles. Further, we find that cut regulates salm expression in flight muscles and mitochondrial network configuration in leg muscles. These data indicate cell type-specific regulation of muscle mitochondrial network organization through evolutionarily conserved transcription factors cut , salm , and H15 . Mitochondrial networks are carefully positioned to facilitate energy distribution within muscle cells. Here they show that energetic demands and conserved transcription factors regulate mitochondrial network organization and contractile phenotypes independently in Drosophila.

Sustained muscle contraction occurs through interactions between actin and myosin filaments within sarcomeres and requires a constant supply of adenosine triphosphate (ATP) from nearby mitochondria. However, it remains unclear how different physical configurations between sarcomeres and mitochondria alter the energetic support for contractile function. Here, we show that sarcomere cross-sectional area (CSA) varies along its length in a cell type-dependent manner where the reduction in Z-disk CSA relative to the sarcomere center is closely coordinated with mitochondrial network configuration in flies, mice, and humans. Further, we find myosin filaments near the sarcomere periphery are curved relative to interior filaments with greater curvature for filaments near mitochondria compared to sarcoplasmic reticulum. Finally, we demonstrate variable myosin filament lattice spacing between filament ends and filament centers in a cell type-dependent manner. These data suggest both sarcomere structure and myofilament interactions are influenced by the location and orientation of mitochondria within muscle cells. How different physical configurations between sarcomeres and mitochondria alter energetic support for contractile function of skeletal muscle is not clear. Here the authors use advanced 3D imaging and analysis techniques to show how space is made for mitochondria within the tightly packed sarcomere networks of striated muscle cells.

Skeletal muscles play a central role in human movement through forces transmitted by contraction of the sarcomere. We recently showed that mammalian sarcomeres are connected through frequent branches forming a singular, mesh-like myofibrillar matrix. However, the extent to which myofibrillar connectivity is evolutionarily conserved as well as mechanisms which regulate the specific architecture of sarcomere branching remain unclear. Here, we demonstrate the presence of a myofibrillar matrix in the tubular, but not indirect flight (IF) muscles within Drosophila melanogaster . Moreover, we find that loss of transcription factor H15 increases sarcomere branching frequency in the tubular jump muscles, and we show that sarcomere branching can be turned on in IF muscles by salm -mediated conversion to tubular muscles. Finally, we demonstrate that neurochondrin misexpression results in myofibrillar connectivity in IF muscles without conversion to tubular muscles. These data indicate an evolutionarily conserved myofibrillar matrix regulated by both cell-type dependent and independent mechanisms. Recent work has shown that mammalian muscle cells are comprised of multiple branching sarcomeres, though how this connectivity is regulated has remained unknown. Here the authors show three different mechanisms which regulate connectivity of the muscle contractile apparatus.

High-resolution 3D images of organelles are of paramount importance in cellular biology. Although light microscopy and transmission electron microscopy (TEM) have provided the standard for imaging cellular structures, they cannot provide 3D images. However, recent technological advances such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM) provide the tools to create 3D images for the ultrastructural analysis of organelles. Here, we describe a standardized protocol using the visualization software, Amira, to quantify organelle morphologies in 3D, thereby providing accurate and reproducible measurements of these cellular substructures. We demonstrate applications of SBF-SEM and Amira to quantify mitochondria and endoplasmic reticulum (ER) structures.

In the original publication [...]