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MicroRNAs (miRNAs) are small noncoding endogenous RNAs, typically 21-23 nucleotides long, that regulate gene expression, usually post-transcriptionally, by binding to the 3'-UTR of target mRNA, thus blocking translation. The expression of several miRNAs is significantly altered during cardiac hypertrophy, myocardial ischemia, fibrosis, heart failure, and other cardiac myopathies. Recent studies have implicated miRNA-9 (miR-9) in myocardial hypertrophy. However, a detailed mechanism remains obscure. In this study, we have addressed the roles of miR-9 in muscle development and function using a genetically tractable model system, the indirect flight muscles (IFMs) of Drosophila melanogaster Bioinformatics analysis identified 135 potential miR-9a targets, of which 27 genes were associated with Drosophila muscle development. Troponin-T (TnT) was identified as major structural gene target of miR-9a. We show that flies overexpressing miR-9a in the IFMs have abnormal wing position and are flightless. These flies also exhibit a loss of muscle integrity and sarcomeric organization causing an abnormal muscle condition known as "hypercontraction." Additionally, miR-9a overexpression resulted in the reduction of TnT protein levels while transcript levels were unaffected. Furthermore, muscle abnormalities associated with miR-9a overexpression were completely rescued by overexpression of TnT transgenes which lacked the miR-9a binding site. These findings indicate that miR-9a interacts with the 3'-UTR of the TnT mRNA and downregulates the TnT protein levels by translational repression. The reduction in TnT levels leads to a cooperative downregulation of other thin filament structural proteins. Our findings have implications for understanding the cellular pathophysiology of cardiomyopathies associated with miR-9 overexpression.
Sexual dimorphism exists in energy balance, but the underlying mechanisms remain unclear. Here we show that the female mice have more pro-opiomelanocortin (POMC) neurons in the arcuate nucleus of hypothalamus than males, and female POMC neurons display higher neural activities, compared to male counterparts. Strikingly, deletion of the transcription factor, TAp63, in POMC neurons confers "male-like" diet-induced obesity (DIO) in female mice associated with decreased POMC neural activities; but the same deletion does not affect male mice. Our results indicate that TAp63 in female POMC neurons contributes to the enhanced POMC neuron functions and resistance to obesity in females. Thus, TAp63 in POMC neurons is one key molecular driver for the sexual dimorphism in energy homeostasis.
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.
The Sorting and Assembly Machinery (SAM) Complex is responsible for assembling β-barrel proteins in the mitochondrial membrane. Comprising three subunits, Sam35, Sam37, and Sam50, the SAM complex connects the inner and outer mitochondrial membranes by interacting with the mitochondrial contact site and cristae organizing system (MICOS) complex. Sam50, in particular, stabilizes the mitochondrial intermembrane space bridging (MIB) complex, which is crucial for protein transport, respiratory chain complex assembly, and regulation of cristae integrity. While the role of Sam50 in mitochondrial structure and metabolism in skeletal muscle remains unclear, this study aims to investigate its impact. Serial block-face-scanning electron microscopy (SBF-SEM) and computer-assisted 3D renderings were employed to compare mitochondrial structure and networking in Sam50-deficient myotubes from mice and humans with wild-type (WT) myotubes. Furthermore, autophagosome 3D structure was assessed in human myotubes. Mitochondrial metabolic phenotypes were assessed using Gas Chromatography-Mass Spectrometry-based metabolomics to explore differential changes in WT and Sam50-deficient myotubes. The results revealed increased mitochondrial fragmentation and autophagosome formation in Sam50-deficient myotubes compared to controls. Metabolomic analysis indicated elevated metabolism of propanoate and several amino acids, including ß-Alanine, phenylalanine, and tyrosine, along with increased amino acid and fatty acid metabolism in Sam50-deficient myotubes. Furthermore, impairment of oxidative capacity was observed upon Sam50 ablation in both murine and human myotubes, as measured with the XF24 Seahorse Analyzer. Collectively, these findings support the critical role of Sam50 in establishing and maintaining mitochondrial integrity, cristae structure, and mitochondrial metabolism. By elucidating the impact of Sam50-deficiency, this study enhances our understanding of mitochondrial function in skeletal muscle.
Voltage-gated Cav Ca2+ channels play crucial roles in regulating gene transcription, neuronal excitability, and synaptic transmission. Natural or pathological variations in Cav channels have yielded rich insights into the molecular determinants controlling channel function. Here, we report the consequences of a natural, putatively disease-associated mutation in the CACNA1D gene encoding the pore-forming Cav1.3 α1 subunit. The mutation causes a substitution of a glutamine residue that is highly conserved in the extracellular S1-S2 loop of domain II in all Cav channels with a histidine and was identified by whole-exome sequencing of an individual with moderate hearing impairment, developmental delay, and epilepsy. When introduced into the rat Cav1.3 cDNA, Q558H significantly decreased the density of Ca2+ currents in transfected HEK293T cells. Gating current analyses and cell-surface biotinylation experiments suggested that the smaller current amplitudes caused by Q558H were because of decreased numbers of functional Cav1.3 channels at the cell surface. The substitution also produced more sustained Ca2+ currents by weakening voltage-dependent inactivation. When inserted into the corresponding locus of Cav2.1, the substitution had similar effects as in Cav1.3. However, the substitution introduced in Cav3.1 reduced current density, but had no effects on voltage-dependent inactivation. Our results reveal a critical extracellular determinant of current density for all Cav family members and of voltage-dependent inactivation of Cav1.3 and Cav2.1 channels.
Dynamic changes in mitochondrial shape and size are vital for mitochondrial health and for tissue development and function. Adult Drosophila indirect flight muscles contain densely packed mitochondria. We show here that mitochondrial fusion is critical during early muscle development (in pupa) and that silencing of the outer mitochondrial membrane fusion gene, Marf, in muscles results in smaller mitochondria that are functionally defective. This leads to abnormal muscle development resulting in muscle dysfunction in adult flies. However, post-developmental silencing of Marf has no obvious effects on mitochondrial and muscle phenotype in adult flies, indicating the importance of mitochondrial fusion during early muscle development.
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.
Neurons that co-express agouti-related peptide (AgRP) and neuropeptide Y (NPY) are indispensable for normal feeding behavior. Firing activities of AgRP/NPY neurons are dynamically regulated by energy status and coordinate appropriate feeding behavior to meet nutritional demands. However, intrinsic mechanisms that regulate AgRP/NPY neural activities during the fed-to-fasted transition are not fully understood. We found that AgRP/NPY neurons in satiated mice express high levels of the small-conductance calcium-activated potassium channel 3 (SK3) and are inhibited by SK3-mediated potassium currents; on the other hand, food deprivation suppresses SK3 expression in AgRP/NPY neurons, and the decreased SK3-mediated currents contribute to fasting-induced activation of these neurons. Genetic mutation of SK3 specifically in AgRP/NPY neurons leads to increased sensitivity to diet-induced obesity, associated with chronic hyperphagia and decreased energy expenditure. Our results identify SK3 as a key intrinsic mediator that coordinates nutritional status with AgRP/NPY neural activities and animals' feeding behavior and energy metabolism.
Hypothalamic neurons expressing the anorectic peptide Pro-opiomelanocortin (Pomc) regulate food intake and body weight. Here, we show that Steroid Receptor Coactivator-1 (SRC-1) interacts with a target of leptin receptor activation, phosphorylated STAT3, to potentiate Pomc transcription. Deletion of SRC-1 in Pomc neurons in mice attenuates their depolarization by leptin, decreases Pomc expression and increases food intake leading to high-fat diet-induced obesity. In humans, fifteen rare heterozygous variants in SRC-1 found in severely obese individuals impair leptin-mediated Pomc reporter activity in cells, whilst four variants found in non-obese controls do not. In a knock-in mouse model of a loss of function human variant (SRC-1L1376P), leptin-induced depolarization of Pomc neurons and Pomc expression are significantly reduced, and food intake and body weight are increased. In summary, we demonstrate that SRC-1 modulates the function of hypothalamic Pomc neurons, and suggest that targeting SRC-1 may represent a useful therapeutic strategy for weight loss.
Background Nuclear-to-mitochondrial communication regulating gene expression and mitochondrial function is a critical process following cardiac ischemic injury. In this study, we determined that cyclin C, a component of the Mediator complex, regulates cardiac and mitochondrial function in part by modifying mitochondrial fission. We tested the hypothesis that cyclin C functions as a transcriptional cofactor in the nucleus and a signaling molecule stimulating mitochondrial fission in response to stimuli such as cardiac ischemia. Methods and Results We utilized gain- and loss-of-function mouse models in which the CCNC (cyclin C) gene was constitutively expressed (transgenic, CycC cTg) or deleted (knockout, CycC cKO) in cardiomyocytes. The knockout and transgenic mice exhibited decreased cardiac function and altered mitochondria morphology. The hearts of knockout mice had enlarged mitochondria with increased length and area, whereas mitochondria from the hearts of transgenic mice were significantly smaller, demonstrating a role for cyclin C in regulating mitochondrial dynamics in vivo. Hearts from knockout mice displayed altered gene transcription and metabolic function, suggesting that cyclin C is essential for maintaining normal cardiac function. In vitro and in vivo studies revealed that cyclin C translocates to the cytoplasm, enhancing mitochondria fission following stress. We demonstrated that cyclin C interacts with Cdk1 (cyclin-dependent kinase 1) in vivo following ischemia/reperfusion injury and that, consequently, pretreatment with a Cdk1 inhibitor results in reduced mitochondrial fission. This finding suggests a potential therapeutic target to regulate mitochondrial dynamics in response to stress. Conclusions Our study revealed that cyclin C acts as a nuclear-to-mitochondrial signaling factor that regulates both cardiac hypertrophic gene expression and mitochondrial fission. This finding provides new insights into the regulation of cardiac energy metabolism following acute ischemic injury.
Isolation of skeletal muscles allows for the exploration of many complex diseases. Fibroblasts and myoblast play important roles in skeletal muscle morphology and function. However, skeletal muscles are complex and made up of many cellular populations and validation of these populations is highly important. Therefore, in this article, we discuss a comprehensive method to isolate mice skeletal muscle, create satellite cells for tissue culture, and use immunofluorescence to validate our approach.
Isolation of skeletal muscles allows for the exploration of many complex diseases. Here, we present a protocol for isolating mice skeletal muscle myoblasts and myotubes that have been differentiated through antibody validation. We describe steps for collecting and preparing murine skeletal tissue, myoblast cell maintenance, plating, and cell differentiation. We then detail procedures for cell incubation, immunostaining, slide preparation and storage, and imaging for immunofluorescence validation.
The mechanisms of cell cycle exit by myoblasts during skeletal muscle development are poorly understood. Cell cycle arrest is known to be a prerequisite for myoblast fusion and subsequent differentiation. Despite tremendous knowledge on myoblast fusion and differentiation, tissue-specific factors that spatio-temporally regulate the cell cycle exit are not well known. In this paper, we show that the transcriptional factor/co-activator "Erect wing" (Ewg) synchronises myoblast cell cycle exit with that of the fusion process. Ewg-null myoblasts show delayed temporal development of dorsal longitudinal muscles (DLMs), a group of indirect flight muscles (IFMs), which culminates to abnormal and asymmetric muscle pattern. A role for Ewg in cell cycle exit at G1/S stage is also shown. Reducing Cyclin E dose in Ewg-null mutant rescues the lack of IFMs and flight ability. Thus, we show that Ewg repression of Cyclin E expression is required for the arrest of myoblast proliferation and initiate myoblast fusion and terminal differentiation.
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.
Current postdoctoral fellows have a range of career options; however, following a career path into academia can be daunting. Here, we discuss essential elements needed to transition the postdoctoral position into faculty candidates. Furthermore, we provide critical hacks to help postdoctoral fellows to be well prepared to navigate the application and interview processes.
Proximity ligation assays (PLA) use specific antibodies to detect endogenous protein-protein interactions. PLA is a highly useful biochemical technique that allows two proteins within close proximity to be visualized with fluorescent probes amplified by PCR. While this technique has gained prominence, the use of PLA in mouse skeletal muscle (SkM) is novel. In this article, we discuss how the PLA method can be used in SkM to study the protein-protein interactions within mitochondria-endoplasmic reticulum contact sites (MERCs).
Mitochondria are required for energy production and even give brown adipose tissue (BAT) its characteristic color due to their high iron content and abundance. The physiological function and bioenergetic capacity of mitochondria are connected to the structure, folding, and organization of its inner-membrane cristae. During the aging process, mitochondrial dysfunction is observed, and the regulatory balance of mitochondrial dynamics is often disrupted, leading to increased mitochondrial fragmentation in aging cells. Therefore, we hypothesized that significant morphological changes in BAT mitochondria and cristae would be present with aging. We developed a quantitative three-dimensional (3D) electron microscopy approach to map cristae network organization in mouse BAT to test this hypothesis. Using this methodology, we investigated the 3D morphology of mitochondrial cristae in adult (3-month) and aged (2-year) murine BAT tissue via serial block face-scanning electron microscopy (SBF-SEM) and 3D reconstruction software for manual segmentation, analysis, and quantification. Upon investigation, we found increases in mitochondrial volume, surface area, and complexity and decreased sphericity in aged BAT, alongside significant decreases in cristae volume, area, perimeter, and score. Overall, these data define the nature of the mitochondrial structure in murine BAT across aging.
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.
Mitochondria and peroxisomes are dynamic signaling organelles that constantly undergo fission, driven by the large GTPase dynamin-related protein 1 (DRP1; encoded by DNM1L). Patients with de novo heterozygous missense mutations in DNM1L present with encephalopathy due to defective mitochondrial and peroxisomal fission (EMPF1) - a devastating neurodevelopmental disease with no effective treatment. To interrogate the mechanisms by which DRP1 mutations cause cellular dysfunction, we used human-derived fibroblasts from patients who present with EMPF1. In addition to elongated mitochondrial morphology and lack of fission, patient cells display lower coupling efficiency, increased proton leak and upregulation of glycolysis. Mitochondrial hyperfusion also results in aberrant cristae structure and hyperpolarized mitochondrial membrane potential. Peroxisomes show a severely elongated morphology in patient cells, which is associated with reduced respiration when cells are reliant on fatty acid oxidation. Metabolomic analyses revealed impaired methionine cycle and synthesis of pyrimidine nucleotides. Our study provides insight into the role of mitochondrial dynamics in cristae maintenance and the metabolic capacity of the cell, as well as the disease mechanism underlying EMPF1.
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