Three-dimensional mitochondria reconstructions of murine cardiac muscle changes in size across aging.

With sparse treatment options, cardiac disease remains a significant cause of death among humans. As a person ages, mitochondria breakdown and the heart becomes less efficient. Heart failure is linked to many mitochondria-associated processes, including endoplasmic reticulum stress, mitochondrial bioenergetics, insulin signaling, autophagy, and oxidative stress. The roles of key mitochondrial complexes that dictate the ultrastructure, such as the mitochondrial contact site and cristae organizing system (MICOS), in aging cardiac muscle are poorly understood. To better understand the cause of age-related alteration in mitochondrial structure in cardiac muscle, we used transmission electron microscopy (TEM) and serial block facing-scanning electron microscopy (SBF-SEM) to quantitatively analyze the three-dimensional (3-D) networks in cardiac muscle samples of male mice at aging intervals of 3 mo, 1 yr, and 2 yr. Here, we present the loss of cristae morphology, the inner folds of the mitochondria, across age. In conjunction with this, the three-dimensional (3-D) volume of mitochondria decreased. These findings mimicked observed phenotypes in murine cardiac fibroblasts with CRISPR/Cas9 knockout of Mitofilin, Chchd3, Chchd6 (some members of the MICOS complex), and Opa1, which showed poorer oxidative consumption rate and mitochondria with decreased mitochondrial length and volume. In combination, these data show the need to explore if loss of the MICOS complex in the heart may be involved in age-associated mitochondrial and cristae structural changes.NEW & NOTEWORTHY This article shows how mitochondria in murine cardiac changes, importantly elucidating age-related changes. It also is the first to show that the MICOS complex may play a role in outer membrane mitochondrial structure.

Imaging the rapid yet transient accumulation of regulatory lipids, lipid kinases, and protein kinases during membrane fusion, at sites of exocytosis of MMP-9 in MCF-7 cells.

BACKGROUND: The regulation of exocytosis is physiologically vital in cells and requires a variety of distinct proteins and lipids that facilitate efficient, fast, and timely release of secretory vesicle cargo. Growing evidence suggests that regulatory lipids act as important lipid signals and regulate various biological processes including exocytosis. Though functional roles of many of these regulatory lipids has been linked to exocytosis, the dynamic behavior of these lipids during membrane fusion at sites of exocytosis in cell culture remains unknown. METHODS: Total internal reflection fluorescence microscopy (TIRF) was used to observe the spatial organization and temporal dynamics (i.e. spatial positioning and timing patterns) of several lipids, and accessory proteins, like lipid kinases and protein kinases, in the form of protein kinase C (PRKC) associated with sites of exocytosis of matrix metalloproteinase-9 (MMP-9) in living MCF-7 cancer cells. RESULTS: Following stimulation with phorbol myristate acetate (PMA) to promote exocytosis, a transient accumulation of several distinct regulatory lipids, lipid kinases, and protein kinases at exocytic sites was observed. This transient accumulation centered at the time of membrane fusion is followed by a rapid diffusion away from the fusion sites. Additionally, the synthesis of these regulatory lipids, degradation of these lipids, and the downstream effectors activated by these lipids, are also achieved by the recruitment and accumulation of key enzymes at exocytic sites (during the moment of cargo release). This includes key enzymes like lipid kinases, protein kinases, and phospholipases that facilitate membrane fusion and exocytosis of MMP-9. CONCLUSIONS: This work suggests that these regulatory lipids and associated effector proteins are locally synthesized and/or recruited to sites of exocytosis, during membrane fusion and cargo release. More importantly, their enrichment at fusion sites serves as an important spatial and temporal organizing "element" defining individual exocytic sites.

Differential sensitivity to methylated DNA by ETS-family transcription factors is intrinsically encoded in their DNA-binding domains.

Transactivation by the ETS family of transcription factors, whose members share structurally conserved DNA-binding domains, is variably sensitive to methylation of their target genes. The mechanism by which DNA methylation controls ETS proteins remains poorly understood. Uncertainly also pervades the effects of hemi-methylated DNA, which occurs following DNA replication and in response to hypomethylating agents, on site recognition by ETS proteins. To address these questions, we measured the affinities of two sequence-divergent ETS homologs, PU.1 and Ets-1, to DNA sites harboring a hemi- and fully methylated CpG dinucleotide. While the two proteins bound unmethylated DNA with indistinguishable affinity, their affinities to methylated DNA are markedly heterogeneous and exhibit major energetic coupling between the two CpG methylcytosines. Analysis of simulated DNA and existing co-crystal structures revealed that hemi-methylation induced non-local backbone and groove geometries that were not conserved in the fully methylated state. Indirect readout of these perturbations was differentially achieved by the two ETS homologs, with the distinctive interfacial hydration in PU.1/DNA binding moderating the inhibitory effects of DNA methylation on binding. This data established a biophysical basis for the pioneering properties associated with PU.1, which robustly bound fully methylated DNA, but not Ets-1, which was substantially inhibited.

Pharmacologic efficacy of PU.1 inhibition by heterocyclic dications: a mechanistic analysis.

Heterocyclic dications are receiving increasing attention as targeted inhibitors of transcription factors. While many dications act as purely competitive inhibitors, some fail to displace protein efficiently at drug concentrations expected to saturate their DNA target. To achieve a mechanistic understanding of these non-competitive effects, we used a combination of dications, which are intrinsically fluorescent and spectrally-separated fluorescently labeled DNA to dissect complex interactions in multi-component drug/DNA/protein systems. Specifically, we interrogated site-specific binding by the transcription factor PU.1 and its perturbation by DB270, a furan-bisbenzimidazole-diamidine that strongly targets PU.1 binding sites yet poorly inhibits PU.1/DNA complexes. By titrating DB270 and/or cyanine-labeled DNA with protein or unlabeled DNA, and following the changes in their fluorescence polarization, we found direct evidence that DB270 bound protein independently of their mutual affinities for sequence-specific DNA. Each of the three species competed for the other two, and this interplay of mutually dependent equilibria abrogated DB270's inhibitory activity, which was substantively restored under conditions that attenuated DB270/PU.1 binding. PU.1 binding was consistent with DB270's poor inhibitory efficacy of PU.1 in vivo, while its isosteric selenophene analog (DB1976), which did not bind PU.1 and strongly inhibited the PU.1/DNA complex in vitro, fully antagonized PU.1-dependent transactivation in vivo.

Ester-prodrugs of ethambutol control its antibacterial activity and provide rapid screening for mycobacterial hydrolase activity.

M. tuberculosis contains an unusually high number of serine hydrolases by proteome percentage compared to other common bacteria or humans. This letter describes a method to probe the global substrate specificity of mycobacterial serine hydrolases with ester-protected prodrugs of ethambutol, a first-line antibiotic treatment for TB. These compounds were synthesized directly from ethambutol using a selective o-acylation to yield products in high yield and purity with minimal workup. A library of derivatives was screened against M. smegmatis, a non-infectious model for M. tuberculosis, which displayed significantly lowered biological activity compared to ethambutol. Incubation with a general serine hydrolase reactivated each derivative to near-ethambutol levels, demonstrating that esterification of ethambutol should provide a simple screen for mycobacterial hydrolase activity.

Is space the final frontier for mitochondrial study?

Tweetable abstract This perspective considers several avenues for future research on mitochondrial dynamics, stress, and DNA in outer space.

Considerations for developing mitochondrial transplantation techniques for individualized medicine.

Tweetable abstract Mitochondrial transplantation has been used to treat various diseases associated with mitochondrial dysfunction. Here, we highlight the considerations in quality control mechanisms that should be considered in the context of mitochondrial transplantation.

Protocol for isolating mice skeletal muscle myoblasts and myotubes via differential antibody validation.

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.

Serial Block Face-Scanning Electron Microscopy as a Burgeoning Technology.

Serial block face scanning electron microscopy (SBF-SEM), also referred to as serial block-face electron microscopy, is an advanced ultrastructural imaging technique that enables three-dimensional visualization that provides largerx- and y-axis ranges than other volumetric EM techniques. While SEM is first introduced in the 1930s, SBF-SEM is developed as a novel method to resolve the 3D architecture of neuronal networks across large volumes with nanometer resolution by Denk and Horstmann in 2004. Here, the authors provide an accessible overview of the advantages and challenges associated with SBF-SEM. Beyond this, the applications of SBF-SEM in biochemical domains as well as potential future clinical applications are briefly reviewed. Finally, the alternative forms of artificial intelligence-based segmentation which may contribute to devising a feasible workflow involving SBF-SEM, are also considered.

In the Age of Machine Learning Cryo-EM Research is Still Necessary: A Path toward Precision Medicine.

Machine learning has proven useful in analyzing complex biological data and has greatly influenced the course of research in structural biology and precision medicine. Deep neural network models oftentimes fail to predict the structure of complex proteins and are heavily dependent on experimentally determined structures for their training and validation. Single-particle cryogenic electron microscopy (cryoEM) is also advancing the understanding of biology and will be needed to complement these models by continuously supplying high-quality experimentally validated structures for improvements in prediction quality. In this perspective, the significance of structure prediction methods is highlighted, but the authors also ask, what if these programs cannot accurately predict a protein structure important for preventing disease? The role of cryoEM is discussed to help fill the gaps left by artificial intelligence predictive models in resolving targetable proteins and protein complexes that will pave the way for personalized therapeutics.

Creating Optimal Conditions for OPA1 Isoforms by Western Blot in Skeletal Muscle Cells and Tissue.

OPA1 is a dynamin-related GTPase that modulates various mitochondrial functions and is involved in mitochondrial morphology. There are eight different isoforms of OPA1 in humans and five different isoforms in mice that are expressed as short or long-form isoforms. These isoforms contribute to OPA1's ability to control mitochondrial functions. However, isolating OPA1 all long and short isoforms through western blot has been a difficult task. To address this issue, we outline an optimized western blot protocol to isolate 5 different isoforms of OPA1 on the basis of different antibodies. This protocol can be used to study changes in mitochondrial structure and function.

Optimizing In Situ Proximity Ligation Assays for Mitochondria, ER, or MERC Markers in Skeletal Muscle Tissue and Cells.

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).

Components of Isolated Skeletal Muscle Differentiated Through Antibody Validation.

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.

Project Strengthen: An STEMM-focused career development workshop to prepare underrepresented minority students for graduate school.

Maximizing Access to Research Careers (MARC) programs are aimed to increase diversity in science, technology, engineering, math, and medicine (STEMM) fields. However, limited programs and eligibility requirements limit the students who may apply to similar programs. At Winston-Salem State University, we piloted a series of workshops, collectively termed Project Strengthen, to emulate some of the key aspects of MARC programs. Following the workshop, Project Strengthen students showed a significant increase in their understanding of essential educational development skills, such as writing personal statements, applying to graduate school, studying for the GRE, and seeking summer internships. This suggests Project Strengthen may be a potential lower cost comparable option than MARC to make up for current deficiencies in preparedness for graduate school. We also provide educational materials from Project Strengthen, including a clear framework for this seminar series, six ready-made PowerPoints to share with trainees that have been demonstrated to be effective.

Organizing 'Elements': Facilitating Exocytosis and Promoting Metastasis.

For metastasis to occur, cancer cells must exocytose proteases, like matrix metalloproteinases (MMPs), that are key in extracellular matrix (ECM) degradation. Growing evidence suggests that cancer cells use distinct spatial and temporal clustering patterns or organizing 'elements' that facilitate secretory vesicle fusion and the subsequent exocytosis of proteins that contribute to metastasis.

Spatiotemporal organization and protein dynamics involved in regulated exocytosis of MMP-9 in breast cancer cells.

Altered regulation of exocytosis is an important mechanism controlling many diseases, including cancer. Defects in exocytosis have been implicated in many cancer cell types and are generally attributed to mutations in cellular transport, trafficking, and assembly of machinery necessary for exocytosis of secretory vesicle cargo. In these cancers, up-regulation of trafficking and secretion of matrix metalloproteinase-9 (MMP-9), a proteolytic enzyme, is responsible for degrading the extracellular matrix, a necessary step in tumor progression. Using TIRF microscopy, we identified proteins associated with secretory vesicles containing MMP-9 and imaged the local dynamics of these proteins at fusion sites during regulated exocytosis of MMP-9 from MCF-7 breast cancer cells. We found that many regulators of exocytosis, including several Rab GTPases, Rab effector proteins, and SNARE/SNARE modulator proteins, are stably assembled on docked secretory vesicles before exocytosis. At the moment of fusion, many of these components are quickly lost from the vesicle, while several endocytic proteins and lipids are simultaneously recruited to exocytic sites at precisely that moment. Our findings provide insight into the dynamic behavior of key core exocytic proteins, accessory proteins, lipids, and some endocytic proteins at single sites of secretory vesicle fusion in breast cancer cells.