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The Sorting and Assembly Machinery (SAM) Complex functions in the assembly of β-barrel in the mitochondrial membrane. The SAM complex is made up of three subunits, Sam35, Sam37, and Sam50. While both Sam35 and Sam37 are peripheral membrane proteins that are not required for survival, Sam50 interacts with the MICOS complex to connect the inner and outer mitochondrial membranes and forms the mitochondrial intermembrane space bridging (MIB) complex. Specifically, Sam50 stabilizes the MIB complex for protein transport, respiratory chain complex assembly, and cristae integrity regulation. To structurally form and sustain the cristae, the MICOS complex assembles at the cristae junction and binds directly to Sam50. However, the role of Sam50 in overall mitochondrial structure and metabolism in skeletal muscle remains unclear. Here, we use SBF-SEM and Amira software perform 3D renderings of mitochondria and autophagosomes in human myotubes. Beyond this, Gas Chromatography-Mass Spectrometry-based metabolomics was utilized to interrogate differential changes of the metabolites in wild-type (WT) andSam50deficient myotubes. Ablation ofSam50, revealed increases in ß-Alanine, propanoate, and phenylalanine, and tyrosine metabolism. Additionally, we observed that mitochondrial fragmentation and autophagosome formation was increased inSam50-deficient myotubes compared to control myotubes. Beyond this, the metabolomic analysis revealed an increase in amino acid metabolism and fatty acid metabolism. XF24 Seahorse Analyzer shows that oxidative capacity is further impaired upon ablation ofSam50in both murine and human myotubes. Together, these data suggest Sam50 is critical for establishing and maintaining mitochondria, mitochondrial cristae structure, and mitochondrial metabolism.

SummaryOPA1 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 that are expressed as short or long-form isoforms. These isoforms contribute to OPA1’s ability to control mitochondrial functions. However, isolating OPA1 isoforms through western blot has been a difficult task. To address this issue, we outline an optimized western blot protocol to isolate different isoforms of OPA1 on the basis of different antibodies. This protocol can be used to study changes in mitochondrial structure and function.Tweetable AbstractWestern blot protocol optimization to visualize OPA1 isoforms.HighlightsProtocol for isolating OPA1 isoforms in skeletal muscle tissueSteps for running isolated skeletal muscle cells from muscle tissue on a gelHow to collect samples in preparation for western blottingDetection of OPA1 isoformsGraphical AbstractKey Resources TableMethod SummarySamples for western blot analysis are isolated from lysed cells, loaded onto a gel, and ran using optimized conditions to better isolate OPA1 isoforms. Samples are transferred to a membrane to for incubation and protein detection using OPA1 antibodies.

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.Novel imaging technology is increasing our ability to visualize and analyze cellular organelles and compartments.Tweetable AbstractProper antibody validation of cellular populations within isolated skeletal muscle can lead to better elucidation of skeletal muscle structure and function, and their roles in complex diseases.Graphical AbstractMethod SummaryMyoblasts are isolated from mouse limb muscles. They are plated for immunofluorescence-based validation using confocal microscopy. This method demonstrates the need for reliable antibodies to correctly determine and differentiate between cellular populations within isolated skeletal muscles.

SummaryProximity 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).Tweetable AbstractProximity Ligation Assays can be used in skeletal muscle tissue and myoblasts to explore the protein-protein interactions involved in MERC sites.HighlightsSkeletal muscle tissue and cells are plated on glass coverslips for evaluation by proximity ligation assay (PLA).Following fixation, cells are probed and stained for Mfn1, Mfn2, mitochondria, and ER and imaged using fluorescence confocal microscopy.This method shows that PLA can be used in mouse SkM and is adaptable to other models.Protocol for detection of protein-protein interactions using PLA.Graphical AbstractKey Resources Table

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 the morphological changes in BAT mitochondria and cristae observed across aging would reflect alterations to energy dynamics. 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) and3D 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. Cristae examination revealed decreased cristae volume, perimeter, and complexity, with cristate appearing more heterogeneous. Overall, these data define the nature of the mitochondrial structure in BAT, showing loss of cristae definition across aging, suggesting alterations in functionality and bioactivity.Graphical AbstractWorkflow overview of serial block facing-scanning electron microscopy (SBF-SEM), data segmentation, and 3D analysis of mitochondria using Amira software in murine BAT.

Choosing a mentor requires a certain level of introspection for both the mentor and the mentee. The dynamics of mentorship may change depending on the academic status of the mentee. Regardless, mentors should help their trainees grow both academically and professionally. The success of an individual in the fields of science, technology, engineering, mathematics, and medicine (STEMM) depends on more than intellectual capacity; a holistic view encompassing all factors that contribute to scientific achievement is all-important. Specifically, one new method scientists can adopt is quotients, which are scales and techniques that can be used to measure aptitude in a specific area. In this paper, we focus on these factors and how to grow one's adversity quotient (AQ), social quotient (SQ), and Personal Growth Initiative Scale (PGIS). We also look at how mentors can better understand the biases of their trainees. In addressing this, mentors can help trainees become more visible and encourage other trainees to become allies while increasing the visibility of their peers.

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.

Disability remains an underacknowledged and underdiscussed topic in science, technology, engineering, mathematics, and medicine (STEMM). Social stigma and fear of negative outcomes have resulted in a consistent lack of disclosure. Disabilities cause social and professional difficulties for those that have them. While some faculty can be allies, past literature shows that steps must be taken to make disabilities visible in STEMM at both student and faculty levels. Here, we offer suggestions to better support faculty and students in enhancing the outcomes of individuals who have invisible disabilities. Critically, techniques such as abolishing stigma, universal learning, and better mentoring may improve the challenges faced by those who self-identify as an individual with a disability.

SummaryMitochondria and endoplasmic reticulum (ER) contact sites (MERCs) are protein- and lipid-enriched hubs that mediate intracellular communications, contributing to the dynamic transfer of Ca2+, lipid, and other metabolites between these organelles. Defective MERCs are associated with cellular oxidative stress, neurodegenerative disease, and cardiac and skeletal muscle pathology via mechanisms that are poorly understood. We previously demonstrated that skeletal muscle-specific knockdown (KD) of the mitochondrial fusion mediator optic atrophy 1 (OPA1) induced ER stress and upregulated Mitofusin-2, a known MERC protein. In the present study, we tested the hypothesis that Opa1 downregulation in skeletal muscle cells induces MERC formation using multiple experimental models, including mice, Drosophila, and primary myotubes. Our results revealed that OPA1 deficiency increased MERC tethering and activated the integrated stress response (ISR) pathway effector, activating transcription factor 4 (ATF4). Loss of OPA1, reduced mitochondria calcium uptake and caffeine-induced calcium release by the ER, which was associated with changes in expression of mediators of calcium exchange IP3R3, GRP75, VDAC, at MERCs. Reducing Atf4 expression in Opa1-deficient muscle cells altered MERC ultrastructure changes and reestablished cytosolic calcium homeostasis. These data identify a role for ATF4 in the regulation of MERCs distance and mitochondrial calcium exchange.

Many studies in mice have demonstrated that cardiac-specific innate immune signaling pathways can be reprogrammed to modulate inflammation in response to myocardial injury and improve outcomes. While the echocardiography standard parameters of left ventricular (LV) ejection fraction, fractional shortening, and end-diastolic diameter, and others, are used to assess cardiac function, their dependency on loading conditions somewhat limit their utility in completely reflecting the contractile function and global cardiovascular efficiency of the heart. A true measure of global cardiovascular efficiency should include of the interaction between the ventricle and the aorta (ventriculo-vascular coupling, VVC) as well as measures of aortic impedance and pulse wave velocity. We measured cardiac Doppler velocities, blood pressures, along with VVC, aortic impedance, and pulse wave velocity to evaluate global cardiac function in mouse model of cardiac-restricted low levels TRAF2 overexpression that conferred cytoprotection in the heart. While previous studies reported that response to myocardial infraction and reperfusion was improved in the TRAF2 overexpressed mice, we found that TRAF2 mice had significantly lower cardiac systolic velocities and accelerations, diastolic atrial velocity, lower aortic pressures and rate-pressure product, lower LV contractility and relaxation, and lower stroke work when compared to littermate control mice. Also, we found significantly longer aortic ejection time, isovolumic contraction and relaxation times, and significantly higher mitral early/atrial ratio, myocardial performance index, and ventricular vascular coupling in the TRAF2 overexpression mice compared to their littermate controls. We found no significant differences in the aortic impedance and pulse wave velocity. While the reported tolerance to ischemic insults in TRAF2 overexpression mice may suggest enhanced cardiac reserve, our results indicate a diminished cardiac function in these mice.

Scientists at all career stages can benefit from building diverse mentoring networks that transcend boundaries and promote inclusion. In this piece, we define mentoring networks, describe examples of how mentoring networks can reinforce scientific identity, and help minority scientists overcome unique challenges to achieve their goals in cancer research.

Despite efforts to increase diversity, a glaring underrepresentation of minorities (URM) persists in the fields of science, technology, engineering, and mathematics (STEM). Graduate school can be a stressful step in the STEM pipeline, especially for students previously unaware of the structure and challenges of postgraduate education. To promote successful minority participation in STEM and prepare prospective students for the impending challenges of applying for and attending graduate school, we developed a workshop based on the mentoring and fostering of a champion-oriented mindset entitled, “The Trials and Tribulations of Graduate School: How Do You Make an Impact?.” Students from the HBCU Winston-Salem State University attended the workshop, and a pre/post—a 10-point Likert scale-based survey was administered. The questions used in this seminar were newly designed by the authors as program evaluations. The results suggest that the workshop was well-received by the students and provided information that they considered helpful to help navigate the graduate school process.

BackgroundAbdominal and thoracic aortic aneurysms (AAA; TAA) remain a large cause of deaths worldwide. This is in part a result of the lack of prognostic markers or early warning signs, leading to undiagnosed aortic aneurysms. Sox6 has been found to function as a regulator of renin expression controlling the rate limiting step in the renin angiotensin aldosterone system. We hypothesized that the transcription factor Sox6 may serve as an important regulator of mechanisms contributing to hypertension induced aortic aneurysms.MethodsOur approach includes mRNA analysis, immunohistology staining, and protein expression studies in human samples from patients affected with AAA and TAA. In vivo, we use Angiotensin (II) to induce AAA in mice with a tamoxifen inducible Cre to specifically knock out Sox6 in smooth muscle cells. Additionally, we utilize large-scale biobank data linking de-identified medical records with genotype information to perform phenotype and laboratory-wide association scans to assess the effects of SOX6 expression in a clinical cohort.ResultsIn a large biobank population, SOX6 gene expression is associated with aortic aneurysm in humans of European ancestry. Protein expression of Sox6 and TNFα was upregulated in tissue from patients affected by AAA and TAA. Moreover, we found that knocking out Sox6 in smooth muscle cells protected mice from hypertension-induced AAA, suggesting that Sox6 may be a molecular target in aortic aneurysms.ConclusionsThe data presented here suggest that the transcription factor Sox6 functions in the development of abdominal aortic aneurysms, and hypertension-induced rupture.Clinical PerspectiveUsing electronic health records and biobank samples, we found that the transcription factor SOX6 is associated with abdominal and thoracic aortic aneurysm and its expression is upregulated in tissue from patients affected by those diseases.Laboratory-wide association study (LabWAS) provides several clinical laboratory measurements associated with aortic aneurysm diagnosis that may be potential biomarkers for the disease.Mice with smooth muscle-specific Sox6 knock out attenuated hypertension-induced abdominal aortic aneurysm. These novel mice may be useful tools to elucidate the mechanisms associated with abdominal aortic aneurysm.

Cardiac disease remains a significant cause of death among humans and therapies to treat the disease are lacking. Importantly, heart failure has also been linked to factors including endoplasmic reticulum stress, mitochondrial bioenergetics, insulin signaling, autophagy, and oxidative stress 1, which are all factors the mitochondria plays a role in. Critically, mitochondria break down across aging and heart also decrease in efficiency in aging. Key factors implicated in mitochondria morphology, such as the mitochondrial contact site and cristae organizing system (MICOS), and its role across aging remains to be seen in cardiac muscle. To better understand the relationship between mitochondria in cardiac muscle, we used transmission electron microscopy (TEM) and serial block facing-scanning electron microscopy (SBF-SEM) to quantitatively analyze the 3D networks in cardiac muscle samples of mice at aging intervals. In studying cristae, the inner folds of mitochondria, we observed a loss of morphology across aging. This mimicked what was observed upon CRISPR/Cas9 knockdown of Mitofilin, Chchd3, Chchd6 (some members of the MICOS complex) and Opa1 which showed poorer quality cristae and fragmented mitochondria, while mitochondria length and volume decreased. We are the first to examine mitochondria changes in cardiac muscle across aging. In combination, these data suggest that, in the heart, loss of the MICOS complex may be implicated in the loss of function in mitochondria that is seen across aging.

We identify problematic areas throughout the Science, Technology, Engineering and Mathematics (STEM) pipeline that perpetuate racial disparities in academia. Distinct ways to curtail these disparities include early exposure and access to resources, supportive mentoring networks and comprehensive training programs specifically for racially minoritized students and trainees at each career stage. These actions will revitalize the STEM pipeline.

Skeletal muscle gradually loses mass, strength, endurance, and oxidative capacity during aging. While mitochondrial aging is associated with endoplasmic reticulum stress, fragmented mitochondria, and decreased mitochondrial capacity, the genes associated with morphological changes in mitochondria during aging still requires further elucidation. Further, it is not completely understood how 3D mitochondrial networks and the specialization of mitochondria are altered during aging. We measured changes in mitochondrial morphology and mitochondrial connectivity during the aging of the mouse gastrocnemius muscle through serial block facing-scanning electron microscopy and 3D reconstruction. We found changes in mitochondrial network configuration, nanotunneling, size, shape, number, contact sites, cristae organizing system (MICOS) dynamics and gene expression in skeletal muscle during aging. We also found an association of OPA-1 and the MICOS complex in the gastrocnemius with mitochondrial aging. Further, the loss of the MICOS complex was linked with decreased oxidative capacity and altered mitochondrial metabolism. MICOS proteins decreased with age and mitochondrial morphology was similar between aged skeletal muscle and that of young mice with MICOS protein loss. In tandem, our data suggest a relationship between the MICOS complex and aging, which could be potentially linked to disease states with additional 3D reconstruction.Graphical Abstract

Despite an increase in programming to promote persons excluded by their ethnicity or race (PEER) scholars, minorities remain underrepresented in many STEM programs. The academic pipeline is largely leaky for underrepresented minority (URM) scholars due to a lack of effective mentorship. Many URM students experience microaggressions and discrimination from their mentors due to a lack of quality mentorship training. In this workshop, we provide a framework to show trainees what effective mentoring looks like. Mentees, especially URM trainees, can flourish in effective mentoring environments where they feel welcomed and can comfortably develop new ideas without feeling threatened by external factors. Effective mentoring environments provide motivational support, empathy, cultural competency, and training. This workshop explains facets of effective mentoring to students, as well as highlights to URM trainees why mentors can serve as valuable resources.

Experimental evolution with Drosophila melanogaster has been used extensively for decades to study aging and longevity. In recent years, the addition of DNA and RNA sequencing to this framework has allowed researchers to leverage the statistical power inherent to experimental evolution study the genetic basis of longevity itself. Here we incorporated metabolomic data into to this framework to generate even deeper insights into the physiological and genetic mechanisms underlying longevity differences in three groups of experimentally evolved D. melanogaster populations with different aging and longevity patterns. Our metabolomic analysis found that aging alters mitochondrial metabolism through increased consumption of NAD+ and increased usage of the TCA cycle. Combining our genomic and metabolomic data produced a list of biologically relevant candidate genes. Among these candidates, we found significant enrichment for genes and pathways associated with neurological development and function, and carbohydrate metabolism. While we do not explicitly find enrichment for aging canonical genes, neurological dysregulation and carbohydrate metabolism are both known to be associated with accelerated aging and reduced longevity. Taken together, our results provide plausible genetic mechanisms for what might be driving longevity differences in this experimental system. More broadly, our findings demonstrate the value of combining multiple types of omic data with experimental evolution when attempting to dissect mechanisms underlying complex and highly polygenic traits like aging.

Mitochondria and peroxisomes are both dynamic signaling organelles that constantly undergo fission. While mitochondrial fission is known to coordinate cellular metabolism, proliferation, and apoptosis, the physiological relevance of peroxisome dynamics and the implications for cell fate are not fully understood. DRP1 (dynamin-related protein 1) is an essential GTPase that executes both mitochondrial and peroxisomal fission. Patients with de novo heterozygous missense mutations in the gene that encodes DRP1, DNM1L (Dynamin 1 Like), present with encephalopathy due to defective mitochondrial and peroxisomal fission (EMPF1). EMPF1 is 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 with mutations in DRP1 who present with EMPF1. As expected, patient cells display elongated mitochondrial morphology and lack of fission. Patient cells display a lower coupling efficiency of the electron transport chain, increased proton leak, and upregulation of glycolysis. In addition to these metabolic abnormalities, mitochondrial hyperfusion results in aberrant cristae structure and hyperpolarized mitochondrial membrane potential, both of which are tightly linked to the changes in metabolism. Peroxisome structure is also severely elongated in patient cells and results in a potential functional compensation of fatty acid oxidation. Understanding the mechanism by which DRP1 mutations cause these metabolic changes will give 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.

IntroductionWorking with multiple mentors is a critical way for students to expand their network, gain opportunities, and better prepare for future scholastic or professional ventures. However, students from underrepresented groups (UR) are less likely to be mentored or have access to mentors, particularly in science, technology, engineering, and mathematics (STEM) fields. We developed and implemented a workshop, to provide the necessary foundation for students to be better prepared for establishing future mentorships throughout graduate and professional school.MethodsFaculty well-versed in the area of effective mentorship from multiple universities developed and delivered a 1.5-hour workshop to address the roles of a mentor, especially when it comes to UR students, and how students may effectively work with multiple mentors. This workshop was delivered to a group of students from the HBCU Winston Salem State University, and a pre/post-Likert scale-based survey was administered.ResultsWe analyzed the raw data with nonparametric tests for comparison within paired samples. Wilcoxon matched-pairs and signed-rank tests showed statistically significant growth in student self-ratings related to the workshop learning objectives.ConclusionsThe “How to Handle More than One Mentor to Achieve Excellence” workshop was well received as a component of pre-graduate and pre-professional training. Incorporating workshops like this may increase student preparedness around developing and cultivating healthy mentorship relationships throughout STEM training.EDUCATIONAL OBJECTIVESBy the end of this workshop, learners will be able to:Describe the role of mentors in developing the next generation of trainees.Describe current research on mentorship among underrepresented populations.Apply skills on effective communication needed in the development of successful mentorship relationships.Work with multiple mentors at one time while maintaining solid professional and personal relationships.

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Mentoring success is derived from active and respectful listening and the willingness to learn and accept opportunities for personal growth. Mentoring shapes every trainee and their career path in science, technology, engineering, and mathematics (STEM). Productive mentoring relationships cultivate rapport, stimulate moments of introspection, and provide constructive feedback. Effective mentoring in STEM allows trainees, especially underrepresented minorities (URMs), to flourish in welcoming and supportive environments. However, URM trainees often experience inadequate mentoring due to their mentor’s inexperience with URM groups, poor mentorship training, or a lack of understanding of their mentee’s journey. To promote diversity, equity, and inclusion in STEM education and research, it is essential for mentors and mentees to work together with creativity, authenticity, and networking. In this workshop, we will focus on mentees’ perspective on how mentors can enhance their training, professional and career development, and improve their focus. We analyzed data on feedback obtained from students interested in pursuing graduate education who attended a recent workshop. Our results show that despite low initial expectations for the workshop, many students were satisfied with the knowledge they learned. The future of increasing the URM representation in STEM lies in providing adequate community support and mentorship throughout the careers of URM professionals.

Despite an increase in programming to promote persons excluded by their ethnicity or race (PEER) scholars, minorities remain underrepresented in many STEM programs. The academic pipeline is largely leaky for underrepresented minority (URM) scholars due to a lack of effective mentorship. Many URM students experience microaggressions and discrimination from their mentors due to a lack of quality mentorship training. In this workshop, we provide a framework for how to be an effective mentor to URM trainees. Mentees, especially URM trainees, can flourish in effective mentoring environments where they feel welcomed and can comfortably develop new ideas without feeling threatened by external factors. Effective mentoring environments provide motivational support, empathy, cultural competency, and training.

Despite efforts to increase diversity, a glaring underrepresentation of minorities (URM) persists in the fields of science, technology, engineering, and mathematics (STEM). Graduate school can be a stressful step in the STEM pipeline, especially for students previously unaware of the structure and challenges of post-graduate education. To promote successful minority participation in STEM and prepare prospective students for the impending challenges of graduate school, we developed a workshop based on the mentoring and fostering of a champion-oriented mindset entitled, “The Trials and Tribulations of Graduate School: How Do You Make an Impact?”. We administered the workshop to a cohort of university undergraduates and conducted pre- and post-workshop surveys to measure students’ perceived need for instruction on specific workshop topics. The results suggest that the workshop was well received by the students and provided information that they considered helpful to help navigate the graduate school process.

Autophagosomes and lysosomes work in tandem to conduct autophagy, an intracellular degradation system which is crucial for cellular homeostasis. Altered autophagy contributes to the pathophysiology of various diseases, including cancers and metabolic diseases. Although many studies have investigated autophagy to elucidate disease pathogenesis, specific identification of the various components of the cellular degradation machinery remains difficult. The goal of this paper is to describe an approach to reproducibly identify and distinguish subcellular structures involved in autophagy. We provide methods that avoid common pitfalls, including a detailed explanation for how to distinguish lysosomes and lipid droplets and discuss the differences between autophagosomes and inclusion bodies. These methods are based on using transmission electron microscopy (TEM), capable of generating nanometer-scale micrographs of cellular degradation components in a fixed sample. In addition to TEM, we discuss other imaging techniques, such as immunofluorescence and immunogold labeling, which can be utilized for the reliable and accurate classification of cellular organelles. Our results show how these methods may be employed to accurately quantify the cellular degradation machinery under various conditions, such as treatment with the endoplasmic reticulum stressor thapsigargin or the ablation of dynamin-related protein 1. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=164 HEIGHT=200 SRC="FIGDIR/small/461841v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@17f3517org.highwire.dtl.DTLVardef@768c3corg.highwire.dtl.DTLVardef@f0d93aorg.highwire.dtl.DTLVardef@45aad0_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

Mentoring is a developmental experience intended to increase the willingness to learn and establish credibility while building positive relationships through networking. In this commentary, we focus on intentional mentoring for underrepresented mentees, including individuals that belong to minority racial, ethnic and gender identity groups in Science, Technology, Engineering, Mathematics and Medicine (STEMM) fields. Intentional mentoring is the superpower action necessary for developing harmony and comprehending the purpose and value of the mentor/mentee relationship. Regardless of a mentor's career stage, we believe the strategies discussed may be used to create a supportive and constructive mentorship environment; thereby improving the retention rates of underrepresented mentees within the scientific community., This article discusses how to be an intentional mentor in the 21st Century.

2ABSTRACTTransmission electron microscopy (TEM) is a scientific research standard for producing nanometer-resolution ultrastructural images of subcellular components within cells and tissues. Mitochondria, endoplasmic reticulum (ER), lysosomes, and autophagosomes are organelles of particular interest to those investigating metabolic disorders. However, there is no clear consensus amongst regarding the best methods for quantifying the features of organelles in TEM images. In this protocol, we propose a standardized approach to accurately measure the morphology of these important subcellular structures using the free program ImageJ, developed by the National Institutes of Health (NIH). Specifically, we detail procedures for obtaining mitochondrial length, width, area, and circularity, in addition to assessing cristae morphology. We further provide methods for measuring interactions between the mitochondria and ER and measuring the length and width of lysosomes and autophagosomes. This standardized method can be used to quantify key features of organelle morphology, allowing investigators to produce accurate and reproducible measurements of organelle structures in their experimental samples.1SUMMARYWe discuss a standardized method for measuring and quantifying organelle features using transmission electron microscopy and accessing for interactions between subcellular structures; organelles of focus include mitochondria, endoplasmic reticulum, lysosomes, and autophagosomes.

While it is commonly thought that microaggressions are isolated incidents, microaggressions are ingrained throughout the academic research institution (Young, Anderson and Stewart 2015; Lee et al. 2020). Persons Excluded from science because of Ethnicity and Race (PEERs) frequently experience microaggressions from various academicians, including graduate students, postdocs and faculty (Asai 2020; Lee et al. 2020). Here, we elaborate on a rationale for concrete actions to cope with and diminish acts of microaggressions that may otherwise hinder the inclusion of PEERs. We encourage Science, Technology, Engineering and Mathematics (STEM) departments and leadership to affirm PEER scholar identities and promote allyship by infusing sensitivity, responsiveness and anti-bias awareness., This article focuses on how mentors can be allies against microaggressions in STEM.

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.

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.

Voltage-gated Ca(v) Ca(2+) channels play crucial roles in regulating gene transcription, neuronal excitability, and synaptic transmission. Natural or pathological variations in Ca(v) 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 Ca(v)1.3 α(1) 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 Ca(v) 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 Ca(v)1.3 cDNA, Q558H significantly decreased the density of Ca(2+) 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 Ca(v)1.3 channels at the cell surface. The substitution also produced more sustained Ca(2+) currents by weakening voltage-dependent inactivation. When inserted into the corresponding locus of Ca(v)2.1, the substitution had similar effects as in Ca(v)1.3. However, the substitution introduced in Ca(v)3.1 reduced current density, but had no effects on voltage-dependent inactivation. Our results reveal a critical extracellular determinant of current density for all Ca(v) family members and of voltage-dependent inactivation of Ca(v)1.3 and Ca(v)2.1 channels.