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Abstract Proximity ligation assays (PLAs) use specific antibodies to detect endogenous protein‐protein interactions. PLAs are a highly useful biochemical technique that allow two proteins within proximity to be visualized with fluorescent probes amplified by PCR. While this technique has gained prominence, the use of a 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). © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Proximity ligation assay for skeletal muscle tissue and myoblast for MERC proteins 

Abstract OPA1 is a dynamin‐related GTPase that modulates mitochondrial dynamics and cristae integrity. Humans carry eight different isoforms of OPA1 and mice carry five, all of which are expressed as short‐ or long‐form isoforms. These isoforms contribute to OPA1's ability to control mitochondrial energetics and DNA maintenance. However, western blot isolation of all long and short isoforms of OPA1 can be difficult. To address this issue, we developed an optimized western blot protocol based on improving running time to isolate five different isoforms of OPA1 in mouse cells and tissues. This protocol can be applied to study changes in mitochondrial structure and function. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Western Blot Protocol for Isolating OPA1 Isoforms in Mouse Primary Skeletal Muscle Cells 

Abstract While some established undergraduate summer programs are effective across many institutions, these programs may only be available to some principal investigators or may not fully address the diverse needs of incoming undergraduates. This article outlines a 10‐week science, technology, engineering, mathematics, and medicine (STEMM) education program designed to prepare undergraduate students for graduate school through a unique model incorporating mentoring dyads and triads, cultural exchanges, and diverse activities while emphasizing critical thinking, research skills, and cultural sensitivity. Specifically, we offer a straightforward and adaptable guide that we have used for mentoring undergraduate students in a laboratory focused on mitochondria and microscopy, but which may be customized for other disciplines. Key components include self‐guided projects, journal clubs, various weekly activities such as mindfulness training and laboratory techniques, and a focus on individual and cultural expression. Beyond this unique format, this 10‐week program also seeks to offer an intensive research program that emulates graduate‐level experiences, offering an immersive environment for personal and professional development, which has led to numerous achievements for past students, including publications and award‐winning posters. 

ABSTRACT Age‐related skeletal muscle atrophy, known as sarcopenia, is characterized by loss of muscle mass, strength, endurance, and oxidative capacity. Although exercise has been shown to mitigate sarcopenia, the underlying governing mechanisms are poorly understood. Mitochondrial dysfunction is implicated in aging and sarcopenia; however, few studies explore how mitochondrial structure contributes to this dysfunction. In this study, we sought to understand how aging impacts mitochondrial three‐dimensional (3D) structure and its regulators in skeletal muscle. We hypothesized that aging leads to remodeling of mitochondrial 3D architecture permissive to dysfunction and is ameliorated by exercise. Using serial block‐face scanning electron microscopy (SBF‐SEM) and Amira software, mitochondrial 3D reconstructions from patient biopsies were generated and analyzed. Across five human cohorts, we correlate differences in magnetic resonance imaging, mitochondria 3D structure, exercise parameters, and plasma immune markers between young (under 50 years) and old (over 50 years) individuals. We found that mitochondria are less spherical and more complex, indicating age‐related declines in contact site capacity. Additionally, aged samples showed a larger volume phenotype in both female and male humans, indicating potential mitochondrial swelling. Concomitantly, muscle area, exercise capacity, and mitochondrial dynamic proteins showed age‐related losses. Exercise stimulation restored mitofusin 2 (MFN2), one such of these mitochondrial dynamic proteins, which we show is required for the integrity of mitochondrial structure. Furthermore, we show that this pathway is evolutionarily conserved, as Marf, the MFN2 ortholog inDrosophila, knockdown alters mitochondrial morphology and leads to the downregulation of genes regulating mitochondrial processes. Our results define age‐related structural changes in mitochondria and further suggest that exercise may mitigate age‐related structural decline through modulation of mitofusin 2. 

Abstract A first‐generation college student is typically defined as a student whose biological parent(s) or guardian(s) never attended college or who started but did not finish college. However, “first‐generation” can represent diverse family education situations. The first‐generation student community is a multifaceted, and intersectional group of individuals who frequently lack educational/financial resources to succeed and, consequently, require supportive environments with rigorous mentorship. However, first‐generation students often do not make their identity as first‐generation students known to others due to several psychosocial and academic factors. Therefore, they are often “invisible minorities” in higher education. In this paper, we describe the diverse family situations of first‐generation students, further define “first‐generation,” and suggest five actions that first‐generation trainees at the undergraduate/graduate stages can engage in to succeed in an academic climate. We also provide suggestions for mentors to accommodate first‐generation students' unique experiences and equip them with tools to deliver intentional mentoring practices. We hope that this paper will help promote first‐generation student success throughout the academic pipeline. 

Abstract The physical characteristics of brown adipose tissue (BAT) are defined by the presence of multilocular lipid droplets (LDs) within the brown adipocytes and a high abundance of iron‐containing mitochondria, which give it its characteristic color. Normal mitochondrial function is, in part, regulated by organelle‐to‐organelle contacts. For example, the contact sites that mediate mitochondria–LD interactions are thought to have various physiological roles, such as the synthesis and metabolism of lipids. Aging is associated with mitochondrial dysfunction, and previous studies show that there are changes in mitochondrial structure and the proteins that modulate organelle contact sites. However, how mitochondria–LD interactions change with aging has yet to be fully clarified. Therefore, we sought to define age‐related changes in LD morphology and mitochondria–lipid interactions in BAT. We examined the three‐dimensional morphology of mitochondria and LDs in young (3‐month) and aged (2‐year) murine BAT using serial block face‐scanning electron microscopy and the Amira program for segmentation, analysis, and quantification. Our analyses showed reductions in LD volume, area, and perimeter in aged samples in comparison to young samples. Additionally, we observed changes in LD appearance and type in aged samples compared to young samples. Notably, we found differences in mitochondrial interactions with LDs, which could implicate that these contacts may be important for energetics in aging. Upon further investigation, we also found changes in mitochondrial and cristae structure for the mitochondria interacting with LDs. Overall, these data define the nature of LD morphology and organelle–organelle contacts during aging and provide insight into LD contact site changes that interconnect biogerontology with mitochondrial function, metabolism, and bioactivity in aged BAT.