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.

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.

Mitochondria contain connexins, a family of proteins that is known to form gap junction channels. Connexins are synthesized in the endoplasmic reticulum and oligomerized in the Golgi to form hemichannels. Hemichannels from adjacent cells dock with one another to form gap junction channels that aggregate into plaques and allow cell–cell communication. Cell–cell communication was once thought to be the only function of connexins and their gap junction channels. In the mitochondria, however, connexins have been identified as monomers and assembled into hemichannels, thus questioning their role solely as cell–cell communication channels. Accordingly, mitochondrial connexins have been suggested to play critical roles in the regulation of mitochondrial functions, including potassium fluxes and respiration. However, while much is known about plasma membrane gap junction channel connexins, the presence and function of mitochondrial connexins remain poorly understood. In this review, the presence and role of mitochondrial connexins and mitochondrial/connexin-containing structure contact sites will be discussed. An understanding of the significance of mitochondrial connexins and their connexin contact sites is essential to our knowledge of connexins’ functions in normal and pathological conditions, and this information may aid in the development of therapeutic interventions in diseases linked to mitochondria.

Background: Sickle cell disease (SCD) is a hereditary disorder affecting red blood cells’ shape and functional capacity. Individuals with SCD report relatively high co-occurrence of neurodevelopmental disorders (NDDs). In addition, these children also have higher rates of enuresis (incontinence) and pica, disorders prevalent in children with developmental delays. Both enuresis and pica can have negative effects on mental health, but their pathophysiology, especially in SCD, remains unclear. Objectives: The objective of this study was to determine the rates of pica and enuresis in a pediatric SCD clinic to compare the co-occurrence of NDDs and enuresis/pica. Methods: To do so, we performed a cross-sectional explanatory retrospective chart review of 275 pediatric SCD patients. Results: Our SCD cohort had a 27% prevalence of enuresis, 9% prevalence of pica, and 24% prevalence of one or more NDDs. We noted significant inter-group overlap between pica/enuresis and other risk SCD severity factors. NDDs were approximately twice as frequent in SCD patients with pica or enuresis compared to those without. While pica was associated with HbSβ⁺, it was not linked to disease severity indicators. Enuresis was associated with hydroxyurea usage (66.7% vs. 42.6%, p = 0.001) and reticulocyte counts, indicative of higher disease severity. Conclusions: Clinically, these results are the first to show co-occurrence between pica, enuresis, and NDDs in SCD. We suggest that the occurrence of pica or enuresis may serve as an indicator for previously unknown NDD risk. Together, these results underscore the need for targeted screenings of pica and enuresis in SCD populations.