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The endomembrane system is essential for healthy cell function, with the various compartments carrying out a large number of specific biochemical reactions. To date, almost all of our understanding of the endomembrane system has come from the study of cultured cells growing as monolayers. However, monolayer-grown cells only poorly represent the environment encountered by cells in the human body. As a first step to address this disparity, we have developed a platform that allows us to investigate and quantify changes to the endomembrane system in three-dimensional (3D) cell models, in an automated and highly systematic manner. HeLa Kyoto cells were grown on custom-designed micropatterned 96-well plates to facilitate spheroid assembly in the form of highly uniform arrays. Fully automated high-content confocal imaging and analysis were then carried out, allowing us to measure various spheroid-, cellular- and subcellular-level parameters relating to size and morphology. Using two drugs known to perturb endomembrane function, we demonstrate that cell-based assays can be carried out in these spheroids, and that changes to the Golgi apparatus and endosomes can be quantified from individual cells within the spheroids. We also show that image texture measurements are useful tools to discriminate cellular phenotypes. The automated platform that we show here has the potential to be scaled up, thereby allowing large-scale robust screening to be carried out in 3D cell models. [ABSTRACT FROM AUTHOR]

Proteins are biological units whose essence is defined by their functional relationships with other proteins or biomolecules such as RNA, DNA, lipids, or carbohydrates. These functions encompass enzymatic, structural, regulatory, or physical interaction roles. The STRING database (Nucleic Acids Research, 8 Jan 2021;49(D1): D605-12) provides an index that defines the functional interaction networks between proteins in model organisms. To facilitate the identification, visualization, and evaluation of potential functional networks across organisms from different phylogenetic lineages, we have developed PhyloString (https://biocomputo.ibt.unam.mx/phylostring/), a web server that utilizes the indices of the STRING database. PhyloString decomposes these functional networks into modules, representing cohesive units of proteins grouped based on their similarity of STRING values and the phylogenetic origins of their respective organisms. This study presents and thoroughly discusses examples of such functional networks and their modules identified using PhyloString. [ABSTRACT FROM AUTHOR]

The environment within the Endoplasmic Reticulum (ER) influences Insulin biogenesis. In particular, ER stress may contribute to the development of Type 2 Diabetes (T2D) and Cystic Fibrosis Related Diabetes (CFRD), where evidence of impaired Insulin processing, including elevated secreted Proinsulin/Insulin ratios, are observed. Our group has established the role of a novel ER chaperone ERp29 (ER protein of 29 kDa) in the biogenesis of the Epithelial Sodium Channel, ENaC. The biogenesis of Insulin and ENaC share may key features, including their potential association with COP II machinery, their cleavage into a more active form in the Golgi or later compartments, and their ability to bypass such cleavage and remain in a less active form. Given these similarities we hypothesized that ERp29 is a critical factor in promoting the efficient conversion of Proinsulin to Insulin. Here, we confirmed that Proinsulin associates with the COP II vesicle cargo recognition component, Sec24D. When Sec24D expression was decreased, we observed a corresponding decrease in whole cell Proinsulin levels. In addition, we found that Sec24D associates with ERp29 in co-precipitation experiments and that ERp29 associates with Proinsulin in co-precipitation experiments. When ERp29 was overexpressed, a corresponding increase in whole cell Proinsulin levels was observed, while depletion of ERp29 decreased whole cell Proinsulin levels. Together, these data suggest a potential role for ERp29 in regulating Insulin biosynthesis, perhaps in promoting the exit of Proinsulin from the ER via Sec24D/COPII vesicles. [ABSTRACT FROM AUTHOR]

Proteins and lipids are known to be transported to targeted cytosolic compartments in vesicles. A similar system in chloroplasts is suggested to transfer lipids from the inner envelope to the thylakoids. However, little is known about both possible cargo proteins and the proteins required to build a functional vesicle transport system in chloroplasts. A few components have been suggested, but only one (CPSAR1) has a verified location in chloroplast vesicles. This protein is localized in the donor membrane (envelope) and vesicles, but not in the target membrane (thylakoids) suggesting it plays a similar role to a cytosolic homologue, Sar1, in the secretory pathway. Thus, we hypothesized that there may be more similarities, in addition to lipid transport, between the vesicle transport systems in the cytosol and chloroplast, i.e. similar vesicle transport components, possible cargo proteins and receptors. Therefore, using a bioinformatics approach we searched for putative chloroplast components in the model plant Arabidopsis thaliana, corresponding mainly to components of the cytosolic vesicle transport system that may act in coordination with previously proposed COPII chloroplast homologues. We found several additional possible components, supporting the notion of a fully functional vesicle transport system in chloroplasts. Moreover, we found motifs in thylakoid-located proteins similar to those of COPII vesicle cargo proteins, supporting the hypothesis that chloroplast vesicles may transport thylakoid proteins from the envelope to the thylakoid membrane. Several putative cargo proteins are involved in photosynthesis, thus we propose the existence of a novel thylakoid protein pathway that is important for construction and maintenance of the photosynthetic machinery. [ABSTRACT FROM AUTHOR]