Proteins often show alterations in their subcellular localization with changing environmental conditions; transcription factors enter the nucleus or are actively removed from the nucleus; some even bind to endo-membranes by conditional membrane anchors; and other proteins and mRNA arrange in RNA granules. These are some examples of the complex regulation of subcellular localization, which often depends on posttranslational modifications and is triggered by environmental stressors. The challenge is the precise identification of the compartments, the quantitative analysis of proteins, which reside in multiple compartments, and their transport dynamics. Therefore, appropriate compartment markers and routines for a reproducible quantitative workflow are required.

Mitochondria-associated membranes (MAMs) are regions where the endoplasmic reticulum (ER) interacts with mitochondria and regulate lipid trafficking, calcium signaling, ER stress, and inflammation activation. Isolation of MAMs from endothelial cells is vital for studying insight into the immune regulation of many inflammatory diseases. Endothelial cells (ECs) are critical innate immune cells due to their paracrine function of secreting interleukins, chemokines, cytokines, and growth factors, as well as expressing levels of pattern recognition receptors including toll-like receptors (TLRs). Furthermore, ECs regulate and recruit monocytes by expressing adhesion molecules including vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), P-selectin, and E-selectin, to facilitate monocyte diapedesis in areas of damage and inflammation. This protocol consists of step-by-step instructions on isolating pure MAMs and other subcellular fractions from endothelial cells, which is critical to understanding ER and mitochondria crosstalks in endothelial functions in health and disease.

The phthalate ester, dibutyl phthalate (DBP), is one of the endocrine-disrupting chemicals detected in various aquatic environments. Previous research has found multiple toxic effects of DBP in aquatic organisms; however, the neurotoxic effects of the compound are surprisingly scanty. The purpose of this study was aimed to evaluate the role of oxidative stress in the induction of neurotoxicity in the brain tissue of the fish Pseudetroplus maculatus. The fish were exposed to the sublethal concentration of DBP (200 µg L-1) for 1, 4, 7, and 15 days along with control and vehicle control groups. The induction of oxidative stress in the brain subcellular fractions was proved by alterations in the activities of superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase along with the reduction in the total antioxidant capacity. Meanwhile, the levels of hydrogen peroxide and lipid peroxidation were increased. Neurotransmitters such as acetylcholine, dopamine, adrenaline, noradrenaline, and serotonin were altered in all subcellular fractions suggesting the disruption of the neurotransmitter system in the fish brain. These results indicate that DBP induces oxidative stress, which correlates with neurotoxicity in Pseudetroplus maculatus brain tissue.

Bafilomycin A1 inhibits V-type H+ ATPases on the molecular level, which acidifies endo-lysosomes. The main objective of the study was to assess the effect of bafilomycin A1 on Ca2+ content, NAADP-induced Ca2+ release, and ATPase activity in rat hepatocytes and human colon cancer samples. Chlortetracycline (CTC) was used for a quantitative measure of stored calcium in permeabilized rat hepatocytes. ATPase activity was determined by orthophosphate content released after ATP hydrolysis in subcellular post-mitochondrial fraction obtained from rat liver as well as from patients\' samples of colon mucosa and colorectal cancer samples. In rat hepatocytes, bafilomycin A1 decreased stored Ca2+ and prevented the effect of NAADP on stored Ca2+. This effect was dependent on EGTA-Ca2+ buffers in the medium. Bafilomycin A1 significantly increased the activity of Ca2+ ATPases of endoplasmic reticulum (EPR), but not plasma membrane (PM) Ca2+ ATPases in rat liver. Bafilomycin A1 also prevented the effect of NAADP on these pumps. In addition, bafilomycin A1 reduced Na+/K+ ATPase activity and increased basal Mg2+ ATPase activity in the subcellular fraction of rat liver. Concomitant administration of bafilomycin A1 and NAADP enhanced these effects. Bafilomycin A1 increased the activity of the Ca2+ ATPase of EPR in the subcellular fraction of normal human colon mucosa and also in colon cancer tissue samples. In contrast, it decreased Ca2+ ATPase PM activity in samples of normal human colon mucosa and caused no changes in colon cancer. Bafilomycin A1 decreased Na+/K+ ATPase activity and increased basal Mg2+ ATPase activity in normal colon mucosa samples and in human colon cancer samples. It can be concluded that bafilomycin A1 targets NAADP-sensitive acidic Ca2+ stores, effectively modulates ATPase activity, and assumes the link between acidic stores and EPR. Bafilomycin A1 may be useful for cancer therapy.

The disruption of mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) plays a relevant role in Alzheimer\'s disease (AD). MAMs have been implicated in neuronal dysfunction and death since it is associated with impairment of functions regulated in this subcellular domain, including lipid synthesis and trafficking, mitochondria dysfunction, ER stress-induced unfolded protein response (UPR), apoptosis, and inflammation. Since MAMs play an important role in lipid metabolism, in this study we characterized and investigated the lipidome alterations at MAMs in comparison with other subcellular fractions, namely microsomes and mitochondria, using an in vitro model of AD, namely the mouse neuroblastoma cell line (N2A) over-expressing the APP familial Swedish mutation (APPswe) and the respective control (WT) cells. Phospholipids (PLs) and fatty acids (FAs) were isolated from the different subcellular fractions and analyzed by HILIC-LC-MS/MS and GC-MS, respectively. In this in vitro AD model, we observed a down-regulation in relative abundance of some phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and lysophosphatidylethanolamine (LPE) species with PUFA and few PC with saturated and long-chain FA. We also found an up-regulation of CL, and antioxidant alkyl acyl PL. Moreover, multivariate analysis indicated that each organelle has a specific lipid profile adaptation in N2A APPswe cells. In the FAs profile, we found an up-regulation of C16:0 in all subcellular fractions, a decrease of C18:0 levels in total fraction (TF) and microsomes fraction, and a down-regulation of 9-C18:1 was also found in mitochondria fraction in the AD model. Together, these results suggest that the over-expression of the familial APP Swedish mutation affects lipid homeostasis in MAMs and other subcellular fractions and supports the important role of lipids in AD physiopathology.

REAP+ is an enhanced version of the rapid, efficient, and practical (REAP) method designed for the isolation of nuclear fractions. This improved version, REAP+, enables fast and effective extraction of mitochondria, cytoplasm, and nuclei. The mechanical cell disruption process has been optimized to cerebral tissues, snap-frozen liver, and HT22 cells with remarkable fraction enrichment. REAP+ is well-suited for samples containing minimal protein quantities, such as mouse hippocampal slices. The method was validated by Western blot and marker enzyme activities, such as LDH and G6PDH for the cytoplasmic fraction and succinate dehydrogenase and cytochrome c oxidase for the mitochondrial fraction. One of the outstanding features of this method is its rapid execution, yielding fractions within 15 min, allowing for simultaneous preparation of multiple samples. In essence, REAP+ emerges as a swift, efficient, and practical technique for the concurrent isolation of nuclei, cytoplasm, and mitochondria from various cell types and tissues. The method would be suitable to study the multicompartment translocation of proteins, such as metabolic enzymes and transcription factors migrating from cytosol to the mitochondria and nuclei. Moreover, its compatibility with small samples, such as hippocampal slices, and its potential applicability to human biopsies, highlights the potential application in medical research.

Although the subcellular dynamics of RNA and proteins are key determinants of cell homeostasis, their characterization is still challenging. Here we present an integrative framework to simultaneously interrogate the dynamics of the transcriptome and proteome at subcellular resolution by combining two methods: localization of RNA (LoRNA) and a streamlined density-based localization of proteins by isotope tagging (dLOPIT) to map RNA and protein to organelles (nucleus, endoplasmic reticulum and mitochondria) and membraneless compartments (cytosol, nucleolus and cytosolic granules). Interrogating all RNA subcellular locations at once enables system-wide quantification of the proportional distribution of RNA. We obtain a cell-wide overview of localization dynamics for 31,839 transcripts and 5,314 proteins during the unfolded protein response, revealing that endoplasmic reticulum-localized transcripts are more efficiently recruited to cytosolic granules than cytosolic RNAs, and that the translation initiation factor eIF3d is key to sustaining cytoskeletal function. Overall, we provide the most comprehensive overview so far of RNA and protein subcellular localization dynamics.

Protein is one of the primary biochemical macromolecular regulators in the compartmental cellular structure, and the subcellular locations of proteins can therefore provide information on the function of subcellular structures and physiological environments. Recently, data-driven systems have been developed to predict the subcellular location of proteins based on protein sequence, immunohistochemistry (IHC) images, or immunofluorescence (IF) images. However, the research on the fusion of multiple protein signals has received little attention. In this study, we developed a dual-signal computational protocol by incorporating IHC images into protein sequences to learn protein subcellular localization. Three major steps can be summarized as follows in this protocol: first, a benchmark database that includes 281 proteins sorted out from 4722 proteins of the Human Protein Atlas (HPA) and Swiss-Prot database, which is involved in the endoplasmic reticulum (ER), Golgi apparatus, cytosol, and nucleoplasm; second, discriminative feature operators were first employed to quantitate protein image-sequence samples that include IHC images and protein sequence; finally, the feature subspace of different protein signals is absorbed to construct multiple sub-classifiers via dimensionality reduction and binary relevance (BR), and multiple confidence derived from multiple sub-classifiers is adopted to decide subcellular location by the centralized voting mechanism at the decision layer. The experimental results indicated that the dual-signal model embedded IHC images and protein sequences outperformed the single-signal models with accuracy, precision, and recall of 75.41%, 80.38%, and 74.38%, respectively. It is enlightening for further research on protein subcellular location prediction under multi-signal fusion of protein.

Ionic liquids (ILs) have been considered eco-friendly alternatives to conventional organic solvents. However, several studies have reported that ILs exert toxicity towards aquatic invertebrates. Applying in vitro methodology, the aim of the present study was to evaluate the potential effect of three ILs on the biochemical performance of exposed Mytilus galloprovincialis digestive gland and gills cellular fractions. Carboxylesterase might be involved in the derived toxicity mechanism of ILs as activity levels increased significantly in digestive gland exposed fractions. This group of ILs did not seem to induce genotoxicity, except in gills cellular fractions exposed to 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide. In the literature, in vitro methodology has been suggested as an important complement to animal testing and in silico studies. The present research underlines its efficacy as a quick pre-screening before in vivo testing, particularly with heterogenic groups of substances with high variability in composition, such as ILs and deep eutectic solvents.

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