Biochemical Separation of Cytoplasmic and Nuclear Fraction for Downstream Molecular Analysis.

Biochemical fractionation is a technique used to isolate and separate distinct cellular compartments, critical for dissecting cellular mechanisms and molecular pathways. Herein we outline a biochemical fraction methodology for isolation of ultra-pure nuclei and cytoplasm. This protocol utilizes hypotonic lysis buffer to suspend cells, coupled with a calibrated centrifugation strategy, for enhanced separation of cytoplasm from the nuclear fraction. Subsequent purification steps ensure the integrity of the isolated nuclear fraction. Overall, this method facilitates accurate protein localization, essential for functional studies, demonstrating its efficacy in separating cellular compartments. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Biochemical fractionation Support Protocol 1: Protein quantification using Bradford assay Support Protocol 2: SDS/PAGE and Western blotting.

Size-Selective Capturing of Exosomes Using DNA Tripods.

Fractionating and characterizing target samples are fundamental to the analysis of biomolecules. Extracellular vesicles (EVs), containing information regarding the cellular birthplace, are promising targets for biology and medicine. However, the requirement for multiple-step purification in conventional methods hinders analysis of small samples. Here, we apply a DNA origami tripod with a defined aperture of binders (e.g., antibodies against EV biomarkers), which allows us to capture the target molecule. Using exosomes as a model, we show that our tripod nanodevice can capture a specific size range of EVs with cognate biomarkers from a broad distribution of crude EV mixtures. We further demonstrate that the size of captured EVs can be controlled by changing the aperture of the tripods. This simultaneous selection with the size and biomarker approach should simplify the EV purification process and contribute to the precise analysis of target biomolecules from small samples.

Bacterial Envelope Fractionation.

Numerous bioinformatics tools allow predicting the localization of membrane proteins in the outer or inner membrane of Escherichia coli with high precision. Nevertheless, it might be desirable to experimentally verify such predictions or to assay the correct localization of recombinant or mutated variants of membrane proteins. Here we describe two methods (preferential detergent solubilization and sucrose-gradient fractionation) that allow to fractionate Gram-negative bacterial membranes and subsequently to enrich inner or outer membrane proteins.

REAP+: A single preparation for rapid isolation of nuclei, cytoplasm, and mitochondria.

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.

Separation of Single Core and Multicore Lytic Granules by Subcellular Fractionation and Immunoisolation.

Subcellular fractionation is an important tool used to separate intracellular organelles, structures or proteins. Here, we describe a stepwise protocol to isolate two types of lytic granules, multicore (MCG), and single core (SCG), from primary murine CTLs. We used cell disruption by nitrogen cavitation followed by separation of organelles via discontinuous sucrose density gradient centrifugation. Immunoisolation with a Synaptobrevin 2 antibody attached to magnetic beads was then used to harvest Synaptobrevin 2 positive granules for immunoblotting, mass spectrometry, electron, and light microscopy.

Isolation of Mammalian Peroxisomes by Density Gradient Centrifugation.

Sophisticated organelle fractionation strategies were the workhorse of early peroxisome research and led to the characterization of the principal functions of the organelle. However, even in the era of molecular biology and "omics" technologies, they are still of importance to unravel peroxisome-specific proteomes, confirm the localization of still uncharacterized proteins, analyze peroxisome metabolism or lipid composition, or study their protein import mechanism. To isolate and analyze peroxisomes for these purposes, density gradient centrifugation still represents a highly reliable and reproducible technique. This article describes two protocols to purify peroxisomes from either liver tissue or the HepG2 hepatoma cell line. The protocol for liver enables purification of peroxisome fractions with high purity (95%) and is therefore suitable to study low-abundant peroxisomal proteins or analyze their lipid composition, for example. The protocol presented for HepG2 cells is not suitable to gain highly pure peroxisomal fractions but is intended to be used for gradient profiling experiments and allows easier manipulation of the peroxisomal compartment, e.g., by gene knockdown or protein overexpression for functional studies. Both purification methods therefore represent complementary tools to be used to analyze different aspects of peroxisome physiology. Please note that this is an updated version of a protocol, which has been published in a former volume of Methods in Molecular Biology.

Analysis of Yeast Peroxisomes via Spatial Proteomics.

Peroxisomes are ubiquitous organelles with essential functions in numerous cellular processes such as lipid metabolism, detoxification of reactive oxygen species, and signaling. Knowledge of the peroxisomal proteome including multi-localized proteins and, most importantly, changes of its composition induced by altering cellular conditions or impaired peroxisome biogenesis and function is of paramount importance for a holistic view on peroxisomes and their diverse functions in a cellular context. In this chapter, we provide a spatial proteomics protocol specifically tailored to the analysis of the peroxisomal proteome of baker's yeast that enables the definition of the peroxisomal proteome under distinct conditions and to monitor dynamic changes of the proteome including the relocation of individual proteins to a different cellular compartment. The protocol comprises subcellular fractionation by differential centrifugation followed by Nycodenz density gradient centrifugation of a crude peroxisomal fraction, quantitative mass spectrometric measurements of subcellular and density gradient fractions, and advanced computational data analysis, resulting in the establishment of organellar maps on a global scale.

Characterization of Yeast Peroxisomes: Enrichment of Peroxisomal Fractions and Analysis of ß-Oxidation Activity.

Subcellular fractionation approaches have allowed for the identification of various functionally distinct organelles including peroxisomes. The methods enable enrichment of organelles and combined with downstream assays allow for the identification of biochemical functions, composition, and structural characteristics of these compartments. In this chapter, we describe the methods for differential centrifugation and Nycodenz gradients in the yeast Saccharomyces cerevisiae and describe assays for fatty acid ß-oxidation in intact cells and in peroxisomal fractions.

A general method for quantitative fractionation of mammalian cells.

Subcellular fractionation in combination with mass spectrometry-based proteomics is a powerful tool to study localization of key proteins in health and disease. Here we offered a reliable and rapid method for mammalian cell fractionation, tuned for such proteomic analyses. This method proves readily applicable to different cell lines in which all the cellular contents are accounted for, while maintaining nuclear and nuclear envelope integrity. We demonstrated the method's utility by quantifying the effects of a nuclear export inhibitor on nucleoplasmic and cytoplasmic proteomes.

Isolation of Functional Mitochondria and Pure mtDNA from Murine Tissues.

Detailed analysis of mitochondrial function cannot be achieved without good quality preparations of isolated mitochondria. Ideally, the isolation protocol should be quick, while producing a reasonably pure pool of mitochondria that are still intact and coupled. Here, we describe a fast and simple method for the purification of mammalian mitochondria relying on isopycnic density gradient centrifugation. We describe specific steps that should be taken into consideration when functional mitochondria from different tissues should be isolated. This protocol is suitable for the analysis of many aspects of the organelle's structure and function.

Isolation and Quality Control of Yeast Mitochondria.

The isolation of organelles devoid of other cellular compartments is crucial for studying organellar proteomes and the localization of newly identified proteins, as well as for assessing specific organellar functions. Here, we describe a protocol for the isolation of crude and highly pure mitochondria from Saccharomyces cerevisiae and provide methods for testing the functional integrity of the isolated organelles.

Isolation of Neuronal Synaptic Membranes by Sucrose Gradient Centrifugation.

Sucrose gradient centrifugation is a very useful technique for isolating specific membrane types based on their size and density. This is especially useful for detecting fatty acids and lipid molecules that are targeted to specialized membranes. Without fractionation, these types of molecules could be below the levels of detection after being diluted out by the more abundant lipid molecules with a more ubiquitous distribution throughout the various cell membranes. Isolation of specific membrane types where these lipids are concentrated allows for their detection and analysis. We describe herein our synaptic membrane isolation protocol that produces excellent yield and clear resolution of five major membrane fractions from a starting neural tissue homogenate: P1 (nuclear), P2 (cytoskeletal), P3 (neurosynaptosomal), PSD (post-synaptic densities), and SV (synaptic vesicle).

Rapid fractionation of mitochondria from mouse liver and heart reveals in vivo metabolite compartmentation.

The compartmentation and distribution of metabolites between mitochondria and the rest of the cell is a key parameter of cell signalling and pathology. Here, we have developed a rapid fractionation procedure that enables us to take mouse heart and liver from in vivo and within ~ 30 s stabilise the distribution of metabolites between mitochondria and the cytosol by rapid cooling, homogenisation and dilution. This is followed by centrifugation of mitochondria through an oil layer to separate mitochondrial and cytosolic fractions for subsequent metabolic analysis. Using this procedure revealed the in vivo compartmentation of mitochondrial metabolites and will enable the assessment of the distribution of metabolites between the cytosol and mitochondria during a range of situations in vivo.

Subcellular Fractionation for the Isolation of Synaptic Components from the Murine Brain.

Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological disorders. The characterization of synaptic sub-compartments by biochemical isolation is, therefore, a powerful method to elucidate the molecular bases of synaptic processes, both in health and disease. This protocol describes the isolation of synaptic terminals and synaptic sub-compartments from mouse brains by subcellular fractionation. First, sealed synaptic terminal structures, known as synaptosomes, are isolated following brain tissue homogenization. Synaptosomes are neuronal pre- and post-synaptic compartments with pinched-off and sealed membranes. These structures retain a metabolically active state and are valuable for studying synaptic structure and function. The synaptosomes are then subjected to hypotonic lysis and ultracentrifugation to obtain synaptic sub-compartments enriched for synaptic vesicles, synaptic cytosol, and synaptic plasma membrane. Fraction purity is confirmed by electron microscopy and biochemical enrichment analysis for proteins specific to sub-synaptic compartments. The presented method is a straightforward and valuable tool for studying the structural and functional characteristics of the synapse and the molecular etiology of various brain disorders.

Subcellular fractionation of brain tumor stem cells.

Brain tumor stem cells (BTSCs) are a rare population of self-renewing stem cells that are cultured as spheres and are often slow growing compared to other mammalian cell lines. Analysis of BTSC proteome requires careful handling as well as techniques that can be applied to small quantities of cell material. Subcellular fractionation is a widely used technique to assess protein localization. Although proteins are often destined to a defined cell compartment via a signal peptide such as mitochondrial or nuclear localization signals, the recruitment of a protein from one compartment to another can occur as a result of post-translational modification and/or structural variations in response to intracellular and extracellular stimuli. These events assign different functions to a protein making the study of protein localization a useful approach for better understanding of its role in disease progression. Sequential centrifugation remains a simple and versatile fractionation method for proteomic analysis. It can also be applied for diverse downstream applications such as multi-omics using pure nuclear fractions or metabolomic studies on isolated mitochondria. In this chapter, we describe our optimized protocol for subcellular fractionation of BTSC spheres in which we use a commercially available kit with additional centrifugation steps. We provide details on BTSC maintenance and handling, fractionation protocol and evaluation of fraction purity.

An optimized method for tissue glycogen quantification.

Mobilization of glycogen, the short-term storage form of glucose, is the body's first defense against hypoglycemia and is critical for energy provision during high intensity exercise. Therefore, to advance metabolic research, it is critical to be able to accurately measure glycogen concentrations, including during a prolonged fast and other glycogen-modulating interventions. Unfortunately, prior enzymatic methods of glycogen detection have been plagued by poor detection in the lower range, high sample mass requirements, and complicated and/or expensive protocols which introduce substantial technical variability into the measured glycogen concentrations. To address these limitations, here we report a streamlined and versatile glycogen extraction protocol coupled with an optimized phenol-sulfuric acid quantification protocol. With this method, we demonstrate how glycogen can be extracted from only 20 mg of tissue with one centrifugation step and quantified with a highly precise (Intra-assay %CV ranges from 5-10%) and sensitive (proportionality constant for glycogen = 0.07279 A.U./µg) assay. The cost of all materials equates to ~10 cents per sample. Therefore, this method represents an attractive means of assessing ex vivo tissue glycogen content including at the extremes of glycogen concentrations.

Impact of high platelet turnover on the platelet transcriptome: Results from platelet RNA-sequencing in patients with sepsis.

BACKGROUND: Sepsis is associated with high platelet turnover and elevated levels of immature platelets. Changes in the platelet transcriptome and the specific impact of immature platelets on the platelet transcriptome remain unclear. Thus, this study sought to address whether and how elevated levels of immature platelets affect the platelet transcriptome in patients with sepsis. METHODS: Blood samples were obtained from patients with sepsis requiring vasopressor therapy (n = 8) and from a control group of patients with stable coronary artery disease and otherwise similar demographic characteristics (n = 8). Immature platelet fraction (IPF) was determined on a Sysmex XE 2100 analyser and platelet function was tested by impedance aggregometry. RNA from leukocyte-depleted platelets was used for transcriptome analysis by Next Generation Sequencing integrating the use of unique molecular identifiers. RESULTS: IPF (median [interquartile range]) was significantly elevated in sepsis patients (6.4 [5.3-8.7] % vs. 3.6 [2.6-4.6] %, p = 0.005). Platelet function testing revealed no differences in adenosine diphosphate- or thrombin receptor activating peptide-induced platelet aggregation between control and sepsis patients. Putative circular RNA transcripts were decreased in platelets from septic patients. Leukocyte contamination defined by CD45 abundance levels in RNA-sequencing was absent in both groups. Principal component analysis of transcripts showed only partial overlap of clustering with IPF levels. RNA sequencing showed up-regulation of 524 and down-regulation of 118 genes in platelets from sepsis patients compared to controls. Upregulated genes were mostly related to catabolic processes and protein translation. Comparison to published platelet transcriptomes showed a large overlap of changes observed in sepsis and COVID-19 but not with reticulated platelets from healthy donors. CONCLUSIONS: Patients with sepsis appear to have a less degraded platelet transcriptome as indicated by increased levels of immature platelets and decreased levels of putative circular RNA transcripts. The present data suggests that increased protein translation is a characteristic mechanism of systemic inflammation.

Production of human translation-competent lysates using dual centrifugation.

Protein synthesis is a central process in gene expression and the development of efficient in vitro translation systems has been the focus of scientific efforts for decades. The production of translation-competent lysates originating from human cells or tissues remains challenging, mainly due to the variability of cell lysis conditions. Here we present a robust and fast method based on dual centrifugation that allows for detergent-free cell lysis under controlled mechanical forces. We optimized the lysate preparation to yield cytoplasm-enriched extracts from human cells that efficiently translate mRNAs in a cap-dependent as well as in an IRES-mediated way. Reduction of the phosphorylation state of eIF2α using recombinant GADD34 and 2-aminopurine considerably boosts the protein output, reinforcing the potential of this method to produce recombinant proteins from human lysates.

A method for rapid flow-cytometric isolation of endothelial nuclei and RNA from archived frozen brain tissue.

Endothelial cells are important contributors to brain development, physiology, and disease. Although RNA sequencing has contributed to the understanding of brain endothelial cell diversity, bulk analysis and single-cell approaches have relied on fresh tissue digestion protocols for the isolation of single endothelial cells and flow cytometry-based sorting on surface markers or transgene expression. These approaches are limited in the analysis of the endothelium in human brain tissues, where fresh samples are difficult to obtain. Here, we developed an approach to examine endothelial RNA expression by using an endothelial-specific marker to isolate nuclei from abundant archived frozen brain tissues. We show that this approach rapidly and reliably extracts endothelial nuclei from frozen mouse brain samples, and importantly, from archived frozen human brain tissues. Furthermore, isolated RNA transcript levels are closely correlated with expression in whole cells from tissue digestion protocols and are enriched in endothelial markers and depleted of markers of other brain cell types. As high-quality RNA transcripts could be obtained from as few as 100 nuclei in archived frozen human brain tissues, we predict that this approach should be useful for both bulk analysis of endothelial RNA transcripts in human brain tissues as well as single-cell analysis of endothelial sub-populations.

Distinct small non-coding RNA landscape in the axons and released extracellular vesicles of developing primary cortical neurons and the axoplasm of adult nerves.

Neurons have highlighted the needs for decentralized gene expression and specific RNA function in somato-dendritic and axonal compartments, as well as in intercellular communication via extracellular vesicles (EVs). Despite advances in miRNA biology, the identity and regulatory capacity of other small non-coding RNAs (sncRNAs) in neuronal models and local subdomains has been largely unexplored.We identified a highly complex and differentially localized content of sncRNAs in axons and EVs during early neuronal development of cortical primary neurons and in adult axons invivo. This content goes far beyond miRNAs and includes most known sncRNAs and precisely processed fragments from tRNAs, sno/snRNAs, Y RNAs and vtRNAs. Although miRNAs are the major sncRNA biotype in whole-cell samples, their relative abundance is significantly decreased in axons and neuronal EVs, where specific tRNA fragments (tRFs and tRHs/tiRNAs) mainly derived from tRNAs Gly-GCC, Val-CAC and Val-AAC predominate. Notably, although 5'-tRHs compose the great majority of tRNA-derived fragments observed invitro, a shift to 3'-tRNAs is observed in mature axons invivo.The existence of these complex sncRNA populations that are specific to distinct neuronal subdomains and selectively incorporated into EVs, equip neurons with key molecular tools for spatiotemporal functional control and cell-to-cell communication.