细胞生物学

Exosomes are a subpopulation of the heterogenous pool of extracellular vesicles that are secreted to the extracellular space. Exosomes have been purported to play a role in intercellular communication and have demonstrated utility as biomarkers for a variety of diseases. Despite broad interest in exosome biology, the conditions that regulate their secretion are incompletely understood. The goal of this procedure is to biochemically reconstitute exosome secretion in Streptolysin O (SLO)-permeabilized mammalian cells. This protocol describes the reconstitution of lyophilized SLO, preparation of cytosol and SLO-permeabilized cells, assembly of the biochemical reconstitution reaction, and quantification of exosome secretion using a sensitive luminescence-based assay. This biochemical reconstitution reaction can be utilized to characterize the molecular mechanisms by which different gene products regulate exosome secretion.

Key features

• This protocol establishes a functional in vitro system to reconstitute exosome secretion in permeabilized mammalian cells upon addition of cytosol, ATP, GTP, and calcium (Ca²⁺).

Graphical overview

Circular RNA (circRNA) is an intriguing class of non-coding RNA that exists as a continuous closed loop. With the improvements in high throughput sequencing, biochemical analysis, and bioinformatic algorithms, studies on circRNA expression became abundant in recent years. However, functional studies of circRNA are still limited. Subcellular localization of circRNA may provide some clues in elucidating its biological functions by performing subcellular fractionation assay. Notably, circRNAs that are predominantly found in the cytoplasm are more likely to be involved in post-transcriptional gene regulation, e.g., acting as micoRNA sponge, whereas nuclear-retained circRNAs are predicted to play a role in transcriptional regulation. Subcellular fractionation could help researchers to narrow down and prioritize downstream experiments. The majority of the currently available protocols describe the steps for subcellular fractionation followed by western blot analysis for protein molecules. Here, we present a protocol for the subcellular fractionation of cells to detect circRNA via RT-qPCR with divergent primers. Moreover, detailed steps for the generation of specific circRNAs-enriched cDNA included in this protocol will enhance the amplification and detection of low-abundance circRNAs. This will be useful for researchers studying low-abundance circRNAs.

Key features

• This protocol builds upon the method developed by Gagnon et al. (2014) and extends its application to circRNA study.

• Protocol for amplification of low levels of circRNA expression.

• Analysis takes into consideration the ratio of cytoplasmic RNA concentration to nuclear RNA concentration.

Graphical overview

The subfractionation of the endoplasmic reticulum (ER) is a widely used technique in cell biology. However, current protocols present limitations such as low yield, the use of large number of dishes, and contamination with other organelles. Here, we describe an improved method for ER subfractionation that solves other reported methods' main limitations of being time consuming and requiring less starting material. Our protocol involves a combination of different centrifugations and special buffer incubations as well as a fine-tuned method for homogenization followed by western blotting to confirm the purity of the fractions. This protocol contains a method to extract clean ER samples from cells using only five (150 mm) dishes instead of over 50 plates needed in other protocols. In addition, in this article we not only propose a new cell fractionation approach but also an optimized method to isolate pure ER fractions from one mouse liver instead of three, which are commonly used in other protocols. The protocols described here are optimized for time efficiency and designed for seamless execution in any laboratory, eliminating the need for special/patented reagents.

Key features

• Subcellular fractionation from cells and mouse liver.

• Uses only five dishes (150 mm) or one mouse liver to extract highly enriched endoplasmic reticulum without mitochondrial-associated membrane contamination.

• These protocols require the use of ultracentrifuges, dounce homogenizers, and/or Teflon Potter Elvehjem.

• As a result, highly enriched/clean samples are obtained.

Graphical overview

Cilia and flagella are microtubule-based hair-like organelles protruding from the surface of most eukaryotic cells, and play essential roles in cell locomotion, left-right asymmetry, embryo development, and tissue homeostasis. With isolated cilia and flagella, great progress has been made in understanding the composition, structure, and function of cilia. However, the current cilia/flagella isolation methods are deficient in the integrity or productivity of purified cilia when applied to mammalian motile cilia. Here, we describe a new protocol that isolates cilia shafts from mouse ependymal cells, by horizontal shear force and mild detergent. This method enables the production of virtually integral cilia with high yields and less cell body contamination. It is suitable for immunostaining, puromycin labeling assay, and proximity ligation assay of mammalian motile cilia.

Graphical abstract:

Lysosome isolation is a preresiquite for identifying lysosomal protein composition by mass spectroscopic analysis, to reveal lysosome functions, and their involvement in some diseases. Magnetic nanoparticle-based fractionation has received great attention for lysosome isolation, owing to its high efficiency, purity, and preservation of lysosomal structures. Understanding the intracellular trafficking of magnetic probes is the key point of this technique, to determine the appropriate time for magnetic isolation of lysosomes, because this parameter changes depending on different cell lines used. The traditional magnetic probes, such as superparamagnetic iron oxide nanoparticles (SPIONs), require surface modification by fluorescent dyes to enable the investigation of their intracellular trafficking, which has some disadvantages, including the possible alternation of their bio-interaction, and the instability of fluorescence properties in the lysosomal environment. To overcome those limitations, we present a protocol that employs magnetic-plasmonic nanoparticles (MPNPs) to investigate intracellular trafficking using their intrinsic imaging capability, followed by quick lysosome isolation using a magnetic column. This protocol can be easily applied to isolate the intact lysosomes of any adherent cell lines.

Graphical abstract:

Extracellular vesicles (EVs), such as exosomes, are produced by all known eukaryotic cells, and constitute essential means of intercellular communication. Recent studies have unraveled the important roles of EVs in migrating to specific sites and cells. Functional studies of EVs using in vivo and in vitro systems require tracking these organelles using fluorescent dyes or, alternatively, transfected and fluorescent-tagged proteins, located either intravesicularly or anchored to the EV bilayer membrane. Due to design simplicity, the fluorescent dye might be a preferred method if the cells are difficult to modify by transfection or when the genetic alteration of the mother cells is not desired. This protocol describes techniques to label cultured cell-derived EVs, using lipophilic DiR [DiIC18(7) (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine Iodide)] fluorophore. This technique can be used to study the cellular uptake and intracellular localization of EVs, and their biodistribution in vivo, which are crucial evaluations of any isolated EVs.

Endosomal recycling is essential for the appropriate function of the endosome. During this process, endosomal coat complexes (i.e., retromer, and Mvp1) are recruited to the endosome, and deform its membrane to form recycling vesicles. To further analyze this, we developed a protocol for the immunoisolation of recycling vesicles from budding yeast. This method is a powerful way to characterize endosomal recycling pathways.

The plant nucleus is an important subcellular organelle that contains the genome, ribosomal RNA, and regulatory proteins, and performs a central role in the functioning and metabolism of the cell. Fractionation of intact nuclei is a crucial process to elucidate the function of nuclear proteins. Here, we present a simple method for the fractionation of crude nuclei and extraction of nuclear proteins, based on previously established methods. This protocol provides an easy and quick method to isolate crude nuclei and extract nuclear proteins from Arabidopsis seedlings, which is useful for the research on the nuclear proteins, without requirement for high-purity nuclei.

Graphic abstract:

Schematic procedure for the isolation of crude nuclei and extraction of nuclear proteins from Arabidopsis seedlings.

Throughout their life cycle, bacteria shed portions of their outermost membrane comprised of proteins, lipids, and a diversity of other biomolecules. These biological nanoparticles have been shown to have a range of highly diverse biological activities, including pathogenesis, community regulation, and cellular defense (among others). In recent publications, we have isolated and characterized membrane vesicles (MVs) from several species of Lactobacilli, microbes classified as commensals within the human gut microbiome (Dean et al., 2019 and 2020). With increasing scientific understanding of host-microbe interactions, the gut-brain axis, and tailored probiotics for therapeutic or performance increasing applications, the protocols described herein will be useful to researchers developing new strategies for gut community engineering or the targeted delivery of bio-active molecules.

Graphic abstract:

Figure 1. Atomic force microscopic image of Lactobacillus casei ATCC 393 bacteria margins (white arrows) and membrane vesicles (black arrows)

Mitochondria are essential organelles containing approximately 1,500 proteins. Only approximately 1% of these proteins are synthesized inside mitochondria, whereas the remaining 99% are synthesized as precursors on cytosolic ribosomes and imported into the organelle. Various tools and techniques to analyze the import process have been developed. Among them, in vitro reconstituted import systems are of importance to study these processes in detail. These experiments monitor the import reaction of mitochondrial precursors that were previously radiolabeled in a cell-free environment. However, the methods described have been mostly performed in mitochondria isolated from S. cerevisiae. Here, we describe the adaptation of this powerful assay to import proteins into crude mitochondria isolated from human tissue culture cells.

Graphic abstract:

Overview of the assay to monitor protein import into mitochondria isolated from human cells