细胞生物学

Understanding protein–protein interactions is crucial for unravelling subcellular protein distribution, contributing to our understanding of cellular organisation. Moreover, interaction studies can reveal insights into the mechanisms that cover protein trafficking within cells. Although various techniques such as Förster resonance energy transfer (FRET), co-immunoprecipitation, and fluorescence microscopy are commonly employed to detect protein interactions, their limitations have led to more advanced techniques such as the in situ proximity ligation assay (PLA) for spatial co-localisation analysis. The PLA technique, specifically employed in fixed cells and tissues, utilises species-specific secondary PLA probes linked to DNA oligonucleotides. When proteins are within 40 nm of each other, the DNA oligonucleotides on the probes interact, facilitating circular DNA formation through ligation. Rolling-circle amplification then produces DNA circles linked to the PLA probe. Fluorescently labelled oligonucleotides hybridise to the circles, generating detectable signals for precise co-localisation analysis. We employed PLA to examine the co-localisation of dynein with the Kv7.4 channel protein in isolated vascular smooth muscle cells from rat mesenteric arteries. This method enabled us to investigate whether Kv7.4 channels interact with dynein, thereby providing evidence of their retrograde transport by the microtubule network. Our findings illustrate that PLA is a valuable tool for studying potential novel protein interactions with dynein, and the quantifiable approach offers insights into whether these interactions are changed in disease.

Recent advancements in tissue-clearing techniques and volumetric imaging have greatly facilitated visualization and quantification of biomolecules, organelles, and cells in intact organs or even entire organisms. Generally, there are two types of clearing methods: hydrophobic and hydrophilic (i.e., clearing with organic or aqueous solvents, respectively). The popular iDISCO approach and its modifications are hydrophobic methods that involve dehydration, delipidation, decolorization (optional), decalcification (optional), and refractive-index (RI) matching steps. Cleared samples are often stored for a relatively long period of time and imaged repeatedly. However, cleared tissues can become opaque over time, which prevents accurate reimaging. We reasoned that the resurgent haziness is likely due to rehydration, residual lipids, and uneven RI deep inside those tissue samples. For rescue, we have developed a simple procedure based on iDISCO. Beginning with a methanol dehydration, samples are delipidated using dichloromethane, followed by RI matching with dibenzyl ether (DBE). This simple method effectively re-clears mouse brains that have turned opaque during months of storage, allowing the user to effectively image immunolabeled samples over longer periods of time.

Key features

• This simple protocol rescues previously cleared tissue that has turned opaque.

• The method does not cause detectable loss of immunofluorescence from previously stained samples.

Graphical overview

Autophagy is a conserved homeostatic mechanism involved in cellular homeostasis and many disease processes. Although it was first described in yeast cells undergoing starvation, we have learned over the years that autophagy gets activated in many stress conditions and during development and aging in mammalian cells. Understanding the fundamental mechanisms underlying autophagy effects can bring us closer to better insights into the pathogenesis of many disease conditions (e.g., cardiac muscle necrosis, Alzheimer’s disease, and chronic lung injury). Due to the complex and dynamic nature of the autophagic processes, many different techniques (e.g., western blotting, fluorescent labeling, and genetic modifications of key autophagy proteins) have been developed to delineate autophagy effects. Although these methods are valid, they are not well suited for the assessment of time-dependent autophagy kinetics. Here, we describe a novel approach: the use of DAPRed for autophagic flux measurement via live cell imaging, utilizing A549 cells, that can visualize and quantify autophagic flux in real time in single live cells. This approach is relatively straightforward in comparison to other experimental procedures and should be applicable to any in vitro cell/tissue models.

Key features

• Allows real-time qualitative imaging of autophagic flux at single-cell level.

• Primary cells and cell lines can also be utilized with this technique.

• Use of confocal microscopy allows visualization of autophagy without disturbing cellular functions.

Intracellular bacterial pathogens have evolved to be adept at manipulating host cellular function for the benefit of the pathogen, often by means of secreted virulence factors that target host pathways for modulation. The lysosomal pathway is an essential cellular response pathway to intracellular pathogens and, as such, represents a common target for bacterial-mediated evasion. Here, we describe a method to quantitatively assess bacterial pathogen–mediated suppression of host cell trafficking to lysosomes, using Salmonella enterica serovar Typhimurium infection of epithelial cells as a model. This live-cell imaging assay involves the use of a BODIPY TR-X conjugate of BSA (DQ-Red BSA) that traffics to and fluoresces in functional lysosomes. This method can be adapted to study infection with a broad array of pathogens in diverse host cell types. It is capable of being applied to identify secreted virulence factors responsible for a phenotype of interest as well as domains within the bacterial protein that are important for mediating the phenotype. Collectively, these tools can provide invaluable insight into the mechanisms of pathogenesis of a diverse array of pathogenic bacteria, with the potential to uncover virulence factors that may be suitable targets for therapeutic intervention.

Key features

• Infection-based analysis of bacterial-mediated suppression of host trafficking to lysosomes, using Salmonella enterica serovar Typhimurium infection of human epithelial cells as a model.

• Live microscopy–based analysis allows for the visualization of individually infected host cells and is amenable to phenotype quantification.

• Assay can be adapted to a broad array of pathogens and diverse host cell types.

• Assay can identify virulence factors mediating a phenotype and protein domains that mediate a phenotype.

The Plasmodium parasites that cause malaria undergo an obligate, asymptomatic developmental stage in the host liver before initiating the symptomatic blood-stage infection. The parasite liver stage is a key intervention point for antimalarial chemoprophylaxis: successful targeting of liver-stage parasites prevents disease development in individuals and can help to reduce parasite transmission in populations, as the gametocyte forms that transmit infection to mosquitos are exclusively found in the blood stage. Antimalarial drugs that can target multiple parasite stages are thus highly desirable, and one emerging cellular target for such multistage active compounds is the process of protein synthesis or translation. Quantitative study of liver stage translation, and thus mechanistic evaluation of translation inhibitors against liver stage parasites, is not amenable to the methods allowing quantification of asexual blood stage translation, such as radiolabeled amino acid incorporation or lysate-based translation of reporter transcripts. Here, we present a method using o-propargyl puromycin (OPP) labeling of host and parasite nascent proteomes in the P. berghei-HepG2 infection model, followed by automated confocal image acquisition and computational separation of P. berghei vs. H. sapiens nascent proteome signals to allow simultaneous readout of the effects of translation inhibitors on both host and parasite. This protocol details our HepG2 cell culture and infected monolayer handling optimized for microscopy, our OPP labeling workflow, and our approach to automated confocal imaging, image processing, and data analysis.

Key features

• Uses the o-propargyl puromycin labeling technique developed by Liu et al. to quantitatively analyze protein synthesis in Plasmodium berghei liver-stage parasites in actively translating hepatoma cells.

• This quantitative approach should be adaptable for other puromycin-sensitive intracellular pathogens residing in actively translating host cells.

• The P. berghei–infected HepG2 recovery and reseeding protocol presented here is of use in applications beyond nascent proteome labeling and quantification.

Graphical overview

Dopaminergic (DAergic) neurodegeneration in the substantia nigra pars compacta of the human brain is the pathological feature associated with Parkinson’s disease (PD). Drosophila also exhibits mobility defects and diminished levels of brain dopamine on exposure to neurotoxicants mimicking PD. Our laboratory demonstrated in a Drosophila model of sporadic PD that there is no decrease in DAergic neuronal number; instead, there is a significant reduction in tyrosine hydroxylase (TH) fluorescence intensity (FI). Here, we present a sensitive assay based on the quantification of FI of the secondary antibody (ab). As the FI is directly proportional to the amount of TH synthesis, its reduction under PD conditions denotes the decrease in the TH synthesis, suggesting DAergic neuronal dysfunction. Therefore, FI quantification is a refined and sensitive method to understand the early stages of DAergic neurodegeneration. FI quantification is performed using the ZEN 2012 SP2 single-user software; a license must be acquired to utilize the imaging system to interactively control image acquisition, image processing, and analysis. This method will be of good use to biologists, as it can also be used with little modification to characterize the extent of degeneration and changes in the level of degeneration in response to drugs in different cell types. Unlike the expensive and cumbersome confocal microscopy, the present method will be an affordable option for fund-constrained neurobiology laboratories.

Key features

• Allows characterizing the incipient DAergic and other catecholaminergic neurodegeneration, even in the absence of loss of neuronal cell body.

• Great alternative for the fund-constrained neurobiology laboratories in developing countries to utilize this method in different cell types and their response to drugs/nutraceuticals.

Graphical overview

Vertebrate embryogenesis is a highly dynamic process involving coordinated cell and tissue movements that generate the final embryonic body plan. Many of these movements are difficult to image at high resolution because they occur deep within the embryo along the midline, causing light scattering and requiring longer working distances. Here, we present an explant-based method to image transverse cross sections of living zebrafish embryos. This method allows for the capture of all cell movements at high-resolution throughout the embryonic trunk, including hard-to-image deep tissues. This technique offers an alternative to expensive or computationally difficult microscopy methods.

Key features

• Generates intact zebrafish explants with minimal tissue disturbance.

• Allows for live imaging of deep tissues normally obscured by common confocal microscopy techniques.

• Immobilizes tissues for extended periods required for time-lapse imaging.

• Utilizes readily available reagents and tools, which can minimize the time and cost of the procedure.

Graphical overview

All living organisms require the division of a cell into daughter cells for their growth and maintenance. During cell division, both genetic and cytoplasmic contents are equally distributed between the two daughter cells. At the end of cell division, cytoplasmic contents and the plasma membrane are physically separated between the two daughter cells via a process known as cytokinesis. Hundreds of proteins and lipids involved in the cytokinetic process have been identified; however, much less is known about the mechanisms by which these molecules regulate cytokinesis, being therefore an intense area of current research. Male meiotic cytokinesis in Drosophila melanogaster testes has been shown to be an excellent model to study cytokinesis in vivo. Currently, several excellent protocols are available to study cytokinesis in Drosophila testes. However, improved methods are required to study cytokinesis under in vitro and ex vivo conditions. Here, we demonstrate a simple method to perform live imaging on individual spermatocyte cysts isolated from adult testes. We evaluate amenability of this in vitro method for treatment with pharmacological agents. We show that cytokinesis is strongly inhibited upon treatment with Dynasore, a dynamin inhibitor known to block clathrin-mediated endocytosis. In addition, we also demonstrate an ex vivo method to perform live imaging on whole mount adult testes on gas permeable membrane chambers. We believe the protocols described here are valuable tools to study cytokinetic mechanisms under various genetic and treatment conditions.

Key features

• In vitro method to study male meiotic cytokinesis in dissected spermatocyte cysts.

• In vitro method allows acute treatment with various pharmacological agents to study cytokinesis.

• Ex vivo method to image male meiosis cytokinesis in intact adult testes.

• Requires 15–60 min to set up and could be imaged up to 6–12 h.

Graphical overview

In vitro and ex vivo live imaging of male meiotic cytokinesis in adult Drosophila testes

Capillary density in skeletal muscles is key to estimate exercise capacity in healthy individuals, athletes, and those with muscle-related pathologies. Here, we present a step-by-step, high-throughput semi-automated method for quantifying capillary density from whole human skeletal muscle cross-sections, in areas of the muscle occupied by myofibers. We provide a detailed protocol for immunofluorescence staining, image acquisition, processing, and quantification. Image processing is performed in ImageJ, and data analysis is conducted in R. The provided protocol allows high-throughput quantification of capillary density.

Key features

• This protocol builds upon the method and results described in Abbassi-Daloii et al. (2023b).

• It includes step-by-step details on image acquisition and image processing of the entire muscle section.

• It enables high-throughput and semi-automated image quantification of capillary density.

• It provides a robust analysis for determining capillary density over the entire muscle cross section.

Graphical overview

In vitro differentiation of human pluripotent stem cell (hPSC) model systems has furthered our understanding of human development. Techniques used to elucidate gene function during early development have encountered technical challenges, especially when targeting embryonic lethal genes. The introduction of CRISPRoff by Nuñez and collaborators provides an opportunity to heritably silence genes during long-term differentiation. We modified CRISPRoff and sgRNA Sleeping Beauty transposon vectors that depend on tetracycline-controlled transcriptional activation to silence the expression of embryonic lethal genes at different stages of differentiation in a stable manner. We provide instructions on how to generate sgRNA transposon vectors that can be used in combination with our CRISPRoff transposon vector and a stable hPSC line. We validate the use of this tool by silencing MCL-1, an anti-apoptotic protein, which results in pre-implantation embryonic lethality in mice; this protein is necessary for oligodendrocyte and hematopoietic stem cell development and is required for the in vitro survival of hPSCs. In this protocol, we use an adapted version of the differentiation protocol published by Douvaras and Fossati (2015) to generate oligodendrocyte lineage cells from human embryonic stem cells (hESCs). After introduction of the CRISPRoff and sgRNAs transposon vectors in hESCs, we silence MCL-1 in committed oligodendrocyte neural precursor cells and describe methods to measure its expression. With the methods described here, users can design sgRNA transposon vectors targeting MCL-1 or other essential genes of interest to study human oligodendrocyte development or other differentiation protocols that use hPSC model systems.

Key features

• Generation of an inducible CRISPRoff Sleeping Beauty transposon system.

• Experiments performed in vitro for generation of inducible CRISPRoff pluripotent stem cell line amenable to oligodendrocyte differentiation.

• Strategy to downregulate an essential gene at different stages of oligodendrocyte development.

Graphical overview

Workflow for generating inducible CRISPRoff stem cell line and assessing knockdown phenotype in stem cell–derived committed oligodendrocyte neural precursor cells