UNASSIGNED: Our research aimed to investigate the therapeutic effects of Tubastatin-A, a glucocorticoid receptor (GR) mitochondrial translocation inhibitor, and mitoquinone (MitoQ), an antioxidant, on attenuating dexamethasone (DEX)-induced macrophage apoptosis.
UNASSIGNED: We treated RAW264.7 macrophages with different combinations of DEX and either Tubastatin-A or MitoQ. Parameters such as mitochondrial GR translocation, mitochondrial reactive oxygen species levels, mitochondrial membrane potential, mitochondrial permeability transition pore opening, cytochrome C efflux to the cytosol, and apoptosis were subsequently evaluated in the different treatment groups via qRT-PCR, western blotting, and immunofluorescence assays.
UNASSIGNED: DEX intervention increased the translocation of GRs into the mitochondria, while reducing the expression of the mitochondrial gene MT-CO1 and the activity of mitochondrial respiratory chain complex IV in macrophages. In addition, DEX administration increased mtROS levels, mitochondrial permeability transition pore opening, and mitochondrial cytochrome C release in macrophages, which promoted their apoptosis. We found that Tubastatin-A inhibited mitochondrial GR translocation and reversed the DEX-induced increase in GR levels within the mitochondria. Furthermore, Tubastatin-A mitigated various mitochondrial changes induced by DEX, including reducing the efflux of mitochondrial cytochrome C and inhibiting macrophage apoptosis. Similarly, MitoQ exerted its effects on macrophage apoptosis by reducing mtROS levels through the mitochondrial pathway.
UNASSIGNED: The DEX-mediated translocation of GR into mitochondria disrupts the mitochondrial function of macrophages, which induces their apoptosis. By inhibiting mitochondrial translocation of GR and reducing mtROS levels, Tubastatin-A and MitoQ can effectively attenuate macrophage apoptosis, which has clinical implications for reducing the notable side effects associated with glucocorticoid use.

All but a few mitochondrial proteins are translated into the cytosol and imported in via complicated and varied pathways. These processes occur over short time frames and, as such, are difficult to monitor with classical approaches such as Western blotting or autoradiography that require sample collection at discrete time points. The development of an assay based on a split version of the small luciferase-Mitoluc-has allowed us to monitor the import of proteins into mitochondria in high resolution and real time (Pereira et al., J Mol Biol 431:1689-1699, 2019). Luminescence measurements are acquired using a plate reader in the order of seconds. This allows scores of experiments to be conducted in parallel in a single multi-well plate and permits kinetic analysis yielding information about import mechanisms (Ford et al., Elife 11:e75426, 2022).

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

In the present study, we present callus grafting, comprising a method for reproducibly generating tissue chimeras from callus cultures of Arabidopsis thaliana. In this way, callus cultures of different genetic backgrounds may be co-cultivated such that cell-to-cell connectivity is achieved as a chimeric tissue is formed. To track intercellular connectivity and transport between non-clonal callus cells, we used transgenic lines expressing fluorescently tagged mobile and non-mobile fusion constructs. Using fluorescently-labelled reporter lines that label plasmodesmata, we show that secondary complex plasmodesmata are present at the cell walls of connected cells. We use this system to investigate cell-to-cell transport across the callus graft junction and show that different proteins and RNAs are mobile between non-clonal callus cells. Finally, we take advantage of the callus culture system to probe intercellular connectivity of grafted leaf and root calli and the effect of different light regimes of cell-to-cell transport. Taking advantage of the ability of callus to be cultivated in the complete absence of light, we show that the rate of silencing spread is significantly decreased in chimeric calli cultivated in total darkness. We propose that callus grafting is a fast and reliable method for analysing the capacity of a macromolecule to be exchanged between cells independent of the vasculature.

Primary cilia provide a specialized subcellular environment favoring ordered and timely interaction and modification of signaling molecules, necessary for the sensing and transduction of extracellular signals and environmental conditions. Crucial to the understanding of ciliary function is the knowledge of the signaling molecules composing the ciliary compartment. While proteomes of primary cilia have been published recently, the selective isolation of primary cilia from specific cell types and whole tissue still proves difficult, and many laboratories instead resort to the analysis of cultured cells, which may introduce experimental artifacts. Here we present a flow cytometry-based method to isolate and characterize primary cilia from the murine ventricular-subventricular zone. After deciliation, primary cilia are immunolabeled with antibodies against ciliary markers. As an example, we here use a double-staining with acetylated tubulin, which stains the ciliary axoneme, and ciliary membrane protein ADP-ribosylation-like factor 13b (Arl13b); additionally, we triple-labeled primary cilia using the ciliary marker adenylate cyclase 3 (AC3). Besides analysis at the single particle level, fluorescence activated cell sorting (FACS) allows collection of pure preparations of primary cilia suited for subsequent proteomic analyses like mass spectrometry or western blot. As an example of analytical application, we performed triple immunostaining and FACS analysis to reveal cilia heterogeneity. Thus, our cilia isolation method, which can readily be applied to other tissues or cell culture, will facilitate the study of this key cellular organelle and shed light on its role in normal conditions and disease.

The fusing of a protein of interest to a fluorescent protein followed by fluorescence microscopy is a very common method of determining protein localization and dynamics. However even small fluorescent proteins can be large enough to affect protein folding and localization, therefore the ability to use a smaller tag but still be able to detect a fluorescent signal in live cell imaging experiments is extremely valuable. The self-assembling split sfGFPOPT system allows the fusion of the protein of interest with the 11th β-strand of super-folder GFP (sfGFP11) which is only 13 amino acids long. When this construct is delivered into protoplasts made from transgenic plants expressing sfGFP1-10 (sfGFP1-10OPT) targeted to the desired compartment, the two parts assemble and fluorescence is reconstituted that can be detected by confocal laser scanning microscopy. Here, we present the application of this method for protein targeting to plant peroxisomes using Catalase (CAT2 of Arabidopsis thaliana) as an example. As peroxisomes are able to import folded and oligomeric proteins, careful consideration of appropriate controls is also required to ensure correct interpretation of the results.

Mitochondria play central roles in cellular energy production and metabolism. Most proteins required to carry out these functions are synthesized in the cytosol and imported into mitochondria. A growing number of metabolic disorders arising from mitochondrial dysfunction can be traced to errors in mitochondrial protein import. The mechanisms underlying the import of precursor proteins are commonly studied using radioactively labeled precursor proteins imported into purified mitochondria. Here, we establish a fluorescence-based import assay to analyze protein import into mitochondria. We show that fluorescently labeled precursors enable import analysis with similar sensitivity to those using radioactive precursors, yet they provide the advantage of quantifying import with picomole resolution. We adapted the import assay to a 96-well plate format allowing for fast analysis in a screening-compatible format. Moreover, we show that fluorescently labeled precursors can be used to monitor the assembly of the F1 F0 ATP synthase in purified mitochondria. Thus, we provide a sensitive fluorescence-based import assay that enables quantitative and fast import analysis.

Interorgan communication networks are key regulators of organismal homeostasis, and their dysregulation is associated with a variety of pathologies. While mass spectrometry proteomics identifies circulating proteins and can correlate their abundance with disease phenotypes, the tissues of origin and destinations of these secreted proteins remain largely unknown. In vitro approaches to study protein secretion are valuable, however, they may not mimic the complexity of in vivo environments. More recently, the development of engineered promiscuous BirA* biotin ligase derivatives has enabled tissue-specific tagging of cellular secreted proteomes in vivo. The use of biotin as a molecular tag provides information on the tissue of origin and destination, and enables the enrichment of low-abundance hormone proteins. Therefore, promiscuous protein biotinylation is a valuable tool to study protein secretion in vivo.

Seeds are an attractive platform for the production of recombinant proteins because of their excellent storage properties and their well-developed endomembrane system, which allows accumulation of the product within specialized storage organelles. Due to the presence of these additional organelles and the resulting complexity of intracellular protein trafficking it is interesting to investigate the transport and storage of a recombinant protein within seed tissues, its interactions with endogenous reserve proteins and its impact on the ultrastructure of the endomembrane system. Possible approaches include sequential extraction procedures, subcellular fractionation and 2D as well as 3D electron microscopy techniques such as electron tomography (ET) and serial block face scanning electron microscopy (SBF-SEM), which are described and discussed in this chapter.

Synthetic protein-binding tools based on anti-green fluorescent protein (GFP) nanobodies have recently emerged as useful resources to study developmental biology. By fusing GFP-targeting nanobodies to well-characterized protein domains residing in discrete sub-cellular locations, it is possible to directly and acutely manipulate the localization of GFP-tagged proteins-of-interest in a predictable manner. Here, we describe a detailed protocol for the application of nanobody-based GFP-binding tools, namely Morphotrap and GrabFP, to study the localization and function of extracellular and intracellular proteins in the Drosophila wing imaginal disc. Given the generality of these methods, they are easily applicable for use in other tissues and model organisms.