0 Q&A 271 Views Jan 5, 2023

Skeletal muscle, one of the most abundant tissue in the body, is a highly regenerative tissue. Indeed, compared to other tissues that are not able to regenerate after injury, skeletal muscle can fully regenerate upon mechanically, chemically, and infection-induced trauma. Several injury models have been developed to thoroughly investigate the physiological mechanisms regulating skeletal muscle regeneration. This protocol describes how to induce muscle regeneration by taking advantage of a cardiotoxin (CTX)-induced muscle injury model. The overall steps include CTX injection of tibialis anterior (TA) muscles of BL6N mice, collection of regenerating muscles at different time points after CTX injury, and histological characterization of regenerating muscles. Our protocol, compared with others such as those for freeze-induced injury models, avoids laceration or infections of the muscles since it involves neither surgery nor suture. In addition, our protocol is highly reproducible, since it causes homogenous myonecrosis of the whole muscle, and further reduces animal pain and stress.

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0 Q&A 263 Views Jan 5, 2023

Utilizingresources available from the mother's body to guarantee healthy offspring growth is the fundamental reproductive strategy. Recently, we showed that a class of the largest extracellular vesicles known as exophers, which are responsible for the removal of neurotoxic components from neurons (Melentijevic et al., 2017) and damaged mitochondria from cardiomyocytes (Nicolás-Ávila et al., 2020), are released by the Caenorhabditis elegans hermaphrodite body wall muscles (BWM), to support embryonic growth (Turek et al., 2021). Employing worms expressing fluorescent reporters in BWM cells, we found that exopher formation (exophergenesis) is sex-specific and fertility-dependent. Moreover, exophergenesis is regulated by the developing embryo in utero, and exophers serve as transporters for muscle-generated yolk proteins, which can be used to nourish the next generation. Given the specific regulation of muscular exophergenesis, and the fact that muscle-generated exophers are much larger than neuronal ones and have different targeting, their identification and quantification required a modified approach from that designed for neuronal-derived exophers (Arnold et al., 2020). Here, we present a methodology for assessing and quantifying muscle-derived exophers that can be easily extended to determine their function and regulation in various biological contexts.

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0 Q&A 409 Views Dec 20, 2022

Mitochondria are cellular organelles essential for the function and survival of eukaryotic cells. Nearly all mitochondrial proteins are nuclear-encoded and require mitochondrial import upon their synthesis in the cytosol. Various approaches have been described to study mitochondrial protein import, such as monitoring the entry of radiolabeled proteins into purified mitochondria or quantifying newly synthesized proteins within mitochondria by proteomics. Here, we provide a detailed protocol for a commonly used and straightforward assay that quantitatively examines mitochondrial protein import by monitoring the co-localization of mitochondrially targeted enhanced green fluorescent protein (eGFP) with the mitochondrial fluorescence dye MitoTracker TM Deep Red FM by live cell imaging. We describe the preparation and use of a stable mammalian cell line inducibly expressing a mitochondrial targeting sequence (MTS)-eGFP, followed by quantitative image analysis using an open-source ImageJ-based plugin. This inducible expression system avoids the need for transient transfection while enabling titration of MTS-eGFP expression and thereby avoiding protein folding stress. Overall, the assay provides a simple and robust approach to assess mitochondrial import capacity of cells in various disease-related settings.

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0 Q&A 482 Views Dec 5, 2022

Cryo-electron tomography (cryo-ET) is a formidable technique to observe the inner workings of vitrified cells at a nanometric resolution in near-native conditions and in three-dimensions. One consequent drawback of this technique is the sample thickness, for two reasons: i) achieving proper vitrification of the sample gets increasingly difficult with sample thickness, and ii) cryo-ET relies on transmission electron microscopy (TEM), requiring thin samples for proper electron transmittance (<500 nm). For samples exceeding this thickness limit, thinning methods can be used to render the sample amenable for cryo-ET. Cryo-focused ion beam (cryo-FIB) milling is one of them and despite having hugely benefitted the fields of animal cell biology, virology, microbiology, and even crystallography, plant cells are still virtually unexplored by cryo-ET, in particular because they are generally orders of magnitude bigger than bacteria, viruses, or animal cells (at least 10 μm thick) and difficult to process by cryo-FIB milling. Here, we detail a preparation method where abaxial epidermal onion cell wall peels are separated from the epidermal cells and subsequently plunge frozen, cryo-FIB milled, and screened by cryo-ET in order to acquire high resolution tomographic data for analyzing the organization of the cell wall.

0 Q&A 323 Views Dec 5, 2022

Entosis is a process where a living cell launches an invasion into another living cell’s cytoplasm. These inner cells can survive inside outer cells for a long period of time, can undergo cell division, or can be released. However, the fate of most inner cells is lysosomal degradation by entotic cell death. Entosis can be detected by imaging a combination of membrane, cytoplasmic, nuclear, and lysosomal staining in the cells. Here, we provide a protocol for detecting entosis events and measuring the kinetics of entotic cell death by time-lapse imaging using tetramethylrhodamine methyl ester (TMRM) staining.

0 Q&A 675 Views Nov 20, 2022

During an animal's development, a large number of cells undergo apoptosis, a suicidal form of death. These cells are promptly phagocytosed by other cells and degraded inside phagosomes. The recognition, engulfment, and degradation of apoptotic cells is an evolutionarily conserved process occurring in all metazoans. Recently, we discovered a novel event in the nematode Caenorhabditis elegans: the double-membrane autophagosomes are recruited to the surface of phagosomes; subsequently, the outer membrane of an autophagosome fuses with the phagosomal membrane, allowing the inner vesicle to enter the phagosomal lumen and accumulate there over time. This event facilitates the degradation of the apoptotic cell inside the phagosome. During this study, we developed a real-time imaging protocol monitoring the recruitment and fusion of autophagosomes to phagosomes over two hours during embryonic development. This protocol uses a deconvolution-based microscopic imaging system with an optimized setting to minimize photodamage of the embryo during the recording period for high-resolution images. Furthermore, acid-resistant fluorescent reporters are chosen to label autophagosomes, allowing the inner vesicles of an autophagosome to remain visible after entering the acidic phagosomal lumen. The methods described here, which enable high sensitivity, quantitative measurement of each step of the dynamic incorporation in developing embryos, are novel since the incorporation of autophagosomes to phagosomes has not been reported previously. In addition to studying the degradation of apoptotic cells, this protocol can be applied to study the degradation of non-apoptotic cell cargos inside phagosomes, as well as the fusion between other types of intracellular organelles in living C. elegans embryos. Furthermore, its principle of detecting the membrane fusion event can be adapted to study the relationship between autophagosomes and phagosomes or other intracellular organelles in any biological system in which real-time imaging can be conducted.

0 Q&A 481 Views Nov 20, 2022

Sphingolipids are important structural components of cellular membranes. They also function as prominent signaling molecules to control a variety of cellular events, such as cell growth, differentiation, and apoptosis. Impaired sphingolipid metabolism, particularly defects in sphingolipid degradation, has been associated with many human diseases. Fluorescence sphingolipid analogs have been widely used as efficient probes to study sphingolipid metabolism and intracellular trafficking in living mammalian cells. Compared with nitrobenzoxadiazole fluorophores (NBD FL), the boron dipyrromethene difluoride fluorophores (BODIPY FL) have much higher absorptivity and fluorescence quantum. These features allow more intensive labeling of cells for fluorescence microscopy imaging and flow cytometry analysis. Here, we describe a protocol employing BODIPY FL-labeled sphingolipid analogs to elucidate sphingolipid internalization, trafficking, and endocytosis in mouse embryonic stem cells.

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0 Q&A 868 Views Oct 20, 2022

Depending on its local concentration, hydrogen peroxide (H2O2) can serve as a cellular signaling molecule but can also cause damage to biomolecules. The levels of H2O2 are influenced by the activity of its generator sites, local antioxidative systems, and the metabolic state of the cell. To study and understand the role of H2O2 in cellular signaling, it is crucial to assess its dynamics with high spatiotemporal resolution. Measuring these subcellular H2O2 dynamics has been challenging. However, with the introduction of the super sensitive pH-independent genetically encoded fluorescent H2O2 sensor HyPer7, many limitations of previous measurement approaches could be overcome. Here, we describe a method to measure local H2O2 dynamics in intact human cells, utilizing the HyPer7 sensor in combination with a microscopic multi-mode microplate reader.

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Overview of HyPer7 sensor function and measurement results.

0 Q&A 1071 Views Sep 20, 2022

Understanding the molecular and structural mechanisms that govern the assembly and organization of higher-order actin architecture requires the use of in vitro actin binding and bundling assays. Crosslinking of actin filaments into bundles can be monitored in vitro via several techniques, including negative staining/electron microscopy, low-speed co-sedimentation assay/SDS-PAGE, and fluorescence staining/confocal microscopy. We and others have previously characterized the N-BAR domain of ASAP1, an ADP-ribosylation factor GTPase-activating protein, as an actin-bundling module; we further identified key lysine residues responsible for actin cross-linking. Here, we use the ASAP1 BAR domain as an example and describe a detailed procedure for observing the actin bundle formation by confocal microscopy. This protocol requires small reaction volumes and takes advantage of bright commercially available fluorescent phalloidins, making it an ideal choice for medium-throughput screening of mutants or domain truncations in their ability to bundle actin.

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0 Q&A 925 Views Sep 5, 2022

In the human cell cycle, complete replication of DNA is a fundamental process for the maintenance of genome integrity. Replication stress interfering with the progression of replication forks causes difficult-to-replicate regions to remain under-replicated until the onset of mitosis. In early mitosis, a homology-directed repair DNA synthesis, called mitotic DNA synthesis (MiDAS), is triggered to complete DNA replication. Here, we present a method to detect MiDAS in human U2OS 40-2-6 cells, in which repetitive lacO sequences integrated into the human chromosome evoke replication stress and concomitant incomplete replication of the lacO array. Immunostaining of BrdU and LacI proteins is applied for visualization of DNA synthesis in early mitosis and the lacO array, respectively. This protocol has been established to easily detect MiDAS at specific loci using only common immunostaining methods and may be optimized for the investigation of other difficult-to-replicate regions marked with site-specific binding proteins.