Cell Biology


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0 Q&A 617 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 1029 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 873 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.

0 Q&A 757 Views Sep 5, 2022

Mitochondrial dysfunction is associated with perturbations in the cellular oxidative status, changes in energy production and metabolic rate, and the onset of pathological processes. Classic methods of assessing mitochondrial dysfunction rely on indirect measures, such as evaluating mitochondrial DNA copy numbers, or direct but more costly and skilled techniques, such as electron microscopy. The protocol presented here was recently implemented to evaluate mitochondrial dysfunction in response to insecticide exposure in Drosophila melanogaster larvae, and it relies on the use of a previously established MitoTimer mutant strain. MitoTimer is a genetically engineered mitochondrial protein that shows green fluorescence when newly synthetized, irreversibly turning into red as mitochondria age. The protocol described here allows for the easy and direct assessment of shifts in mitochondrial turnover, with tissue-specific accuracy. This protocol can be adapted to assess changes in mitochondrial turnover in response to drugs, rearing conditions, and/or mutations in larva, pupa, or adult fruit flies.

0 Q&A 1072 Views Aug 20, 2022

Autophagy is an evolutionarily conserved intracellular degradation process. During autophagy, a set of autophagy-related (ATG) proteins orchestrate the formation of double-bound membrane vesicles called autophagosomes to engulf cytoplasmic material and deliver it to the vacuole for breakdown. Among ATG proteins, the ATG8 is the only one decorating mature autophagosomes and therefore is regarded as a bona fide autophagic marker; colocalization assays with ATG8 are wildly used as a reliable method to identify the components of autophagy machinery or autophagic substrates. Here, we describe a colocalization assay with fluorescent-tagged ATG8 using a tobacco (Nicotiana benthamiana)-based transient expression system.

0 Q&A 1059 Views Jun 5, 2022

Live labelling of active transcription sites is critical to our understanding of transcriptional dynamics. In the most widely used method, RNA sequence MS2 repeats are added to the transcript of interest, on which fluorescently tagged Major Coat Protein binds, and labels transcription sites and transcripts. Here we describe another strategy, using the Argonaute protein NRDE-3, repurposed as an RNA-programmable RNA binding protein. We label active transcription sites in C. elegans embryos and larvae, without editing the gene of interest. NRDE-3 is programmed by feeding nematodes with double-stranded RNA matching the target gene. This method does not require genome editing and is inexpensive and fast to apply to many different genes.


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0 Q&A 2782 Views Mar 5, 2022

Asymmetric cell division (ACD) is fundamental for balancing cell proliferation and differentiation in metazoans. During active neurogenesis in the developing zebrafish forebrain, radial glia progenitors (RGPs) mainly undergo ACD to produce one daughter with high activity of Delta/Notch signaling (proliferative cell fate) and another daughter with low Delta/Notch signaling (differentiative cell fate). The cell polarity protein partitioning-defective 3 (Par-3) is critical for regulating this process. To understand how polarized Par-3 on the cell cortex can lead to differential Notch activity in the nuclei of daughter cells, we combined an anti-Delta D (Dld) -atto 647N antibody uptake assay with label retention expansion microscopy (LR-ExM), to obtain high resolution immunofluorescent images of Par-3, dynein light intermediate chain 1 (Dlic1), and Dld endosomes in mitotic RGPs. We then developed a protocol for analyzing the colocalization of Par-3, Dlic1, and endosomal DeltaD, using JACoP (Just Another Co-localization Plugin) in ImageJ software (Bolte and Cordelières, 2006). Through such analyses, we have shown that cytosolic Par-3 is associated with Dlic1 on Dld endosomes. Our work demonstrates a direct involvement of Par-3 in dynein-mediated polarized transport of Notch signaling endosomes. This bio-protocol may be generalizable for analysis of protein co-localization in any cryosectioned and immunostained tissue samples.

0 Q&A 5417 Views Mar 5, 2022

The CRISPR/Cas9 technology has transformed our ability to edit eukaryotic genomes. Despite this breakthrough, it remains challenging to precisely knock-in large DNA sequences, such as those encoding a fluorescent protein, for labeling or modifying a target protein in post-mitotic cells. Previous efforts focusing on sequence insertion to the protein coding sequence often suffer from insertions/deletions (INDELs) resulting from the efficient non-homologous end joining pathway (NHEJ). To overcome this limitation, we have developed CRISPR-mediated insertion of exon (CRISPIE). CRISPIE circumvents INDELs and other editing errors by inserting a designer exon flanked by adjacent intron sequences into an appropriate intronic location of the targeted gene. Because INDELs at the insertion junction can be spliced out, “CRISPIEd” genes produce precisely edited mRNA transcripts that are virtually error-free. In part due to the elimination of INDELs, high-efficiency labeling can be achieved in vivo. CRISPIE is compatible with both N- and C-terminal labels, and with all common transfection methods. Importantly, CRISPIE allows for later removal of the protein modification by including exogenous single-guide RNA (sgRNA) sites in the intronic region of the donor module. This protocol provides the detailed CRISPIE methodology, using endogenous labeling of β-actin in human U-2 OS cells with enhanced green fluorescent protein (EGFP) as an example. When combined with the appropriate gene delivery methods, the same methodology can be applied to label post-mitotic neurons in culture and in vivo. This methodology can also be readily adapted for use in other gene editing contexts.

0 Q&A 2078 Views Mar 5, 2022

Mitochondria are relatively small, fragmented, and abundant in the large embryos of Drosophila, Xenopus and zebrafish. It is essential to study their distribution and dynamics in these embryos to understand the mechanistic role of mitochondrial function in early morphogenesis events. Photoactivation of mitochondrially tagged GFP (mito-PA-GFP) is an attractive method to highlight a specific population of mitochondria in living embryos and track their distribution during development. Drosophila embryos contain large numbers of maternally inherited mitochondria, which distribute differently at specific stages of early embryogenesis. They are enriched basally in the syncytial division cycles and move apically during cellularization. Here, we outline a method for highlighting a population of mitochondria in discrete locations using mito-PA-GFP in the Drosophila blastoderm embryo, to follow their distribution across syncytial division cycles and cellularization. Photoactivation uses fluorophores, such as PA-GFP, that can change their fluorescence state upon exposure to ultraviolet light. This enables marking a precise population of fluorescently tagged molecules of organelles at selected regions, to visualize and systematically follow their dynamics and movements. Photoactivation followed by live imaging provides an effective way to pulse label a population of mitochondria and follow them through the dynamic morphogenetic events during Drosophila embryogenesis.

1 Q&A 2500 Views Dec 5, 2021

Primary cilia are microtubule-based sensory organelles surrounded by membrane. They can detect mechanical and chemical stimuli. The last few years have uncovered cilia as unique signaling hubs that host a number of receptors and effector molecules. Thus, defining how specific proteins localize and are distributed along the cilium is critical to understanding its function.


Quantitative immunofluorescence can be used to accurately assess the localization of receptors and signaling molecules within the primary cilia. However, image analysis can be time consuming, and there are limited programs that can accurately determine staining intensity along the cilia. To overcome these issues, we developed a series of MATLAB scripts to accurately measure staining intensity along the length of the cilia, in both a semi-automated and automated fashion. Here, we describe the scripts and include a protocol for image analysis for each. With these scripts, the protocols can be used to analyze the distribution of any ciliary protein using immunofluorescence images.





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