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Past Issue in 2023
Volume: 13, Issue: 8
Biochemistry
A Modified Acyl-RAC Method of Isolating Retinal Palmitoyl Proteome for Subsequent Detection through LC-MS/MS
Palmitoylation is a unique and reversible posttranslational lipid modification (PTM) that plays a critical role in many cellular events, including protein stability, activity, membrane association, and protein–protein interactions. The dynamic nature of palmitoylation dictates the efficient sorting of various retinal proteins to specific subcellular compartments. However, the underlying mechanism through which palmitoylation supports efficient protein trafficking in the retina remains unclear. Recent studies show that palmitoylation can also function as a signaling PTM, underlying epigenetic regulation and homeostasis in the retina. Efficient isolation of retinal palmitoyl proteome will pave the way to a better understanding of the role(s) for palmitoylation in visual function. The standard methods for detecting palmitoylated proteins employ 3H- or 14C-radiolabeled palmitic acid and have many limitations, including poor sensitivity. Relatively recent studies use thiopropyl Sepharose 6B resin, which offers efficient detection of palmitoylated proteome but is now discontinued from the market. Here, we describe a modified acyl resin–assisted capture (Acyl-RAC) method using agarose S3 high-capacity resin to purify palmitoylated proteins from the retina and other tissues, which is greatly compatible with downstream processing by LC-MS/MS. Unlike other palmitoylation assays, the present protocol is easy to perform and cost-effective.Graphical overview
Biological Engineering
Synthetic Promoter Screening Using Poplar Mesophyll Protoplast Transformation
Plant protoplasts are useful to study both transcriptional regulation and protein subcellular localization in rapid screens. Protoplast transformation can be used in automated platforms for design-build-test cycles of plant promoters, including synthetic promoters. A notable application of protoplasts comes from recent successes in dissecting synthetic promoter activity with poplar mesophyll protoplasts. For this purpose, we constructed plasmids with TurboGFP driven by a synthetic promoter together with TurboRFP constitutively controlled by a 35S promoter, to monitor transformation efficiency, allowing versatile screening of high numbers of cells by monitoring green fluorescent protein expression in transformed protoplasts. Herein, we introduce a protocol for poplar mesophyll protoplast isolation followed by protoplast transformation and image analysis for the selection of valuable synthetic promoters.Graphical overview
Biophysics
In vitro Reconstitution of Phase-separated p62 Bodies on the Arp2/3-derived Actin Network
In cells, p62/SQSTM1 undergoes liquid–liquid phase separation (LLPS) with poly-ubiquitin chains to form p62 bodies that work as a hub for various cellular events, including selective autophagy. Cytoskeleton components such as Arp2/3-derived branched actin network and motor protein myosin 1D have been shown to actively participate in the formation of phase-separated p62 bodies. Here, we describe a detailed protocol on the purification of p62 and other proteins, the assembly of the branched actin network, and the reconstitution of p62 bodies along with cytoskeletal structures in vitro. This cell-free reconstitution of p62 bodies vividly mimics the phenomenon in which low concentrations of protein in vivo rely on cytoskeleton dynamics to increase the local concentration to reach the threshold for phase separation. This protocol provides an easily implemented and typical model system to study cytoskeleton-involved protein phase separation.
Cancer Biology
Analysis of RNA Polymerase II Chromatin Binding by Flow Cytometry
RNA polymerase II (RNAPII) transcribes DNA into mRNA and thereby plays a critical role in cellular protein production. In addition, RNAPII plays a central role in DNA damage responses. Measurements of RNAPII on chromatin may thus give insight into several essential processes in eukaryotic cells. During transcription, the C-terminal domain of RNAPII becomes post-translationally modified, and phosphorylation on serine 5 and serine 2 can be used as markers for the promoter proximal and productively elongating forms of RNAPII, respectively. Here, we provide a detailed protocol for the detection of chromatin-bound RNAPII and its serine 5– and serine 2–phosphorylated forms in individual human cells through the cell cycle. We have recently shown that this method can be used to study the effects of ultraviolet DNA damage on RNAPII chromatin binding and that it can even be used to reveal new knowledge about the transcription cycle itself. Other commonly used methods to study RNAPII chromatin binding include chromatin immunoprecipitation followed by sequencing or chromatin fractionation followed by western blotting. However, such methods are frequently based on lysates made from a large number of cells, which may mask population heterogeneity, e.g., due to cell cycle phase. With strengths such as single-cell analysis, speed of use, and accurate quantitative readouts, we envision that our flow cytometry method can be widely used as a complementary approach to sequencing-based methods to study effects of different stimuli and inhibitors on RNAPII-mediated transcription.Graphical overview
Cell Biology
Visualizing the Cisternal Organization of Golgi Ministacks in HeLa Cells by Side-averaging
The mammalian Golgi complex consists of laterally connected Golgi stacks, each comprising close-packed and flattened membrane sacks called cisternae. However, the convoluted spatial organization of Golgi stacks and limited resolution of light microscopy prevent us from resolving the cisternal organization of the Golgi. Here, we describe our recently developed side-averaging approach coupled with Airyscan microscopy to visualize the cisternal organization of nocodazole-induced Golgi ministacks. First, the nocodazole treatment greatly simplifies the organization of Golgi stacks by spatially separating the crowded and amorphous Golgi complex into individual disk-shaped ministacks. The treatment also makes it possible to identify en face and side-views of Golgi ministacks. Next, after manually selecting the side-view Golgi ministack images, they are transformed and aligned. Finally, the resulting images are averaged to enhance the common structural features and suppress the morphological variations among individual Golgi ministacks. This protocol describes how to image and analyze the intra-Golgi localization of giantin, GalT-mCherry, GM130, and GFP-OSBP in HeLa cells by side-averaging.Graphical overview
Immunology
Anti-tumor Efficacy of CD19 CAR-T in a Raji B Cell Xenografted Mouse Model
Chimeric antigen receptor (CAR)-T therapy launched a new era for cancer treatments, displaying outstanding effectiveness in relapsed or refractory B-cell malignancies. Demonstrating the tumor-killing ability of CAR-Ts in mouse xenograft models serves as a golden criterium in preclinical research. Here, we describe a detailed method for evaluating CAR-T’s function in immune-deficient mice bearing Raji B cell–induced tumors. It includes generating CD19 CAR-T cells from healthy donors, injecting tumor cells and CAR-T cells into mice, and monitoring tumor growth and CAR-T state. This protocol provides a practical guide to evaluate CAR-T’s function in vivo within eight weeks.Graphical overview
Microbiology
A Quick DNA Extraction Method for High Throughput Screening in Gram-positive Bacteria
In this study, a sonication-based DNA extraction method was developed, in which the whole process can be finished within 10 min. This method is almost zero cost and time-saving, which is useful for high throughput screening, especially in the screening of mutants generated in random mutagenesis. This method is effective in genomic DNA extraction for PCR amplification in several Gram-positive bacteria, including Bacillus cereus, Bacillus thuringiensis, Bacillus subtilis, and Listeria monocytogenes.
Development of a Gene Replacement Method for the Filamentous Bacterium Leptothrix cholodnii SP-6
Genetic strategies such as gene disruption and fluorescent protein tagging largely contribute to understanding the molecular mechanisms of biological functions in bacteria. However, the methods for gene replacement remain underdeveloped for the filamentous bacteria Leptothrix cholodnii SP-6. Their cell chains are encased in sheath composed of entangled nanofibrils, which may prevent the conjugation for gene transfer. Here, we describe a protocol optimized for gene disruption through gene transfer mediated by conjugation with Escherichia coli S17-1 with details on cell ratio, sheath removal, and loci validation. The obtained deletion mutants for specific genes can be used to clarify the biological functions of the proteins encoded by the target genes.Graphical overview
Molecular Biology
Application of a Spacer-nick Gene-targeting Approach to Repair Disease-causing Mutations with Increased Safety
The CRISPR/Cas9 system is a powerful tool for gene repair that holds great potential for gene therapy to cure monogenic diseases. Despite intensive improvement, the safety of this system remains a major clinical concern. In contrast to Cas9 nuclease, Cas9 nickases with a pair of short-distance (38–68 bp) PAM-out single-guide RNAs (sgRNAs) preserve gene repair efficiency while strongly reducing off-target effects. However, this approach still leads to efficient unwanted on-target mutations that may cause tumorigenesis or abnormal hematopoiesis. We establish a precise and safe spacer-nick gene repair approach that combines Cas9D10A nickase with a pair of PAM-out sgRNAs at a distance of 200–350 bp. In combination with adeno-associated virus (AAV) serotype 6 donor templates, this approach leads to efficient gene repair with minimal unintended on- and off-target mutations in human hematopoietic stem and progenitor cells (HSPCs). Here, we provide detailed protocols to use the spacer-nick approach for gene repair and to assess the safety of this system in human HSPCs. The spacer-nick approach enables efficient gene correction for repair of disease-causing mutations with increased safety and suitability for gene therapy.Graphical overview
Stem Cell
Establishing Bipotential Human Lung Organoid Culture System and Differentiation to Generate Mature Alveolar and Airway Organoids
A robust in vitro model of the human respiratory epithelium, including the alveolar and the airway epithelium, is essential for understanding the biology and pathology of the human respiratory system. We previously described a protocol to derive human lung organoids from primary lung tissues. We now describe a protocol to induce bidirectional differentiation to generate mature alveolar or airway organoids. The lung organoids are consecutively expanded for over one year with high stability, while the differentiated alveolar and airway organoids morphologically and functionally simulate the human alveolar and airway epithelium to a near-physiological level. Thus, we establish a robust organoid culture system of the entire human respiratory epithelium, the first two-phase bipotential organoid culture system that enables long-term expansion and bidirectional differentiation of respiratory epithelial cells. The long-term expandable lung organoids and differentiated organoids generate a stable and renewable source of respiratory epithelial cells, enabling scientists to reconstruct and expand the human respiratory epithelium in culture dishes. The respiratory organoid system provides a unique and physiologically active in vitro model of the human respiratory epithelium for various applications, including studying respiratory viral infection, disease modeling, drug screening, and pre-clinical testing.Graphical overview