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1. Gene Delivery and Analysis of Optogenetic Induction of Lytic Cell Death

   https://doi.org/10.1002/cpz1.1023  

   Oh, Teak‐Jung; Gworek, Bryan; Mehfooz, Amna; Zhang, Kai (April 2024, Current Protocols)

   Abstract Necroptosis is a form of inflammatory lytic cell death involving active cytokine production and plasma membrane rupture. Progression of necroptosis is tightly regulated in time and space, and its signaling outcomes can shape the local inflammatory environment of cells and tissues. Pharmacological induction of necroptosis is well established, but the diffusive nature of chemical death inducers makes it challenging to study cell‐cell communication precisely during necroptosis. Receptor‐interacting protein kinase 3, or RIPK3, is a crucial signaling component of necroptosis, acting as a crucial signaling node for both canonical and non‐canonical necroptosis. RIPK3 oligomerization is crucial to the formation of the necrosome, which regulates plasma membrane rupture and cytokine production. Commonly used necroptosis inducers can activate multiple downstream signaling pathways, confounding the signaling outcomes of RIPK3‐mediated necroptosis. Opsin‐free optogenetic techniques may provide an alternative strategy to address this issue. Optogenetics uses light‐sensitive protein‐protein interaction to modulate cell signaling. Compared to chemical‐based approaches, optogenetic strategies allow for spatiotemporal modulation of signal transduction in live cells and animals. We developed an optogenetic system that allows for ligand‐free optical control of RIPK3 oligomerization and necroptosis. This article describes the sample preparation, experimental setup, and optimization required to achieve robust optogenetic induction of RIPK3‐mediated necroptosis in colorectal HT‐29 cells. We expect that this optogenetic system could provide valuable insights into the dynamic nature of lytic cell death. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Production of lentivirus encoding the optogenetic RIPK3 system Support Protocol: Quantification of the titer of lentivirus Basic Protocol 2: Culturing, chemical transfection, and lentivirus transduction of HT‐29 cells Basic Protocol 3: Optimization of optogenetic stimulation conditions Basic Protocol 4: Time‐stamped live‐cell imaging of HT‐29 lytic cell death Basic Protocol 5: Quantification of HT‐29 lytic cell death 

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2. Fluorescence-based Methods for the Study of Protein-Protein Interactions Modulated by Ligand Binding

   https://doi.org/10.2174/1381612826666201116120934  

   Stoneman, Michael R.; Raicu, Naomi; Biener, Gabriel; Raicu, Valerică (December 2020, Current Pharmaceutical Design)

   null (Ed.)

   Background: The growing evidence that G protein-coupled receptors (GPCRs) not only form oligomersbut that the oligomers also may modulate the receptor function provides a promising avenue in the area ofdrug design. Highly selective drugs targeting distinct oligomeric sub-states offer the potential to increase efficacywhile reducing side effects. In this regard, determining the various oligomeric configurations and geometricsub-states of a membrane receptor is of utmost importance. Methods: In this report, we have reviewed two techniques that have proven to be valuable in monitoring thequaternary structure of proteins in vivo: Fӧrster resonance energy transfer (FRET) spectrometry and fluorescenceintensity fluctuation (FIF) spectrometry. In FRET spectrometry, distributions of pixel-level FRET efficiencyare analyzed using theoretical models of various quaternary structures to determine the geometry andstoichiometry of protein oligomers. In FIF spectrometry, spatial fluctuations of fluorescent molecule intensitiesare analyzed to reveal quantitative information on the size and stability of protein oligomers. Results: We demonstrate the application of these techniques to a number of different fluorescence-based studiesof cells expressing fluorescently labeled membrane receptors, both in the presence and absence of variousligands. The results show the effectiveness of using FRET spectrometry to determine detailed information regardingthe quaternary structure receptors form, as well as FIF and FRET for determining the relative abundanceof different-sized oligomers when an equilibrium forms between such structures. Conclusion: FRET and FIF spectrometry are valuable techniques for characterizing membrane receptor oligomers,which are of great benefit to structure‐based drug design. 

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3. Immunofluorescence Microscopy

   https://doi.org/10.1002/cpz1.842  

   Galati, Domenico F.; Asai, David J. (August 2023, Current Protocols)

   Abstract Visualizing fluorescence‐tagged molecules is a powerful strategy that can reveal the complex dynamics of the cell. One robust and broadly applicable method is immunofluorescence microscopy, in which a fluorescence‐labeled antibody binds the molecule of interest and then the location of the antibody is determined by fluorescence microscopy. The effective application of this technique includes several considerations, such as the nature of the antigen, specificity of the antibody, permeabilization and fixation of the specimen, and fluorescence imaging of the cell. Although each protocol will require fine‐tuning depending on the cell type, antibody, and antigen, there are steps common to nearly all applications. This article provides protocols for staining the cytoskeleton and organelles in two very different kinds of cells: flat, adherent fibroblasts and thick, free‐swimmingTetrahymenacells. Additional protocols enable visualization with widefield, laser scanning confocal, and eSRRF super‐resolution fluorescence microscopy. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Immunofluorescence staining of adherent cells such as fibroblasts Basic Protocol 2: Immunofluorescence of suspension cells such asTetrahymena Basic Protocol 3: Visualizing samples with a widefield fluorescence microscope Alternate Protocol 1: Staining suspension cells adhered to poly‐l‐lysine‐coated coverslips Alternate Protocol 2: Visualizing samples with a laser scanning confocal microscope Alternate Protocol 3: Generating super‐resolution images with SRRF microscopy 

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4. Identification and Profiling of Histone Acetyltransferase Substrates by Bioorthogonal Labeling

   https://doi.org/10.1002/cpz1.497  

   Song, Jiabao; Han, Zhen; Zheng, Y. George (July 2022, Current Protocols)

   Abstract Histone acetyltransferases (HATs, also known as lysine acetyltransferases, KATs) catalyze acetylation of their cognate protein substrates using acetyl‐CoA (Ac‐CoA) as a cofactor and are involved in various physiological and pathological processes. Advances in mass spectrometry‐based proteomics have allowed the discovery of thousands of acetylated proteins and the specific acetylated lysine sites. However, due to the rapid dynamics and functional redundancy of HAT activities, and the limitation of using antibodies to capture acetylated lysines, it is challenging to systematically and precisely define both the substrates and sites directly acetylated by a given HAT. Here, we describe a chemoproteomic approach to identify and profile protein substrates of individual HAT enzymes on the proteomic scale. The approach involves protein engineering to enlarge the Ac‐CoA binding pocket of the HAT of interest, such that a mutant form is generated that can use functionalized acyl‐CoAs as a cofactor surrogate to bioorthogonally label its protein substrates. The acylated protein substrates can then be chemoselectively conjugated either with a fluorescent probe (for imaging detection) or with a biotin handle (for streptavidin pulldown and chemoproteomic identification). This modular chemical biology approach has been successfully implemented to identify protein substrates of p300, GCN5, and HAT1, and it is expected that this method can be applied to profile and identify the sub‐acetylomes of many other HAT enzymes. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Labeling HAT protein substrates with azide/alkyne‐biotin Alternate Protocol: Labeling protein substrates of HATs with azide/alkyne‐TAMRA for in‐gel visualization Support Protocol 1: Expression and purification of HAT mutants Support Protocol 2: Synthesis of Ac‐CoA surrogates Basic Protocol 2: Streptavidin enrichment of biotinylated HAT substrates Basic Protocol 3: Chemoproteomic identification of HAT substrates Basic Protocol 4: Validation of specific HAT substrates with western blotting 

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5. Production of CRISPR‐Cas9 Transgenic Cell Lines for Knocksideways Studies

   https://doi.org/10.1002/cpz1.965  

   Wagenbach, Michael; Vicente, Juan Jesus; Wagenbach, Wren; Wordeman, Linda (December 2023, Current Protocols)

   Abstract Protein activity is generally functionally integrated and spatially restricted to key locations within the cell. Knocksideways experiments allow researchers to rapidly move proteins to alternate or ectopic regions of the cell and assess the resultant cellular response. Briefly, individual proteins to be tested using this approach must be modified with moieties that dimerize under treatment with rapamycin to promote the experimental spatial relocalizations. CRISPR technology enables researchers to engineer modified protein directly in cells while preserving proper protein levels because the engineered protein will be expressed from endogenous promoters. Here we provide straightforward instructions to engineer tagged, rapamycin‐relocalizable proteins in cells. The protocol is described in the context of our work with the microtubule depolymerizer MCAK/Kif2C, but it is easily adaptable to other genes and alternate tags such as degrons, optogenetic constructs, and other experimentally useful modifications. Off‐target effects are minimized by testing for the most efficient target site using a split‐GFP construct. This protocol involves no proprietary kits, only plasmids available from repositories (such as addgene.org). Validation, relocalization, and some example novel discoveries obtained working with endogenous protein levels are described. A graduate student with access to a fluorescence microscope should be able to prepare engineered cells with spatially controllable endogenous protein using this protocol. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Choosing a target site for gene modification Basic Protocol 2: Design of gRNA(s) for targeted gene modification Basic Protocol 3: Split‐GFP test for target efficiency Basic Protocol 4: Design of the recombination template and analytical primers Support Protocol 1: Design of primers for analytical PCR Basic Protocol 5: Transfection, isolation, and validation of engineered cells Support Protocol 2: Stable transfection of engineered cells with binding partners 

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