细胞生物学

Immune tolerance and response are both largely driven by the interactions between the major histocompatibility complex (MHC) expressed by antigen presenting cells (APCs), T-cell receptors (TCRs) on T-cells, and their cognate antigens. Disordered interactions cause the pathogenesis of autoimmune diseases such as type 1 diabetes. Therefore, the identification of antigenic epitopes of autoreactive T-cells leads to important advances in therapeutics and biomarkers. Next-generation sequencing methods allow for the rapid identification of thousands of TCR clonotypes from single T-cells, and thus there is a need to determine cognate antigens for identified TCRs. This protocol describes a reporter system of T-cell activation where the fluorescent reporter protein ZsGreen-1 is driven by nuclear factor of activated T-cells (NFAT) signaling and read by flow cytometry. Reporter T-cells also constitutively express additional pairs of fluorescent proteins as identifiers, allowing for multiplexing of up to eight different reporter T-cell lines simultaneously, each expressing a different TCR of interest and distinguishable by flow cytometry. Once TCR expression cell lines are made they can be used indefinitely for making new T-cell lines with just one transduction step. This multiplexing system permits screening numbers of TCR-antigen interactions that would otherwise be impractical, can be used in a variety of contexts (i.e., screening individual antigens or antigen pools), and can be applied to study any T-cell-MHC-antigen trimolecular interaction.

Stress granules (SGs) are membrane-less organelles that form in the cytoplasm through phase separation, in response to diverse stressors. SGs contain translationally stalled mRNAs, proteins involved in translation, and various RNA-binding proteins (RBPs). Due to the high local concentration of aggregation-prone RBPs, SGs might act as condensation sites for aberrant phase transitions of RBPs and could favor formation of solid protein aggregates underlying the pathological cytoplasmic inclusions found in numerous neurodegenerative diseases. Most assays aiming at studying the recruitment of RBPs into SGs are based on overexpression and SG recruitment of RBPs in intact cells. These approaches are, however, often limited by the predominantly nuclear localization of many RBPs, which precludes cytoplasmic RBP concentrations sufficient for SG localization, and does not address RBP recruitment independent of SG formation. Here, we present a quantitative method to assess recruitment of recombinant RBPs into pre-formed SGs, independent of the RBP’s nuclear localization, using semi-permeabilized cells and fluorescence microscopy. In this assay, SGs are firstly induced by a stressor, and then the plasma membrane of the stressed cells is subsequently selectively permeabilized to provide access of the recombinant protein to SGs. Nuclear import of the protein-of-interest is prevented by blocking nuclear pores with wheat germ agglutinin. This assay allows one to study the molecular mechanisms underlying recruitment of RBPs into SGs quantitatively, in absence of their nuclear import and under controlled conditions. The method allows for a direct comparison of wildtype, mutant or posttranslationally modified RBPs, for addressing the influence of other proteins’ preventing or promoting SG association of RBPs, and is also applicable to synthetic peptides.

Graphic abstract


Workflow overview for analysis of SG recruitment of recombinant proteins or peptides in semi-permeabilized cells

G-protein coupled receptors (GPCRs) remain at the forefront of drug discovery efforts. Detailed assessment of features contributing to GPCR ligand engagement in a physiologically relevant environment is imperative to the development of new therapeutics with improved efficacy. Traditionally, binding properties such as affinity and kinetics were obtained using biochemical radioligand binding assays. More recently, the high specificity of resonance energy transfer has been leveraged toward the development of homogeneous cell-based proximity assays with capacity for real-time kinetic measurements. This suite of ligand binding protocols couples the specificity of bioluminescent resonance energy transfer (BRET) with the sensitivity afforded by the luminescent HiBiT peptide. The BRET format is used to quantify dynamic interactions between ligands and their cognate HiBiT-tagged GPCRs through competitive binding with fluorescent Tracers. At the same time, high affinity complementation of HiBiT with the cell impermeable LgBiT limits the bright bioluminescence donor signal to the cell surface and eliminates luminescence background from unoccupied receptors present in intracellular compartments.

Protein-protein interactions play key roles in nuclear processes including transcription, replication, DNA damage repair, and recombination. Co-immunoprecipitation (Co-IP) followed by western blot or mass spectrometry is an invaluable approach to identify protein-protein interactions. One of the challenges in the Co-IP of a protein localized to nucleus is the extraction of nuclear proteins from sub-nuclear fractions without losing physiologically relevant protein interactions. Here we describe a protocol for native Co-IP, which was originally used to successfully identify previously known as well novel topoisomerase 1 (TOP1) interacting proteins. In this protocol, we first extracted nuclear proteins by sequentially increasing detergent and salt concentrations, the extracted fractions were then diluted, pooled, and used for Co-IP. This protocol can be used to identify protein-interactome of other chromatin-associated proteins in a variety of mammalian cells.

Genetically encoded biosensors are powerful tools for quantitative visualization of ions and metabolites in vivo. Design and optimization of such biosensors typically require analyses of large numbers of variants. Sensor properties determined in vitro such as substrate specificity, affinity, response range, dynamic range, and signal-to-noise ratio are important for evaluating in vivo data. This protocol provides a robust methodology for in vitro binding assays of newly designed sensors. Here we present a detailed protocol for purification and in vitro characterization of genetically encoded sensors, exemplified for the His affinity-tagged GO-(Green-Orange) MatryoshCaMP6s calcium sensor. GO-Matryoshka sensors are based on single-step insertion of a cassette containing two nested fluorescent proteins, circularly permutated fluorescent green FP (cpGFP) and Large Stoke Shift LSSmOrange, within the binding protein of interest, producing ratiometric sensors that exploit the analyte-triggered change in fluorescence of a cpGFP.

Cryo-Electron Tomography (cryo-ET) is a method that enables resolving the structure of macromolecular complexes directly in the cellular environment. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-Focused Ion Beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. The PIE-scope can be retrofitted on existing microscopes, although the drawings we provide are meant to work on ThermoFisher DualBeams with small mechanical modifications those can be adapted on other brands.

Numerous experimental approaches exist to study interactions between two subunits of a large macromolecular complex. However, most methods do not provide spatial and temporal information about binding, which are critical for dissecting the mechanism of assembly of nanosized complexes in vivo. While recent advances in super-resolution microscopy techniques have provided insights into biological structures beyond the diffraction limit, most require extensive expertise and/or special sample preparation, and it is a challenge to extend beyond binary, two color experiments. Using HyVolution, a super-resolution technique that combines confocal microscopy at sub-airy unit pinhole sizes with computational deconvolution, we achieved 140 nm resolution in both live and fixed samples with three colors, including two fluorescent proteins (mTurquoise2 and GFP) with significant spectral overlap that were distinguished by means of shifting the excitation wavelength away from common wavelengths. By combining HyVolution super-resolution fluorescence microscopy with bimolecular fluorescence complementation (SRM-BiFC), we describe a new assay capable of visualizing protein-protein interactions in vivo at sub-diffraction resolution. This method was used to improve our understanding of the ordered assembly of the Saccharomyces cerevisiae spindle pole body (SPB), a ~1 giga-Dalton heteromeric protein complex formed from 18 structural components present in multiple copies. We propose that SRM-BiFC is a powerful tool for examination of direct interactions between protein complex subunits at sub-diffraction resolution in live cells.

Studying protein-protein and protein-lipid interactions in their native environment is highly desirable, yet, the heterogeneity and complexity of cellular systems limits the repertoire of experimental methods available. In cells, interactions are often taking place in confined microenvironments where factors such as avidity, hindered diffusion, reduced dimensionality, crowding etc. strongly influence the binding kinetics and therefore it can be problematic to equate binding affinities obtained by bulk in-solution methods (e.g., Fluorescence Polarization, Isothermal titration calorimetry, Microscale thermophoresis) with those occurring in real cellular environments. The Supported Cell Membrane Sheet method presented here, addresses these issues by allowing access to the inner leaflet of the apical plasma membrane. The method is a highly versatile, near-native platform for both qualitative and quantitative studies of protein-protein and protein-lipid interactions occurring directly in or on the plasma membrane.