Single-Cell and Single-Molecule Analysis of Gene Expression Regulation

Abstract

Recent advancements in single-cell and single-molecule imaging technologies have resolved biological processes in time and space that are fundamental to understanding the regulation of gene expression. Observations of single-molecule events in their cellular context have revealed highly dynamic aspects of transcriptional and post-transcriptional control in eukaryotic cells. This approach can relate transcription with mRNA abundance and lifetimes. Another key aspect of single-cell analysis is the cell-to-cell variability among populations of cells. Definition of heterogeneity has revealed stochastic processes, determined characteristics of under-represented cell types or transitional states, and integrated cellular behaviors in the context of multicellular organisms. In this review, we discuss novel aspects of gene expression of eukaryotic cells and multicellular organisms revealed by the latest advances in single-cell and single-molecule imaging technology.

Keywords: gene expression; mRNA localization; single cell; single-molecule imaging; transcription; translation.

Figure 1

Single-cell and single-molecule imaging technologies to analyze gene expression regulation. (a) Tools to achieve single-molecule resolution in cells. Visualizing single mRNA: schematic of an mRNA molecule with repetitions of MS2 or PP7 stem-loops genetically inserted in the 3′ untranslated region (3′ UTR). Tandem dimers of MCP (MS2 capsid protein) or PCP (PP7 capsid protein) (gray circles) fused to two molecules of green fluorescent protein (GFP) (green crystal structure) bind to each loop to label mRNAs with two GFP molecules per stem-loop in live cells. Multiple smFISH (single-molecule fluorescence in situ hybridization) probes labeled with a fluorophore bind to their complementary sequence in the mRNA, allowing detection of single mRNA molecules in fixed cells. Visualization of single proteins: schematic of a translating mRNA genetically modified with multiple GCN4 epitopes (SunTag) or Flag and HA epitopes (Spaghetti monster) sequences. As GCN4 or Flag and HA sequences are translated, they are recognized by scFv (SunTag) or Fab (Spaghetti monster) antibodies fused to GFP. Each protein is labeled with multiple GFP molecules at its N terminus. (b) Techniques to analyze single-molecule interactions and dynamics in live cells. Fluorescence correlation spectroscopy (FCS) uses a focused laser to excite fluorescently tagged molecules as they pass through the femtoliter volume. On the basis of the brightness correlation between two spectrally different fluorophores, their molecular interaction is inferred. Single-particle tracking (SPT) follows molecules from frame to frame, allowing the indicated measurements. Fluorescence recovery after photobleaching (FRAP) measures the rate of fluorescence intensity recovery and can be used to determine the kinetics of transcription and translation. In the case of transcription (left panel), mRNAs are tagged in the 5′ UTR with the MS2-MCP system, and in the case of translation (right panel), the proteins are tagged at the N terminus with the SunTag system. The plot represents measurements of fluorescence intensity before and over time after photobleaching, which can be used to calculate the mRNA or protein synthesis rate. (c) Schematic of a eukaryotic cell to illustrate how each step of gene expression is studied at the single-molecule level (starting from the top left and moving clockwise). (i) Transcription and mRNA splicing. The residence time of a fluorescently labeled transcription factor (TF) on DNA can be quantified using super-resolution and SPT techniques. By labeling nascent transcripts, the fluorescence intensity of the transcription site (TS) can be monitored over time to define the frequency, intensity, and amplitude of transcription. Cotranscriptional splicing can be visualized by labeling exons and introns on a nascent mRNA with spectrally different fluorophores. A plot of different probes’ intensities allows us to measure the number of mRNAs per TS, calculate the transcription elongation rate, and visualize mRNA splicing. (ii) Nuclear export. SPT of single mRNAs as they exit the nucleus indicates three distinct stages of nuclear export through the nuclear pore complex. (iii) Translation. Simultaneous imaging of mRNAs and proteins (red and green dots, respectively) reveals the dynamics of translation. Magnification of a site of translation (orange dot indicates the colocalization and comovement of an mRNA and more than one peptide). Magnification shows a schematic of a translating transcript genetically modified with the MS2-MCP [bound to red fluorescent protein (RFP)] system in the 3′ UTR and the SunTag system (scFv or Spaghetti monster bound to GFP) at the N terminus. A plot of the protein signal (GFP) describes the dynamics of translation. Labeling of mRNAs and ribosomes or RNA-binding proteins and comovement analysis or FCS reveals the dynamics of polysomes and the interaction of mRNA-binding proteins with mRNAs. (iv) mRNA decay. Correlation of the number of mRNAs with the cell cycle reveals a time-dependent switch in the mRNA’s half-life.

Figure 2

Examples of single-cell and single-molecule analysis of transcription, mRNA localization, and translation. (a) smFISH (single-molecule fluorescence in situ hybridization) for the c-Fos gene in a U2OS cell at 30 min after serum induction. The signal in proximity of the transcription site (TS) appears saturated because of scaling to show individual mature mRNAs. The surface plot (not to scale) shows the area indicated with a red dashed line. Detected mature mRNAs are shown as green spots on top of the DAPI image. Panel a adapted from Senecal et al. (123). (b) Specific expression and localization of ASH1 mRNA in Saccharomyces cerevisiae. The representative image shows smFISH of ASH1 (red signal) in fixed cells in an asynchronous population (different stages of the cell cycle are indicated in white). The ASH1 gene is exclusively expressed during anaphase of mitosis (M phase). ASH1transcripts (red signal) localize at the bud tip and are degraded before G1, the next stage of the cell cycle. Nuclei stained with DAPI are shown in blue. This unpublished image was kindly provided by Evelina Tutucci. (c) Translation sites in neurons visualized by an smFISH-immunofluorescence (IF) experiment on hippocampal neurons. The three boxes on the far right are magnifications of the same area. IF (green) recognizes the scFv-sfGFP bound to the 24 copies of the GCN4 sequence (SunTag system). smFISH (red) recognizes the transcript coding for the GCN4 polypeptide. The asterisk indicates a single mRNA translating several nascent peptides. The translation site is more intense than single proteins because several nascent peptides are being synthesized from the same single mRNA. This unpublished image was kindly provided by Carolina Eliscovich.