Targeting and imaging single biomolecules in living cells by complementation-activated light microscopy with split-fluorescent proteins

Abstract

Single-molecule (SM) microscopy allows outstanding insight into biomolecular mechanisms in cells. However, selective detection of single biomolecules in their native environment remains particularly challenging. Here, we introduce an easy methodology that combines specific targeting and nanometer accuracy imaging of individual biomolecules in living cells. In this method, named complementation-activated light microscopy (CALM), proteins are fused to dark split-fluorescent proteins (split-FPs), which are activated into bright FPs by complementation with synthetic peptides. Using CALM, the diffusion dynamics of a controlled subset of extracellular and intracellular proteins are imaged with nanometer precision, and SM tracking can additionally be performed with fluorophores and quantum dots. In cells, site-specific labeling of these probes is verified by coincidence SM detection with the complemented split-FP fusion proteins or intramolecular single-pair Förster resonance energy transfer. CALM is simple and combines advantages from genetically encoded and synthetic fluorescent probes to allow high-accuracy imaging of single biomolecules in living cells, independently of their expression level and at very high probe concentrations.

Fig. 1.

Complementation of GFP 1–10 with synthetic M3 peptides and in vitro SM imaging. (A) Schematic of self-complementation between nonfluorescent (Upper) and fluorescent (Lower) synthetic M3 peptides and GFP 1–10. (B) Native gel electrophoresis of biotin-M3 peptide (lane 1), GFP 1–10 (lane 2), and complementation reaction of biotin-M3 with GFP 1–10 (lane 3). (C) Native gel shift of free Alexa 647 (A647; lane 1), unreacted M3-Alexa 647 conjugate (M3-A647; lane 2), and M3-A647 reaction with GFP 1–10 in the absence (lane 3) or presence (lane 4) of a competing excess of biotin-M3 peptides. (D) Sequential photobleaching of complemented and purified individual GFP-biotin nonspecifically bound to a glass coverslip and imaged by TIRF (Movie S1). GFP diffraction-limited spots are intentionally expanded to facilitate visualization. (Scale bar: 2 μm.) (E) Fluorescence intensity distribution for 152 single-complemented GFP-biotin molecules and background fluorescence from coverslips. (F) Fluorescence time traces of four single split-GFPs complemented on M3 peptide-coated coverslips. Single-step photobleaching and blinking events are observed. (G) Representative TIRF fields of view taken at different incubation times before and after addition of GFP 1–10 to M3 peptide-coated coverslips. The number of individual-complemented GFP-biotin per fields of view increases with increasing incubation times. GFP diffraction-limited spots are intentionally expanded to facilitate visualization. (Scale bar: 2 μm.)

Fig. 2.

CALM imaging in living cells. (A) Schematic representation of plasma membrane split-GFP fusions used in this work. (B) Wide-field fluorescence imaging of GFP 1–10-CD4 expression and complementation in U2OS, COS-7, and HEK cells. Expressing cells (+) are detected with a fluorescent anti-GFP antibody. When incubated with M3 peptides (+biotin-M3 or +FCC-M3), GFP 1–10-CD4 proteins are activated into bright GFP-CD4 proteins, and expressing cells become fluorescent (overlay). No GFP signal is seen in the absence of peptides (−biotin-M3) or for nonexpressing cells (−). Binding of the complementary biotin-M3 peptides on the cell surface is verified by staining with fluorescent streptavidin (SAV-A647). (Scale bar: 20 μm.) (C) Wide-field fluorescence imaging of GFP 1–10_(h)-GPI expression and complementation in U2OS and COS-7 cells. (Scale bar: 10 μm.) (D) Fluorescence confocal, wide-field, and TIRF imaging of cav1-GFP 1–10_(h) expression and intracellular complementation in U2OS cells. (Upper) Ventral plasma membrane confocal images of fixed cells immunolabeled for endogenous cav1 (anti-cav1) and cav1-GFP 1–10_(h) (anti-GFP) showing cav1-GFP 1–10_(h) colocalization with endogenous cav1 (overlays and Insets). 3D reconstructions of cells are available in Movie S4. (Scale bar: 20 μm.) (Lower) Fluorescence wide-field imaging of live U2OS cells coexpressing cav1-GFP 1–10_(h) and ABP-mCherry (+). A cell microinjected with M3 peptides (star; ∼25 μM final intracellular M3 peptide concentration) and imaged after 45 min incubation at 37 °C shows a perinuclear pool of complemented cav1-GFP_(h) (arrows) and a plasma membrane pool of caveolae-associated cav1-GFP_(h) (arrowheads). The typical punctuated pattern of complemented caveolae is better seen by TIRF imaging of the plasma membrane (white square). The TIRF image is a pixel-based maximum intensity projection (ΣI_max) overlay image for all frames of the dual-color Movie S5. (Scale bar: wide-field, 10 μm; TIRF, 5 μm.)

Fig. 3.

SM imaging and tracking of extracellular GFP 1–10-CD4 proteins by CALM. (A) A region of interest (black square) in the plasma membrane of a U2OS cell stably expressing GFP 1–10-CD4 is imaged by TIRF before and after complementation with biotin-M3 peptides at different incubation times. Diffraction-limited single GFP-CD4 spots appear and diffuse in the plasma membrane within minutes of M3 peptide addition. Single GFP-CD4 spots are intentionally expanded to facilitate visualization. (Scale bar: 5 μm.) (B) Pixel-based maximum intensity projections (ΣI_max) TIRF images of all complemented GFP-CD4 detected during 20-min complementation with 1.8 or 18 μM biotin-M3 peptides (Movie S6). The field of view corresponds to the bright field image in A. (Scale bar: 5 μm.) (C) 3D rendering of raw and Gaussian-fitted diffraction-limited spots corresponding to individual complemented GFP-CD4 in the cell plasma membrane (white square in A). (D) Representative trajectories from single GFP-CD4 diffusing in the plasma membrane of U2OS cells during CALM imaging.

Fig. 4.

SM imaging and tracking of intracellular cav1-GFP 1–10_(h) proteins by CALM. (A) A U2OS cell coexpressing cav1-GFP 1–10_(h) and the nucleus-localized CFP-LacI-NLS coexpression marker (+) are microinjected with biotin-M3 peptides (∼5 μM final intracellular concentration) together with a biotin-Alexa 647 injection marker (star). A region of interest (white square) is then imaged for GFP fluorescence by TIRF microscopy. (Scale bar: 10 μm.) (B) Pixel-based maximum intensity projection (ΣI_max) TIRF images of all complemented cav1-GFP_(h) detected at the ventral intracellular plasma membrane for the region of interest in A 3, 5, and 10 min after injection of M3 peptides (Movie S8). When overlaid with the wide-field fluorescence image, the cumulative 3- to 10-min maximum intensity projection image shows the high specificity of complementation. (Scale bar: 5 μm.) (C) 3D rendering of raw diffraction-limited spots corresponding to membrane-associated single cav1-GFP_(h) proteins (white square in B). (D) Representative trajectories from single cav1-GFP_(h) diffusing in the cytoplasmic side of the plasma membrane of U2OS cells during CALM imaging.

Fig. 5.

Addressable live cell targeting and tracking of single fluorophores and qdots by CALM. (A) Schematic of CALM with fluorescent M3 peptide conjugates and M3 peptide-coated qdots. (B) Labeling of a GFP 1–10-CD4–expressing U2OS cells (+) with M3-A647 peptide conjugates. (Scale bar: 10 μm.) (C) Coincident dual-color detection of single diffusing A647-GFP-CD4 proteins by TIRF in U2OS cells. Colocalizing diffraction-limited spots are simultaneously detected in the GFP and M3-A647 channels (colored asterisks). During diffusion, GFP photobleaches in a single step (frames 6 and 11), but A647-GFP-CD4 proteins can still be tracked in the M3-A647 channel before disappearing on M3-A647 photobleaching (frame 13). The diffraction-limited spots are intentionally expanded to facilitate visualization (Movie S10). (Scale bar: 1 μm.) (D) Wide-field fluorescence imaging of peptide-coated CdSe/ZnS M3-qdots specifically targeted to U2OS cells expressing GFP 1–10-CD4 fusion proteins (+). The M3-qdot image is a pixel-based maximum intensity projection of diffusing M3-qdots (ΣI_max) for all frames of Movie S11. (Scale bar: 15 μm.) (E) Representative trajectories from qdot tracking of single complemented qdot-GFP-CD4 proteins diffusing in the plasma membrane of U2OS cells.

Fig. 6.

Cell imaging and single biomolecule tracking by complementation-induced intramolecular spFRET. (A) In vitro native gel shift assay of M3-A647 peptide binding to soluble GFP 1–10 (+) or in TGN buffer (−). The gel is sequentially imaged for M3-A647, GFP, and A647-GFP intramolecular FRET emission. (B) Live cell imaging of GFP 1–10-CD4 proteins complemented with fluorescent M3-A647 (Left) or nonfluorescent M3-biotin (Right) peptides and imaged by dual-color TIRF microscopy using only 488-nm excitation. Images are pixel-based maximum intensity projections of diffusing A647-GFP-CD4 proteins (ΣI_max) for all frames of Movie S12. (Scale bar: 5 μm.) (C) GFP (green) and M3-A647 fluorescence time traces (red) along the diffusion path (Right) of individual A647-GFP-CD4 proteins showing intramolecular spFRET. Fluorescence background traces (gray) are taken in the immediate vicinity of the trajectories. The single-step photobleaching of GFP (Upper, green arrow) or M3-A647 (Lower, red arrow) induces an arrest of intramolecular spFRET (Movie S13). (D) Live cell TIRF imaging and tracking of individual A647-GFP-CD4 proteins by spFRET at high M3-A647 concentrations (0.7 μM) without washing. A cell is sequentially imaged using direct M3-A647 excitation at 638 nm (Left) and then, indirect spFRET excitation at 488-nm laser (Right). Individual A647-GFP-CD4 proteins diffusing in the plasma membrane can be tracked in the M3-A647 channel using indirect spFRET excitation (red trajectories and white squares) but are lost in the saturating surrounding fluorescent signal when directly excited at 638 nm (red squares). Three representative examples of A647-GFP-CD4 trajectories are presented (from white squares). (Scale bar: 10 μm.)

Fig. P1.

Complementation-activated light microscopy (CALM) imaging. A protein of interest is fused to the large fragment of a nonfluorescent split-GFP, expressed in cells and detected by exogenously providing synthetic versions of the peptide fragment complementary to the split-GFP. The activation of GFP depends on the stochastic binding of the complementary fragment and the chromophore maturation time. Adjusting peptide concentrations and incubation times allows for only a subset of the fusion proteins to be switched on (green circles), despite their high expression in cells (gray circles). Thousands of individual proteins can be progressively highlighted and continuously detected with minimal background. This allows for a high-resolution localization of their position by Gaussian fitting and nanometer precision tracking of their diffusion in living cells. Using split-GFP as Förster resonance energy transfer (FRET) donors and fluorescently labeled complementary peptides, specific targeting and high-resolution tracking of individual fusion proteins was also performed in the low-background far-red region of the light spectrum (red circles) even at fluorophore concentrations that normally prevent SM imaging (direct detection).