Specific protein labeling with caged fluorophores for dual-color imaging and super-resolution microscopy in living cells

Abstract

We present new fluorophore-conjugates for dual-color photoactivation and super-resolution imaging inside live mammalian cells. These custom-designed, photo-caged Q-rhodamines and fluoresceins are cell-permeable, bright and localize specifically to intracellular targets. We utilized established orthogonal protein labeling strategies to precisely attach the photoactivatable fluorophores to proteins with subsequent activation of fluorescence by irradiation with UV light. That way, diffusive cytosolic proteins, histone proteins as well as filigree mitochondrial networks and focal adhesion proteins were visualized inside living cells. We applied the new photoactivatable probes in inverse fluorescence recovery after photo-bleaching (iFRAP) experiments, gaining real-time access to protein dynamics from live biological settings with resolution in space and time. Finally, we used the caged Q-rhodamine for photo-activated localization microscopy (PALM) on both fixed and live mammalian cells, where the superior molecular brightness and photo-stability directly resulted in improved localization precisions for different protein targets.

Scheme 1. Schematic representation of live cell dual-color photoactivation of caged-fluorescein and caged-Q-rhodamine conjugates, localized to different cellular compartments, using the Halo- and eDHFR-tag as orthogonal protein labeling strategies.

Fig. 1. Photoactivation of (A) TMP–PA-Q-Rh (5 μM) and (B) TMP–PA-Fl (5 μM) by UV-light (λ = 365 nm, 8 W) in aqueous solution. Irradiation intervals are represented by the following color code: 0–20 s in 10 s-intervals (black), 20–60 s in 20 s-intervals (blue), 60 s–2.30 min in 1.5 min interval (cyan), 2.5–10 min in 2.5 min-intervals (green), 10–30 min in 5 min intervals (red), 30–80 min in 10 min-intervals (orange). The fluorescence intensities at the peak maxima are plotted as a function of activation time to obtain activation curves (inserts). (C) Structures of the TMP–PA-Fl-conjugate and the TMP–PA-Q-Rh and Halo–PA-Q-Rh-conjugates before and after activation by UV-light.

Fig. 2. Mono- and dual color photoactivation inside living cells. Cells are depicted as bright field recordings, as well as composite red-green images, pre- and post-photoactivation. (A) Mono- (i–iii) and dual-color photoactivation (iv) of nuclear and mitochondrial targets inside live COS-7 and NIH 3T3 cells. Co-overexpressed, Halo- and eDHFR-tagged H2B and Tom20 were stained with TMP- and Halo-(photoactivatable) fluorescent conjugates: (i) Tom20–Halo with Halo–TMR, H2B–eDHFR with TMP–PA-Fl; (ii) Tom20–Halo with Halo–diAcFl, H2B–eDHFR with TMP–PA-Q-Rh; (iii) H2B–Halo with Halo–TMR, Tom20–eDHFR with TMP–PA-Fl, (iv) H2B–eDHFR with TMP–PA-Fl, Tom20–Halo with Halo–PA-Q-Rh. Non-PA dyes were applied in 1 μM, caged fluorophores in 5 μM concentration. Photoactivation was conducted using the DAPI-channel: (352–402 nm, 14.5 mW cm^–2). Scale bars: 10 μm. (B) Dual-color photoactivation of histone proteins inside live COS-7 cells. Halo- and eDHFR-tagged histones H2A and H2B were transiently co-overexpressed and stained with TMP–PA-Fl and Halo–PA-Q-Rh. Caged fluorophore conjugates were applied in 5 μM concentration. Photoactivation was conducted using DAPI-filtered UV-light (352–402 nm, 14.5 W cm^–2). Scale bars: 10 μm. (C) Photoactivation of focal adhesion sites inside live MEF Vcl^–/– cells after overexpression of Halo-tagged vinculin–EGFP and staining with Halo–PA-Q-Rh. Recordings of post-activation images are shown in the red channel and as composite images. The green channel (lower left) shows the signal from EGFP. The composite image shows the co-localization of green (EGFP) and red (Q-Rh) fluorescent signals, after photoactivation at 405 nm (laser power: 60 mW cm^–2. Scale bars: 5 μm).

Fig. 3. Monitoring protein dynamics in iFRAP experiments using photoactivatable fluorophores to target proteins. (A) HeLa Kyoto cells expressing Halo-tagged lymphocyte-specific protein tyrosine kinase (Halo-Lck), with Lck localizing the fusion protein at the inside of the PM. Cells were stained with Halo–PA-Q-Rh (5 μM). Activation was conducted in a pre-defined ROI (diameter: 10 μm, indicated by dashed white circle) at 405 nm (60 mW cm^–2), which resulted in an immediate stepped increase of the fluorescence intensity. Scale bars: 5 μm. (B) Diffusion dynamics with a half-residence time of 7.8 s and an equilibration time of 47.4 s were determined from the exponentially decrease of the fluorescence signal. The depicted curve represents the means ± SD of 3 independent cells.

Fig. 4. PALM on Halo- (A) and eDHFR- (B) tagged H2B in live NIH 3T3 cells. NIH 3T3 cells were transiently transfected with H2B–Halo (A) or H2B–eDHFR (B) and stained with Halo–PA-Q-Rh (A), respective TMP–PA-Q-Rh (B). Acquired images are represented as diffraction-limited reconstruction (left) and single-molecule localization (super-resolution, middle). Close-up representations of the selected regions are shown on the right. Scale bars: 10 μm and 1 μm (zoomed view).