SiR-XActin: A fluorescent probe for imaging actin dynamics in live cells

Abstract

Imaging actin-dependent processes in live cells is important for understanding numerous biological processes. However, currently used natural-product based fluorescent probes for actin filaments affect the dynamics of actin polymerization and can induce undesired cellular phenotypes. Here, we introduce SiR-XActin, a simplified jasplakinolide-based, far-red fluorescent probe that enables bright and photostable staining in various cell types without requiring genetic modifications. Due to its relatively weak binding affinity, the probe exhibits minimal cytotoxicity and labels actin filaments without significantly altering actin dynamics. Furthermore, SiR-XActin is suitable for time-resolved, live-cell super-resolution STED microscopy. Exchanging the SiR fluorophore in SiR-XActin for other fluorophores yields probes in different colors. All these properties make SiR-XActin and its analogs powerful tools for studying actin dynamics using live-cell fluorescence microscopy.

Figure 1.. SiR-XActin is a hybrid natural product with reduced cytotoxicity and perturbation of cellular dynamics:

(A) Scheme of actin polymerization/depolymerization machinery and binding cleft of jasplakinolide-like natural products. Cryo-EM structure of jasplakinolide-stabilized F-actin (PDB: 6T24) was used for generating cartoon of jasplakinolide binding to F-actin. (B) Structures of natural product jasplakinolide (1), cell permeable F-actin labeling tool SiR-actin (2), non-toxic hybrid natural product (3). (C) Synthesized analogs and cytotoxicity profiling. (D) Structure of SiR-XActin (8). (E) Comparison of SiR-actin and SiR-XActin influence on migration of HeLa cells in wound healing assay, 500 nM dye concentration used. Shown curves represent normalized wound gap area over time. Average values and standard deviation of five independent measurements.

Figure 2.. Labeling performance of SiR-XActin in fixed and live cells:

(A) i: Fixed-cell no-wash CLSM images of U-2 OS cells. 652 nm excitation with 0.4% (SiR-actin) or 2% (SiR-XActin) laser power. ii: Fluorescence signal reduction in fixed U-2 OS cells labelled with 500 nM dye after medium exchange. The signal was normalized to the no-wash fluorescence intensity of each replicate. iii: Fluorescence intensity bleaching curve of SiR-actin and SiR-XActin (500 nM) in U-2 OS cells. 652 nm excitation (0.1% laser power) used for imaging, 652 nm (100% laser power) used for bleaching. The signal was normalized to the initial fluorescence intensity of each measurement. (B) i: U-2 OS and ii: HeLa cells stained with SiR-actin and SiR-XActin at the given dye concentrations in presence and absence of verapamil (10 μM), 4 h of incubation. (C) i: Median fluorescence signal in U-2 OS and HeLa cells (condition described in B-i and B-ii, V - verapamil). Average values and standard deviation from three independent measurements. ii: Live-cell labeling kinetics of SiR-actin and SiR-XActin (both 500 nM) in HeLa cells, signal was normalised to signal intensity measured after 4 h of incubation. Average values and standard deviation from three biological replicates / independent measurements. iii: Live-cell bleaching curves for SiR-actin and SiR-XActin (both 500 nM) in HeLa cells, incubation time 4 h, 652 nm excitation (0.1% laser power) used for imaging, 652 nm (100% laser power) used for bleaching. Red Hot lookup - pixel intensities were scaled between 0 (black) and 65535 (bright yellow) by Fiji. Scale bars 20 μm.

Figure 3.. SiR-XActin is a tool to study F-actin in live cells by STED microscopy:

(A) Phenotypic comparison of SiR-actin and SiR-XActin stainings of live HeLa cells. Cells were stained with 1 μM actin probes for 2 h, no-wash imaging. Representative CLSM and STED images. Pixel intensities were scaled between dark and bright colors according to the reference bars. Scale bars: 10 μm. (B) Magnification of blue ROIs from A. Scale bars: 1 μm. (C) Line-scan profiles shown in B. (STED) perpendicular to actin filaments. Staining with SiR-actin reveals increased number and thickness of stress fibres in the centre and a fewer filopodia on the cell’s edges in comparison to SiR-XActin staining. (D) Normalized fluorescence intensity of multi-frame STED imaging in a 10x10 μm ROI. U-2 OS cells were stained with 1 μM SiR-actin or SiR-XActin supplemented with 10 μM Verapamil for 2 h. After an initial bleaching phase, SiR-XActin reveals improved apparent photostability. (E) Combination of SiR-XActin with exchangeable HaloTag Ligands (xHTL) for multi-color STED. Hippocampal rat neurons were transduced with rAAVs with TOM20-HaloTag7 at seven DIV and imaged at twelve DIV. Exchangeable HaloTag Ligand (JF₅₈₅-S5, 500 nM) and SiR-XActin (1 μM) were co-imaged with the same depletion laser (775 nm). Scale bars: 10 μm (overview), 1 μm (magnification).

Figure 4.. XActin as a conjugate with other fluorophores to cover the whole imaging spectral window:

(A) Labeling of fixed HeLa cells, (B) labeling of live U-2 OS cells or (C) HeLa cells with XActin probes, 4 h incubation, with and without 10 μM verapamil. All conditions no-wash, 500 nM. Laser settings live cells: MAP500-XActin 0.3%, MAP555-XActin 0.3%, MAP620-XActin 0.1%, SiR700-XActin 0.3% laser power. Two Line Average. For Laser settings in fixed cells see Figure S19. Red Hot lookup - pixel intensities were scaled between 0 (black) and 65535 (bright yellow) by Fiji. Scale bars: 20 μm.

Figure 5.. Labeling actin dynamic structures in live CAD cells:

(A) Representative images of actin imaged with SiR-XActin, SPY650-FastAct, or Lifeact-GFP. Scale bars = 10 μm. (B) Colocalization analysis of SiR-XActin and SPY650-FastAct with Lifeact-GFP. n = 20 cells, unpaired t-test. (C) Representative kymographs of actin imaged with SiR-XActin and Lifeact-GFP. Scale bars, vertical = 1 μm and scale bars, horizontal = 10 seconds. (D) Scatter plot of retrograde flow measurements of Lifeact-GFP and SiR-XActin. Each point represents the average measurement of ten kymographs from each cell. n.s. = not significant, Mann-Whitney test. (E) Representative images of cells containing both Lifeact-GFP and SiR-XActin (above) or SPY650-FastAct (below). Scale bar = 10 μm.