Efflux pump insensitive rhodamine-jasplakinolide conjugates for G- and F-actin imaging in living cells

Chromatin Labeling and Imaging Group, Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077, Göttingen, Germany. Facility for Synthetic Chemistry, Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077, Göttingen, Germany. Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077, Göttingen, Germany. Optical Microscopy Facility, Max Planck Institute for Medical Research, Jahnstraße 29, 69120 Heidelberg, Germany Advanced Light Microscopy Core Facility, EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany


of probes by staining fixed and permeabilized U-2 OS cells
The cells were fixed with formaldehyde, stained and imaged as described in the Methods section. Actin staining was quantified using an automated pipeline described in Figure S3. Background was defined as median pixel intensity in the areas not covered by cells. Background corrected images are shown. Scale bars 100 µm. The experiments were repeated 3 times, and the quantification of data is represented as mean ± SD. The fitting line is shown and the derived EC50 is indicated.  Hela cells were grown in the presence of the indicated probes for 24h and processed according to NucleoCounter® NC-3000™ (Chemometec) two-step cell cycle analysis protocol as previously described 1 . In each sample DNA content of ~10 000 cells in total were measured and the obtained cell cycle histograms were analysed with ChemoMetec NucleoView NC-3000 software, version 2.1.25.8. All experiments were repeated three times and the results are presented as means with standard deviations. Probe toxicity thereshold is defined as a minimum concentration at which increase in SubG1 subpopulation can be detected. These values are marked with red arrows and listed in the main text Table 1.
Imaging was performed without washing after labeling for 2h at 37°C in DMEM containing 10% FBS. Scale bar 5 µm.
Cells were stained for 1h at 37°C in DMEM containing 10% FBS and imaged after washing once with the same medium. Four panels represent raw and deconvolved (SVI Huygens) images acquired on microscope operating in confocal and STED modes. Scale bar 2 µm. Table S1. Optical properties of the probes.

Determination of Quantum Yields
Relative quantum yields of the probes bound to actin were calculated by recording absorbance and fluorescence spectra using a multiwell plate reader Spark® 20M and glass bottom 96-well plates (MatTek, Cat. No. PBK96G-1.5-5-F) at room temperature (25 °C). To account for background due to light scattering, spectra of the solutions containing no probes, but equivalent amount of DMSO, were acquired and subtracted from the respective probe spectra. Absolute quantum yields of probes were measured using Quantaurus-QY absolute PL quantum yield spectrometer for samples containing 1 µM probe and 0.1 % SDS in PBS. Samples were incubated for 1 h at room temperature before measurement. The experiment was repeated three times in triplicates and the obtained QY probe SDS are presented as mean ± SD in Supplementary  Table S2. Relative quantum yields (QYactin) of the probes bound to the target were calculated in a|e -UV-Vis-IR Spectral Software v2.2 (FluorTools) using the following formula (1): where

Preparation of G-actin
Lyophilized rabbit skeletal muscle actin (unlabeled and pyrene-labeled) was obtained from Cytoskeleton Inc (Cat. No. AKL99). For preparing G-actin, 1 mg of actin was dissolved in 0.5 ml General Actin Buffer (5 mM Tris·HCl, 0.2 mM CaCl2), supplemented with 0.2 mM ATP, and incubated on ice for 1 h, in order to depolymerize the oligomers that might have formed during storage. Any remaining oligomers or aggregates were removed by centrifugation at 14000 rpm for 30 min. at 4°C. The solution was transferred into a new tube and kept on ice. This gave 46.5 µM actin stock solution.

Polymerization of pyrene-actin
4 µM pyrene-actin was prepared in General Actin Buffer, supplemented with 0.2 mM ATP and 0.5 mM DTT containing 10 nM SiR-actin or 610CP-JAS probes or 0.1% DMSO. Aliquots of 50 µl were added to a black 96well half-area plate and the fluorescence was read for 3 min. at 37°C, taking one read every 30 s. Where required, 5 µl of 10× actin polymerization buffer was quickly added and the measurements were continued for 60 min. Pyrene fluorescence was measured with Tecan Spark20M plate reader in a top-reading mode, using excitation of 360 nm (bandwidth 20 nm) and recording emission at 410 nm (bandwidth 10 nm).

General experimental information for organic synthesis
All standard chemicals and solvents were purchased from commercial suppliers (Sigma-Aldrich, Merck, VWR) and used without further purification. Boc-jasplakinolide was synthesized via solid-phase-synthesis according to the previously described procedure by Milroy et al. 4 . All powder reagents were weighted using high precision Mettler Toledo XPE206DR balances.
Analysis was performed on a ThermoScientific Ultimate 3000 system with a diode array detector using a Kinetex 2,6µm C18 (4,6 x 75 mm) column at 20 °C. 2.0 mg of dye-COOH were dissolved under argon atmosphere in an oven dried pear shaped flask in 400 µl abs. DMSO. 26 eq of N,N-Diisopropylethylamine and 1.2 eq of TSTU were added and the reaction mixture was stirred for 5 minutes under inert gas. After in situ formation of the NHS-ester, 3.8 eq of 6aminohexanoic acid were added, the reaction mixture was sonicated for 15 minutes. After addition of 20 µl ultrapure water, stirring at room temperature was continued for 15 minutes and the conversion monitored by analytical HPLC. The reaction mixture was quenched, adding 30 eq of formic acid, frozen on dry ice and lyophilized, followed by preparative RP-HPLC, yielding dye-C6-linker in 75-85% yield. 1.5 mg of dye-C6-linker were dissolved under argon atmosphere in an oven dried pear shaped flask in 300 µl of abs. DMSO. 8 eq of N,N-Diisopropylethylamine and 2.6 eq of TSTU were added and the reaction mixture was stirred for 60 minutes under inert gas. The reaction conversion was monitored by analytical HPLC. After full conversion, the reaction mixture was frozen on dry ice, lyophilized and purified via preparate HPLC, yielding dye-C6-linker-NHS in 60-80% yield. 1 mg of dye-C6-linker-NHS were dissolved under argon atmosphere in an oven dried pear shaped flask in 145 µl abs. DMSO. 8 eq of N,N-Diisopropylethylamine and 1 -1.2 eq of jasplakinolide-amine formiate salt were added and the reaction mixture was stirred for 40 minutes under inert gas. The reaction conversion was monitored by analytical HPLC.After full conversion, the reaction mixture was frozen on dry ice, lyophilized and purified via preparative HPLC, yielding dye-C6-linker-jasplakinolide in 40-70% yield.