Streptavidin interfacing as a general strategy to localize fluorescent membrane tension probes in cells

Site-specific labeling with biotinylated mechanophores is probed to address the next challenge toward the imaging of forces in cells.

Column chromatography was carried out on silica gel 60 (SiliaFlash P60, 40-63 µm). Analytical (TLC) and preparative thin layer chromatography (PTLC) were performed on silica gel 60 (Merck, 0.2 mm) and silica gel GF (SiliCycle, 1 mm), respectively. Reverse phase flash chromatography was performed on a Biotage® Isolera Spektra using pre-packed Biotage® SNAP Ultra C18 cartridges. Fluorescence measurements were performed using a FluoroMax-4 spectrofluorometer (Horiba Scientific) equipped with a stirrer and a temperature controller. Fluorescence spectra were corrected using correction factors supplied by the manufacturer. LCMS were recorded using a Thermo Scientific Accela HPLC equipped with a Thermo C18 Hypersil GOLD column (50 × 2.1 mm, 1.9 µm particles size) coupled with a LCQ Tris(hydroxymethyl)aminomethane.
Compound 42 was synthesized and purified according to procedures described in ref. S1.
Compound 43 was purchased from Iris Biotech and used as received. were dissolved in a dichloromethane (2.6 mL) and water (0.6 mL) mixture. The solution was vigorously stirred for 2 h at rt, then water was added (10 mL), the mixture extracted with dichloromethane (2 x 10 mL), the organic phase was dried with Na2SO4, filtered and evaporated under reduced pressure. The crude red residue was dissolved in DMSO (2 mL) and purified on reverse phase flash chromatography Compound 45 was synthesized and purified according to procedures described in ref. S2.

Streptavidin complexes
To a solution of wild-type streptavidin (37.8 µM of tetramer) in buffer (10 mM Tris, 100 mM NaCl, pH 7.4, 25 ºC) was added probe 3 (37.8 µM, from 1 mM stock solution in DMSO) and the resulting solution was gently shaken at 4 °C for 2 h. Then, the next biotinylated ligand (biotin-X-DHPE 9, biotin 11, desthiobiotin 12, or probe 3) was added (113.4 µM) and the solution was shaken again at 4 °C for 2 h. The resulting stock solution was used as such for the fluorescence spectroscopy and imaging experiments without further purification.  buffer solution) was attached to DPPC vesicles containing 5% of lipid 9 and was measured as described above. Then, biotinylated insulin (1 mM solution in buffer solution) was added in different amounts (1 equiv.: 2 µL, 2 equiv.: 4 µL; 10 equiv.: 20 µL). The resulting solution was gently stirred for 5 min at rt before spectra measurement. Obtained spectra were background subtracted and corrected using the correction function supplied by the manufacturer.  (0.25 µM) recorded fifteen minutes after addition to DPPC LUVs with 0 (purple, solid), 2 (purple, dotted), 5 (red, dotted), 10 (red, solid) and 20 mol% biotin-X DHPE 9 (green, dashed).

Preparation of GUVs
GUVs were prepared by electroformation method using a Nanion Vesicles Prep Pro device made of two ITO electrodes. S8

Studies in GUVs
Confocal laser scanning microscopy. GUVs stock solutions resulting from the procedure reported above were diluted (10 µL in 190 µL) before being deposed on a 35 mm glass bottom dish. The probe was added in order to reach the desired concentration and the liquid was very carefully mixed with a pipette. For the experiments involving the biotinylated probe 3 or any streptavidin complex, an argon laser (λex = 488 nm, 30% laser power) was used and the fluorescence was collected between 550 and 650 nm. For dual dye experiments, images were obtained from sequential scans using a white laser at 70% laser power (λex1 = 488 nm, collection between 550 and 650 nm, 50% laser power, and λex2 = 584 nm, collection between 600 and 700 nm, 20% laser power).
Fluorescence lifetime imaging microscopy. Samples were prepared as for CLSM imaging. Laser at 485 nm (PicoQuant, LDH-D-C-485) was used and the fluorescence was collected between 550 and 650 nm. FLIM images were analyzed using SymPhoTime 64 software from PicoQuant.

Studies in HeLa cells
The cells were grown 72 h in DMEM in 35 mm glass bottom dishes. Then, the medium was removed and the cells were washed with PBS and twice with Leibovitz's medium. Leibovitz's medium containing the dye at the desired concentration was then added to the cells before imaging. Similar FLIM images were analyzed using SymPhoTime 64 software from PicoQuant.
Hypertonic osmotic shock. The cells were imaged in 2 mL of Leibovitz's media. Then, 1 mL of media was removed using a 1 mL micropipette and replaced by 1 mL of 1 M sucrose solution. The cells were imaged again right after the shock.