Turning Cell Adhesions ON or OFF with High Spatiotemporal Precision Using the Green Light Responsive Protein CarH

Abstract Spatiotemporal control of integrin‐mediated cell adhesions to extracellular matrix regulates cell behavior with has numerous implications for biotechnological applications. In this work, two approaches for regulating cell adhesions in space and time with high precision are reported, both of which utilize green light. In the first design, CarH, which is a tetramer in the dark, is used to mask cRGD adhesion‐peptides on a surface. Upon green light illumination, the CarH tetramer dissociates into its monomers, revealing the adhesion peptide so that cells can adhere. In the second design, the RGD motif is incorporated into the CarH protein tetramer such that cells can adhere to surfaces functionalized with this protein. The cell adhesions can be disrupted with green light, due to the disassembly of the CarH‐RGD protein. Both designs allow for photoregulation with noninvasive visible light and open new possibilities to investigate the dynamical regulation of cell adhesions in cell biology.

rpm overnight and harvested the next day by centrifugation at 6000 rpm, 4 ºC for 8 min (Beckman Coulter Avanti J-26S XP, JA-10 rotor). The bacteria pellet was resuspended in 20 mL buffer A (300 mM NaCl, 50 mM Tris, pH = 7.4) supplemented with 1 mM protease inhibitor phenylmethane sulfonyl fluoride (PMSF) and 1 mM DL-dithiothreitol (DTT). The bacteria were lysed by sonication and the lysate was cleared by centrifugation at 12000 rpm (Beckman Coulter Avanti J-26S XP, JA-25.50 rotor) for 30 min, followed by filtration through a 0.45 μm filter twice. The lysate was loaded onto a 5 mL Ni 2+ -NTA agarose column (HisTrap TM HP, column volume 5 mL). The column was washed with 50 mL Buffer A with 25 mM imidazole and 1 mM DTT and the protein was eluted with 10 mL Buffer A with 250 mM imidazole and 1 mM DTT. The purified proteins were dialyzed against 2 L Buffer A with 1 mM DTT twice for at least 6 h at 4 ºC. The CarH-RGD tetramer complex was assembled upon addition of a four-fold molar excess of AdoB12 to the monomer solution in the dark and incubation at 4 °C overnight. The obtained tetramer was further purified utilizing a size exclusion column (GE Healthcare, HiLoad 16/600, Superdex 200 pg) with Buffer A ( Figure S3). The photoresponse of the CarH-RGD tetramer upon green light illumination (green LED, 515 nm -520 nm) was followed with UV-Vis spectroscopy (Tecan infinite M200, Tecan, Männedorf, Switzerland) over 1 min ( Figure S4).

Preparation of protein functionalized glass surface:
Glass slides (20 × 20 mm) were cleaned with freshly prepared Piranha solution (3:1 (v/v) concentrated H2SO4:H2O2 (30%)) for 1 h, rinsed 3 times with Milli-Q water and dried in an N2 stream. For the PEGylation reaction, surfaces were immersed in a solution of PEG3000-azide (10 mg PEG3000-azide, MW = 3500 g/mol) and 200 μl dry triethylamine in dry toluene and kept at 80 ºC overnight under a N2 atmosphere. The surfaces were first washed with ethyl acetate for 5 min by sonication, then with methanol for 5 min by sonication and dried in a N2 stream. Each PEG-azide coated surface was incubated with 100 μl of reaction solution containing 100 mM L-ascorbic acid, 100 mM Tris HCl (pH 9.0), 150 μM of NTA-alkyne, (0.3 μM of cRGD-alkyne was added additionally for the GREEN-ON design) and 1 mM CuSO4 in a moisture chamber for 2 h. The surfaces were incubated with the following solutions to obtain PEG-Ni 2+ -NTA functionalities on the surfaces: (1) 50 mM EDTA (pH 7.4) for 5 min; (2) Buffer A (50 mM Tris-HCl, 300 mM NaCl, pH 7.4) twice for 5 min; (3) 0.1 M NiCl2 in water for 5 min; (4) Buffer A twice for 5 min.
Afterward the surfaces were incubated first with 5 μM purified protein for 30 min and the washed with Buffer A for 10 min. Surfaces for the GREEN-ON and GREEN-OFF designs CarH and CarH-RGD were used as proteins, respectively.

Quantification of cell adhesion: One set of surfaces functionalized with CarH and cRDG
(GREEN-ON) or CarH-RGD (GREEN-OFF) was illuminated with green light for 5 min (15 W), the other set was kept in the dark. A PEG-Ni 2+ -NTA functionalized surface without protein functionalization was used as a negative control. The surfaces were placed into the 6-well cell culture plates and were washed with phosphate buffered saline 1x (PBS). Subsequently, 2 x 10 5 MCF-7 cells were seeded per well in 2 ml Dulbecco's modified Eagle's medium (DMEM) (Gibco BRL) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Sigma Aldrich) and 1% penicillin/streptomycin (P/S) (Gibco BRL) and incubated at 37 ºC, 5% CO2 in the dark. For the GREEN-ON and the GREEN-OFF design the cells were incubated on the surfaces overnight and for 2 hours, respectively. Subsequently, the surfaces were washed twice with PBS and the cells were fixed with 2% paraformaldehyde at room temperature for 20 min. After fixation, the surfaces were washed twice with PBS, permeabilized with 0.1% v/v Triton X-100 in PBS at room temperature for 5 min and stained with 0.5 μg/mL phalloidin-TRITC in PBS at room temperature for 30 min for actin staining. The cells were then mounted with Moviol-488 containing 1 μg/ml DAPI (Sigma Aldrich) for nucleus staining. Fluorescent images were acquired with an inverted fluorescence microscope (DMi8, Leica) in the DAPI and TRITC channel with a 10x objective for an area of 5 mm 2 per sample. The number of cells was quantified using the DAPI staining and the cell spreading area was the analyzed based on the actin staining (100 single cells were analyzed per condition) using Fiji ImageJ. Each experiment was performed in biological triplicates. In the box plots, the box represents the 25-75% percentile and the whiskers are the 10-90% percentiles. Statistical significance is evaluated using the One Way ANOVA test. p-value *** <0.001.

Metabolic Activity Test:
The influence of light on the metabolic activity of cells was determined with the 3-(4,5-Dimethylthiozol-2-yl)-2,5-diphenaltetrazolium bromide (MTT) assay. Therefore, MCF-7 cells were seeded in a density of 50000 cells/cm 2 in a 96 well plate in 100 µL complete growth medium (DMEM + 10% FBS + 1% P/S). The cells were incubated at 37 °C exposed to green light (532 nm, Flora LED Module) at different intensities for 2 h. The control was kept meanwhile in the dark. Afterwards 75 µL medium per well were removed and 10 µL MTT (saturated solution in PBS) was added. The cells were incubated at 37 °C in dark for 4 h. 50 µL DMSO per well was added and incubated at room temperature in dark for 10 min. The absorption at 540 nm was measured with a Tecan plate reader. The lowest tested light intensity at 12.5 μM/m 2 s was used in all cell experiments, to assure no influence of the cells.

Cell spatial and temporal control of cell adhesions: To pattern cells on GREEN-ON surfaces,
surfaces modified with CarH and cRGD-alkyne peptide were illuminated in a desired pattern using the FRAP function with green light at 552 nm (argon laser, 10% intensity) for 1 min under a confocal laser scanning microscopy (Leica TCS SP8) through a 10x objective. Then, the surfaces were incubated with 1×10 6 MCF-7 cells in the 2 ml DMEM supplemented with 10% FBS and 1% P/S and 300 nM SiR-actin for actin staining at 37 ºC, 5% CO2 overnight.
Subsequently, 1 μg/ml Hoechest 33342 was added to stain the cell nuclei for 10 min and surfaces were washed with PBS before acquiring images of the cells on the surfaces in the actin and nuclear stain channels.       were first acquired in the dark (t = 0 min) and the cell spreading was monitored over time after