Optogenetic Tuning of Protein-protein Binding in Bilayers Using LOVTRAP.

Modern microscopy methods are powerful tools for studying live cell signaling and biochemical reactions, enabling us to observe when and where these reactions take place from the level of a cell down to single molecules. With microscopy, each cell or molecule can be observed both before and after a given perturbation, facilitating better inference of cause and effect than is possible with destructive modes of signaling quantitation. As many inputs to cell signaling and biochemical systems originate as protein-protein interactions near the cell membrane, an outstanding challenge lies in controlling the timing, location and the magnitude of protein-protein interactions in these unique environments. Here, we detail our procedure for manipulating such spatial and temporal protein-protein interactions in a closed microscopy system using a LOVTRAP-based light-responsive protein-protein interaction system on a supported lipid bilayer. The system responds in seconds and can pattern details down to the one micron level. We used this technique to unlock fundamental aspects of T cell signaling, and this approach is generalizable to many other cell signaling and biochemical contexts.

how cells sense and respond to their environments, we need new techniques to control stimuli in both space and time.
Cells commonly sense their environment through protein-protein interactions at the cell membrane.
In this protocol, we mimic these cell-presented ligands using a light-gated protein-protein interaction system on a supported lipid bilayer (Figure 1). Light controllable protein-protein interactions are also powerful for studying biochemical reactions that occur close to a membrane, such as the phosphoinositol cycle or Ras signaling (Toettcher et al., 2011 and2013).
Our approach for manipulating signals in bilayers is based on LOVTRAP, a light-induced protein dissociation technique (Wang et al., 2016) that consists of a naturally light-sensitive protein (the LOV2 domain of Avena sativa phototropin I) and an engineered binding partner, Zdk. In the ground state, Zdk binds to LOV2 with high affinity. When LOV2 is excited by blue light, it changes conformation, causing Zdk to dissociate. Active LOV2 slowly relaxes to the ground state, allowing Zdk to rebind and reset the system. By controlling when and where blue light is delivered, micron-sized patterns of a protein-protein interaction can be generated and altered in seconds. Because LOV2 is insensitive to red and infra-red light, this system is compatible with biosensors in these channels for microscopy-based quantification of the perturbation and cell response. protein LOV2 is biochemically purified and attached to a supported lipid bilayer (SLB). Its binding partner, Zdk, binds to LOV2 in the dark and dissociates upon illumination with blue light. Zdk can be free in solution (as diagrammed here) or attached to a cell surface receptor to control cell signaling (as was done in Tischer and Weiner, 2019). In biochemical reconstitution, Zdk can be fused to a protein whose activity depends on membrane proximity, such as a lipid kinase or GTPase regulator. 3 www.bio-protocol.org/e3745 We previously leveraged our optogenetic approach to stimulate the T cell signaling with precise temporal control, revealing that T cells measure the dynamics of ligand binding in their decision to activate (Tischer and Weiner, 2019). Ligands with longer binding half-lives signal disproportionately better than ligands with short binding half-lives, even when controlling for receptor occupancy. Such a conclusion was only possible because the optogenetic system allowed us to directly manipulate the variable of interest: protein-protein interaction half-life. This direct manipulation was not possible with existing experimental techniques. Because of the general nature of protein-protein interactions in initiating cell signaling, we detail our procedure here in the hopes that our approach will enable a powerful interrogation of other signaling systems.  10. Transfer the loaded column to an AKTA and wash with IMAC binding buffer until the A280 of the effluent is less than 60 mAu. Elute the protein with a 10-column volume gradient (going from IMAC binding buffer to IMAC elution buffer) and collect in 1.5 ml fractions in a deep-well 96-well plate. After elution, equilibrate the column in IMAC binding buffer.

Materials and Reagents
11. Pool visibly yellow fractions in the 96-well block and then buffer exchange into IMAC binding buffer using a HiPrep 26/10 desalting column.
Note: TEV protease was purified in house, but can be obtained commercially.
13. Recirculate the digested LOV2 mixture over a HiTrap Chelating column (this can be the one used previously to elute LOV2 after equilibrating with IMAC binding buffer) with a peristaltic pump as before to remove the TEV protease, free His-tags, and any uncut LOV2. Recirculating at least twice over the column.
14. Collect the flow-through and exchange into HEPES buffered saline (HBS) with the desalting column.
15. Concentrate to approximately 4 mg/ml with a Vivaspin protein concentrator. 9. Add the following components in order to the Coplin jar: 38 ml warmed ddH2O, 9.5 ml 37% HCl and 12.6 ml 30% hydrogen peroxide.
10. Incubate in the water bath for 12 min as before. The solution will bubble similarly to the base solution, but slightly less strongly.
11. Remove the Coplin jar from the water bath, decant the acid solution, and wash 5 times with the warmed ddH2O. Cover the coverslips with warmed ddH2O, add the Coplin jar lid, and store at room temperature for up to one week.

H. Forming SLBs
While drying and preparing the glass coverslips, take care to only handle the edges and avoid touching any part near the center.
1. Use forceps to remove a glass coverslip from the ddH2O in the Coplin jar. Hold it in one gloved hand by pinching between the two long sides and immediately blow dry with compressed nitrogen (or compressed air or other inert gas). The reflection off the coverslip should be spotless.
2. Stack a sheet of microscopy lens paper on top of two paper towels and place the coverslip on top.
3. Firmly press a six-well Ibidi sticky chamber onto the coverslip. Use very firm pressure to ensure contact, especially between the channels and between the ends of the channels and the sides of the coverslip.  Antifade Reagent 1:100 into the solution in which microscopy will be performed and let it incubate at room temp for at least 90 min for the dissolved oxygen to be depleted. It is useful to begin the oxygen scavenger treatment before functionalizing the SLBs so that the solution is ready by the time the SLBs are fully functionalized.

J. Microscope
A microscope capable of TIRF (total internal reflection fluorescence) microscopy is necessary for quantifying Zdk recruitment and release from a LOV2-functionalized bilayer and is a natural choice for imaging a resulting change in cell signaling or of a biochemical reaction.
We imaged using an Eclipse Ti inverted microscope (Nikon) with two tiers of dichroic turrets to allow simultaneous fluorescence imaging and optogenetic stimulation. The microscope was also equipped with a motorized laser TIRF illumination unit, a 60x Apochromat TIRF 1.49 NA objective Before performing experiments, it is important to confirm that your bilayers are functionalized with LOV2 and fluid. This is easily done with a fluorescence recovery after photobleaching (FRAP) experiment. One can use almost any fluorescent imaging modality (e.g., TIRF, confocal, wide field) to check that the bilayer is functionalized with LOV2.
1. Load an SLB functionalized with a saturating amount of fluorescently labeled LOV2 onto the microscope and use a 60x or 100x objective to focus at the glass-water interface. It may be useful to dope in a small concentration of fluorescent beads to make locating this interface easier.
2. Close down the episcopic field diaphragm so that only part of the field of view is illuminated.
There should be an obvious increase in fluorescence inside the illuminated region if the bilayer is functionalized with fluorescently labeled LOV2.

3.
A qualitative FRAP measurement is usually all that is needed to assess bilayer fluidity. While it is useful to have a proper FRAP system or high power TIRF laser, it is sometimes possible to use a lower-powered light source (such as a wide field epifluorescence light source) if neither is available. In such a case, it is useful to label the LOV2 with a fluorophore like FITC (fluorescein isothiocyanate) that is easy to bleach. Not treating the imaging buffer with an oxygen scavenger also makes fluorophores easier to bleach. 4. Bleach a small region of the field of view with the method of your choice. If you are not using a proper FRAP system (e.g., using a TIRF laser or wide field epifluorescence light source instead), close down the episcopic field diaphragm so only a small portion of the field of view is illuminated. 14 www.bio-protocol.org/e3745 If using a TIRF laser, point it straight up. For non-FRAP systems, the power will likely need to be increased to its maximum. The time it takes to bleach the area of interest will vary depending on the intensity of the light source, the small molecule fluorophore attached to LOV2, and the imaging media, among other factors. Bleaching times longer than 30 seconds are generally not effective, as diffusion in the bilayer (if it is fluid) will equilibrate LOV2 between the illuminated and non-illuminated regions.

L. Testing LOV2 functionality
The most direct way to test LOV2 functionality on the bilayer is to use TIRF to measure the recruitment and release of fluorescently labeled Zdk. This test should be performed after you have confirmed the bilayers are functionalized with LOV2 and fluid.
1. Functionalize an SLB with saturating amounts of LOV2 and flow in imaging media containing 250 nM Zdk that has been treated with the oxygen scavenger. It is best to use the same media you intend to perform your experiments in, but DPBS can serve as a good starting point. Make sure the Zdk is labeled with a dye that is excited by light with a wavelength longer than 560 nm so that you can continuously image it without activating LOV2.
2. Set up a 10 min time course where Zdk is imaged with TIRF every 5 s. Every 2 min, expose the LOV2 to blue light of around 470 nm for 1 s. Any sort of GFP imaging setting usually works well.
Depending on the intensity of blue light, Zdk should be rapidly released from the bilayer in ~1-10 s and then slowly recover over the following 2 min. Similar cycles of release and recruitment should be observed over the next 10 min (Figure 4). If more than a 50% reduction in Zdk recruitment occurs over 10 min, the most likely reason is that the oxygen scavenger treatment was not effective. For protocols using Zdk on a cell surface receptor to initiate signaling, we refer the interested reader to our previous publication (Tischer and Weiner, 2019). For our previous work with T cell signaling, we were able to make low-dozens of single cell measurements from a single field of view.
With three biological replicates, we recorded on the order of ~50-100 single cell measurements per condition, which provided sufficient numbers for statistical significance in our experiments.