Dynamic in Situ Confinement Triggers Ligand-Free Neuropeptide Receptor Signaling

Membrane receptor clustering is fundamental to cell–cell communication; however, the physiological function of receptor clustering in cell signaling remains enigmatic. Here, we developed a dynamic platform to induce cluster formation of neuropeptide Y2 hormone receptors (Y2R) in situ by a chelator nanotool. The multivalent interaction enabled a dynamic exchange of histidine-tagged Y2R within the clusters. Fast Y2R enrichment in clustered areas triggered ligand-independent signaling as determined by an increase in cytosolic calcium and cell migration. Notably, the calcium and motility response to ligand-induced activation was amplified in preclustered cells, suggesting a key role of receptor clustering in sensitizing the dose response to lower ligand concentrations. Ligand-independent versus ligand-induced signaling differed in the binding of arrestin-3 as a downstream effector, which was recruited to the clusters only in the presence of the ligand. This approach allows in situ receptor clustering, raising the possibility to explore different receptor activation modalities.

homogeneous pressure onto a clean epoxy-coated glass substrate (Schott Nexterion Slide E) and incubated overnight at 4 °C. The next day, the stamp was stripped from the glass with a forceps, and the microstructured glass was bonded to a 96-well plastic casting using an adhesive tape (3M) and closed with an appropriate lid. Cell culture. HeLa Flp-In TM T-Rex TM Y2R cells (His6-Y2R mEGFP or Y2R mEGFP ) were generated and cultured at 37 °C, 5% CO2, and 95% humidity 3 . For culturing the stable cell line, high glucose Dulbecco's modified Eagle's medium (DMEM) (Gibco/Thermo Fisher Scientific) was supplemented with 10% tetracycline-free fetal calf serum (FCS, Bio&Sell), blasticidin S HCl (1 µg/ml, Thermo Fisher Scientific), and hygromycin B (50 µg/ml, Thermo Fisher Scientific).
To induce receptor expression the cell medium was replaced with fresh medium containing tetracycline (0.1 µg/ml, Fluka) 18 h before imaging. The same concentration of tetracycline resulting in an efficient plasma membrane targeting was used for all the experiments. The cells were regularly tested for mycoplasma contamination.
Receptor confinement in real-time by trisNTA PEG12-B . Cells expressing Y2R (His6-Y2R mEGFP or Y2R mEGFP ) were trypsinized and allowed to adhere to SA pre-structured matrices for 3 h or overnight. 15-18 h prior to the experiment, the cell medium was replaced with fresh medium containing tetracycline (0.1 µg/ml) to induce receptor expression. The cells were visualized by CLSM in live-cell imaging solution (LCIS, Thermo Fisher Scientific) at 37 °C. Cells were subsequently incubated with nickel-loaded trisNTA PEG12-B (final concentration 100 nM) in LCIS for 10-15 min at 37 °C. For reversibility experiments, micropatterned cells were incubated with histidine (5 mM) in LCIS for 2 to 10 min followed by washing with LCIS. To avoid photo-bleaching in our recordings, we recorded an image every minute after adding the nanotool. We applied control of the focus by the 'definite focus' tool, which allowed to always image the same z-position by re-focusing before taking every image. We generated a monoclonal HeLa cell line, which was established for low expression of the Y2 receptor. This led to a more homogeneous cell population with respect to receptor expression. Slight cell-to-cell variations were still observed. Experiments were performed in biological replicas (N=4).

Receptor confinement on antibody-micropatterned matrices.
Wells pre-structured with BSA were subsequently incubated with biotin-BSA (0.1 mg/ml), SA (1 µM), and a biotinylated anti-His6 antibody (1 µM) (ab106261, Abcam) in PBS for 1 h at RT. Wells were washed thoroughly with PBS to remove unbound antibody. Cells expressing Y2R were trypsinized and seeded onto the antibody patterns. After 3 h, cells were visualized by CLSM in LCIS at 37 °C. Experiments were performed in biological replicas (N=5).
Time-lapse calcium imaging. 18 h after seeding the cells onto pre-structured SA-matrices, cells were incubated with BioTracker 609 Red Ca 2+ AM dye (3 µM, Merck Millipore) in fresh medium for 30 min. The cell-membrane permeable dye is de-esterified by cellular esterases and remains trapped in the cytosol. After incubation with the Ca 2+ dye, cells were rinsed three times with PBS and imaged by CLSM in LCIS at 37 °C. For investigation of Ca 2+ signal, time-lapse images were taken (5 slices z-stacks, 45-s interval) before and after addition of trisNTA PEG12-B . Fluorescence intensity (lex/em 590/609 nm) of the dye changes depending on the intracellular Ca 2+ concentration.
Maximum intensity projections of single channels were analyzed. The ImageJ ROI tool was used to define the areas of the image to be analyzed. We consider a ROI covering the complete cell contour. Mean gray values (F) were background subtracted and normalized to the fluorescence in cells before F0. Experiments were performed in biological replicas (N=3).
Plasma membrane staining. Live-cell membrane staining was performed directly after receptor assembly in living cells grown on pre-structured matrices. CellMask TM deep red plasma membrane stain (Thermo Fisher Scientific) was used according to manufacturer's instruction. 1 µl of the stock solution (1000x dilution) was dissolved in 1 ml of warm LCIS (final concentration 5 µg/ml) and subsequently added to the cells, incubated for 5 min at 37 °C, and washed with LCIS before visualization. Experiments were performed in biological replicas (N=3).  Image analysis. Fluorescence images were processed with Zen blue, ImageJ, and Fiji software. 4,5 All images were background subtracted. Integrated density, mean gray value and cell area were obtained with ImageJ. Data were plotted with OriginPro. were bleached within 10 s with high laser intensities. Fluorescence recovery was monitored by repetitively imaging an area containing the photobleached region at 0.1 frame/s for ∼150 s. For the analysis, a simulation approach that allows computation of diffusion coefficients regardless of bleaching geometry used in the FRAP series was applied. 6 The method is based on fitting a computer-simulated recovery to actual recovery data of a FRAP series. The algorithm accepts a multiple-frame TIFF file, representing the experiment as input, and simulates the diffusion of the fluorescent probes (2D random walk) starting with the first post-bleach frame of the actual data.
Once the simulated recovery is finished, the algorithm fits the simulated data to the real one and extracts the diffusion coefficient. The algorithm iteratively creates a series of simulated images, where each frame corresponds to a single iteration. The intensity values are extracted from the (user indicated) bleached area of the simulated frames, thus determining the general shape of the recovery curve. The "time" axis at this stage is in arbitrary units (iterations). To extract the diffusion coefficient, the simulated recovery curve needs to be fitted to the real recovery curve, by appropriately stretching the "time" axis. The time between frames in the actual data set is obviously known, thus once overlapping optimally the simulated curve with the real one, the duration of one iteration, in real-time units, is determined. The diffusion coefficient of the simulated series is then calculated according to eq. 1, where ! is the simulation-extracted diffusion coefficient, is the step of a molecule in each iteration of the simulation, corresponding to one pixel in the image (the pixel size is calibrated previously, by imaging a known calibration sample), and " is the time interval between steps (determined as explained). (1) The simulation proceeds until a plateau is reached (equilibration of the fluorescence intensity in the bleached area). The number of data points in the simulated recovery is typically different (larger) than the number of experimental points. In addition, the real experimental data may not have been acquired until equilibration of fluorescence. To determine " , the algorithm scans a range of possible values for the total duration represented by the simulation and calculates a value # for the goodness-of-fit between the simulated data and the real FRAP data. Total simulation duration is selected as the one that produces the minimal # . Experiments were performed in biological replicas (N=3). applied to compare the diffusion coefficients for the different conditions. All datasets were tested for normality using the Kolmogorov-Smirnov test (α = 0.05). Significance was assigned as follows: p > 0.05 no significant difference between populations (n.s.), p < 0.05 significant difference (*), p < 0.01 significant difference (**), and p < 0.001 significant difference (***). Two-dimensional maps of diffusion coefficients were generated also in OriginPro. Diffusion coefficients were color-coded from light yellow to dark red in the range of 0 to 0.5 µm²/s. Pixels that yielded correlation curves with diffusion coefficients higher than 0.5 µm²/s are presented in black. Pixels that yielded

Contrast quantification and statistical analyses.
Contrast analysis was performed as described previously. 2,11,12 Initial imaging recording was supported by the Nikon NIS Elements software. Images were exported as TIFF frames and fluorescence contrast analysis was performed using the Spotty framework. 13 The fluorescence contrast <c> was calculated as <c> = (F + -F − )/(F + -Fbg), where F + denotes the intensity of the inner pixels of the pattern. F − shows the intensity of the surrounding pixels of the micropattern, and Fbg the intensity of the global background. Data are expressed as the means ± SEM. Comparisons of more than two different groups were performed using one-way ANOVA, which was followed by Tukey's multiple comparisons test in GraphPad Prism software (version 9.1.2).

Supplementary Text 1
NPY ligand plays an important role in the nervous, immune, and endocrine systems [14][15][16] and can affect the proliferation, apoptosis, differentiation, and migration of different cell types 17 . NPY is a potent angiogenic factor in vivo and the NPY-induced angiogenic response is mediated by the Y2 receptor. In knock-out mice lacking the Y2 receptor, skin wound repair was significantly delayed. Thus, NPY may play an important role in regulation of angiogenesis and angiogenesisdependent physiological and pathological processes 18 .
Further, NPY has recently been found to play a role in the progression of diverse types of cancer and diseases such as brain cancer, bone cancer, breast cancer and osteoporosis 19,20 . A rapid increase in bone mass was observed in adult mice after central Y2R deletion suggesting that Y2R may represent a promising new target for the prevention and treatment of osteoporosis.
Recently, NPY or analogous small peptide agonists were tested as potential new strategies for the diagnosis or treatment of breast cancer or osteoporosis. However, these applications remained primarily in the research phase of animal testing 20 . In addition, an antinociceptive effect of spinally administrated NPY was recently observed in a cancer-induced bone pain model, mediated through both Y1R and Y2R, suggesting also that NPY might be a promising target for the development of future treatments for cancer-induced bone pain 21 .

Supplementary Text 2
To corroborate the specificity of the interaction, clustering was evaluated in cells expressing Y2Rs without the His6-tag (Y2R mEGFP ) cultured on SA-matrices. Cells showed no receptor clustering upon addition of trisNTA PEG12-B (Figure S2), demonstrating the specificity of the His6tag/trisNTA interaction.
With the aim to compare the nanotool-induced clustering with approaches using patterned antibodies, we utilized an anti-His6 antibody. Ten minutes after receptor clustering by the multivalent nanotool, the Y2R enrichment resulted in an integrated receptor density equivalent to that of cells cultured on matrices functionalized with anti-His6 antibodies. However, a 10-fold higher antibody concentration (1 µM final) was required compared to the multivalent nanotool, demonstrating its efficacy in capturing His6-tagged Y2 receptors ( Figure S3). In addition, the nanotool-induced 3 µm clusters presented a 9-fold increase in integrated density compared to 1 µm arrays, consistent with the increase in pattern area.

Video 1
Time-lapse after photobleaching of a region of interest containing Y2R clusters induced by the nanotool. A fast recovery is observed within the first minutes indicating a high dynamic behavior for these clusters.

Video 2
Time-lapse after photobleaching of a region of interest containing Y2R clusters induced by an anti-His6 tag antibody immobilized on the pre-structured matrices. There is no recovery of the clusters indicating a high degree of immobilization.

Video 3
Time-lapse immediately after the addition of the nanotool to Y2R expressing cells over SA prestructured matrices show fast enrichment of the receptors in real-time.