Dynamic in Situ Confinement Triggers Ligand-Free Neuropeptide Receptor Signaling

Abstract

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.

Keywords: G protein-coupled receptors; membrane organization; phase separation; receptor condensates; receptor dynamics.

Figure 1

In situ ligand-free receptor confinement. (a) Rationale of the experimental design for ligand-free receptor clustering. Matrices prestructured with BSA are stepwise functionalized with biotin-BSA and SA. Upon the addition of the multivalent nanotool trisNTA^PEG12-B, His₆-tagged receptors in HeLa cells are captured in the prestructured regions via multivalent His-tag/trisNTA interaction. (b) Chemical structure of the trisNTA^PEG12-B. (c) Protein patterns of variable size enerated by functionalization of SA matrices with the nanotool followed by incubation with His₆-GFP (0.1 μM, 20 min). Images were acquired by confocal laser scanning microscopy (CLSM). (d) Intensity profile of the 1 μm pattern (white line in c) reflects high specificity of the interaction. (e) Large-scale cell patterning in living cells occurred 10 min after incubation with the nanotool (trisNTA^PEG12-B 100 nM final, 10 min). Y₂R-expressing cells were allowed to adhere to the functionalized matrix for 3 h and immediately imaged by CLSM in live-cell imaging solution (LCIS) at 37 °C. (f) Customized Y₂R assembly on 3 and 1 μm SA-prestructured matrices. (g) Intensity profile of the 1 μm pattern (white line in f) showed an intensity comparable to that of a soluble His₆-tagged protein. Scale bars: 10 μm.

Figure 2

Decrease of receptor mobility in confined regions. (a) FRAP analyses upon Y₂R clustering induced either by the nanotool in situ or by an anti-His₆ antibody (αHis₆ Ab). Y₂R-expressing cells were allowed to adhere to SA- or -αHis₆ Ab matrices for 3 h and immediately imaged by CLSM in LCIS at 37 °C. The trisNTA^PEG12-B nanotool was added to a final concentration of 100 nM. Insets represent the bleached ROIs. Fast recovery of the clusters can be detected in the case of the multivalent nanotool. (b) Quantification of the receptor mobile fraction for cell patterning by the trisNTA^PEG12-B and anti-His₆ antibody demonstrated an unchanged receptor mobile fraction for the nanotool, suggesting a high receptor exchange. The mean ± SD is shown. Nine cells before, 11 cells after trisNTA^PEG12-B addition (45 × 1 μm diameter ROIs), and five cells on anti-His₆ antibody matrices (13 × 1 μm diameter ROIs) were analyzed. ***p ≤ 0.001 for Tukey test. (c) imFCS correlates fluorescence intensity fluctuations in single camera pixels, providing diffusion coefficients with high spatial and temporal resolution. (d) Widefield image of an ROI at the plasma membrane of a living cell upon addition of the nanotool analyzed by imFCS (left). The analyses of numerous pixels simultaneously provide two-dimensional diffusion data that draw a picture of the mobility of membrane receptors and reveal local differences in the diffusion (right). (e) Both techniques demonstrated a decrease in the lateral diffusion of the receptor at the plasma membrane after addition of the chelator nanotool. Analysis of 1 μm clusters within the entire ROI led to a further decrease in the lateral diffusion coefficient. For imFCS analyses, two-sample t tests (α = 0.05) were applied to compare the diffusion coefficients for the different conditions. The mean ± SD is shown. 36 and 24 cells for the conditions before and after addition of trisNTA^PEG12-B were analyzed. For FRAP, the mean ± SD is shown. Nine cells before and 11 cells after trisNTA^PEG12-B addition (41 × 1 μm diameter ROIs) were analyzed. ***p ≤ 0.001 for Tukey test. Scale bar: 10 μm (a), 1 μm (d).

Figure 3

In situ receptor clustering with high spatiotemporal resolution. (a) Time-lapse imaging of Y₂R assembly. Y₂R-expressing HeLa cells were allowed to adhere to prestructured SA matrices for 3 h and were visualized by CLSM in LCIS at 37 °C. Time-lapse images were recorded for 20 min immediately after the addition of trisNTA^PEG12-B (100 nM). Scale bar: 20 μm. (b) Receptor-integrated density in the patterned regions increased monoexponentially, leading to an assembly rate of 0.35 ± 0.05 min^–1 and τ_1/2 = 3 min (50–200 × 1 μm ROIs per experiment were analyzed from a total of 30 cells from three independent experiments, 10 cells per experiment). (c) Reversal of the interaction and disassembly of the clusters is demonstrated upon the addition of histidine. Y₂R-expressing cells were allowed to adhere to the SA matrices for 3 h, and then receptor confinement was induced by the addition of trisNTA^PEG12-B (100 nM). Subsequently, cells were incubated with histidine (5 mM) for 2 to 10 min followed by washing. Scale bar: 10 μm.

Figure 4

Receptor clustering amplifies the cell response induced by ligand activation. (a) Confocal microscopy images of cells expressing Y₂R exposed to different conditions. Y₂R-expressing HeLa cells were allowed to adhere to prestructured SA matrices for 3 h and visualized by CLSM in LCIS at 37 °C. Cells were visualized and imaged for 20 min after the addition of trisNTA^PEG12-B or pNPY or both, first trisNTA^PEG12-B and subsequently pNPY (20 min incubation time, each). Scale bar: 20 μm. (b) Cell area analysis before and 20 min after the addition of pNPY (10 nM) showed a 20% area increase, confirming an effect of ligand activation on cell motility. Values for cell area were normalized with respect to the highest value. The mean ± SD (13 cells) is shown. **p ≤ 0.01 for Tukey test. (c) Cell area analysis before and 20 min after the addition of trisNTA^PEG12-B (100 nM) and subsequent addition of pNPY (1, 5, and 10 nM, one well for each concentration) showed a dose-dependent area increase, demonstrating an amplification effect of receptor clustering in combination with pNPY. Cell area values were normalized with respect to the highest value. The mean ± SD (42 cells before, 21 cells after trisNTA^PEG12-B and 14, 7, 19 for pNPY 1, 5, and 10 nM, respectively) is shown. **p ≤ 0.01 and ***p ≤ 0.001 for the Tukey test. (d) Quantification of receptor intensity in the nanotool-induced patterned regions showed a significant increase in pattern intensity after the addition of pNPY (10 nM), the concentration that had the largest effect on cell motility. The mean ± SD is shown (19–39 cells and 50–220 × 1 μm ROI, were analyzed). ***p ≤ 0.001 for the Tukey test.

Figure 5

Arrestin-3 recruitment upon ligand-induced receptor activation. (a) Schematic representation of the experimental setup. Cells coexpressing Y₂R and Arr3 were allowed to adhere to SA-prestructured matrices for 3 h and visualized by TIRF microscopy in LCIS at 37 °C. (b) Representative TIRF images of cells before and upon addition of trisNTA^PEG12-B (100 nM, 30 min) and subsequent incubation with pNPY (10 nM) and histidine (5 mM) in LCIS for 30 min at 37 °C. All concentrations mentioned are final concentrations in the wells. Scale bar: 5 μm. (c) Quantification of the fluorescence contrast in the patterned regions for Y₂R confirmed receptor enrichment upon the addition of trisNTA^PEG12-B (2-fold with respect to the basal signal before, 100 nM, 30 min), which further increased 4-fold upon the addition of pNPY (10 nM, 30 min). Histidine addition led to a decrease in the signal (1.7-fold decrease compared to pNPY, 5 mM, 30 min). Data were normalized with respect to the fluorescence intensity before clustering and are displayed as the means ± SEM (60 cells for each condition were analyzed). Tukey’s multiple comparison test was applied (***p ≤ 0.001). (d) Fluorescence contrast analysis demonstrated no significant recruitment of Arr3 upon trisNTA^PEG12-B (1.4-fold with respect to the basal signal before, 100 nM, 30 min). Addition of pNPY increased the Arr3 signal (3.6-fold, 10 nM, 30 min), confirming copatterning of the downstream signaling molecules. Subsequent addition of histidine led to a decrease in the signal (2.3-fold, 5 mM, 30 min). Data were normalized with respect to the fluorescence intensity before clustering, and it is expressed as the means ± SEM (60 cells for each condition were analyzed). Tukey’s multiple comparison test was applied (***p ≤ 0.001).