Ratiometric Tension Probes for Mapping Receptor Forces and Clustering at Intermembrane Junctions

Abstract

Short-range communication between cells is required for the survival of multicellular organisms. One mechanism of chemical signaling between adjacent cells employs surface displayed ligands and receptors that only bind when two cells make physical contact. Ligand-receptor complexes that form at the cell-cell junction and physically bridge two cells likely experience mechanical forces. A fundamental challenge in this area pertains to mapping the mechanical forces experienced by ligand-receptor complexes within such a fluid intermembrane junction. Herein, we describe the development of ratiometric tension probes for direct imaging of receptor tension, clustering, and lateral transport within a model cell-cell junction. These probes employ two fluorescent reporters that quantify both the ligand density and the ligand tension and thus generate a tension signal independent of clustering. As a proof-of-concept, we applied the ratiometric tension probes to map the forces experienced by the T-cell receptor (TCR) during activation and showed the first direct evidence that the TCR-ligand complex experiences sustained pN forces within a fluid membrane junction. We envision that the ratiometric tension probes will be broadly useful for investigating mechanotransduction in juxtacrine signaling pathways.

Keywords: Receptor clustering; T-cell; artificial antigen presenting cell; immunological synapse; intermembrane junction; molecular tension probe.

Figure 1.

A) Illustration of the contact zone between a biotinylated microparticle and a DNA tension probe surface anchored to a supported lipid membrane. B) Representative brightfield, RICM, Cy3B, A488, and tension density images showing the contact zone of a microparticle (diameter = 5 μm) that binds to SLB tension probes (F_1/2 = 4.7 pN). Scale bar = 5 μm. C) Plot displaying line scan of Cy3B and A488 channels for the microparticle shown in B. The intensity is normalized (I) to the background regions lacking the microparticle (I₀). D) Plot overlaying the line scan profile of fluorescence and RICM channels, and demonstrating their spatial colocalization. E) Bar graph showing the tension density of microparticles engaged to tension probe SLBs (F_1/2 = 4.7 pN) and control SLB surfaces decorated using DNA duplexes (n = 20 for each sample, error bar represents S.D. of the data) within the microparticle-SLB contact zone. F) Representative FRAP images showing recovery after 90 s. Scale bars = 10 μm. G) Representative FRAP recovery plots for Cy3B and A488 channels. Solid lines represent the fit made using the following equation I(t) = A(1−e^-tτ). The Lateral diffusion coefficient (D) is calculated by: D = w²/4t_1/2, where w is the radius of the Gaussian bleaching area; t_1/2 is the time for 50% recovery obtained from the fit. The values used for the calculation were: w = 10.4 μm (for both channels); t_1/2 = 26.5 s (Cy3B) and 27.9 s (A488).

Figure 2.

A) Representative time-lapse images (RICM, Cy3B and A488 and tension density) showing the first 15 min of CD4+ T-cell engagement with the CD3-tension probes anchored onto an SLB. B) The kymographs display tension density and the A488 intensity as a function of time within the three regions of interest (lines in the tension density channel from A). Scale bar = 5 μm.

Figure 3.

A) Representative images (RICM, Cy3B, A488 and tension density) of CD4+ T-cells plated on fluid SLBs containing tension probes (upper panel) or control duplexes (lower panel) for a duration of 30 min. B) Scatter plot showing the mean tension density signal generated on tension probes and control duplexes within the cSMAC structure (n = 25 cells). C) Representative images (RICM, Cy3B, A488 and tension density) of CD4+ T-cells plated on the hindered SLB displaying tension probes. The mobility of tension probes was limited due to the high density of streptavidin on the SLB. D) Line profile across the cell (dashed line in 3C) showing differential response in the Cy3B and A488 channels. E) Representative images (RICM, Cy3B, A488 tension density, and zoom in) of T-cells pre-treated with 50 μM blebbistatin (bleb) or without blebbistatin (control) and plated onto the 4.7 pN tension probe surface for a duration of 30 min. Scale bars = 5 μm.

Scheme 1.

Schematic representation of gold nanoparticle-based ratiometric tension probes