Minimum degree of overlap between IL‐9R and IL‐2R on human T lymphoma cells: A quantitative CLSM and FRET analysis

Abstract

The heterodimeric receptor complex of IL-9 consists of the cytokine-specific α-subunit and the common γ c -chain shared with other cytokines, including IL-2, a central regulator of T cell function. We have shown previously the bipartite spatial relationship of IL-9 and IL-2 receptors at the surface of human T lymphoma cells: in addition to common clusters, expression of the two receptor kinds could also be observed in segregated membrane areas. Here we analyzed further the mutual cell surface organization of IL-9 and IL-2 receptors. Complementing Pearson correlation data with co-occurrence analysis of confocal microscopic images revealed that a minimum degree of IL-9R/IL-2R co-localization exists at the cell surface regardless of the overall spatial correlation of the two receptor kinds. Moreover, our FRET experiments demonstrated molecular scale assemblies of the elements of the IL-9/IL-2R system. Binding of IL-9 altered the structure and/or composition of these clusters. It is hypothesized, that by sequestering receptor subunits in common membrane areas, the overlapping domains of IL-9R and IL-2R provide a platform enabling both the formation of the appropriate receptor complex as well as subunit sharing between related cytokines.

IL-2 receptors preassemble and signal in the ER/Golgi causing resistance to antiproliferative anti–IL-2Rα therapies

“…FRET microscopy is a well-established method to report on molecular interactions (39)(40)(41). Earlier, we used this technique to study the interaction between IL-2/9/15 receptor subunits (31,36,37,42,43) or c-Fos and c-Jun transcription factors (44)(45)(46). Here, we used fluorescent ER and Golgi markers, allowing us to study protein-protein interactions in living cells in an organelle-specific manner.…”

Thermogenic Activation Downregulates High Mitophagy Rate in Human Masked and Mature Beige Adipocytes

“…Images were taken in sequential mode to minimize cross-talk between the channels. Images of approximately 1 μm thick optical sections, each containing 512 × 512 pixels (pixel size was ~200 nm), were obtained with a 60× UPLSAPO oil immersion objective (NA 1.35) [ 67 ]. In the case of SGBS cells, the images were acquired on a Nikon A1 Eclipse Ti2 confocal laser-scanning microscope by using a Plan Apo 60× water (numerical aperture (NA) 1.27) objective, with a pixel size of 210 nm.…”

I Am the Alpha and the …Gamma, and the G. Calibration of Intensity‐Based FRET Measurements

“…Its sensitivity to molecular interactions and conformations stems from the fact that its efficiency steeply decreases with the donor-acceptor separation in the 2-10 nm range. Although it is already valuable as a standalone approach, its usefulness can be further increased by combining with other modalities, like confocal microscopy (2). Its flexibility and versatility are enhanced by the generation of sensors, that is, donor-acceptor fluorescence protein constructs connected by a linker, whose conformation responds to certain properties of the environment.…”