Dynamic Submicroscopic Signaling Zones Revealed by Pair Correlation Tracking and Localization Microscopy

Abstract

Unraveling the spatiotemporal organization of signaling complexes within the context of plasma membrane nanodomains has remained a highly challenging task. Here, we have applied super-resolution image correlation based on tracking and localization microscopy (TALM) for probing transient confinement as well as ligand binding and intracellular effector recruitment of the type I interferon (IFN) receptor in the plasma membrane of live cells. Ligand and receptor were labeled with monofunctional quantum dots, thus allowing long-term tracking with very high spatial and temporal resolution without an artificial receptor cross-linking at the cell surface. Dual-color TALM was employed for visualizing protein-protein interactions involved in IFN signaling at both sides of the plasma membrane with high spatial and temporal resolution. By pair correlation analyses based on time-lapse TALM images (pcTALM), complex assembly within dynamic submicroscopic zones was identified. Strikingly, recruitment of the IFN effector protein signal transducer and activator of transcription 2 (STAT2) into these dynamic signaling zones could be observed. The results suggest that confined diffusion zones in the plasma membrane are employed as transient platforms for the assembly of signaling complexes.

Receptor dimer stabilization by hierarchical plasma membrane microcompartments regulates cytokine signaling

“…ISGF3 is formed upon phosphorylation of STAT1 and STAT2 by the IFN signaling complex, resulting in the nuclear translocation of these proteins ( 39 ). Functional studies were performed in HeLa cells, in which IFN downstream signaling is more robust than in the complemented U5A cell line, whereas both cell lines display similar receptor density, diffusion, and signaling dynamics ( 33 , 40 ). In a first step, we quantified IFN-stimulated nuclear translocation of STAT2 by fluorescence microscopy in living cells.…”

“…We therefore explored in more detail the submicroscopic spatiotemporal organization of IFNAR1 and IFNAR2 in the PM. For this purpose, we used TALM ( 45 ), a robust method that we recently developed for identifying and characterizing transient confinement zones ( 40 ). Superresolution TALM images, reconstructed by localizing individual receptor subunits over a period of 25 s, revealed substantial local confinement of the receptors (Fig.…”

Spatiotemporally Controlled Reorganization of Signaling Complexes in the Plasma Membrane of Living Cells

“…The kinetics of spatial reorganization was mainly limited by the mobility of the transmembrane proteins in the plasma membrane, which is strongly limited by the cortical actin cytoskeleton. [ 38,39 ] In situ reorganization of entire signaling complexes was achieved, opening exciting avenues for systematically studying the role of spatial organization of receptors in signal propagation. Surface micropatterning by microcontact printing of PLL-PEG conjugates ensures high biocompatibility of the surface required for effi cient assembly of transmembrane protein complexes by an exogenous ligand as demonstrated for the IFN-receptor complex.…”

Quantum dots for quantitative imaging: from single molecules to tissue

“…Labeling different protein species with spectrally distinct QDs also allows for the direct comparison of protein diffusion or capturing of heterodimer interactions (Low-Nam et al 2011; Steinkamp et al 2014). You et al have used pair correlation of dual-color imaging experiments to monitor QD-labeled IFNa2 binding to its receptor, IFNAR2, and the recruitment of STAT2 to IFNAR2 (You et al 2014). Torreno-Pina et al have used two-color single QD-tracking as part of a study to examine the role of glycans in micropatterning of the plasma membrane.…”