Super-resolution Imaging of the T cell Central Supramolecular Signaling Cluster Using Stimulated Emission Depletion Microscopy.

Sin Tan,Christoph Wülfing,Dominic Alibhai,Harry Thompson,Stephen Cross

doi:10.21769/bioprotoc.3806

Abstract

Supramolecular signaling assemblies are of interest for their unique signaling properties. A µm scale signaling assembly, the central supramolecular signaling cluster (cSMAC), forms at the center interface of T cells activated by antigen presenting cells (APC). The adaptor protein linker for activation of T cells (LAT) is a key cSMAC component. The cSMAC has widely been studied using total internal reflection fluorescence microscopy of CD4+ T cells activated by planar APC substitutes. Here we provide a protocol to image the cSMAC in its cellular context at the interface between a T cell and an APC. Super resolution stimulated emission depletion microscopy (STED) was utilized to determine the localization of LAT, that of its active, phosphorylated form and its entire pool. Agonist peptide-loaded APCs were incubated with TCR transgenic CD4+ T cells for 4.5 min before fixation and antibody staining. Fixed cell couples were imaged using a 100x 1.4 NA objective on a Leica SP8 AOBS confocal laser scanning microscope. LAT clustered in multiple supramolecular complexes and their number and size distributions were determined. Using this protocol, cSMAC properties in its cellular context at the interface between a T cell and an APC could be quantified.

Highlights

[Abstract] Supramolecular signaling assemblies are of interest for their unique signaling properties
Dynamic recruitment of signaling intermediates into microscopically detectable structures at T cell interface is an important element of signal integration. μm scale signaling assemblies were first described at the center and periphery of T cells activated by antigen presenting cells (APC) for the T cell receptor (TCR), PKCθ and LFA-1, talin, respectively, as central and peripheral supramolecular activation clusters (Monks et al, 1997 and 1998; Grakoui et al, 1999). μm scale of assemblies, in particular in the form of supramolecular protein complexes, provides unique biophysical and signaling properties (Li et al, 2012; Banani et al, 2016; Shin and Brangwynne, 2017)
The central supramolecular signaling cluster (cSMAC) has been imaged using confocal microscopy of fixed T cell APC couples (Monks et al, 1997 and 1998; Freiberg et al, 2002), spinning disk confocal live cell imaging of T cell APC couples (Singleton et al, 2009; Clark et al, 2019), and using total internal reflection (TIRF) imaging of T cells activated on planar APC substitutes (Yokosuka et al, 2005; Varma et al, 2006)

Summary

Introduction

[Abstract] Supramolecular signaling assemblies are of interest for their unique signaling properties. The use of planar APC substitutes allows more efficient diffraction limited imaging but does not reflect the membrane topology of T cell APC couples well (Roybal et al, 2015) and, may result in altered structural details of μm scale signaling complexes (Clark et al, 2019). Coverslips (Carl ZeissTM, catalog number: 10474379), store at room temperature 9. Microscope Slide (VWR, SuperFrost Plus, catalog number: 631-0108, store at room temperature) 11.

Results

Conclusion