Spatial association of signaling proteins and F-actin effects on cluster assembly analyzed via photoactivation localization microscopy in T cells.

Chih-Jung Hsu,Tobias Baumgart

doi:10.1371/journal.pone.0023586

Abstract

Recognition of antigens by T cell receptors (TCRs) triggers cellular signaling cascades initiated by recruitment to the plasma membrane of numerous effector molecules to form signaling microclusters (MCs). Here we show that the method of high-resolution photoactivation localization microscopy (PALM) imaging can be used to analyze the spatial correlation between kinase ZAP70 and adaptor SLP76 MCs at the cell periphery and the effects of F-actin on MC assembly. We first determined the photophysical rate constants of Dronpa and tdEos fluorescence probes, which allowed us to optimize our dual-color PALM imaging method. We next analyzed the degrees of spatial association through determination of Mander's colocalization coefficients from PALM images, which revealed increasing spatial segregation of ZAP70 and SLP76 MCs at the cell periphery after initiation of signaling. We showed that this spatial segregation at the cell periphery occurred in parallel with the reduction of MC phosphorylation levels. Furthermore, we used Ripley's K function to analyze spatial randomness, and determined average radii of clusters as a function of activation time. The average radii of SLP76 and LAT MCs were found to decrease, whereas ZAP70 MC radii remained relatively constant. Finally, effects of F-actin depolymerization on MC morphology were studied by determining radial distributions of cluster circularity. Our data suggest that MC morphology is affected by actin polymerization. The quantitative analysis of sub-diffraction PALM images may provide a starting point for a molecular interpretation of cluster-cluster interactions and of the regulation of T cell signaling MCs by the cytoskeleton.

Highlights

The engagement of the T cell antigen receptor (TCR) by antigen induces dynamic molecular rearrangement and leads to the translocation of kinases and adaptors to the plasma membrane of T cells [1,2,3]
Phosphorylation of TCR/CD3 complex is followed by the recruitment of zeta-chain associated protein kinase of 70 kDa (ZAP70) via its tandem Src-homology 2 domains
The phosphotyrosines of SLP76 in the N-terminal domain regulate binding to guaninenucleotide-exchange factor VAV, the adaptor non-catalytic region of tyrosine kinase (NCK), and TEC-family kinase interleukin-2inducible T-cell kinase (ITK) [6,7,8]

Summary

Introduction

The engagement of the T cell antigen receptor (TCR) by antigen induces dynamic molecular rearrangement and leads to the translocation of kinases and adaptors to the plasma membrane of T cells [1,2,3]. Photophysical rate constants of tdEos and Dronpa Dual-color high resolution PALM imaging has previously been performed via a combination of an irreversibly photoswitchable fluorophore and a reversibly photoactivatable fluorescent protein [17]. After the complete depletion of the pool of fluorescent tdEos molecules, Dronpa localization was performed, enabling us to obtain dual-color PALM images (Fig. 2A–2B, and 2D).

Results

Conclusion