Plasma membrane LAT activation precedes vesicular recruitment defining two phases of early T-cell activation

Lakshmi Balagopalan,Tiffany Nguyen,Adam S Harned,Katherine M Mcintire,Lawrence E Samelson,Jason Yi,Kedar Narayan

doi:10.1038/s41467-018-04419-x

Abstract

The relative importance of plasma membrane-localized LAT versus vesicular LAT for microcluster formation and T-cell receptor (TCR) activation is unclear. Here, we show the sequence of events in LAT microcluster formation and vesicle delivery, using lattice light sheet microscopy to image a T cell from the earliest point of activation. A kinetic lag occurs between LAT microcluster formation and vesicular pool recruitment to the synapse. Correlative 3D light and electron microscopy show an absence of vesicles at microclusters at early times, but an abundance of vesicles as activation proceeds. Using TIRF-SIM to look at the activated T-cell surface with high resolution, we capture directed vesicle movement between microclusters on microtubules. We propose a model in which cell surface LAT is recruited rapidly and phosphorylated at sites of T-cell activation, while the vesicular pool is subsequently recruited and dynamically interacts with microclusters.

Highlights

The relative importance of plasma membrane-localized linker for activation of T cells (LAT) versus vesicular LAT for microcluster formation and T-cell receptor (TCR) activation is unclear
The evidence for the first model involving plasma membraneresident LAT comes from transmission electron microscopy (TEM) and super-resolution photoactivated localization microscopy (PALM) studies that propose that cell surface LAT is preclustered at the plasma membrane and cluster sizes increase upon T-cell stimulation[7,8,9]
These results clearly indicate that calcium influx is not required for LAT microcluster formation and argue that calcium-mediated exocytosis of vesicles is not required for LAT microcluster formation

Summary

Introduction

The relative importance of plasma membrane-localized LAT versus vesicular LAT for microcluster formation and T-cell receptor (TCR) activation is unclear. We propose a model in which cell surface LAT is recruited rapidly and phosphorylated at sites of T-cell activation, while the vesicular pool is subsequently recruited and dynamically interacts with microclusters. Interfering with LAT release from vesicular compartments by silencing vesicular fusion machinery such as the calcium sensor synaptotagmin[7], or the vesicular SNARE VAMP7, resulted in decreased LAT phosphorylation and IL-2 production[13, 14] From these results, it was proposed that calcium-dependent exocytosis of vesicular LAT is the primary mechanism by which LAT is recruited to microclusters, phosphorylated, and propagates downstream signals in an activated T cell. To interrogate the relationship between plasma membrane and vesicular pools and how they contribute to microcluster formation, we simultaneously imaged LAT and a vesicular marker VAMP7, which partially colocalizes with intracellular LAT, from the instant a T cell becomes activated. Vesicles travel on microtubules and dynamically interact with microclusters

Methods

Results

Conclusion