Molecular scale dynamics of T cell immunological synapse components by advanced and super-resolution fluorescence microscopy

Abstract

Cortical actin forms a central role in the reorganisation and heterogeneity of plasma membrane (PM) components of T cells. During T cell activation, actin remodelling aids formation of the stable interface between cells known as the immunological synapse; this interface is a dynamic event where actin plays a key role in forming and translocating protein microclusters for sustained signalling. Cortical actin creates a dense meshwork at the synapse periphery, possibly acting as a barrier against molecular trafficking to the synapse interface. Additionally, actin flows in a retrograde manner towards the relatively actin-poor synapse centre, driving transmembrane proteins inwards.
Here, fixed cell single molecule localisation microscopy was used to characterise the actin cortex upon T cell synapse formation. Live-cell structured illumination microscopy (SIM) was quantified using spatiotemporal image correlation spectroscopy (STICS), this demonstrated flow speed and directionality was dependent on balanced actin turnover and membrane order. Building on these results, two-channel SIM imaging was carried out, correlating actin flow with plasma membrane (PM) dynamics, as these systems are known to interact via transmembrane and linker proteins. The PM was shown to correlate with actin flow speeds and direction in an -actinin dependent manner.
Sustained signalling during synapse formation requires protein delivery to the interface, relying on vesicle trafficking via different components of the cytoskeleton. As membrane order modulates protein clustering within the PM, it was investigated using the polarity-sensitive dye di-4-ANEPPDHQ whether this was also true of vesicles. Results here demonstrated vesicle lipid order negatively correlated with microtubule structures.
Finally, vesicle cargo was shown to correlate with different vesicle populations, based on their membrane order, demonstrating vesicle order may mirror the heterogeneous nature of the PM. These results demonstrate the complex biophysical processes that control the T cell immunological synapse are an amalgamation of cytoskeletal organisation, vesicle dynamics and membrane polarity.

Date of Award	2017
Original language	English
Awarding Institution	King's College London
Supervisor	Madeline Parsons (Supervisor) & Dylan Owen (Supervisor)