Dynamics of the Actin Cytoskeleton and Plasma Membrane at the Immunological Synapse Revealed using Live-Cell Super-Resolution Microscopy

Abstract

Filamentous (F)-actin and the plasma membrane (PM) both have a role in regulating molecular organisation and trafficking during cellular processes. In T-cells, this includes cell migration and the formation of the immunological synapse (IS). The IS is a specialised cell-cell junction crucial for antigen recognition. The molecular organisation during IS formation can influence downstream signalling and therefore the effectiveness of the antigen-mediated immune response.Combining Total Internal Reflection Fluorescence (TIRF) with Structured Illumination Microscopy (SIM) provides live-cell super-resolution images and molecular dynamics of sub-diffraction structures using standard fluorophores. Using Spatio-Temporal Image Correlation Spectroscopy (STICS) we can extract directionality and velocity information from this data in the form of vector maps and histograms. We applied TIRF-SIM and STICS to T-cells during the formation of the Immunological Synapse (IS) to study the dynamics of the cortical F-actin meshwork and the PM itself.At resolutions of approximately 100 nm both the cortical F-actin meshwork and the PM itself demonstrate retrograde flow during IS formation and in mature synapses. To establish what could be driving this retrograde flow we applied various drug treatments designed to alter the cytoskeletal meshwork in different ways such as Latrunculin-B and Cytochalasin-D which disrupt F-actin and Jasplakinolide which stabilises fibres. Using these reagents we were able to influence not only actin retrograde flow, but also the flow of the plasma membrane lipids themselves. This suggests F-actin polymerisation and retrograde flow may drive PM flow during IS formation through actin-membrane coupling.