High plasma membrane lipid order imaged at the immunological synapse periphery in live T cells

Abstract

Cholesterol- and glycosphingolipid-enriched membrane lipid microdomains, frequently called lipid rafts, are thought to play an important role in the spatial and temporal organization of immunological synapses. Higher ordering of lipid acyl chains was suggested for these entities and imaging of membrane order in living cells during activation can therefore help to understand the mechanisms responsible for the supramolecular organization of molecules involved in the activation of T cells. Here, we employ the phase-sensitive membrane dye di-4-ANEPPDHQ together with a variety of spectrally-resolved microscopy techniques, including 2-channel ratiometric TIRF microscopy and fluorescence lifetime imaging, to characterize membrane order at the T cell immunological synapse at high spatial and temporal resolution in live cells at physiological temperature. We find that higher membrane order resides at the immunological synapse periphery where proximal signalling through the immunoreceptors and accessory proteins in microclusters has previously been shown to take place. The observed spatial patterning of membrane order in the immunological synapse depends on active receptor signalling.

Keywords::

• Lipid rafts
• membrane lipid order
• the immunological synapse
• T cells

Acknowledgements

We thank Rajat Varma and Etienne Joly for helpful suggestions and critical reading of the manuscript, and Chris Dunsby for helpful comments on the manuscript and assistance with optical tweezing. PMWF and DMD acknowledge a Wolfson Royal Society Research Merit Award. We also acknowledge the Facility for Imaging by Light Microscopy at Imperial College.

Declaration of interest: DMO, SK and SO acknowledge funding from the Engineering and Physical Sciences Research Council via the Chemical Biology Centre. We acknowledge Medical Research Council (MRC) grants G0700771 and G0100471 to AIM and National Institutes of Health grant AI43542 to MLD. DMD acknowledges funding from the MRC. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.