Our study suggests that the TCR resides in ordered plasma membrane domains that are linked to actin filaments and aggregate upon TCR engagement

Our study suggests that the TCR resides in ordered plasma membrane domains that are linked to actin filaments and aggregate upon TCR engagement. Ordered membrane nanodomains, often referred to as lipid rafts, are implicated in immune cell signalling. plasma membrane domains in both Jurkat and main T cells. The TCR colocalised with actin filaments at the plasma membrane in unstimulated Jurkat T cells, consistent with it being localised to ordered membrane domains. The colocalisation was most prominent in cells in G1 phase when the cells are ready to commit to proliferation. At other cell cycle phases the TCR was mainly found at perinuclear membranes. Our study suggests that the TCR resides in ordered plasma membrane domains that are linked to actin filaments and aggregate upon TCR engagement. Ordered membrane nanodomains, often referred to as lipid rafts, are implicated in immune cell signalling. They are considered to form STO-609 acetate by the self-aggregation of cholesterol and sphingolipids1 and are believed to exist STO-609 acetate as liquid ordered (lo) domains, in contrast to the rest of the membrane that exists liquid disordered (ld) domains. However, both are liquid phases and diffusion can take place inside, around as well as into and out of the domains so membrane components constantly shift between domains and their surroundings. T cell signalling is initiated by Src family tyrosine kinases, Lck and Fyn, by phosphorylation of immunoreceptor tyrosine based activation motifs (ITAMs) in the CD3 subunits of the T cell receptor (TCR). Downstream signalling entails the activation of Ras and calcium pathways. All these pathways can be activated by crosslinking different lipid raft components, the ganglioside GM1 or the GPI-anchored protein CD59, suggesting a link between the aggregation of ordered membrane nanodomains and early T cell signalling2,3,4. Cold stress and moderate cholesterol depletion can also lead to lipid raft aggregation and T cell activation5,6. Formation of an immunological synapse (Is usually) occurs after the initial signalling events7 and is accomplished by the transport of microclusters made up of the TCR and signalling proteins along both actin filaments and microtubules to form a central supramolecular activation cluster (cSMAC)8,9,10. The IS in both fixed and live T cells has been shown to contain ordered membrane domains11,12. In addition the IS in fixed cells is usually enriched in lo-domain partitioning signalling molecules13. However, the lipid packing in the TCR-containing microclusters has not been analyzed. Using total internal fluorescence microscopy (TIRF) it has recently been suggested that TCR microclusters exist in resting T cells14 although TCR microclusters are not generally observed in resting T cells using other fluorescence microscopy methods. Whether or not the TCR exists exclusively as monomers or a mix of monomers and dimer/multimer clusters also seems to reflect the choice of methodology15,16. However, super resolution studies suggest that the TCR in resting T cells resides in nanodomains17 which is not incompatible with the TCR existing as monomers since within the nanodomains there could be lipids that preclude direct interaction of individual TCRs but still lead to a TCR density required to respond to scarce agonists15. Remodelling of the actin cytoskeleton is usually integral to T cell activation18,19. Polymerised actin has long been known to accumulate at capping sites of the TCRs20. Polymerised actin also accumulates underneath aggregated lipid rafts21. Moreover, ordered lipid domains form at attachment points between actin filaments and the plasma membrane in a phosphoinositide dependent manner, further strengthening the link between lipid rafts and the cytoskeleton22. In this study, we have used the probe laurdan to assess the plasma membrane order in live Jurkat and main human T cells upon initiation of signalling by antibodies directed at the TCR subunit CD3 in order to address the nature of the lipid environment in TCR nano- and microdomains. Our data provides answers to the questions of whether the TCR is usually a lipid raft resident protein or is usually recruited to lipid rafts upon T cell activation and whether lipid rafts form upon aggregation of the TCR. Results There is consensus that this TCR is found in ordered plasma membrane domains after its engagement/the formation of the immunological synapse but you will find three possible scenarios compatible with this notion. The first is that this TCR is usually recruited to ordered plasma membrane nanodomains upon its engagement, the second that this TCR usually resides in ordered plasma membrane domains and the third that ordered plasma membrane domains form upon engagement of the TCR. The TCR resides in small ordered domains in the plasma membrane of resting T cells To address the outstanding questions whether TCR nanoclusters in resting cells have the expected features of lipid rafts and STO-609 acetate whether Mouse Monoclonal to E2 tag ordered membrane domains form upon TCR ligation, laurdan labelled T cells were imaged live and the generalised polarisation (GP) values, STO-609 acetate a measure of the relative proportion of lo and ld phase in.