Microvilli: The ERM Dependent Activation Hubs of T-Cells

Introduction:

When T cells encounter cognate peptide-MHC complexes on antigen presenting cells, they respond within seconds. How such a fast response is orchestrated in very short periods of time has long been disputed. Interestingly, T-cell surfaces are covered with microvilli, actin-rich and flexible protrusions. Surprisingly, the role of the microvilli in the activation process of T-cells has long been underrated. Here we probe the localization of key surface molecules involved in the initial immune response, and demonstrate their enrichment on microvilli, implication these structures as T-cell activation hubs.

Materials and methods: Variable-angle total internal reflection microscopy was conducted on pre-fixed human T-cells cells residing on a glass surface. The fluorescence intensity measured at each point in an image was converted into the distance of that point from the glass, which permitted us to plot the 3D topography of the membrane surface. We then recorded stochastic localization nanoscopy images of antibody-labelled membrane proteins from the same cells, which allowed us to determine the positions of individual molecules with an accuracy well below the diffraction limit. By superimposing the super-resolved membrane protein map on the 3D membrane topographical map, we were able to characterize the distribution of each protein molecule in relation to microvilli.

Results and discussion: We show that >90% T-cell receptor (TCR) complex molecules TCRαβ and TCRζ, as well as the co-receptor CD4 and the co-stimulatory molecule CD2 reside on microvilli on the T cell surfaces. Furthermore, TCR proximal signaling molecules involved in the initial stages of the immune response, such as the protein tyrosine kinase Lck and the key adaptor molecule LAT, are also enriched on microvilli. Phosphorylated proteins of the ERM (ezrin, radixin, moesin) family colocalize with TCRαβ heterodimers as well as with actin filaments within the microvilli of resting T cells. This finding implies a role for one or more phosphorylated ERMs in linking the TCR complex to the actin cytoskeleton within microvilli. Indeed, expression of a dominant-negative ezrin fragment effectively redistributes TCR molecules over the whole T cell surface.

Conclusion:

Our results establish microvilli as key signaling hubs, on which the TCR complex and its proximal signaling molecules and adaptors are pre-assembled prior to TCR activation. The preformed organization of these actin-binding TCR assemblies on individual microvilli can facilitate the local transmission of TCR signals seconds after TCR occupancy and might also impact the slower subsequent events that lead to the assembly of immunological synapses.