ERM-Dependent Assembly of T-Cell Receptor Signaling and Co-stimulatory Molecules on Microvilli Prior to Activation

Abstract

T-cell surfaces are covered with microvilli, actin-rich and flexible protrusions. We use super-resolution microscopy to show that 90% T-cell receptor (TCR) complex molecules TCR and TCR, as well as the co-receptor CD4 and the co-stimulatory molecule CD2 reside on microvilli of human T cells. 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. Notably, phosphorylated proteins of the ERM (ezrin, radixin, moesin) family colocalize with these 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. Our results establish microvilli as key signaling hubs, in which the TCR complex and its proximal signaling molecules and adaptors are pre-assembled prior to activation in an ERMdependent manner. The preformed positioning of these actin-binding TCR assemblies on individual microvilli can facilitate the local transmission of TCR signals seconds after TCR occupancy and impacts the slower subsequent events that lead to the assembly of immunological synapses.

Pre-organized landscape of T cell surface

“…distribution in relation to T cell microvilli was revealed in a recent study using super-resolution microscopic techniques (59). This study showed colocalization of phosphorylated ERM proteins with TCRs and actin filaments in microvilli, suggesting that ERM proteins may stabilize TCR assemblies within microvilli prior to TCR ligand binding and T cell activation (Figure 3A).…”

“…Another study using super-resolution optical fluctuation imaging (SOFI) (102) also reported high-density CD4 regions on the surface of Jurkat T cells (101). Haran's group also demonstrated that more than 90% of CD4 and CD2 accumulate on the microvilli of human effector T cells and Jurkat T cells (59). In a 3D superresolution imaging study, CD4 was also reported to accumulate at the microvilli tips, while CD45 was excluded from the tips in unstimulated Jurkat T cells (93).…”

“…Similarly, TCR-proximal signaling molecules that propagate TCR triggering intracellularly, such as Lck kinase and the adaptor protein linker for activation of T cells (LAT) (104), are highly enriched on the microvilli of resting human effectors and Jurkat T cells (59). The microvilli-specific fractions of Lck and LAT were lower than that of TCR, CD4, and CD2, but still quite high at 76% and 68%, respectively.…”

“…Schematic illustration of ERM-mediated TCR localization on microvilli (59). (A) A single T cell microvillus containing an actin bundle connected by actin-filament crosslinking proteins is depicted.…”

Clathrin controls bidirectional communication between T cells and antigen presenting cells

“…[92,93] Upon activation, the microvilli rapidly retract as the T cell spreads out across the surface of an APC. [94] This generates a relatively flat surface with reduced membrane tension and high local concentration of DAG, PI3P, and ubiquitinated TCR. This then leads to recruitment of ESCRT-0 which in turn recruits clathrin and subsequent ESCRT machinery and triggers ectocytic release of TCR-loaded vesicles through CEME.…”