Three-dimensional localization of T-cell receptors in relation to microvilli using a combination of superresolution microscopies

Abstract

Leukocyte microvilli are flexible projections enriched with adhesion molecules. The role of these cellular projections in the ability of T cells to probe antigen-presenting cells has been elusive. In this study, we probe the spatial relation of microvilli and T-cell receptors (TCRs), the major molecules responsible for antigen recognition on the T-cell membrane. To this end, an effective and robust methodology for mapping membrane protein distribution in relation to the 3D surface structure of cells is introduced, based on two complementary superresolution microscopies. Strikingly, TCRs are found to be highly localized on microvilli, in both peripheral blood human T cells and differentiated effector T cells, and are barely found on the cell body. This is a decisive demonstration that different types of T cells universally localize their TCRs to microvilli, immediately pointing to these surface projections as effective sensors for antigenic moieties. This finding also suggests how previously reported membrane clusters might form, with microvilli serving as anchors for specific T-cell surface molecules.T-cell receptor | microvilli | superresolution microscopy | membrane protein clusters | total internal reflection microscopy C irculating leukocytes have a distinctive surface topography dominated by finger-like membranous protrusions, the microvilli (1). Unlike the uniform and regular-sized microvilli found in the intestinal brush border, microvilli on immune cells are highly flexible and dynamic (1-3). Although a role for microvilli in leukocyte capture to blood vessel walls has been demonstrated (4, 5), a physiological role for these projections in the immune response of T cells outside of the vasculature has not yet been established.Going beyond morphological studies requires probing receptor distribution on microvilli and other compartments on leukocyte membranes. The largest obstacles in performing such studies are the thin and short dimensions of microvilli, which require higher resolution than that offered by standard fluorescence microscopy. Therefore, studies of protein distribution in relation to immune cell microvilli have heavily relied on electron microscopy (EM) methods. Indeed, several EM studies of immunogold-labeled surface molecules proposed that some membrane proteins are preferentially localized on microvilli in T cells and macrophages (6-8), whereas other proteins were found to be enriched on the cell body between microvilli (9, 10). Although these earlier studies promoted the idea that microvilli serve as distinctive membranal regions on which certain membrane proteins are selectively localized, they suffered from problems inherent to EM such as sample distortion during the preparation process, as well as artifacts arising from the relatively bulky gold particles and their tendency to stick together (11-13). A fluorescence-based method should be able to overcome these problems.T-cell receptors (TCRs) are membrane protein complexes that recognize antigens as part of the primary steps of adaptiv...

Visualizing Dynamic Microvillar Search and Stabilization during Ligand Detection by T Cells

“…Scanning angle interference microscopy ( Fig S5B) of T cells encountering lipid bilayers similarly confirmed that there were significant height variations of membrane for cells engaging lipid bilayers (Fig S5C). Notably bright TCR accumulations (microclusters) were closer to lipid bilayers as compared to the entire pool average, which is consistent with recent variable-angle TIRF studies showing TCR pre-enriched at microvillar tips (21). ICAM-1, a bigger molecule, was found farther away from the bilayer (Fig S5B-D).…”

Three-Dimensional Localization of Single Molecules for Super-Resolution Imaging and Single-Particle Tracking

“…While not a wide-field detection method, this has been used to track the motion of single nanoparticles, one at a time, by using a xyz translation stage in closed-loop operation, permitting the observation of complex cellular transport processes with a large range and high resolution both spatially and temporally. (142–145)…”

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

“…We found that at both temperatures L-selectin was concentrated on microvilli, 1,2 while CD45 was distributed uniformly throughout the membrane. 1,4,5 Next, we stained T cells with Alexa-647 labelled anti human TCRαβ antibodies at the two temperatures. At 4°C the structure of microvilli on almost all the T cells was intact, and the TCRαβ molecules localized on them (Fig.…”