Three-Dimensional Localization of T-Cell Receptors in Relation to Microvilli Using a Combination of Superresolution Microscopies
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Three-dimensional localization of T-cell receptors in relation to microvilli using a combination of superresolution microscopies Yunmin Junga, Inbal Rivena, Sara W. Feigelsonb, Elena Kartvelishvilyc, Kazuo Tohyad, Masayuki Miyasakae,f, Ronen Alonb, and Gilad Harana,1 aDepartment of Chemical Physics, Weizmann Institute of Science, Rehovot 76100, Israel; bDepartment of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel; cChemical Research Support, Weizmann Institute of Science, Rehovot 76100, Israel; dDepartment of Anatomy, Kansai University of Health Sciences, Kumatori, Osaka 590-0482, Japan; eInstitute for Academic Initiatives, Osaka University, Suita, Osaka 565-0871, Japan; and fMediCity Research Laboratory, University of Turku, FI-20521, Turku, Finland Edited by Arthur Weiss, University of California, San Francisco, CA, and approved August 8, 2016 (received for review April 3, 2016) Leukocyte microvilli are flexible projections enriched with adhesion recognition event and its consequences, culminating in the forma- molecules. The role of these cellular projections in the ability of T cells to tion of the immunological synapse (IS) (15–17), have been exten- probe antigen-presenting cells has been elusive. In this study, we probe sively studied. Fluorescence imaging studies using a variety of the spatial relation of microvilli and T-cell receptors (TCRs), the major methods, including superresolution microscopy, have demonstrated molecules responsible for antigen recognition on the T-cell membrane. that, even before activation by an antigen, TCRs are not randomly To this end, an effective and robust methodology for mapping distributed on the cell surface, but exist as preformed microclusters membrane protein distribution in relation to the 3D surface structure of an average size of tens of nanometers (18–20). It was further of cells is introduced, based on two complementary superresolution shown that, upon activation, TCRs assemble into larger clusters microscopies. Strikingly, TCRs are found to be highly localized on while concentrating in the central region of the IS (17, 18, 21, 22). microvilli, in both peripheral blood human T cells and differentiated However, the distribution of TCR microclusters in relation to the effector T cells, and are barely found on the cell body. This is a decisive 3D architecture of the T-cell membrane has not been explored. demonstration that different types of T cells universally localize their In the current study, we aim to amend this deficiency by TCRs to microvilli, immediately pointing to these surface projections as mapping the spatial arrangement of TCRs with respect to the 3D effective sensors for antigenic moieties. This finding also suggests how topography of the membrane in T cells. Our methodology merges previously reported membrane clusters might form, with microvilli serv- together two superresolution fluorescence techniques, variable- ing as anchors for specific T-cell surface molecules. angle total internal reflection microscopy (VA-TIRFM) and stochastic localization nanoscopy (SLN). It obtains similar spatial T-cell receptor | microvilli | superresolution microscopy | membrane protein resolution to immunogold EM, yet does not suffer from this clusters | total internal reflection microscopy method’s problems. The method allows us to observe that the TCRs of both types of T cells are highly enriched on microvilli compared irculating leukocytes have a distinctive surface topography with the cell body. Our results suggest that these dynamic projec- Cdominated by finger-like membranous protrusions, the mi- tions serve as sensors for antigenic moieties and that, once T-cell crovilli (1). Unlike the uniform and regular-sized microvilli found in clones generate stable contacts with the correct antigen-presenting the intestinal brush border, microvilli on immune cells are highly cell (APC) that presents their cognate antigen, these microvilli may flexible and dynamic (1–3). Although a role for microvilli in leu- serve as physical barriers for the diffusion and random mixing of kocyte capture to blood vessel walls has been demonstrated (4, 5), a surface proteins. physiological role for these projections in the immune response of T cells outside of the vasculature has not yet been established. Significance Going beyond morphological studies requires probing receptor distribution on microvilli and other compartments on leukocyte membranes. The largest obstacles in performing such studies are T lymphocytes play a central role in cell-mediated immunity. the thin and short dimensions of microvilli, which require higher Their surfaces are covered by narrow and short protrusions called microvilli. It is not known whether there is a role for microvilli resolution than that offered by standard fluorescence microscopy. in the immune response. To shed light on this question, we Therefore, studies of protein distribution in relation to immune probed the location of T-cell receptors (TCRs), the molecules cell microvilli have heavily relied on electron microscopy (EM) that initiate the immune response of T cells, with respect to methods. Indeed, several EM studies of immunogold-labeled the 3D structure of microvilli. Superresolution optical micros- surface molecules proposed that some membrane proteins are copy showed that TCRs are highly concentrated on microvilli. preferentially localized on microvilli in T cells and macrophages – Previous studies stressed the role of small clusters of TCRs in (6 8), whereas other proteins were found to be enriched on the the immune process; our study provides a natural explanation cell body between microvilli (9, 10). Although these earlier studies as to how these clusters form. promoted the idea that microvilli serve as distinctive membranal regions on which certain membrane proteins are selectively lo- Author contributions: Y.J., R.A., and G.H. designed research; Y.J., S.W.F., E.K., K.T., and calized, they suffered from problems inherent to EM such as M.M. performed research; S.W.F. contributed new reagents/analytic tools; Y.J., I.R., R.A., sample distortion during the preparation process, as well as arti- and G.H. analyzed data; and Y.J., I.R., R.A., and G.H. wrote the paper. facts arising from the relatively bulky gold particles and their The authors declare no conflict of interest. tendency to stick together (11–13). A fluorescence-based method This article is a PNAS Direct Submission. should be able to overcome these problems. Freely available online through the PNAS open access option. T-cell receptors (TCRs) are membrane protein complexes that See Commentary on page 11061. recognize antigens as part of the primary steps of adaptive im- 1To whom correspondence should be addressed. Email: [email protected]. α β munity. A TCR is composed of a heterodimer of -and -subunits and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. a cluster of differentiation 3 (CD3) complex (14). The antigen–TCR 1073/pnas.1605399113/-/DCSupplemental. E5916–E5924 | PNAS | Published online September 19, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1605399113 Downloaded by guest on September 24, 2021 Results Mapping Membrane Proteins in Relation to Microvilli. We developed PNAS PLUS Resting and Differentiated T Cells in Peripheral Blood, as well as T a strategy for determining at nanometer resolution the location Cells Migrating Inside Lymph Nodes, Exhibit Numerous Microvilli. In of membrane molecules with respect to surface structures, using this work, we studied two types of T cells, human resting T cells, a synergistic combination of two types of fluorescence micro- copy: VA-TIRFM resolves the 3D topography of the cell, and and effector T cells differentiated from the blood-derived pools SLN obtains the position of molecules with respect to this of naïve and memory resting T cells following TCR activation and topography. expansion by IL-2 (23). The latter serve as models for antigen- VA-TIRFM (34–36) is based on the physics of the exponen- activated T cells that have differentiated into Th1 and Tc1 subsets tially decaying TIRF illumination as a function of the distance in the presence of IL-2 (24). We first used scanning electron from the glass coverslip (Fig. 2A). The penetration depth of the microscopy (SEM) to image microvilli on these cells. Microvilli TIRF evanescent field [d(θ)] is tunable by changing the angle of SEE COMMENTARY were found to be of a diameter of 70–100 nm and a length that incidence, θ (Table S1). With a known d(θ), the relative intensity varied from 0.1 μm to several micrometers (Fig. 1 A and B,and of fluorescence at each point in the image can be used to cal- Fig. S1). Consistent with the literature (25), effector T cells were culate its distance (δz) from the glass surface. The set of δz values about twice as large in cell diameter as resting T cells. Their mi- can then be used to construct a 3D image of the fluorescently crovilli were found to be longer, although their mean diameter was labeled cell membrane. It is possible to increase the statistical precision of the resulting image by combining measurements at comparable to that of resting T cells. several values of θ (Fig. S2). Interestingly, several EM studies reported well-maintained 3D – – SLN is a form of superresolution fluorescence microscopy in structures of microvilli at T-cell APC (26, 27) or T-cell T-cell which bursts of photons from blinking labeled molecules are interfaces (28) in vitro. Nevertheless, the presence of these used to localize them with accuracy well below the diffraction structures in T cells migrating inside lymph nodes in search for limit (37, 38) (Fig. 2B). We implemented a simple technique that antigen in vivo has been disputed (29–33). We therefore tested enables measuring molecules located on two different planes in whether microvilli are also maintained by T cells within the T the z direction, which we term dual-plane SLN (Fig. S3). In this zones of secondary lymphoid organs. Using transmission electron technique, a piezo stage is used to move the sample up or down microscopy (TEM), we imaged these T-cell zones in murine pe- (Fig.