Scanning immunoelectron microscopy of mouse B and T lymphocytes

Linthicum, D. Scott; Sell, Stewart; Wagner, R; Trefts, Park E.

doi:10.1038/252173a0

Cited by 43 publications

(14 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Nevertheless, the presence of these structures in T cells migrating inside lymph nodes in search for antigen in vivo has been disputed (29)(30)(31)(32)(33). We therefore tested whether microvilli are also maintained by T cells within the T zones of secondary lymphoid organs.…”

Section: Resultsmentioning

confidence: 99%

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

Jung

Riven

Feigelson

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

184

232

View full text Add to dashboard Cite

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...

show abstract

Section: Resultsmentioning

confidence: 99%

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

Jung

Riven

Feigelson

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

184

232

View full text Add to dashboard Cite

show abstract

“…Thus, using the presence or-absence of villi as reliable markers for B and T cells is excluded. Moreover, other controversial SEM findings were recently published stating that either T cells were hairy cells, while B cells were not [23,24], or that both could be hairy [25].…”

Section: Are Villi On Lymphocytes Facts or Artefacts?mentioning

confidence: 99%

The modulation of microprojections on the lymphocyte membrane and the redistribution of membrane‐bound ligands, a correlation

Loor

Hägg²

1975

Eur J Immunol

View full text Add to dashboard Cite

We gratefully thank Pro6 H. lsliker for providing rabbit Fc and pep III, Dr. M. Klein for fragments of human IgC. A . Collk f o r 02microglobulin. The helpful technical assistance o f M. Cristofari and the stimulating discussions with Dr. F.M. Kourilsky were appreciated. Abbreviations: BSA: Bovine serum albumin Con A: Concanavalin A Fab: Monovalent fragments (from papain cleaved Ig) FITC: Fluoresceine isothiocyanate-conjugated IFM: Immunofluoresccnce microscopy S-: sheep-) PHA: Phytohemagglytinin RTO: Room temperature SEM: Scanning electron microscopy TL: TLa-determined antigen TRITC: Tetramethylrhodamine isothiocyanate-conjugated E.M.: F.lectron microscopy CB: Cytochalasin B F(ab')z: Bivalent fragments (from pepsin cleaved Ig) Ig: Immunoglobulins (M-: mouse-; R-: rabbit-; Thy-1-determined antigen T h e binding t o the cell surface of ligands able to cross-link cell m e m b r a n e c o m p o n e n t s is followed by a redistribution phenomenon k n o w n as the ring + spots -+ cap + endocytosis transition. This was first described for lymphocytes but is a general characteristic o f cells with a "fluid" membrane [ 1-51, Although it is ;I real and reproducible p h e n o m e n o n which is very useful for studies o n t h e organization of the cell membrane, m a n y aspects of t h e transition a s it has been described so far might be artefactual, due t o unphysiological experimental conditions. Actual physiological conditions for lymphocytes are o f course not k n o w n , b u t probably two key experimental conditions never occur in uivo: I ) at least part of t h e in vitro cell processing was operated at or near 0 "C, and 2 ) t h e membrane-binding ligand (antigen, antibody, lectin) w a s presented in excess to t h e cells, its concentration was left constant f o r s o m e time and then rapidly brought d o w n to zero b y washing. Eur. J. Immunol. 1975.5: 854-865 Villi on lymphocytes 855

show abstract

“…Several types of markers have been employed so far: hemocyanin (SMITH and REVEL, 1972), polystyrene (RINEHART et al, 1971;LOBUGLIO et al, 1972;LINTHICUM et al, 1974;LINTHICUM and SELL, 1975;AMANO, 1975) or acryic (MOLDAY et al, 1974(MOLDAY et al, , 1975 latex spheres, tobacco mosaic virus (NEMANIC, 1975;NEMANIC et al 1975;HAMMERLING et al, 1975), colloidal gold granules (HORISBERGER, ROSSET and BAUER, 1975) and ferritin (TOKUNAGA and FUJITA, 1973;FUJITA and TOKUNAGA, 1975;LINTHICUM and SELL, 1975;KAY, 1975). These latex and viral particles previously used may be said to be too large and cover the fine surface structures such as micropro jections of lymphocytes and macrophages.…”

Section: Discussionmentioning

confidence: 99%