Co-localization of cell surface receptors at high spatial resolution by single-particle fluorescence imaging

Dual-wavelength single-particle fluorescence imaging has been used to quantify the co-localization of receptors and/or ligands on cells by widefield microscopy. Methods for correction of chromatic aberration and identification of submicroscopic artefacts are presented, with data for the lipopolysaccharide/CD14 and MHC class II/CD74 systems.

Download Full-text

Measurements of associations of cell-surface receptors by single-particle fluorescence imaging

Biochemical Society Transactions ◽

10.1042/bst0311028 ◽

2003 ◽

Vol 31 (5) ◽

pp. 1028-1031 ◽

Cited By ~ 6

Author(s):

R.J. Cherry ◽

I.E.G. Morrison ◽

I. Karakikes ◽

R.E. Barber ◽

G. Silkstone ◽

...

Keyword(s):

Cell Surface ◽

Fluorescence Imaging ◽

Mhc Class Ii ◽

Single Particle ◽

Gaussian Function ◽

Human Leukocyte ◽

Cell Surface Receptors ◽

Intact Cells ◽

Surface Receptors ◽

Hla Dr

SPFI (single-particle fluorescence imaging) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labelled with small fluorescent particles. The images of particles are diffraction-limited spots that are analysed by fitting with a two-dimensional Gaussian function. The spot intensities depend on whether they arise from one or more particles; this provides the basis for determining self-association of cell-surface receptors. We have used this approach to determine dimerization of MHC class II molecules and its disruption by interface peptides. We have also exploited the positional information obtained from SPFI to detect co-localization of cell-surface molecules. This involves labelling two different molecules with different coloured fluorophores and determining their positions separately by dual wavelength imaging. The images are analysed to quantify the overlap of the particle images and hence determine the extent of co-localization of the labelled molecules. The technique provides quantification of the extent of co-localization and can detect whether co-localized molecules occur singly or in clusters. We have obtained preliminary data for co-localization of lipopolysaccharide and CD14 on intact cells. We also show that HLA-DR (human leukocyte antigen-DR) and CD74 are partially co-localized and that interaction between these molecules involves the peptide-binding groove of HLA-DR.

Download Full-text

Lymphoid Tumors ofXenopus laeviswith Different Capacities for Growth in Larvae and Adults

Developmental Immunology ◽

10.1155/1994/37392 ◽

1994 ◽

Vol 3 (4) ◽

pp. 297-307 ◽

Cited By ~ 50

Author(s):

Jacques Robert ◽

Chantal Guiet ◽

Louis Du Pasquier

Keyword(s):

Cell Surface ◽

Tumor Cell ◽

Tumor Cells ◽

Mhc Class I ◽

Mhc Class Ii ◽

Cell Lineage ◽

Cell Types ◽

Class Ii ◽

Class I ◽

Specific Marker

Three new lymphoid tumors offering an assortment of variants in terms of MHC class I expressions, MHC class II expression, and Ig gene transcription have been discovered in the amphibianXenopus. One was developed in an individual of the isogenic LG15 clone (LG15/0), one in a frog of the LG15/40 clone (derived from a small egg recombinant of LG15), and one (ff-2) in a maleffsib of the individual in which MAR1, the first lymphoid tumor in Xenopus was found 2 years ago. These tumors developed primarily as thymus outgrowths and were transplantable in histocompatible tadpoles but not in nonhistocompatible hosts. Whereas LG15/0 and LG15/40 tumor cells also grow in adult LG15 frogs, theff-2 tumor, like the MAR1 cell line, is rejected by adultffanimals. Using flow cytometry with fluorescence-labeled antibodies and immunoprecipitation analysis, we could demonstrate that, like MAR1, these three new tumors express on their cell surface lymphopoietic markers recognized by mAbs FIF6 and RC47, as well as T-cell lineage markers recognized by mAbs AM22 (CD8-1ike) and X21.2, but not by immunologobulin (Ig) nor MHC class II molecules. Another lymphocyte-specific marker AM15 is expressed by 15/0 and 15/40 but notff-2 tumor cells. Theff-2 tumor cell expresses MHC class molecule in association withβ2-microglobulin on the surface, 15/40 cells contain cytoplasmic Iαchain that is barely detected at the cell surface by fluocytometry, and 15/0 cells do not synthesize class Iαchain at all. The three new tumors all produce large amounts of IgM mRNA of two different sizes but no Ig protein on the membrane nor in the cytoplasm. All tumor cell types synthesize large amount of Myc mRNA and MHC class I-like transcripts considered to be non classical.

Download Full-text

Expression of the CD11/CD18 cell surface adhesion glycoprotein family and MHC class II antigen on blood monocytes and alveolar macrophages in interstitial lung diseases

Lung ◽

10.1007/bf00174119 ◽

1992 ◽

Vol 170 (4) ◽

pp. 221-233 ◽

Cited By ~ 8

Author(s):

Henk C. Hoogsteden ◽

Peter Th. W. van Hal ◽

Johanna M. Wijkhuijs ◽

Wim Hop ◽

Chris Hilveringi

Keyword(s):

Cell Surface ◽

Mhc Class Ii ◽

Alveolar Macrophages ◽

Lung Diseases ◽

Class Ii ◽

Interstitial Lung Diseases ◽

Blood Monocytes ◽

Surface Adhesion ◽

Class Ii Antigen ◽

Mhc Class Ii Antigen

Download Full-text

Modulation of Major Histocompatibility Class II Protein Expression by Varicella-Zoster Virus

Journal of Virology ◽

10.1128/jvi.74.4.1900-1907.2000 ◽

2000 ◽

Vol 74 (4) ◽

pp. 1900-1907 ◽

Cited By ~ 94

Author(s):

Allison Abendroth ◽

Barry Slobedman ◽

Eunice Lee ◽

Elizabeth Mellins ◽

Mark Wallace ◽

...

Keyword(s):

Cell Surface ◽

Varicella Zoster Virus ◽

Mhc Class Ii ◽

Class Ii ◽

Northern Blot Hybridization ◽

Major Histocompatibility ◽

Varicella Zoster ◽

Ifn Γ ◽

In Cells

ABSTRACT We sought to investigate the effects of varicella-zoster virus (VZV) infection on gamma interferon (IFN-γ)-stimulated expression of cell surface major histocompatibility complex (MHC) class II molecules on human fibroblasts. IFN-γ treatment induced cell surface MHC class II expression on 60 to 86% of uninfected cells, compared to 20 to 30% of cells which had been infected with VZV prior to the addition of IFN-γ. In contrast, cells that were treated with IFN-γ before VZV infection had profiles of MHC class II expression similar to those of uninfected cell populations. Neither IFN-γ treatment nor VZV infection affected the expression of transferrin receptor (CD71). In situ and Northern blot hybridization of MHC II (MHC class II DR-α) RNA expression in response to IFN-γ stimulation revealed that MHC class II DR-α mRNA accumulated in uninfected cells but not in cells infected with VZV. When skin biopsies of varicella lesions were analyzed by in situ hybridization, MHC class II transcripts were detected in areas around lesions but not in cells that were infected with VZV. VZV infection inhibited the expression of Stat 1α and Jak2 proteins but had little effect on Jak1. Analysis of regulatory events in the IFN-γ signaling pathway showed that VZV infection inhibited transcription of interferon regulatory factor 1 and the MHC class II transactivator. This is the first report that VZV encodes an immunomodulatory function which directly interferes with the IFN-γ signal transduction via the Jak/Stat pathway and enables the virus to inhibit IFN-γ induction of cell surface MHC class II expression. This inhibition of MHC class II expression on VZV-infected cells in vivo may transiently protect cells from CD4+ T-cell immune surveillance, facilitating local virus replication and transmission during the first few days of cutaneous lesion formation.

Download Full-text