Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2001 Aug 6;154(3):645-58.
doi: 10.1083/jcb.200104049.

High resolution mapping of mast cell membranes reveals primary and secondary domains of Fc(epsilon)RI and LAT

B S Wilson  1 , J R Pfeiffer, Z Surviladze, E A Gaudet, J M Oliver
Affiliations

High resolution mapping of mast cell membranes reveals primary and secondary domains of Fc(epsilon)RI and LAT

B S Wilson et al. J Cell Biol. .

Abstract

In mast cells, cross-linking the high-affinity IgE receptor (Fc(epsilon)RI) initiates the Lyn-mediated phosphorylation of receptor ITAMs, forming phospho-ITAM binding sites for Syk. Previous immunogold labeling of membrane sheets showed that resting Fc(epsilon)RI colocalize loosely with Lyn, whereas cross-linked Fc(epsilon)RI redistribute into specialized domains (osmiophilic patches) that exclude Lyn, accumulate Syk, and are often bordered by coated pits. Here, the distribution of Fc(epsilon)RI beta is mapped relative to linker for activation of T cells (LAT), Grb2-binding protein 2 (Gab2), two PLCgamma isoforms, and the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase), all implicated in the remodeling of membrane inositol phospholipids. Before activation, PLCgamma1 and Gab2 are not strongly membrane associated, LAT occurs in small membrane clusters separate from receptor, and PLCgamma2, that coprecipitates with LAT, occurs in clusters and along cytoskeletal cables. After activation, PLCgamma2, Gab2, and a portion of p85 colocalize with Fc(epsilon)RI beta in osmiophilic patches. LAT clusters enlarge within 30 s of receptor activation, forming elongated complexes that can intersect osmiophilic patches without mixing. PLCgamma1 and another portion of p85 associate preferentially with activated LAT. Supporting multiple distributions of PI3-kinase, Fc(epsilon)RI cross-linking increases PI3-kinase activity in anti-LAT, anti-Fc(epsilon)RIbeta, and anti-Gab2 immune complexes. We propose that activated mast cells propagate signals from primary domains organized around Fc(epsilon)RIbeta and from secondary domains, including one organized around LAT.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
PLCγ2 redistributes to FcεRI-rich osmiophilic patches in activated mast cells. Membrane sheets were prepared from RBL-2H3 cells before (A) or after (B and C) cross-linking the FcεRI with DNP-BSA. Sheets were double- labeled with anti-FcεRI β monoclonal antibody conjugated to 10-nm anti–mouse gold particles and anti-PLCγ2 antibody conjugated to 5-nm anti–rabbit gold particles. (A) 10-nm gold particles marking FcεRI are distributed in small clusters and singlets (boxed regions) that do not mix substantially with small clusters of 5-nm gold particles marking PLCγ2 on the membrane (circled regions) and on cytoskeletal cables (arrowheads). (B and C) 5-nm gold particles marking PLCγ2 (arrows) colocalize with 10-nm gold particles specific for FcεRI in osmiophilic membrane patches (boxed regions). *Marks a coated vesicle budding from the patch in B. PLCγ2 label also persists along cytoskeletal cables in activated cells (arrowheads). Bar, 0.1 μm.
Figure 2.
Figure 2.
Actin and myosin are components of the cytoskeletal fibers that associate with membrane sheets, coprecipitate with PLCγ2, and contribute to PLCγ2 activation in suspension cells. Membrane sheets were prepared from untreated RBL-2H3 cells and labeled with 5-nm antimyosin gold particles (A and B) and either 15- (B) or 5-nm (C) avidin-gold conjugated to biotinylated phalloidin to label filamentous actin. Arrowheads (A and B) point to examples of myosin label, typically found at branchpoints in the membrane-associated cytoskeletal meshwork. Arrows (B and C) point to examples of gold particles for actin that decorates, but does not uniformly label, the cytoskeletal cables. Coated vesicles (B and C) are marked (*). Bars, 0.1 μm. (D) PLCγ2 and myosin immune complexes were prepared from pooled fractions of sucrose density gradients, separated by SDS-PAGE, and probed for actin or PLCγ2.
Figure 3.
Figure 3.
PLCγ1 membrane label increases after FcεRI activation, mostly outside of osmiophilic patches. RBL-2H3 membrane sheets were labeled with 5-nm anti-PLCγ1 gold particles and with 10 nm anti-FcεRI β gold particles. In resting cells (A), there are very few 5-nm gold particles marking PLCγ1 (circled regions) and no FcεRI β-PLCγ1 colocalization. In stimulated cells (B), there is an increase in 5-nm gold particles marking PLCγ1 (circled regions), but very few of these particles colocalize with 10-nm gold marking FcεRI in osmiophilic patches (boxed regions). Bar, 0.1 μm.
Figure 4.
Figure 4.
PI3-kinase label colocalizes both with FcεRI in osmiophilic patches and with PLCγ1 outside of these patches in activated cells. Membrane sheets were prepared from RBL-2H3 cells before (A) or after (B and C) FcεRI cross-linking. (A and B) The sheets were labeled with 5-nm anti-p85 gold particles and with 10-nm anti-FcεRI β gold particles. Resting cells have relatively little membrane-bound PI3-kinase (A, circled region). 5-nm gold particles marking p85 are strikingly colocalized with FcεRI β within the osmiophilic patches (boxed regions) of activated cells (B). (C) Sheets were labeled with 5-nm anti-PLCγ1 gold and 10-nm anti-p85 gold. PLCγ1 and p85 colocalize in activated cells in mixed clusters outside of osmiophilic patches (circled regions). Bar, 0.1 μm.
Figure 10.
Figure 10.
Biochemical analyses of protein–protein interactions in activated mast cells. (A) Coprecipitation studies demonstrate associations between signaling proteins. Resting and activated RBL-2H3 cells were solubilized in 0.5% Triton X-100, and the clarified cell lysates were used for immunoprecipitation afterwards by immunoblotting as indicated. Results show constitutive associations of p85 with LAT, Gab2, and FcεRI in adherent cells. They also show constitutive association of PLCγ2 with LAT and an inducible association of PLCγ1 with LAT. (B) Analysis of proteins distributed across sucrose density fractions. RBL-2H3 cells were solubilized in 0.05% Triton X-100 before (left) or after (right) cross-linking the FcεRI for 2 min with DNP-BSA. Cell lysates were loaded onto an 80% sucrose cushion, followed by layers of 35, 25, 20%, 15, and 10% sucrose. After ultracentrifugation, fractions were collected from the top (fraction 1) to the bottom of the gradient (from the lightest to heaviest fractions). Aliquots of the fractions were solubilized in Laemmli buffer, and proteins were resolved by SDS-PAGE, followed by immunoblot analysis as indicated in the key. Results show very little correlation with the protein–protein interactions observed by microscopy.
Figure 5.
Figure 5.
Receptor cross-linking leads to increases in PI3-kinase activity associated with FcεRI, Gab2, and LAT. Immune complexes were prepared from detergent lysates of RBL-2H3 cells before or after 2 min activation with DNP-BSA. To measure associated PI3- kinase activity, immune complexes were incubated with PtdIns and γ[32P]ATP for 30 min at 37°C. Lipids in the reaction were extracted and resolved by TLC, and formation of PtdIns(3)P measured by PhosphorImager analysis. Background levels were established by adding PI3-kinase inhibitors (10 nM Wortmannin or 10 μM LY2940002) to replicate samples 15 min before initiating the reaction. As shown in A–C, there is an increase in PI3-kinase activity associated with FcεRI, Gab2, and LAT after 2 min of activation. Data are representative of at least three separate experiments, each performed in duplicate, and error bars represent SEM. Units are arbitary PhosphorImager values per 1,000. A one-sample t test was performed using Prism software (Graph Pad); all results have significant p-values (<0.03). (D) Shows the comparative levels of PI3-kinase activity in FcεRI, Gab2, and LAT precipitates, relative to total PI3-kinase activity in p85 immune complexes prepared from replicate lysates of antigen-stimulated cells. Data for Gab2 is from seven separate experiments (p-value = 0.00005); data for LAT (p-value = 0.0004) and FcεRI (p-value = 0.0288) are for three separate experiments.
Figure 6.
Figure 6.
Gab2 colocalizes with FcεRI in signaling patches in activated mast cells. Membrane sheets were prepared from RBL-2H3 cells before (A) or after (B) cross-linking the FcεRI for 2 min with DNP-BSA. Membranes were labeled with 5-nm anti-Gab2 gold particles and with 10-nm anti-FcεRI β gold particles. (A) Shows that resting membranes have rather few 5-nm gold particles marking Gab2 (circled regions) and only occasional FcεRI β–Gab2 colocalization (boxed region). (B) Gold particles marking Gab2 (arrows) are strikingly colocalized with FcεRI β in osmiophilic patches of activated cells (boxed regions). Bar, 0.1 μm.
Figure 7.
Figure 7.
LAT rafts are larger after FcεRI cross-linking and can intersect osmiophilic patches. Membrane sheets prepared before (A) or after (B-E) cross-linking the FcεRI for 2 min with DNP-BSA were labeled with 5-nm gold particles for LAT and 10 nm gold particles for FcεRI β. (A) Shows that the numerous clusters of LAT (circled regions) and FcεRI (boxed regions) rarely colocalize in resting membranes. (B–E) Osmiophilic patches that label with FcεRI after 2 min activation are boxed and LAT clusters are outlined. LAT clusters in activated cells are often large (>50 particles). Bar, 0.1 μm.
Figure 8.
Figure 8.
FcεRI cross-linking leads to rapid increases in LAT cluster size. (A) Summary data representing LAT cluster sizes in resting cells (hatched bars) or cells stimulated for 2 min with antigen (solid bars) for four independent experiments. A minimum of 3,000 gold particles were counted for each experimental condition. (B–E) Gold particles marking LAT were scored for cluster size in membrane sheets prepared from resting cells (B) or cells stimulated for 30 s (C), 1 min (D), or 2 min (E) with DNP-BSA. Counts are taken from one of two similar experiments.
Figure 9.
Figure 9.
LAT topography in relation to the distributions of PI3-kinase and PLCγ1. Membrane sheets were prepared from antigen-stimulated RBL-2H3 cells and labeled with 5-nm anti-LAT gold particles (A–C) and with 10-nm gold particles specific for either the p85 regulatory subunit of PI3-kinase (A) or PLCγ1 (B and C). (A) Examples of LAT-PI3- kinase proximity are marked by triangles. Note that several large LAT rafts (circled regions) in an osmiophilic patch in the lower right do not mix extensively with nearby p85 clusters. (B and C) Examples of LAT-PLCγ1 colocalization are marked with triangles; separate LAT rafts are outlined. Note that colocalization is principally outside of osmiophilic patches. Bar, 0.1 μm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Anderson, R.G.W. 1998. The caveolae membrane system. Ann. Rev. Biochem. 67:199–225. - PubMed
    1. Barker, S.A., K.K. Caldwell, J.R. Pfeiffer, and B.S. Wilson. 1998. Wortmannin-sensitive phosphorylation, translocation and activation of PLCγ1, but not PLCγ2, in antigen-stimulated RBL-2H3 mast cells. Mol. Biol. Cell. 9:483–496. - PMC - PubMed
    1. Barker, S.A., D. Lujan, and B.S. Wilson. 1999. Multiple roles for PI3-kinase in the regulation of PLCγ activity and Ca2+ mobilization in antigen-stimulated mast cells. J. Leukoc. Biol. 65:321–329. - PubMed
    1. Berlin, R.D., J.M. Oliver, T.E. Ukena, and H.H. Yin. 1974. Control of cell surface topography. Nature. 247:45–46. - PubMed
    1. Berlin, R.D., J.M. Oliver, T.E. Ukena, and H.H. Yin. 1975. The cell surface. New Engl. J. Med. 292:515–520. - PubMed

Publication types

MeSH terms

-