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

Wilson BS, Pfeiffer JR, Surviladze Z, Gaudet EA, Oliver JM - J. Cell Biol. (2001)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Cancer Research and Treatment Center, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA. bwilson@salud.unm.edu

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.

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Related in: MedlinePlus

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.
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fig3: 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.

Mentions: In contrast to PLCγ2, very little PLCγ1, the minor isoform of PLCγ in RBL-2H3 cells, was bound to the membrane of unstimulated mast cells (Fig. 3 A; Table I).


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

Wilson BS, Pfeiffer JR, Surviladze Z, Gaudet EA, Oliver JM - J. Cell Biol. (2001)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196429&req=5

fig3: 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.
Mentions: In contrast to PLCγ2, very little PLCγ1, the minor isoform of PLCγ in RBL-2H3 cells, was bound to the membrane of unstimulated mast cells (Fig. 3 A; Table I).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Cancer Research and Treatment Center, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA. bwilson@salud.unm.edu

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.

Show MeSH
Related in: MedlinePlus