Limits...
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.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.

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

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196429&req=5

fig10: 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.

Mentions: The p85 subunit of PI3-kinase can be detected in FcεRI immunoprecipitates (see Fig. 10 A); anti-FcεRI β immune complexes from activated cells have ∼3× as much PI3-kinase activity as anti-FcεRI β immune complexes from resting cells (Fig. 5 A). The specificity of the assay was confirmed by use of the PI3-kinase inhibitor, Ly294002. Relative to the levels of activity in anti-p85 immunoprecipitates, this represents <5% of total PI3-kinase activity (Fig. 5 D). The results suggest that a small but significant fraction of p85 forms detergent-stable complexes with FcεRI β, which becomes activated after FcεRI phosphorylation and movement into osmiophilic patches.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

fig10: 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.
Mentions: The p85 subunit of PI3-kinase can be detected in FcεRI immunoprecipitates (see Fig. 10 A); anti-FcεRI β immune complexes from activated cells have ∼3× as much PI3-kinase activity as anti-FcεRI β immune complexes from resting cells (Fig. 5 A). The specificity of the assay was confirmed by use of the PI3-kinase inhibitor, Ly294002. Relative to the levels of activity in anti-p85 immunoprecipitates, this represents <5% of total PI3-kinase activity (Fig. 5 D). The results suggest that a small but significant fraction of p85 forms detergent-stable complexes with FcεRI β, which becomes activated after FcεRI phosphorylation and movement into osmiophilic patches.

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.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.

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