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.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

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196429&req=5

fig9: 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.

Mentions: Because PLCγ1 also redistributes on activated cells to membrane ruffles (Barker et al., 1998) and is dependent on D-3 phosphophoinositides for membrane recruitment, tyrosine phosphorylation and activation (Barker et al., 1998, 1999), it seemed likely that LAT might nucleate secondary signaling domains that include PLCγ1 and PI3-kinase. Analyses of membrane sheets double labeled for p85 and LAT support this hypothesis. Fig. 9 A shows the intersection, but not mixing, of large LAT clusters with p85 in a particularly dramatic osmiophilic patch after 2 min of antigen stimulation. There are also gold particles marking LAT away from the osmiophilic patches. These LAT clusters are larger than those on resting membranes and are strongly colocalized with p85 (Fig. 9 A, triangle regions).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

fig9: 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.
Mentions: Because PLCγ1 also redistributes on activated cells to membrane ruffles (Barker et al., 1998) and is dependent on D-3 phosphophoinositides for membrane recruitment, tyrosine phosphorylation and activation (Barker et al., 1998, 1999), it seemed likely that LAT might nucleate secondary signaling domains that include PLCγ1 and PI3-kinase. Analyses of membrane sheets double labeled for p85 and LAT support this hypothesis. Fig. 9 A shows the intersection, but not mixing, of large LAT clusters with p85 in a particularly dramatic osmiophilic patch after 2 min of antigen stimulation. There are also gold particles marking LAT away from the osmiophilic patches. These LAT clusters are larger than those on resting membranes and are strongly colocalized with p85 (Fig. 9 A, triangle regions).

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