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Visualizing lipid raft dynamics and early signaling events during antigen receptor-mediated B-lymphocyte activation.

ABSTRACT: Recent biochemical evidence indicates that an early event in signal transduction by the B-cell antigen receptor (BCR) is its translocation to specialized membrane subdomains known as lipid rafts. We have taken a microscopic approach to image lipid rafts and early events associated with BCR signal transduction. Lipid rafts were visualized on primary splenic B lymphocytes from wild-type or anti-hen egg lysozyme BCR transgenic mice, and on a mature mouse B-cell line Bal 17 by using fluorescent conjugates of cholera toxin B subunit or a Lyn-based chimeric protein, which targets green fluorescent protein to the lipid raft compartment. Time-lapse imaging of B cells stimulated via the BCR with the antigen hen egg lysozyme, or surrogate for antigen anti-IgM, demonstrated that lipid rafts are highly dynamic entities, which move laterally on the surface of these cells and coalesce into large regions. These regions of aggregated lipid rafts colocalized with the BCR and tyrosine-phosphorylated proteins. Microscopic imaging of live B cells also revealed an inducible colocalization of lipid rafts with the tyrosine kinase Syk and the receptor tyrosine phosphatase CD45. These two proteins play indispensable roles in BCR-mediated signaling but are not detectable in biochemically purified lipid raft fractions. Strikingly, BCR stimulation also induced the formation of long, thread-like filopodial projections, similar to previously described structures called cytonemes. These B-cell cytonemes are rich in lipid rafts and actin filaments, suggesting that they might play a role in long-range communication and/or transportation of signaling molecules during an immune response. These results provide a window into the morphological and molecular organization of the B-cell membrane during the early phase of BCR signaling.

SUBMITTER: Gupta N

PROVIDER: S-EPMC149983 | biostudies-literature | 2003 Feb

REPOSITORIES: biostudies-literature

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Visualizing lipid raft dynamics and early signaling events during antigen receptor-mediated B-lymphocyte activation.

Gupta Neetu N DeFranco Anthony L AL

Molecular biology of the cell 20030201 2

Recent biochemical evidence indicates that an early event in signal transduction by the B-cell antigen receptor (BCR) is its translocation to specialized membrane subdomains known as lipid rafts. We have taken a microscopic approach to image lipid rafts and early events associated with BCR signal transduction. Lipid rafts were visualized on primary splenic B lymphocytes from wild-type or anti-hen egg lysozyme BCR transgenic mice, and on a mature mouse B-cell line Bal 17 by using fluorescent conj ...[more]

PMID: 12589045

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Project description:Glucocorticoid has been widely accepted to induce Alzheimer's disease, but the nongenomic effect of glucocorticoid on amyloid β (Aβ) generation has yet to be studied. Here, we investigated the effect of the nongenomic pathway induced by glucocorticoid on amyloid precursor protein processing enzymes as well as Aβ production using male ICR mice and human neuroblastoma SK-N-MC cells. Mice groups exposed to restraint stress or intracerebroventricular injection of Aβ showed impaired cognition, decreased intracellular glucocorticoid receptor (GR) level, but elevated level of membrane GR (mGR). In this respect, we identified the mGR-dependent pathway evoked by glucocorticoid using impermeable cortisol conjugated to BSA (cortisol-BSA) on SK-N-MC cells. Cortisol-BSA augmented the expression of β-site amyloid precursor protein cleaving enzyme 1 (BACE1), the level of C-terminal fragment β of amyloid precursor protein (C99) and Aβ production, which were maintained even after blocking intracellular GR. We also found that cortisol-BSA enhanced the interaction between mGR and Gαs, which colocalized in the lipid raft. The subsequently activated CREB by cortisol-BSA bound to the CRE site of the BACE1 promoter increasing its expression, which was downregulated by inhibiting CBP. Consistently, blocking CBP attenuated cognitive impairment and Aβ production induced by corticosterone treatment or intracerebroventricular injection of Aβ more efficiently than inhibiting intracellular GR in mice. In conclusion, glucocorticoid couples mGR with Gαs and triggers cAMP-PKA-CREB axis dependent on the lipid raft to stimulate BACE1 upregulation and Aβ generation.SIGNIFICANCE STATEMENT Patients with Alzheimer's disease (AD) have been growing sharply and stress is considered as the major environment factor of AD. Glucocorticoid is the primarily responsive factor to stress and is widely known to induce AD. However, most AD patients usually have impaired genomic pathway of glucocorticoid due to intracellular glucocorticoid receptor deficiency. In this respect, the genomic mechanism of glucocorticoid faces difficulties in explaining the consistent amyloid β (Aβ) production. Therefore, it is necessary to investigate the novel pathway of glucocorticoid on Aβ generation to find a more selective therapeutic approach to AD patients. In this study, we revealed the importance of nongenomic pathway induced by glucocorticoid where membrane glucocorticoid receptor plays an important role in Aβ formation.

Project description:Background: Image-based screening improves the detection of early-stage lung adenocarcinoma (LUAD)but also highlights the issue of high false-positive diagnoses, which puts patients at a risk of unnecessary over-treatment. Therefore, more precise discrimination criteria are required to ensure that patients with early-stage LUAD receive appropriate treatments. Methods: we integrated 158 early-stage LUAD cases from 2 independent cohorts, including 30 matched resected specimens with complete radiological and pathological information, and 128 retrospective pathological pair-samples with partial follow-up data. This integration allowed us to conduct a correlation analysis between clinical phenotype and transcriptome landscape. Immunohistochemistry was performed using tissue microarrays to examine the expression of phospholipid phosphatase 2 (PLPP2) and lipid-raft markers. Lipidomics analysis was used to determine the changes of lipid components in PLPP2-overexpressed cells. To assess the effects of PLPP2 on the malignant phenotypes of LUAD cells, we conducted mice tumor-bearing experiments and in vitro cellular experiments by knocking down PLPP2 and inhibiting lipid raft synthesis with MβCD, respectively. Results: Bioinformatics analysis indicated that the co-occurrence of lipid raft formation and rapid cell proliferation might exhibit synergistic effects in driving oncogenesis from lung preneoplasia to adenocarcinoma. The enhanced activation of the cell cycle promoted the transition from non-invasive to invasive status in early-stage LUAD, which was related to an increase in lipid rafts within LUAD cells. PLPP2 participated in lipid raft formation by altering the component contents of lipid rafts, such as esters, sphingomyelin, and sphingosine. Furthermore, elevated PLPP2 levels were identified as an independent prognostic risk factor for LUAD patients. Further results from in vivo and in vitro experiments confirmed that PLPP2 could induce excessive cell proliferation by enhancing lipid raft formation in LUAD cells. Conclusions: Our study has revealed the characteristics of gene expression profiles in early-stage LUAD patients with the different radiological and pathological subtypes, as well as deciphered transcriptomic evolution trajectory from preneoplasia to invasive LUAD. Furthermore, it suggests that PLPP2-mediated lipid raft synthesis may be a significant biological event in the initiation of early-stage LUAD, offering a potential target for more precise diagnosis and therapy in clinical settings.

Dataset Information

Visualizing lipid raft dynamics and early signaling events during antigen receptor-mediated B-lymphocyte activation.

Publications

Visualizing lipid raft dynamics and early signaling events during antigen receptor-mediated B-lymphocyte activation.

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