Functional roles of stimulated recycling endosome exocytosis in mast cells

肥大细胞中刺激再循环内体胞吐作用的功能作用

基本信息

批准号：
8123286
负责人：
Joshua D Wilson
金额：
$ 4.84万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-26 至 2013-07-25
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8123286
关键词：
Affect Affinity Allergens Allergic Antibodies Antigens Binding Biological Assay Cell membrane Cell physiology Cell surface Cells Cellular biology Cholera Toxin Complex Data Disease Dominant-Negative Mutation Dynamin 2 Endocytosis Endosomes Event Exocytosis Exocytosis Inhibition Fc Receptor GPI Membrane Anchors Ganglioside GM1 Genetic Goals Green Fluorescent Proteins Guanosine Triphosphate Phosphohydrolases Health Hematopoietic Histamine Release Hypersensitivity IgE Immune response Immunology Individual Inflammation Intervention Label Ligands Measurement Mediating Mediator of activation protein Membrane Fusion Membrane Protein Traffic Methods Molecular Monitor Monomeric GTP-Binding Proteins Pattern Phagocytosis Process Protein Secretion Proteins Recycling Regulation Research Role SNAP receptor Secretory Vesicles Seminal Signal Transduction Site Small Interfering RNA Surface Therapeutic Work allergic response cellubrevin chemical release crosslink cytokine endosome membrane imaging modality inhibitor/antagonist knock-down mast cell membrane flux novel protein expression public health relevance release of sequestered calcium ion into cytoplasm research study response spatial relationship trafficking

项目摘要

DESCRIPTION (provided by applicant): The allergic immune response is initiated by crosslinking of immunoglobulin E (IgE) antibodies bound to receptors (Fc?RI) on the surface of mast cells, stimulating a signaling cascade that results in exocytosis of secretory granules (degranulation) to release chemical mediators of allergy and inflammation. IgE crosslinking on mast cells also stimulates exocytosis of recycling endosomes (REs) in a process that, like degranulation, depends on calcium mobilization. The long term goal of this research is to determine the roles of RE exocytosis in mast cell function. To this end: we will define protein components required for antigen-stimulated RE exocytosis; we will develop a imaging method to visualize this process directly; and we will characterize the relationship of this process to other events activated by antigen-mediated IgE crosslinking, including phagocytosis and cytokine secretion. A key objective in this work is to identify proteins important for stimulated RE exocytosis in mast cells, and likely candidates emergent from previous studies will be examined, including small GTPases and SNARE proteins. Both siRNA-mediated knockdown and dominant negative protein expression will be used to assess the roles of specific proteins in RE exocytosis in a novel assay that follows the outward trafficking of green fluorescent protein-labeled glycosylphosphatidylinositol-anchored protein (GFP-GPI). Subsequent studies will use genetic inhibition of RE exocytosis to examine its roles in antigen-mediated phagocytosis and cytokine secretion. In addition, a photoactivatable form of GFP-GPI will be constructed and used to visualize stimulated RE membrane trafficking and fusion in individual cells. Microfabricated arrays of ligands will be used to characterize the spatial relationship between RE exocytosis and activation via IgE-Fc?RI that are concentrated in defined patterns. The proposed studies will advance our understanding of the functional roles of recycling endosomes in mast cells and, in general, of stimulated membrane trafficking in cell biology and immunology. PUBLIC HEALTH RELEVANCE: This research plan is directed towards a molecular understanding of the coordination of functional responses stimulated by allergens during mast cell activation. Elucidation of mast cell mechanisms that operate in inflammation and allergic responses will provide new opportunities for intervention in therapeutic applications and a clearer understanding of hematopoietic cell biology that is seminal to regulation of immune responses in health and disease.

描述（由申请人提供）：过敏性免疫反应是通过与肥大细胞表面受体（FcRI）结合的免疫球蛋白 E（IgE）抗体交联引发的，刺激信号级联，导致分泌颗粒的胞吐作用（脱颗粒））释放过敏和炎症的化学介质。肥大细胞上的 IgE 交联也会刺激回收内体 (RE) 的胞吐作用，这一过程与脱粒一样依赖于钙动员。本研究的长期目标是确定 RE 胞吐作用在肥大细胞功能中的作用。为此：我们将定义抗原刺激的RE胞吐作用所需的蛋白质成分；我们将开发一种成像方法来直接可视化这一过程；我们将描述这一过程与抗原介导的 IgE 交联激活的其他事件的关系，包括吞噬作用和细胞因子分泌。这项工作的一个关键目标是鉴定对肥大细胞中刺激 RE 胞吐作用重要的蛋白质，并且将对先前研究中出现的可能候选蛋白进行检查，包括小 GTP 酶和 SNARE 蛋白。 siRNA 介导的敲低和显性失活蛋白表达将用于评估特定蛋白在 RE 胞吐作用中的作用，该实验遵循绿色荧光蛋白标记的糖基磷脂酰肌醇锚定蛋白 (GFP-GPI) 的向外运输。后续研究将利用 RE 胞吐作用的基因抑制来检查其在抗原介导的吞噬作用和细胞因子分泌中的作用。此外，还将构建可光激活形式的 GFP-GPI，并用于可视化单个细胞中受刺激的 RE 膜运输和融合。微加工的配体阵列将用于表征 RE 胞吐作用和通过 IgE-Fc?RI 激活之间的空间关系，这些 IgE-Fc?RI 集中在定义的模式中。拟议的研究将增进我们对肥大细胞中回收内体的功能作用的理解，以及一般情况下对细胞生物学和免疫学中刺激膜运输的理解。公共健康相关性：该研究计划旨在从分子角度理解肥大细胞激活过程中过敏原刺激的功能反应的协调。阐明在炎症和过敏反应中起作用的肥大细胞机制将为干预治疗应用提供新的机会，并更清楚地了解造血细胞生物学，这对健康和疾病中免疫反应的调节具有重要意义。