Regulation of brush border cortical F-actin by sphingomyelinase

鞘磷脂酶对刷状缘皮质 F-肌动蛋白的调节

• 批准号: 7706653
• 负责人: DAVID E SASLOWSKY
• 金额: $ 8.53万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7706653
• 关键词: Actin-Binding Protein; Actins; Acute; Affect; Agonist; Apical; Award; Binding; Biochemical; Biological; Biological Assay; Brush Border; Cell membrane; Cell model; Cell physiology; Cells; Ceramides; Chimeric Proteins; Cholera Toxin; Cholesterol; Complex; Cytoskeleton; Data; Defect; Down-Regulation; Employee Strikes; Enterocytes; Enzymes; Epithelial Cells; Epithelium; F-Actin; Family; Family member; Ganglioside GM1; Generations; Genetic; Goals; Grant; Guanosine Triphosphate Phosphohydrolases; Human; Hydrolysis; Intestines; Invaded; Lateral; Link; Lipids; Mediating; Membrane; Membrane Lipids; Membrane Microdomains; Membrane Proteins; Microbe; Microfilaments; Molecular; Phenotype; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phosphoproteins; Play; Process; Protein Isoforms; Reaction; Regulation; Reporter; Resistance; Rho-associated kinase; Role; Spatial Distribution; Sphingolipids; Sphingomyelinase; Sphingomyelins; Staging; Stress; Structure; Testing; Transfection; Work; base; brush border membrane; cellular microvillus; design; ezrin; follow-up; in vivo; inhibitor/antagonist; intestinal epithelium; microbial; monolayer; polarized cell; public health relevance; research study; rho; salmonella cholera; small hairpin RNA; small molecule

DESCRIPTION (provided by applicant): The goals of this application are to 1) define the molecular components that mediate the ceramide-induced remodeling of cortical F-actin underlying the apical brush border membrane (BBM) of polarized intestinal epithelia, and 2), test how endogenous sphingomyelinase (SMase) activity at the BBM contributes to the regulation of cytoskeletal dynamics or otherwise affects polarized cellular physiology. Our hypothesis is that acute SMase action at the BBM induces ceramide microdomains that alter the spatial distribution and/or activity of upstream regulators of cortical F-actin structure/function. Such regulation has consequences for pathogenic microbes and microbial products that require F-actin for invasion into host cells. The BBM of intestinal epithelia is highly enriched in sphingomyelin (SM), a sphingolipid that coalesces with cholesterol and other membrane components into microdomains termed lipid rafts, which are linked both structurally and functionally to the actin cytoskeleton. I have discovered that the conversion of SM to ceramide in the BBM of human intestinal epithelia induces a striking reorganization of the cortical actin network. How this occurs is not known. Two complementary approaches are proposed to delineate the mechanism(s) by which ceramide generation in the apical plasma membrane (PM) of polarized epithelia induces structural and functional alterations in the cortical actin cytoskeleton: The first approach will test if SMase treatment impacts F-actin structure and function by altering the localization or turnover of phosphatidylinositol (4,5)-bisphosphate (PIP2, a well-known regulator of actin dynamics). We will examine PIP2 dynamics in vivo using a GFP-PH domain reporter transfected into polarized T84 and Caco2 cell models. The functional requirements of PIP2 for initiating the F-actin phenotype will be investigated by pharmacologically inducing/inhibiting PIP2 accumulation. We will also test if other known regulators of polarized cell cortical actin structure/function are responsible for mediating the observed SMase-induced F-actin phenotype by using small molecule inhibitors and activators. The second approach will explore the role of endogenous SMase in modulating lipid raft and/or cortical actin function at the apical PM of polarized epithelia. I recently found that polarized intestinal T84 cells express an alkaline isoform of SMase (alk-SMase) as an apical ecto-enzyme that uses SM in the outer PM leaflet as substrate under steady-state conditions. Does alk-SMase play a role in modulating BBM lipid raft function including the linkage of these microdomains to cortical F-actin? Initial experiments show that the genetically-tractable Caco2 and MDCK polarized cell models also express alk-SMase, so we will silence alk-SMase expression in these cells by shRNA and assay for alterations or defects in the structure and function both of lipid raft microdomains and the F-actin cytoskeleton. PUBLIC HEALTH RELEVANCE: The proposed experiments in this grant will explore how a certain class of lipid-degrading enzyme (sphingomyelinase) affects host intestinal cell physiology. This is particularly relevant to the intestine as numerous bacterial species inhabiting the human gut also make and secrete this class of enzyme. This work is important because it will increase our understanding of how the regulation of lipids in the membranes of human intestinal cells impacts the machinery that is co-opted by pathogenic microbes and microbial products to invade host cells.

描述(由申请人提供)： 这项应用的目标是1)确定介导神经酰胺诱导的极化肠上皮顶端刷状缘膜(BBM)下皮质F-肌动蛋白重塑的分子成分，以及2)测试BBM的内源性鞘磷脂酶(SMase)活性如何有助于调节细胞骨架动力学或以其他方式影响极化细胞生理。我们的假设是，BBM处的急性SMase作用诱导神经酰胺微域，从而改变皮质F-肌动蛋白结构/功能上游调节因子的空间分布和/或活性。这种调控会对病原微生物和需要F-肌动蛋白侵入宿主细胞的微生物产品产生影响。肠上皮的BBM高度富含鞘磷脂(SM)，这是一种鞘磷脂，它与胆固醇和其他膜成分结合成微域，称为脂筏，在结构和功能上都与肌动蛋白细胞骨架相连。我发现，在人类肠上皮细胞的BBM中，SM转化为神经酰胺会导致皮质肌动蛋白网络的显着重组。目前尚不清楚这种情况是如何发生的。两种互补的方法被用来描述极化上皮顶端质膜神经酰胺的产生引起皮质肌动蛋白细胞骨架结构和功能改变的机制(S)：第一种方法将测试SMASE处理是否通过改变磷脂酰肌醇(4，5)-二磷酸(PIP2，一种众所周知的肌动蛋白动态调节因子)的定位或周转来影响F-肌动蛋白的结构和功能。我们将使用GFP-PH结构域报告基因转染极化的T84和Caco 2细胞模型，在体内检测PIP2的动力学。PIP2启动F-肌动蛋白表型的功能需求将通过药物诱导/抑制PIP2积累来研究。我们还将测试是否有其他已知的极化细胞皮质肌动蛋白结构/功能调节因子通过使用小分子抑制剂和激活剂来调节观察到的sMase诱导的F-肌动蛋白表型。第二种方法将探索内源性sMase在调节极化上皮细胞顶端PM的脂筏和/或皮质肌动蛋白功能中的作用。我最近发现，极化的肠道T84细胞表达一种碱性的sMase亚型(ALK-sMase)，作为一种顶端胞外酶，在稳态条件下使用外PM叶中的SM作为底物。ALK-sMase是否在调节BBM脂筏功能中发挥作用，包括这些微域与皮质F-肌动蛋白的连接？初步实验表明，遗传易感性的Caco 2和MDCK极化细胞模型也表达ALK-sMase，因此我们将通过shRNA和检测脂筏微区和F-肌动蛋白细胞骨架的结构和功能的改变或缺陷来沉默ALK-sMase在这些细胞中的表达。 与公共卫生相关：这项拨款中提议的实验将探索某种脂肪降解酶(鞘磷脂酶)如何影响宿主肠道细胞的生理。这与肠道特别相关，因为人体肠道中的许多细菌也会制造和分泌这种酶。这项工作很重要，因为它将增加我们对人类肠道细胞膜中脂质的调节如何影响病原微生物和微生物产品入侵宿主细胞的机制的理解。