Mechanisms of luminal protein targeting in kidney and hepatic epithelial cells

管腔蛋白靶向肾和肝上皮细胞的机制

• 批准号: 8238756
• 负责人: ANNE MUESCH
• 金额: $ 49.88万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8238756
• 关键词: Ablation; Address; Air; Apical; Bile fluid; Biological Assay; Biotin; Blocking Antibodies; Cell Line; Cell membrane; Cells; Cellular biology; Characteristics; Cholestasis; Chronic; Color; Columnar Cell; Columnar Epithelium; Complementary DNA; Cytomegalovirus Infections; Data; Ensure; Epithelial; Epithelial Cells; Epithelium; Gases; Gatekeeping; Golgi Apparatus; Hepatic; Hepatocyte; Image; Imaging technology; Inflammation; Intestines; Ions; Kidney; Lateral; Life; Liver; Lung; MDCK cell; Malignant Neoplasms; Mammary gland; Mediating; Membrane; Membrane Proteins; Microinjections; Milk; Modeling; Molecular; Molecular Models; Molecular Target; Nutrient; Organ; Pathology; Pathway interactions; Phenotype; Phosphotransferases; Play; Protein-Serine-Threonine Kinases; Proteins; Protocols documentation; Resolution; Role; Route; Site; Sorting - Cell Movement; Surface; Testing; Thick; Tissues; Tube; Urine; Vesicle; Waste Products; base; cell type; cellular microvillus; comparative; fluorescence imaging; innovation; kidney epithelial cell; mathematical model; molecular modeling; novel; novel strategies; protein transport; tool; trafficking; trans-Golgi Network; transcytosis

DESCRIPTION (provided by applicant): Mechanisms of luminal protein targeting in kidney and hepatic epithelial cells Columnar epithelia (e.g. kidney, intestine) and hepatocytes embody the two major organizational phenotypes of non-stratified epithelial cells. They differ morphologically in that columnar epithelia establish their apical domain (AP) and basolateral domains (BL) at opposing poles whereas hepatocytes establish their AP domain, the bile caliculi (BC) in the midst of their BL domains. They also differ drastically in how they establish and maintain their surface domains. Whereas columnar epithelia target their plasma membrane (PM) proteins predominantly from the trans Golgi network (TGN) to the AP domain, hepatocytes target both AP and BL PM proteins from the TGN to the BL surface, from where AP proteins are sorted to BC by transcytosis. Although important progress has been made in understanding the trafficking routes, sorting compartments, and targeting mechanisms of columnar cells, particularly the kidney epithelial cell line MDCK, the trafficking routes, and sorting compartments and mechanisms of hepatocytes remain poorly understood. This is in large part due to the fact that hepatic cell lines are not amenable to trafficking studies with the classical biotin based targeting assays utilized in MDCK cells and are distinctly more difficult to transfect. We propose innovative experimental approaches to identify the trafficking itinerary, subcellular compartments and molecular machinery involved in the trafficking of model AP PM proteins in hepatic WIFB cells, comparatively with MDCK cells. Our hypotheses are: (i) novel imaging technologies and (ii) analysis of the post-exocytic fate of AP proteins provide enough resolution to understand the different sorting and trafficking strategies of hepatocytes and columnar cells and (iii) the serine/threonine kinase Par1b acts as a molecular switch between the direct and transcytotic pathways. We propose the following Specific Aims: Aim 1: To quantitatively assess the extent of vectorial and transcytotic delivery of model apical PM proteins in WIFB and MDCK cells. Aim 2: To define comparatively the site of exocytic sorting of AP and BL markers in WIFB and MDCK cells. Aim 3: To identify the machinery used by AP PM proteins to exit the TGN in WIFB and MDCK cells. With our novel approaches we will set the framework for a long overdue comparative molecular model of protein targeting in hepatic versus columnar cells. This is a question of high priority for epithelial polarity that has remained unanswered for more than 25 years. PUBLIC HEALTH RELEVANCE: Epithelia are tissues of tightly adherent cells that enclose all organs and thus separate and protect them from the surrounding milieu. Yet far from putting up a passive protective wall, epithelial layers act as sophisticated gatekeepers that mediate the controlled traffic of molecules such as gas, ions, nutrients and waste products in and out of the organs they surround, often against a concentration gradient. To fulfill this function, epithelia form polarized membrane domains that restrict the transporters and gated channels responsible for the trafficking of molecules across the cell membrane to either the "inside" or "outside" facing surfaces of the epithelia, thus ensuring the vectorial transport of molecules. Most epithelia, such as those of the kidney, intestinal tract, lungs or mammary gland organize in two cells layers to enclose a central lumen, forming the tubes, ascini or aveoli through which urine, nutrients, milk or air are transported. The surface domain facing the lumen is called apical or luminal surface. Liver epithelia, by contrast, organize in one-cell thick plates and carve out a lumen, the bile canalicular network, by interrupting the lateral domains of neighboring cells. Loss or disorganization of epithelial luminal sufaces is a feature of many cancers. It is also hallmark of other pathologies such as microvilli inclusion disease and chronic inflammation of the liver (cholestasis). Thus, understanding how luminal surfaces are formed and maintained is one of the central questions in epithelial cell biology. This proposal addresses one important aspect of lumen formation. That is: How do kidney and liver epithelial cells target membrane proteins specifically to their luminal domains? It is known that both cell types utilize radically different strategies for luminal proteins targeting, but the molecular mechanisms that distinguish them are not known. We have developed novel tools and approaches to elucidate key features of the luminal protein trafficking machinery characteristic of the two major epithelial phenotypes represented by the kidney and the liver.

描述(由申请人提供)：在肾和肝上皮细胞(如肾、肠)和肝细胞中的鲁米那蛋白靶向机制体现了非复层上皮细胞的两种主要组织表型。它们在形态上的不同之处在于，柱状上皮在相对的两极建立其顶域(AP)和基底外侧域(BL)，而肝细胞则在其BL域的中间建立其AP域，即胆小管(BC)。它们在建立和维护其表面域的方式上也有很大的不同。而柱状上皮主要将质膜(PM)蛋白从跨高尔基网络(TGN)定位到AP结构域，而肝细胞则将AP和BL PM蛋白从TGN定位到BL表面，AP蛋白从那里通过跨细胞作用被分类到BC。尽管对柱状细胞，特别是肾上皮细胞系MDCK的转运途径、分选间隔和靶向机制的了解已经取得了重要进展，但对肝细胞的转运途径、分选间隔和机制仍知之甚少。这在很大程度上是由于这样一个事实，即肝细胞系不适用于在MDCK细胞中使用的经典的基于生物素的靶向分析的转运研究，并且明显更难转染。我们提出了创新的实验方法，以确定与MDCK细胞相比，模型AP PM蛋白在肝WIFB细胞中的转运路线、亚细胞室和分子机制。我们的假设是：(I)新的成像技术和(Ii)对AP蛋白胞吐后命运的分析为理解肝细胞和柱状细胞的不同分选和运输策略提供了足够的分辨率，以及(Iii)丝氨酸/苏氨酸激酶Par1b在直接和跨细胞途径之间起着分子开关的作用。我们提出了以下具体目标：目的1：定量评估模型顶端PM蛋白在WIFB和MDCK细胞中的载体和跨细胞递送程度。目的：比较确定WIFB和MDCK细胞中AP和BL标记的胞外分选部位。目的3：鉴定AP PM蛋白在WIFB和MDCK细胞中退出TGN的机制。通过我们的新方法，我们将为肝脏细胞和柱状细胞中蛋白质靶向的比较分子模型奠定框架。这是上皮极性的一个高度优先的问题，25年多来一直没有答案。 与公共卫生相关：上皮是紧密附着的细胞组织，包围着所有器官，从而将它们与周围环境分开并保护它们。然而，上皮层远没有筑起被动的防护墙，而是充当复杂的守门人，协调气体、离子、营养物质和废物等分子在它们周围的器官之间有控制地进出，通常与浓度梯度背道而驰。为了实现这一功能，上皮细胞形成极化的膜域，限制转运蛋白和门控通道，这些转运蛋白和门控通道负责将分子通过细胞膜运输到上皮细胞面对的内表面或外表面，从而确保分子的矢量运输。大多数上皮，如肾、肠道、肺或乳腺的上皮，组织成两层细胞层，包围中央管腔，形成尿液、营养物质、牛奶或空气通过的管道、阿西尼或阿魏酸。面对管腔的表面域称为根尖或管腔表面。相比之下，肝上皮细胞以单细胞厚板的形式组织起来，通过中断相邻细胞的侧域，形成一个管腔，即胆小管网络。上皮腔表面的丢失或紊乱是许多癌症的一个特征。它也是其他病理的特征，如微绒毛包涵体疾病和慢性肝脏炎症(胆汁淤积症)。因此，了解管腔表面是如何形成和维持的是上皮细胞生物学的中心问题之一。这项建议涉及管腔形成的一个重要方面。也就是说：肾和肝上皮细胞如何将膜蛋白特异性地靶向到它们的管腔区域？众所周知，这两种类型的细胞利用截然不同的策略进行腔蛋白靶向，但区分它们的分子机制尚不清楚。我们开发了新的工具和方法来阐明以肾脏和肝脏为代表的两种主要上皮表型的管腔蛋白运输机制的关键特征。