Mechanisms of luminal protein targeting in kidney and hepatic epithelial cells

描述（由申请人提供）：肾和肝上皮细胞中管腔蛋白靶向的机制柱状上皮（例如肾、肠）和肝细胞体现了非复层上皮细胞的两种主要组织表型。它们在形态学上的不同之处在于柱状上皮在相对的两极建立其顶端域（AP）和基底外侧域（BL），而肝细胞在其BL域中间建立其AP域、胆汁杯（BC）。它们在如何建立和维持表面结构域方面也有很大的不同。柱状上皮细胞主要将其质膜（PM）蛋白从反式高尔基体网络（TGN）靶向AP结构域，而肝细胞则将AP和BL PM蛋白从TGN靶向BL表面，在BL表面，AP蛋白通过转胞吞作用被分选为BC。尽管在了解柱状细胞（特别是肾上皮细胞系MDCK）的运输途径、分选隔室和靶向机制方面取得了重要进展，但对肝细胞的运输途径、分选隔室和机制仍然知之甚少。这在很大程度上是由于肝细胞系不适合使用MDCK细胞中使用的基于经典生物素的靶向测定进行运输研究，并且明显更难以检测。我们提出了创新的实验方法，以确定贩运行程，亚细胞区室和分子机制参与模型AP PM蛋白在肝WIFB细胞中的贩运，与MDCK细胞相比。我们的假设是：（i）新的成像技术和（ii）对AP蛋白的胞吐后命运的分析提供了足够的分辨率来理解肝细胞和柱状细胞的不同分选和运输策略，以及（iii）丝氨酸/苏氨酸激酶Par 1b充当直接途径和胞吞途径之间的分子开关。我们提出了以下具体目的：目的1：定量评估模型顶端PM蛋白在WIFB和MDCK细胞中的载体和转胞吞递送的程度。目的2：比较确定AP和BL标记在WIFB和MDCK细胞中的胞吐分选位点。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，我们将为一个期待已久的蛋白质靶向肝细胞与柱状细胞的比较分子模型建立框架。这是一个高度优先的上皮极性问题，超过25年来一直没有答案。 公共卫生相关性：上皮是紧密粘附的细胞组织，其包围所有器官，从而将它们与周围环境分开并保护它们。然而，上皮层并不是被动的保护墙，而是扮演着复杂的守门人的角色，负责调节气体、离子、营养物质和废物等分子进出它们周围的器官，通常是逆着浓度梯度的。为了实现这一功能，上皮细胞形成极化的膜结构域，其限制负责分子穿过细胞膜运输到上皮细胞的“内部”或“外部”面向表面的转运蛋白和门控通道，从而确保分子的矢量转运。大多数上皮细胞，如肾脏、肠道、肺或乳腺的上皮细胞，组织成两层细胞，以封闭中心腔，形成管、ascini或aveoli，尿液、营养物质、乳汁或空气通过这些管、ascini或aveoli运输。面向管腔的表面域称为顶面或管腔面。相比之下，肝上皮组织在单细胞厚的板中，并通过中断相邻细胞的侧域来切割出管腔，即胆小管网络。上皮管腔表面的丧失或解体是许多癌症的特征。它也是其他病理学的标志，如微绒毛包涵体疾病和肝脏慢性炎症（胆汁淤积）。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核心问题之一。该提议解决了管腔形成的一个重要方面。即：肾脏和肝脏上皮细胞如何将膜蛋白特异性靶向其管腔结构域？已知这两种细胞类型利用完全不同的管腔蛋白靶向策略，但区分它们的分子机制尚不清楚。我们已经开发了新的工具和方法来阐明由肾脏和肝脏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。

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.