High content analysis of apical versus basolateral trafficking

蛋白质需要被送到不同的细胞区室，以便这些不同的区室有效地发挥作用。被称为囊泡的小结构调节蛋白质的分类和运输。目前的研究项目旨在阐明囊泡如何介导极化细胞的分选和胞内转运到质膜。水通道蛋白是在细胞外膜上发现的可透水的膜通道。哺乳动物细胞表达13种水通道蛋白，其中水通道蛋白-0、-1、-2、-4、-5、-6和-8只可渗透到水，而水通道蛋白-3、-7、-9和-10也可渗透到尿素和甘油等小分子。在细胞内，水通道蛋白可以定位到特定的膜域。例如，水通道蛋白4 （AQP4）运输并定位于基底外侧膜（细胞的侧面和底部），而AQP5存在于顶质膜（细胞的顶部）。虽然AQP的表达受到限制，但调控这种转运的分子机制尚未完全阐明。我们最近发现AQP4囊泡在细胞中是主动运输的，但令我们惊讶的是，我们发现囊泡并不指向它们的目的地。相反，我们发现，非极化（没有特定侧面、底部或顶部的细胞）和极化细胞的囊泡动力学是相似的。这表明，运输囊泡“取样”膜目标并融合到适当的靶膜上以定位蛋白质，这是当前思维的重大转变。***在本提案中，我们将阐明将AQP4包装成转运囊泡所需的机制，研究携带AQP的囊泡与靶膜的相互作用机制，并确定细胞在极化（获得侧、底、顶）过程中AQP囊泡动力学是否不同。为了实现我们的研究目标，我们将使用我们的跟踪算法来确定在缺乏各种已知运输决定因素的细胞中囊泡动力学是否被改变，我们将使用生化工具来研究AQP与膜相互作用的特性。由于我们的跟踪算法使我们能够研究数百个细胞和数千个囊泡，因此我们的方法提供的数据具有统计学意义，这与以往AQP转运研究中依赖于蛋白质定位或单幅图像获得的大多数数据有很大不同。***该提案将提供细胞生物学和生物物理学方面的优秀培训。学员将在这些跨学科项目中学习各种方法，如Western blotting、细胞培养、转染、显微镜、Matlab编程和图像分析。***这一建议的成功完成将有助于更好地理解细胞在极化细胞（根尖与基底外侧）中对蛋白质进行分类和转运的机制。* * * * *

Proteins are required to be sent to various cellular compartments in order for those distinct compartments to function efficiently. Small structures called vesicles mediate the sorting and trafficking of proteins. This current research program aims to elucidate how vesicles mediate the sorting and intracellular trafficking to the plasma membrane of polarized cells. Aquaporins are water permeable membrane channels found on the outer membrane of cells. Mammalian cells express 13 aquaporins, with aquaporins-0, -1, -2, -4, -5, -6 and -8 being permeable only to water, while aquaporins-3, -7, -9 and -10 are also permeable to small molecules such as urea and glycerol. Within cells, aquaporins can localize to specific membrane domains. For example, aquaporin-4 (AQP4) traffics and is localized to the basolateral membrane (the side and bottom of cells), while AQP5 is found on the apical plasma membrane (top of the cell). Although AQP expression is restricted, the molecular mechanisms that regulate this trafficking have not been fully elucidated. We have recently shown AQP4 vesicles are actively transported in cells, but to our surprise, found that vesicles are not directed towards their destination. Instead, we found that, vesicle dynamics in non-polarized (cells that do not have specific sides, bottom or top) and polarized cells was similar. This suggested that trafficking vesicles "sample" membrane targets and fuse to the appropriate target membrane to localize proteins, a significant shift away from current thinking.***In this proposal, we will elucidate the machinery required to package AQP4 into trafficking vesicles, study the mechanism of interaction of AQP carrying vesicles with target membranes and determine whether or not AQP vesicle dynamics differs in cells in the process of polarizing (acquiring sides, bottom and top). In order to accomplish our research goals, we will use our tracking algorithms to determine whether or not vesicle dynamics are altered in cell lacking various known trafficking determinants and we will use biochemical tools to study properties of AQP interactions with membranes. Since our tracking algorithm enables us to study hundreds of cells and thousands of vesicles, our methods provide data with statistical significance, which differs greatly from the majority of data obtained in previous studies on AQP trafficking which relied on protein localization or single images.***This proposal will provide excellent training in both cell biology and biophysics. Trainees will learn a variety of methods in these interdisciplinary projects such as Western blotting, cell culture, transfection, microscopy, programming in Matlab and image analysis. ***The successful completion of this proposal will lead to a greater understanding of the mechanisms cells use to sort and traffic proteins to various membrane domains in polarized cells (apical versus basolateral). *** **