High content analysis of apical versus basolateral trafficking

蛋白质需要被送到不同的细胞隔间，以便这些不同的隔间有效地发挥作用。被称为小泡的小结构调节蛋白质的分类和运输。目前的研究计划旨在阐明囊泡如何介导极化细胞的分选和细胞内向质膜的运输。水通道蛋白是存在于细胞外膜上的透水膜通道。哺乳动物细胞表达13个水通道蛋白，其中水通道蛋白-0、-1、-2、-4、-5、-6和-8只对水渗透，水通道蛋白-3、-7、-9和-10也对尿素和甘油等小分子渗透。在细胞内，水通道蛋白可以定位于特定的膜域。例如，水通道蛋白-4(AQP4)通过并定位于基底膜(细胞的侧面和底部)，而AQP5则存在于顶端质膜(细胞顶部)。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。我们最近展示了AQP4囊泡在细胞中的活跃运输，但令我们惊讶的是，发现囊泡并不指向它们的目的地。相反，我们发现，非极化(没有特定侧面的细胞，底部或顶部)和极化细胞的囊泡动力学相似。这表明，运输小泡以膜为靶标并融合到适当的靶膜上以定位蛋白质，这与目前的想法有重大转变。*在这项提议中，我们将阐明将AQP4包装成运输小泡所需的机制，研究携带AQP的小泡与靶膜相互作用的机制，并确定在极化过程中(获取侧、底部和顶部)AQP囊泡动力学是否在细胞中不同。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缺乏各种已知运输决定因素的细胞中的囊泡动力学是否发生了变化，并将使用生化工具来研究水通道蛋白与膜的相互作用的性质。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，我们的方法提供了具有统计学意义的数据，这与以前关于AQP运输的研究中获得的大多数数据有很大不同，这些数据依赖于蛋白质定位或单个图像。*这一建议将在细胞生物学和生物物理学方面提供极好的训练。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、转基因、显微镜、在MatLab中编程和图像分析。*这一提议的成功完成将使人们更好地理解细胞用来分选和运输蛋白质到极化细胞(顶端与基底侧端)中不同膜域的机制。*目标**

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). *** **