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。称为囊泡的小结构介导蛋白质的分选和运输。目前的研究计划旨在阐明如何囊泡介导的分选和细胞内运输到质膜的极化细胞。水通道蛋白是在细胞外膜上发现的水渗透膜通道。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仅对水是可渗透的，而水通道蛋白-3、-7、-9和-10对小分子如尿素和甘油也是可渗透的。在细胞内，水通道蛋白可以定位于特定的膜结构域。例如，水通道蛋白-4（AQP 4）运输并定位于基底外侧膜（细胞的侧面和底部），而AQP 5发现于顶端质膜（细胞的顶部）。虽然水通道蛋白的表达受到限制，但调节这种运输的分子机制尚未完全阐明。我们最近发现AQP 4囊泡在细胞中被积极转运，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，底部或顶部的细胞）和极化细胞中的囊泡动力学是相似的。这表明运输囊泡“采样”膜靶并融合到适当的靶膜上以定位蛋白质，这与当前的想法有很大的不同。在这个建议中，我们将阐明包装成贩运囊泡水通道蛋白4所需的机械，研究水通道蛋白携带囊泡与靶膜的相互作用机制，并确定是否水通道蛋白囊泡动力学不同的细胞在极化过程中（收购双方，底部和顶部）。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囊泡动力学是否在缺乏各种已知的运输决定因素的细胞中改变，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了具有统计学意义的数据，这与以前依赖于蛋白定位或单个图像的AQP运输研究中获得的大多数数据有很大不同。该建议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印迹，细胞培养，转染，显微镜，Matlab编程和图像分析。* 该提案的成功完成将导致更好地理解细胞用于将蛋白质分类和运输到极化细胞（顶侧与基底侧）中的各种膜结构域的机制。*** **

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). *** **