Erfle Holger, Eskova Anastasia, Reymann Jürgen, Starkuviene Vytaute
BioQuant, University of Heidelberg, Heidelberg, Germany.
Methods Mol Biol. 2011;785:277-87. doi: 10.1007/978-1-61779-286-1_19.
Endocytosis is one of the most essential cellular processes, which enables cells to internalise diverse -material. It is crucial for regulation of receptor activity and signalling, cell polarisation, attachment and motility, and a great number of other cellular functions. A number of diverse endocytosis pathways are described by now; however, their specificity for different cellular cargoes is poorly resolved. Only few of endocytosis regulators are well-characterised and even less are attributed to the specific cargo. That is very true for the integrin endocytosis pathway, which is a key process in cell migration, adhesion, and signalling. The recent advent of quantitative fluorescent microscopy and cell arrays opened an exciting possibility to systematically characterise molecules playing a role in this crucially important process. Here, we describe a fluorescent screening microscopy-based assay to identify regulators of integrin α2 internalisation. The experimental procedure is the best suited for a highly parallel screening format, such as cell arrays, albeit can be used in single experiments. We provide protocols for sample preparation, fabrication of cell arrays and quantification of integrin α2 internalisation. The approach can be modified to quantify endocytosis of other cargo, and can be used under the conditions of knock-down and knock-in as well as for chemical screening.
内吞作用是细胞最基本的过程之一，它使细胞能够摄取各种物质。它对于受体活性和信号传导的调节、细胞极化、附着和运动以及许多其他细胞功能至关重要。目前已经描述了多种不同的内吞途径；然而，它们对不同细胞货物的特异性仍未得到很好的解决。只有少数内吞作用调节剂得到了充分的表征，而归因于特定货物的则更少。对于整联蛋白内吞途径来说尤其如此，它是细胞迁移、黏附和信号传导中的关键过程。定量荧光显微镜和细胞阵列的最新出现为系统地表征在这一至关重要的过程中起作用的分子提供了令人兴奋的可能性。在这里，我们描述了一种基于荧光筛选显微镜的测定方法，用于鉴定整联蛋白α2内化的调节剂。该实验程序最适合高度平行的筛选形式，如细胞阵列，尽管也可用于单个实验。我们提供了样品制备、细胞阵列制作和整联蛋白α2内化定量的方案。该方法可以修改以量化其他货物的内吞作用，并且可以在敲低和敲入的条件下以及用于化学筛选。
