Giovannone Adrian J, Reales Elena, Bhattaram Pallavi, Fraile-Ramos Alberto, Weimbs Thomas
Department of Molecular, Cellular, and Developmental Biology and Neuroscience Research Institute, University of California, Santa Barbara, California, USA.
Universidad Complutense de Madrid, Departmento de Biología Celular, Facultad de Medicina, Plaza de Ramoń y Cajal, s/n Ciudad Universitaria, Madrid, Spain.
Bio Protoc. 2018 Feb 5;8(3). doi: 10.21769/BioProtoc.2453.
The uptake and trafficking of cell surface receptors can be monitored by a technique called 'antibody-feeding' which uses an externally applied antibody to label the receptor on the surface of cultured, live cells. Here, we adapt the traditional antibody-feeding experiment to polarized epithelial cells (Madin-Darby Canine Kidney) grown on permeable Transwell supports. By adding two tandem extracellular Myc epitope tags to the C-terminus of the SNARE protein syntaxin 3 (Stx3), we provided a site where an antibody could bind, allowing us to perform antibody-feeding experiments on cells with distinct apical and basolateral membranes. With this procedure, we observed the endocytosis and intracellular trafficking of Stx3. Specifically, we assessed the internalization rate of Stx3 from the basolateral membrane and observed the ensuing endocytic route in both time and space using immunofluorescence microscopy on cells fixed at different time points. For cell lines that form a polarized monolayer containing distinct apical and basolateral membranes when cultured on permeable supports, , MDCK or Caco-2, this protocol can measure the rate of endocytosis and follow the subsequent trafficking of a target protein from either limiting membrane.
细胞表面受体的摄取和运输可以通过一种称为“抗体喂食”的技术进行监测，该技术使用外部施加的抗体标记培养的活细胞表面的受体。在这里，我们将传统的抗体喂食实验应用于生长在可渗透Transwell支架上的极化上皮细胞（Madin-Darby犬肾细胞）。通过在SNARE蛋白 syntaxin 3（Stx3）的C末端添加两个串联的细胞外Myc表位标签，我们提供了一个抗体可以结合的位点，使我们能够在具有不同顶端和基底外侧膜的细胞上进行抗体喂食实验。通过这个过程，我们观察到了Stx3的内吞作用和细胞内运输。具体来说，我们评估了Stx3从基底外侧膜的内化速率，并使用免疫荧光显微镜在不同时间点固定的细胞上观察了随后在时间和空间上的内吞途径。对于在可渗透支架上培养时形成包含不同顶端和基底外侧膜的极化单层的细胞系，如MDCK或Caco-2，该方案可以测量内吞速率，并追踪目标蛋白从任一限制膜的后续运输。
