Cellular and Chemical Biology Unit, Endocytic Trafficking and Intracellular Delivery Team, U1143 INSERM, UMR3666 CNRS, Institut Curie, PSL Research University, Paris Cedex, France.
Methods Mol Biol. 2022;2442:367-390. doi: 10.1007/978-1-0716-2055-7_20.
The GlycoLipid-Lectin (GL-Lect) hypothesis provides a conceptual framework to explain how endocytic pits are built in processes of clathrin-independent endocytosis. According to this hypothesis, oligomeric cellular or pathogenic lectins interact with glycosylated plasma membrane lipids in a way such as to drive the formation of tubular endocytic pits that then detach to generate clathrin-independent endocytic carriers for the cellular uptake of cellular or pathogenic products. This process operates in a complementary manner to the conventional clathrin pathway for biological function linked to cell polarity. Up to date, the premises of the GL-Lect hypothesis have been based on model membrane and cell culture experiments. It has therefore become urgent to extend its exploration to complex organisms. In the current protocol, we describe methods to study the endocytosis and transcytosis of a key driver of the GL-Lect mechanism, the cellular galectin-3, and of one of its cargoes, lactotransferrin, in enterocytes of the intact jejunum of mice. In a step-by-step manner, we present the generation of fluorescent endocytic ligands, tissue preparation for cellular uptake measurements, binding and internalization assays, tissue fixation and preparation for sectioning, light and electron microscopical observations, and quantification of data by image processing. Pitfalls are discussed to optimize the chances of success with the described methods.
糖脂 - 凝集素 (GL-Lect) 假说提供了一个概念框架，用以解释网格蛋白非依赖性内吞作用过程中网格蛋白包被凹窝是如何形成的。根据该假说，寡聚细胞或病原体凝集素与糖基化质膜脂质以某种方式相互作用，从而驱动管状内吞凹窝的形成，然后这些凹窝脱离以产生网格蛋白非依赖性内吞载体，用于细胞摄取细胞或病原体产物。这个过程与传统的网格蛋白途径协同作用，与细胞极性相关的生物学功能相关。到目前为止，GL-Lect 假说的前提是基于模型膜和细胞培养实验。因此，迫切需要将其探索扩展到复杂的生物体。在当前的方案中，我们描述了研究 GL-Lect 机制的关键驱动因素之一，即细胞半乳糖凝集素-3，及其一种货物乳糖转铁蛋白在完整的小鼠空肠上皮细胞中的内吞和转胞吞作用的方法。我们逐步介绍了荧光内吞配体的生成、用于细胞摄取测量的组织准备、结合和内化测定、组织固定和切片准备、光和电子显微镜观察以及图像处理进行数据定量。讨论了陷阱，以优化所述方法成功的机会。
