Generation of a Clta‐Reporter Mouse Model for in vivo imaging of Endocytosis

Abstract

Endocytosis via different pathways is a fundamental cellular process in all eukaryotic organisms and is essential to higher eukaryotic life. One of the first and best characterized pathways is clathrin-mediated endocytosis (CME) that uses clathrin-coated vesicles for the uptake of material from the cell surface. Over the recent years more and more proteins that are part of the CME machinery as well as new cargoes were described. This led to important insights into the whole pathway. However, there is still a lack of a good understanding of how these different components work together in vivo. Especially the differential physiological role of the clathrin light chains a and b in health and disease is still not clear and under debate. Using the CRISPR/Cas9 technique, we generated a Clathrin-eGFP knock-in reporter mouse model to study CME in vivo using intravital microscopy. To prevent overexpression artefacts, the fusion protein is expressed under the control of the endogenous promoter, as eGFP was introduced in-frame in front of the clta stop codon. The Clta-eGFP fusion protein is expressed in all tissues examined and is able to interact with known interaction partners. Two-photon intravital microscopy in kidney proximal tubules of Clta-eGFP reporter mice showed that albumin uptake is not distinguishable from WT littermates.With this reporter mouse model, we are able to visualize the endocytic process in vivo by intravital microscopy and these mice are therefore a suitable and powerful tool for basic research about CME processes in vivo.