An internally eGFP-tagged α-adaptin is a fully functional and improved fiduciary marker for clathrin-coated pit dynamics.

Clathrin mediated endocytosis (CME) has been extensively studied in living cells by quantitative total internal reflection fluorescence microscopy (TIRFM). Fluorescent protein fusions to subunits of the major coat proteins, clathrin light chains or the heterotetrameric adaptor protein (AP2) complexes, have been used as fiduciary markers of clathrin coated pits (CCPs). However, the functionality of these fusion proteins has not been rigorously compared. Here, we generated stable cells lines overexpressing mRuby-CLCa and/or mu 2-eGFP, sigma 2-eGFP, two markers currently in use, or a novel marker generated by inserting eGFP into the unstructured hinge region of the alpha subunit (alpha-eGFP). Using biochemical and TIRFM-based assays, we compared the functionality of the AP2 markers. All of the eGFP-tagged subunits were efficiently incorporated into AP2 and displayed greater accuracy in image-based CCP analyses than mRuby-CLCa. However, overexpression of either mu 2-eGFP or sigma 2-eGFP impaired transferrin receptor uptake. In addition, mu 2-eGFP reduced the rates of CCP initiation and sigma 2-eGFP perturbed AP2 incorporation into CCPs and CCP maturation. In contrast, CME and CCP dynamics were unperturbed in cells overexpressing alpha-eGFP. Moreover, alpha-eGFP was a more sensitive and accurate marker of CCP dynamics than mRuby-CLCa. Thus, our work establishes alpha-eGFP as a robust, fully functional marker for CME.