TY - JOUR
T1 - An internally eGFP-tagged α-adaptin is a fully functional and improved fiduciary marker for clathrin-coated pit dynamics
AU - Mino, Rosa E.
AU - Chen, Zhiming
AU - Mettlen, Marcel
AU - Schmid, Sandra L.
N1 - Funding Information:
We thank Dr. Zuzana Kadlekova for reagents, Dr. Emanuele Cocucci for manuscript comments, and Dr. Ashley Lakoduk for reagents and help with manuscript preparation. We also thank the FACS core facility at the UTSW Children's Research Center, Heather Grossman for help with sample generation, retrovirus production, and analysis, and Aparna Mohanakrishnan for technical support in PCR. We thank Dr. Ashley Lakoduk and Dr. Madhura Bhave for helpful discussion. This work is supported by NIH R01 GM73165 to Sandra L. Schmid and Marcel Mettlen.
Publisher Copyright:
© 2020 The Authors. Traffic published by John Wiley & Sons Ltd.
PY - 2020/9/1
Y1 - 2020/9/1
N2 - 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 μ2-eGFP, σ2-eGFP, two markers currently in use, or a novel marker generated by inserting eGFP into the unstructured hinge region of the α subunit (α-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 μ2-eGFP or σ2-eGFP impaired transferrin receptor uptake. In addition, μ2-eGFP reduced the rates of CCP initiation and σ2-eGFP perturbed AP2 incorporation into CCPs and CCP maturation. In contrast, CME and CCP dynamics were unperturbed in cells overexpressing α-eGFP. Moreover, α-eGFP was a more sensitive and accurate marker of CCP dynamics than mRuby-CLCa. Thus, our work establishes α-eGFP as a robust, fully functional marker for CME.
AB - 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 μ2-eGFP, σ2-eGFP, two markers currently in use, or a novel marker generated by inserting eGFP into the unstructured hinge region of the α subunit (α-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 μ2-eGFP or σ2-eGFP impaired transferrin receptor uptake. In addition, μ2-eGFP reduced the rates of CCP initiation and σ2-eGFP perturbed AP2 incorporation into CCPs and CCP maturation. In contrast, CME and CCP dynamics were unperturbed in cells overexpressing α-eGFP. Moreover, α-eGFP was a more sensitive and accurate marker of CCP dynamics than mRuby-CLCa. Thus, our work establishes α-eGFP as a robust, fully functional marker for CME.
KW - AP2
KW - clathrin-mediated endocytosis
KW - retroviral expression
KW - total internal reflection fluorescence microscopy
KW - transferrin receptor
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U2 - 10.1111/tra.12755
DO - 10.1111/tra.12755
M3 - Article
C2 - 32657003
AN - SCOPUS:85088316983
SN - 1398-9219
VL - 21
SP - 603
EP - 616
JO - Traffic
JF - Traffic
IS - 9
ER -