An internally eGFP-tagged α-adaptin is a fully functional and improved fiduciary marker for clathrin-coated pit dynamics.
AP2
clathrin-mediated endocytosis
retroviral expression
total internal reflection fluorescence microscopy
transferrin receptor
Journal
Traffic (Copenhagen, Denmark)
ISSN: 1600-0854
Titre abrégé: Traffic
Pays: England
ID NLM: 100939340
Informations de publication
Date de publication:
09 2020
09 2020
Historique:
received:
18
05
2020
revised:
05
07
2020
accepted:
06
07
2020
pubmed:
14
7
2020
medline:
28
4
2021
entrez:
14
7
2020
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 32657003
doi: 10.1111/tra.12755
pmc: PMC7495412
mid: NIHMS1619112
doi:
Substances chimiques
Adaptor Protein Complex 2
0
Adaptor Protein Complex alpha Subunits
0
Clathrin
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
603-616Subventions
Organisme : NIGMS NIH HHS
ID : R01 GM073165
Pays : United States
Organisme : NIH HHS
ID : R01 GM73165
Pays : United States
Informations de copyright
© 2020 The Authors. Traffic published by John Wiley & Sons Ltd.
Références
Curr Opin Cell Biol. 2020 Apr 1;65:86-95
pubmed: 32247230
Cell. 2004 Sep 3;118(5):591-605
pubmed: 15339664
J Biol Chem. 2003 Nov 14;278(46):45160-70
pubmed: 12960147
Cold Spring Harb Perspect Biol. 2014 May 01;6(5):a016725
pubmed: 24789820
J Cell Biol. 2017 Jan 2;216(1):167-179
pubmed: 28003333
Cell. 2010 Jun 25;141(7):1220-9
pubmed: 20603002
Dev Cell. 2006 Mar;10(3):329-42
pubmed: 16516836
Traffic. 2020 Sep;21(9):603-616
pubmed: 32657003
EMBO J. 2004 Nov 10;23(22):4371-83
pubmed: 15496985
J Cell Biol. 1995 Nov;131(3):619-30
pubmed: 7593184
EMBO J. 2015 Aug 13;34(16):2132-46
pubmed: 26139537
Nat Cell Biol. 2002 Sep;4(9):691-8
pubmed: 12198492
Nat Rev Mol Cell Biol. 2011 Jul 22;12(8):517-33
pubmed: 21779028
Cold Spring Harb Perspect Biol. 2014 Aug 28;6(12):a017038
pubmed: 25167858
Mol Biol Cell. 2006 Dec;17(12):5298-308
pubmed: 17035630
Cell Rep. 2015 Sep 29;12(12):2121-30
pubmed: 26387943
Cold Spring Harb Perspect Biol. 2013 Nov 01;5(11):a016790
pubmed: 24186068
J Cell Biol. 2010 Mar 22;188(6):919-33
pubmed: 20231386
Nat Rev Mol Cell Biol. 2018 May;19(5):313-326
pubmed: 29410531
Cell. 2012 Aug 3;150(3):495-507
pubmed: 22863004
Elife. 2020 Apr 30;9:
pubmed: 32352376
Annu Rev Biochem. 2018 Jun 20;87:871-896
pubmed: 29661000
PLoS Biol. 2009 Mar 17;7(3):e57
pubmed: 19296720
Dev Cell. 2013 Nov 11;27(3):353-66
pubmed: 24229647
Curr Opin Cell Biol. 2011 Aug;23(4):404-12
pubmed: 21450449
Annu Rev Cell Dev Biol. 1999;15:705-32
pubmed: 10611976
J Biol Chem. 2003 Oct 31;278(44):43411-7
pubmed: 12900408
Annu Rev Cell Biol. 1990;6:151-71
pubmed: 2177341
J Biol Chem. 1995 Dec 29;270(52):31083-90
pubmed: 8537368
Dev Cell. 2017 Feb 6;40(3):278-288.e5
pubmed: 28171750
EMBO J. 1999 May 4;18(9):2489-99
pubmed: 10228163
Proc Natl Acad Sci U S A. 2000 Feb 1;97(3):1096-100
pubmed: 10655490
Mol Biol Cell. 2016 Nov 7;27(22):3418-3435
pubmed: 27535432
Curr Opin Cell Biol. 2001 Aug;13(4):444-53
pubmed: 11454451
J Cell Biol. 1990 Oct;111(4):1419-26
pubmed: 2211818
J Cell Biol. 2003 Sep 1;162(5):909-18
pubmed: 12952941
Nat Cell Biol. 1999 May;1(1):1-7
pubmed: 10559856
Science. 2014 Jul 25;345(6195):459-63
pubmed: 25061211
Nat Commun. 2018 Mar 16;9(1):1109
pubmed: 29549258
Annu Rev Cell Dev Biol. 2001;17:517-68
pubmed: 11687498
Development. 2013 Sep;140(18):3826-37
pubmed: 23924631
Mol Biol Cell. 2015 Jun 1;26(11):2044-53
pubmed: 25851602
Annu Rev Cell Dev Biol. 2004;20:153-91
pubmed: 15473838
J Biol Chem. 2005 May 13;280(19):19270-80
pubmed: 15728179
Dev Cell. 2013 Aug 12;26(3):279-91
pubmed: 23891661
J Biol Chem. 2004 Mar 26;279(13):13190-204
pubmed: 14722064
Cell Rep. 2019 Jun 4;27(10):3049-3061.e6
pubmed: 31167147
Traffic. 2008 Aug;9(8):1250-5
pubmed: 18498437
Cell. 2006 Oct 6;127(1):113-24
pubmed: 17018281
J Biol Chem. 2002 Jul 26;277(30):27433-41
pubmed: 12021271
Nat Methods. 2008 Aug;5(8):695-702
pubmed: 18641657
Traffic. 2015 Nov;16(11):1137-54
pubmed: 26251007
J Cell Biol. 1993 Oct;123(1):67-77
pubmed: 8408206