TY - JOUR
T1 - Modular transient nanoclustering of activated β2-adrenergic receptors revealed by single-molecule tracking of conformation-specific nanobodies
AU - Gormal, Rachel S.
AU - Padmanabhan, Pranesh
AU - Kasula, Ravikiran
AU - Bademosi, Adekunle T.
AU - Coakley, Sean
AU - Giacomotto, Jean
AU - Blum, Ailisa
AU - Joensuu, Merja
AU - Wallis, Tristan P.
AU - Lo, Harriet P.
AU - Budnar, Srikanth
AU - Rae, James
AU - Ferguson, Charles
AU - Bastiani, Michele
AU - Thomas, Walter G.
AU - Pardon, Els
AU - Steyaert, Jan
AU - Yap, Alpha S.
AU - Goodhill, Geoffrey J.
AU - Hilliard, Massimo A.
AU - Parton, Robert G.
AU - Meunier, Frédéric A.
N1 - Funding Information:
ACKNOWLEDGMENTS. We acknowledge the use of the Microscopy Australia Facility at the Centre for Microscopy and Microanalysis at The University of Queensland. We thank Rowan Tweedale for critical appraisal of the manuscript, Jake Carrol and the IT department at the Queensland Brain Institute (QBI), Rumelo Amor, and all past and present members of the Advanced Microscopy and Microanalysis Facility at QBI for their outstanding microscopy support. We thank Daniel Choquet and J. B. Sibarita for invaluable discussions about this project. We thank Professor Mark von Zastrow for providing the Nb80 nanobody, and Associate Professor Brett Collins for thoughtful discussion, providing the GBP, and creating molecular illustrations within Figs. 1 and 2. The work was supported by Australian Research Council (ARC) Discovery Project Grant DP170100125; ARC Linkage Infrastructure Equipment and Facilities Grant LE130100078; National Health and Medical Research Council (NHMRC) Senior Research Fellowship 1155794 to F.A.M., NHMRC Project Grant 1129367 and NHMRC Senior Research Fellowship 1111042 to M.A.H., and NHMRC-ARC Dementia Research Development Fellowship 1108489 to S.C.; Academia of Finland Postdoctoral Research Fellowship 298124 and an ARC Discovery Early Career Researcher Award (DE190100565) to M.J.; University of Queensland Postdoctoral Research Fellowship to P.P.; NHMRC Project Grants 1037320 and 1058565 and an ARC Centre of Excellence in Convergent Bio-Nano Science and Technology to R.G.P.; and NHMRC Investigator Grant 1174145, NHMRC Project Grant 1165850, and a Rebecca L. Cooper Medical Research Project Grant PG2019405 to J.G. Nb80, Nb37, and Nb60 were discovered in the J.S. laboratory. E.P. and J.S. thank INSTRUCT, part of the European Strategy Forum on Research Infrastructures and the Research Foundation–Flanders, for support of the nanobody discoveries.
Publisher Copyright:
© 2020 National Academy of Sciences. All rights reserved.
PY - 2020/12/1
Y1 - 2020/12/1
N2 - None of the current superresolution microscopy techniques can reliably image the changes in endogenous protein nanoclustering dynamics associated with specific conformations in live cells. Single-domain nanobodies have been invaluable tools to isolate defined conformational states of proteins, and we reasoned that expressing these nanobodies coupled to single-molecule imaging-amenable tags could allow superresolution analysis of endogenous proteins in discrete conformational states. Here, we used anti-GFP nanobodies tagged with photoconvertible mEos expressed as intrabodies, as a proof-of-concept to perform single-particle tracking on a range of GFP proteins expressed in live cells, neurons, and small organisms. We next expressed highly specialized nanobodies that target conformation-specific endogenous β2-adrenoreceptor (β2-AR) in neurosecretory cells, unveiling real-time mobility behaviors of activated and inactivated endogenous conformers during agonist treatment in living cells. We showed that activated β2-AR (Nb80) is highly immobile and organized in nanoclusters. The Gαs−GPCR complex detected with Nb37 displayed higher mobility with surprisingly similar nanoclustering dynamics to that of Nb80. Activated conformers are highly sensitive to dynamin inhibition, suggesting selective targeting for endocytosis. Inactivated β2-AR (Nb60) molecules are also largely immobile but relatively less sensitive to endocytic blockade. Expression of single-domain nanobodies therefore provides a unique opportunity to capture highly transient changes in the dynamic nanoscale organization of endogenous proteins.
AB - None of the current superresolution microscopy techniques can reliably image the changes in endogenous protein nanoclustering dynamics associated with specific conformations in live cells. Single-domain nanobodies have been invaluable tools to isolate defined conformational states of proteins, and we reasoned that expressing these nanobodies coupled to single-molecule imaging-amenable tags could allow superresolution analysis of endogenous proteins in discrete conformational states. Here, we used anti-GFP nanobodies tagged with photoconvertible mEos expressed as intrabodies, as a proof-of-concept to perform single-particle tracking on a range of GFP proteins expressed in live cells, neurons, and small organisms. We next expressed highly specialized nanobodies that target conformation-specific endogenous β2-adrenoreceptor (β2-AR) in neurosecretory cells, unveiling real-time mobility behaviors of activated and inactivated endogenous conformers during agonist treatment in living cells. We showed that activated β2-AR (Nb80) is highly immobile and organized in nanoclusters. The Gαs−GPCR complex detected with Nb37 displayed higher mobility with surprisingly similar nanoclustering dynamics to that of Nb80. Activated conformers are highly sensitive to dynamin inhibition, suggesting selective targeting for endocytosis. Inactivated β2-AR (Nb60) molecules are also largely immobile but relatively less sensitive to endocytic blockade. Expression of single-domain nanobodies therefore provides a unique opportunity to capture highly transient changes in the dynamic nanoscale organization of endogenous proteins.
KW - Single-particle−tracking superresolution microscopy | nanobodies | β2-adrenoreceptor
UR - http://www.scopus.com/inward/record.url?scp=85097211198&partnerID=8YFLogxK
U2 - 10.1073/pnas.2007443117
DO - 10.1073/pnas.2007443117
M3 - Article
C2 - 33214152
AN - SCOPUS:85097211198
VL - 117
SP - 30476
EP - 30487
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 48
ER -