TY - JOUR
T1 - Four-color single-molecule imaging with engineered tags resolves the molecular architecture of signaling complexes in the plasma membrane
AU - Sotolongo Bellón, Junel
AU - Birkholz, Oliver
AU - Richter, Christian P.
AU - Eull, Florian
AU - Kenneweg, Hella
AU - Wilmes, Stephan
AU - Rothbauer, Ulrich
AU - You, Changjiang
AU - Walter, Mark R.
AU - Kurre, Rainer
AU - Piehler, Jacob
N1 - Funding Information:
We thank Gabriele Hikade for technical support. This project was supported by funding to J.P., R.K., and C.Y. from the DFG ( SFB 944 , projects P8 and Z, PI405/10-1 , the DFG Facility iBiOs, PI 405/14-1 and YO 166/1-1) and to M.R.W. (LRI, NIH R01 AI143554-4). S.W. was supported by a long-term EMBO fellowship ( ALTF 454-2017 ).
Publisher Copyright:
© 2022 The Authors
PY - 2022/2/28
Y1 - 2022/2/28
N2 - Localization and tracking of individual receptors by single-molecule imaging opens unique possibilities to unravel the assembly and dynamics of signaling complexes in the plasma membrane. We present a comprehensive workflow for imaging and analyzing receptor diffusion and interaction in live cells at single molecule level with up to four colors. Two engineered, monomeric GFP variants, which are orthogonally recognized by anti-GFP nanobodies, are employed for efficient and selective labeling of target proteins in the plasma membrane with photostable fluorescence dyes. This labeling technique enables us to quantitatively resolve the stoichiometry and dynamics of the interferon-γ (IFNγ) receptor signaling complex in the plasma membrane of living cells by multicolor single-molecule imaging. Based on versatile spatial and spatiotemporal correlation analyses, we identify ligand-induced receptor homo- and heterodimerization. Multicolor single-molecule co-tracking and quantitative single-molecule Förster resonance energy transfer moreover reveals transient assembly of IFNγ receptor heterotetramers and confirms its structural architecture.
AB - Localization and tracking of individual receptors by single-molecule imaging opens unique possibilities to unravel the assembly and dynamics of signaling complexes in the plasma membrane. We present a comprehensive workflow for imaging and analyzing receptor diffusion and interaction in live cells at single molecule level with up to four colors. Two engineered, monomeric GFP variants, which are orthogonally recognized by anti-GFP nanobodies, are employed for efficient and selective labeling of target proteins in the plasma membrane with photostable fluorescence dyes. This labeling technique enables us to quantitatively resolve the stoichiometry and dynamics of the interferon-γ (IFNγ) receptor signaling complex in the plasma membrane of living cells by multicolor single-molecule imaging. Based on versatile spatial and spatiotemporal correlation analyses, we identify ligand-induced receptor homo- and heterodimerization. Multicolor single-molecule co-tracking and quantitative single-molecule Förster resonance energy transfer moreover reveals transient assembly of IFNγ receptor heterotetramers and confirms its structural architecture.
KW - cell surface labeling
KW - cytokine receptor
KW - live-cell imaging
KW - multicolor imaging
KW - plasma membrane dynamics
KW - receptor dimerization
KW - single molecule tracking
KW - single-molecule fluorescence microscopy
KW - single-molecule FRET
KW - type II interferon receptor
UR - http://www.scopus.com/inward/record.url?scp=85124900005&partnerID=8YFLogxK
U2 - 10.1016/j.crmeth.2022.100165
DO - 10.1016/j.crmeth.2022.100165
M3 - Article
C2 - 35474965
AN - SCOPUS:85124900005
VL - 2
JO - Cell Reports Methods
JF - Cell Reports Methods
IS - 2
M1 - 100165
ER -