Single-molecule FRET and tracking of transfected biomolecules: multi-dimensional protein dynamics in living cells

Proteins in cells exhibit conformational dynamics, equally influenced by dynamic interactions with other biomolecules and their spatial variations, which can be induced by the protein’s compartment. Altogether this multi-dimensional dynamic is difficult to measure in cellula, because of limitations in instrumentation, fluorescence methodologies and the difficulty to track freely diffusing molecules. Here, we present a bottom-up engineering approach, which allows us to track transfected proteins in cellula and analyze time-resolved single-molecule FRET efficiencies. This has been achieved by alternating laser excitation (ALEX) based three-channel (donor, acceptor and FRET intensity) tracking with a live-cell HILO microscope. Unexpectedly, we find that the heat shock protein Hsp90 shows different conformational populations in vitro and in cellula. Moreover, Hsp90’s conformational states depend on the localization within the cell, which is demonstrated by comparing a physical (microinjection) and a biological (SLO) transfection method. FRET-TTB (Tracking of Transfected Biomolecules) opens the path to study protein conformational dynamics of transfected and native biomolecules in cellula, including time-resolved cellular localization.

[1]  Timothy D. Craggs,et al.  Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins , 2022, bioRxiv.

[2]  C. Mirkin,et al.  Protein transfection via spherical nucleic acids , 2022, Nature Protocols.

[3]  Janett Göhring,et al.  Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes. , 2021, Journal of visualized experiments : JoVE.

[4]  A. Sieron,et al.  An Overview of Methods and Tools for Transfection of Eukaryotic Cells in vitro , 2021, Frontiers in Bioengineering and Biotechnology.

[5]  Daniel S. Terry,et al.  Single-molecule FRET imaging of GPCR dimers in living cells , 2021, Nature Methods.

[6]  M. Sattler,et al.  The Charged Linker Modulates the Conformations and Molecular Interactions of Hsp90 , 2020, Chembiochem : a European journal of chemical biology.

[7]  W. Houry,et al.  Heat shock protein 90 kDa (Hsp90) from Aedes aegypti has an open conformation and is expressed under heat stress. , 2020, International journal of biological macromolecules.

[8]  G. Schütz,et al.  Temporal analysis of T-cell receptor-imposed forces via quantitative single molecule FRET measurements , 2020, Nature Communications.

[9]  S. Hubbard,et al.  Mechanism of homodimeric cytokine receptor activation and dysregulation by oncogenic mutations , 2020, Science.

[10]  Y. Sako,et al.  In-cell single-molecule FRET measurements reveal three conformational state changes in RAF protein. , 2020, Biochimica et biophysica acta. General subjects.

[11]  Peter van Baarlen,et al.  Visualisation of dCas9 target search in vivo using an open-microscopy framework , 2019, Nature Communications.

[12]  Daniel J. Muller,et al.  Tau protein liquid–liquid phase separation can initiate tau aggregation , 2018, The EMBO journal.

[13]  T. Hugel,et al.  Effects of inhibitors on Hsp90’s conformational dynamics, cochaperone and client interactions , 2018, bioRxiv.

[14]  T. Hugel,et al.  Cooperative Nucleotide Binding in Hsp90 and Its Regulation by Aha1. , 2017, Biophysical journal.

[15]  Andreas Plückthun,et al.  A quantitative comparison of cytosolic delivery via different protein uptake systems , 2017, Scientific Reports.

[16]  Nam Ki Lee,et al.  Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study , 2017, Nature Methods.

[17]  Sang Hak Lee,et al.  Labeling proteins inside living cells using external fluorophores for microscopy , 2016, eLife.

[18]  Thorsten Hugel,et al.  Single-Molecule Analysis beyond Dwell Times: Demonstration and Assessment in and out of Equilibrium. , 2016, Biophysical journal.

[19]  Ji Yu,et al.  Single-Molecule Studies in Live Cells. , 2016, Annual review of physical chemistry.

[20]  A. Kapanidis,et al.  Stable end-sealed DNA as robust nano-rulers for in vivo single-molecule fluorescence† †Electronic supplementary information (ESI) available: Experimental methods, data analysis routines and Fig. S1–S9. See DOI: 10.1039/c6sc00639f , 2016, Chemical science.

[21]  Andreas Plückthun,et al.  Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells , 2015, Nature Methods.

[22]  Arkajit Dey,et al.  Inferring transient particle transport dynamics in live cells , 2015, Nature Methods.

[23]  M. Rief,et al.  The charged linker of the molecular chaperone Hsp90 modulates domain contacts and biological function , 2014, Proceedings of the National Academy of Sciences.

[24]  V. Rotello,et al.  Promises and Pitfalls of Intracellular Delivery of Proteins , 2014, Bioconjugate chemistry.

[25]  D. Bolon,et al.  Designed Hsp90 heterodimers reveal an asymmetric ATPase-driven mechanism in vivo. , 2014, Molecular cell.

[26]  J. Buchner,et al.  Structure, Function and Regulation of the Hsp90 Machinery , 2013, Biomedical journal.

[27]  G. Timp,et al.  Using a nanopore for single molecule detection and single cell transfection. , 2012, The Analyst.

[28]  J. Buchner,et al.  The Hsp90 chaperone machinery: conformational dynamics and regulation by co-chaperones. , 2012, Biochimica et biophysica acta.

[29]  M. Corrotte,et al.  Toxin Pores Endocytosed During Plasma Membrane Repair Traffic into the Lumen of MVBs for Degradation , 2012, Traffic.

[30]  L. Neckers,et al.  Charged linker sequence modulates eukaryotic heat shock protein 90 (Hsp90) chaperone activity , 2012, Proceedings of the National Academy of Sciences.

[31]  J. Eberwine,et al.  Mammalian cell transfection: the present and the future , 2010, Analytical and bioanalytical chemistry.

[32]  K. Weninger,et al.  Detecting the conformation of individual proteins in live cells , 2010, Nature Methods.

[33]  Yongxiang Gao,et al.  Accurate detection and complete tracking of large populations of features in three dimensions. , 2009, Optics express.

[34]  M. Tokunaga,et al.  Highly inclined thin illumination enables clear single-molecule imaging in cells , 2008, Nature Methods.

[35]  Shimon Weiss,et al.  Photobleaching pathways in single-molecule FRET experiments. , 2007, Journal of the American Chemical Society.

[36]  L. Pearl,et al.  Crystal structure of an Hsp90–nucleotide–p23/Sba1 closed chaperone complex , 2006, Nature.

[37]  T. Laurence,et al.  Retention of transcription initiation factor sigma70 in transcription elongation: single-molecule analysis. , 2005, Molecular cell.

[38]  N. Shimizu,et al.  Tracking of microinjected DNA in live cells reveals the intracellular behavior and elimination of extrachromosomal genetic material , 2005, Nucleic acids research.

[39]  Nam Ki Lee,et al.  Alternating-laser excitation of single molecules. , 2005, Accounts of chemical research.

[40]  Nam Ki Lee,et al.  Accurate FRET measurements within single diffusing biomolecules using alternating-laser excitation. , 2005, Biophysical journal.

[41]  Klaus Suhling,et al.  Time-resolved fluorescence microscopy , 2007, SPIE Optics East.

[42]  Nam Ki Lee,et al.  Fluorescence-aided molecule sorting: Analysis of structure and interactions by alternating-laser excitation of single molecules , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[43]  Akihiro Kusumi,et al.  Single-molecule imaging analysis of Ras activation in living cells. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[44]  T. Ha,et al.  Single-molecule fluorescence resonance energy transfer. , 2001, Methods.

[45]  K. Aktories,et al.  Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[46]  S. Simon,et al.  Tracking single proteins within cells. , 2000, Biophysical journal.

[47]  Toshio Yanagida,et al.  Single-molecule imaging of EGFR signalling on the surface of living cells , 2000, Nature Cell Biology.

[48]  A. Verkman,et al.  Direct Measurement of trans-Golgi pH in Living Cells and Regulation by Second Messengers (*) , 1995, The Journal of Biological Chemistry.

[49]  A. Verkman,et al.  Second messengers regulate endosomal acidification in Swiss 3T3 fibroblasts , 1992, The Journal of cell biology.

[50]  J. Lakowicz,et al.  Fluorescence lifetime imaging. , 1992, Analytical biochemistry.

[51]  T. Hugel,et al.  A Multicolor Single-Molecule FRET Approach to Study Protein Dynamics and Interactions Simultaneously. , 2016, Methods in enzymology.

[52]  S. E. Stewart,et al.  Assembly of streptolysin O pores assessed by quartz crystal microbalance and atomic force microscopy provides evidence for the formation of anchored but incomplete oligomers. , 2015, Biochimica et biophysica acta.

[53]  E. Peterman,et al.  Single Molecule Analysis , 2011, Methods in Molecular Biology.

[54]  M. Sloan,et al.  The Present and the Future , 1984 .