Large-scale single-molecule imaging aided by artificial intelligence.

Single-molecule imaging analysis has been applied to study the dynamics and kinetics of molecular behaviors and interactions in living cells. In spite of its high potential as a technique to investigate the molecular mechanisms of cellular phenomena, single-molecule imaging analysis has not been extended to a large scale of molecules in cells due to the low measurement throughput as well as required expertise. To overcome these problems, we have automated the imaging processes by using computer operations, robotics and artificial intelligence (AI). AI is an ideal substitute for expertise to obtain high-quality images for quantitative analysis. Our automated in-cell single-molecule imaging system, AiSIS, could analyze 1600 cells in 1 day, which corresponds to ∼ 100-fold higher efficiency than manual analysis. The large-scale analysis revealed cell-to-cell heterogeneity in the molecular behavior, which had not been recognized in previous studies. An analysis of the receptor behavior and downstream signaling was accomplished within a significantly reduced time frame and revealed the detailed activation scheme of signal transduction, advancing cell biology research. Furthermore, by combining the high-throughput analysis with our previous finding that a receptor changes its behavioral dynamics depending on the presence of a ligand/agonist or inhibitor/antagonist, we show that AiSIS is applicable to comprehensive pharmacological analysis such as drug screening. This AI-aided automation has wide applications for single-molecule analysis.

[1]  Tomonobu M. Watanabe,et al.  Single-molecule analysis of chemoattractant-stimulated membrane recruitment of a PH-domain-containing protein , 2006, Journal of Cell Science.

[2]  G. Carpenter,et al.  Receptors for epidermal growth factor and other polypeptide mitogens. , 1987, Annual review of biochemistry.

[3]  Seunghoon Hong,et al.  Learning Deconvolution Network for Semantic Segmentation , 2015, 2015 IEEE International Conference on Computer Vision (ICCV).

[4]  A. Sorkin,et al.  Interaction of EGF receptor and Grb2 in living cells visualized by fluorescence resonance energy transfer (FRET) microscopy , 2000, Current Biology.

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

[6]  M. Ueda,et al.  Intracellular Protein-Labeling Probes for Multicolor Single-Molecule Imaging of Immune Receptor-Adaptor Molecular Dynamics. , 2017, Journal of the American Chemical Society.

[7]  Y. Sako,et al.  Microenvironments and different nanoparticle dynamics in living cells revealed by a standard nanoparticle. , 2012, Journal of controlled release : official journal of the Controlled Release Society.

[8]  Masahiro Ueda,et al.  Asymmetric PTEN Distribution Regulated by Spatial Heterogeneity in Membrane-Binding State Transitions , 2013, PLoS Comput. Biol..

[9]  A. Citri,et al.  EGF–ERBB signalling: towards the systems level , 2006, Nature Reviews Molecular Cell Biology.

[10]  F. White,et al.  Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases , 2016, eLife.

[11]  Michio Hiroshima,et al.  Automated single-molecule imaging in living cells , 2018, Nature Communications.

[12]  M. Ueda,et al.  Single-molecule diffusion-based estimation of ligand effects on G protein–coupled receptors , 2018, Science Signaling.

[13]  T. Yanagida,et al.  Activation Kinetics of RAF Protein in the Ternary Complex of RAF, RAS-GTP, and Kinase on the Plasma Membrane of Living Cells , 2011, The Journal of Biological Chemistry.

[14]  Yongbao Sun,et al.  Experimental implementation of fully controlled dephasing dynamics and synthetic spectral densities , 2017, Nature Communications.

[15]  Tetsushi Sakuma,et al.  Modification of single-nucleotide polymorphism in a fully humanized CYP3A mouse by genome editing technology , 2017, Scientific Reports.

[16]  K. Lidke,et al.  supplementary figures , 2018 .

[17]  A. Harris,et al.  Quantitative assays of epidermal growth factor receptor in human breast cancer: Cut‐off points of clinical relevance , 1988, International journal of cancer.

[18]  Y. Sako,et al.  Single-molecule fluorescence imaging of RalGDS on cell surfaces during signal transduction from Ras to Ral , 2017, Biophysics and physicobiology.

[19]  Jae-Hoon Kim,et al.  Crystal Structure of the Complex of Human Epidermal Growth Factor and Receptor Extracellular Domains , 2002, Cell.

[20]  H. Akaike.  A new look at the statistical model identification , 1974 .

[21]  M. Ueda,et al.  Statistical analysis of lateral diffusion and multistate kinetics in single-molecule imaging. , 2009, Biophysical journal.

[22]  Masato Yasui,et al.  PTEN Hopping on the Cell Membrane Is Regulated via a Positively-Charged C2 Domain , 2014, PLoS Comput. Biol..

[23]  A. Ullrich,et al.  The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling , 1992, Cell.

[24]  Hiroaki Takagi,et al.  Multiple-state reactions between the epidermal growth factor receptor and Grb2 as observed by using single-molecule analysis , 2007, Proceedings of the National Academy of Sciences.

[25]  Edouard C. Nice,et al.  Crystal Structure of a Truncated Epidermal Growth Factor Receptor Extracellular Domain Bound to Transforming Growth Factor α , 2002, Cell.

[26]  T. Yanagida,et al.  Single-Molecule Analysis of Chemotactic Signaling in Dictyostelium Cells , 2001, Science.

[27]  R. Cherry,et al.  Single particle tracking of cell-surface HLA-DR molecules using R-phycoerythrin labeled monoclonal antibodies and fluorescence digital imaging. , 1996, Journal of cell science.

[28]  A. Chalk,et al.  Activation of PTHrP-cAMP-CREB1 signaling following p53 loss is essential for osteosarcoma initiation and maintenance , 2016, eLife.

[29]  M. Ueda,et al.  Transient Acceleration of Epidermal Growth Factor Receptor Dynamics Produces Higher-Order Signaling Clusters. , 2018, Journal of molecular biology.

[30]  Y. Sako,et al.  Mutation-Specific Mechanisms of Hyperactivation of Noonan Syndrome SOS Molecules Detected with Single-molecule Imaging in Living Cells , 2017, Scientific Reports.

[31]  Y. Teramura,et al.  Single‐molecule analysis of epidermal growth factor binding on the surface of living cells , 2006, EMBO Journal.

[32]  W. Sellers,et al.  Tumor suppressor PTEN acts through dynamic interaction with the plasma membrane. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[33]  J. Schlessinger,et al.  Cell Signaling by Receptor Tyrosine Kinases , 2000, Cell.