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Single-Cell Approaches to Dissect Cellular Signaling Networks

  • Chapter
Regulatory Networks in Stem Cells

Part of the book series: Stem Cell Biology and Regenerative Medicine ((STEMCELL))

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Abstract

Progress in understanding signal transduction, especially in rare and heterogeneous stem cell populations, is dependent on advances in single-cell assays. Newly developed techniques based on flow cytometry, capillary electrophoresis and live-cell imaging have enabled researchers to study kinase activity at the single-cell level. Since kinase activation is central to the regulation of virtually all cellular functions, single-cell kinase assays promise to help elucidating the molecular mechanisms controlling stem cell fate decisions.

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References

  1. Callard R, George AJ, Stark J. Cytokines, chaos and complexity. Immunity. 1999;11(5):507–13.

    Article  PubMed  CAS  Google Scholar 

  2. Ferrell Jr. JE, Machleder EM. The biochemical basis of an all-or-none cell fate switch in Xenopus oocytes. Science. 1998;280: 895–8.

    Article  PubMed  CAS  Google Scholar 

  3. Krutzik PO, Irish JM, Nolan GP, et al. Analysis of protein phosphorylation and cellular signaling events by flow cytometry: techniques and clinical applications. Clin Immunol. 2004;110: 206–21.

    Article  PubMed  CAS  Google Scholar 

  4. Krutzik PO, Nolan GP. Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events. Cytometry. 2003;55A:61–70.

    Article  CAS  Google Scholar 

  5. Muller S, Demotz S, Bulliard C, et al. Kinetics and extent of protein tyrosine kinase activation in individual T cells upon antigenic stimulation. Immunology. 1999;97(2):287–93.

    Article  PubMed  CAS  Google Scholar 

  6. Chow S, Patel H, Hedley DW. Measurement of MAP kinase activation by flow cytometry using phospho-specific antibodies to MEK and ERK: potential for pharmacodynamic monitoring of signal transduction inhibitors. Cytometry. 2001;46:72–8.

    Article  PubMed  CAS  Google Scholar 

  7. Perez OD, Nolan GP. Simultaneous measurement of multiple active kinase states using polychromatic flow cytometry. Nat Biotechnol. 2002;20:155–62.

    PubMed  CAS  Google Scholar 

  8. Irish JM, Hovland R, Krutzik PO, et al. Single cell profiling of potentiated phospho-protein networks in cancer cells. Cell. 2004;118:217–28.

    Article  PubMed  CAS  Google Scholar 

  9. Irish JM, Kotecha N, Nolan GP. Mapping normal and cancer cell signalling networks: towards single-cell proteomics. Nat Rev Cancer. 2006;6(2):146–55.

    Article  PubMed  CAS  Google Scholar 

  10. 10. Nolan GP. Deeper insights into hematological oncology disorders via single-cell phospho-signaling analysis. Hematology. 2006: 123–7.

    Google Scholar 

  11. Sims CE, Allbritton NL. Single-cell kinase assays: opening a window onto cell behavior. Curr Opin Biotechnol. 2003;14(1):23–8.

    Article  PubMed  CAS  Google Scholar 

  12. Babu SC, Song EJ, Babar SM, et al. Capillary electrophoresis of signaling molecules. Electrophoresis. 2007;21(9):890–7.

    Google Scholar 

  13. Meredith GD, Sims CE, Soughayer JS, et al. Measurement of kinase activation in single mammalian cells. Nat Biotechnol. 2000;18:309–12.

    Article  PubMed  CAS  Google Scholar 

  14. Brown RB, Audet J. Sampling efficiency of a single-cell capillary electrophoresis system. Cytometry A. 2007;71(10):882–8.

    PubMed  Google Scholar 

  15. Li H, Sims CE, Kaluzova M, et al. A quantitative single-cell assay for protein kinase B revealls important insights into the biochemical behavior of an intracellular substrate peptide. Biochemistry. 2004;43(6):1599–608.

    Article  PubMed  CAS  Google Scholar 

  16. Manceur A, Wu A, Audet J. Flow cytometric screening of cell-penetrating peptides for their uptake into embryonic and stem cells. Anal Biochem. 2007;364(1):51–9.

    Article  PubMed  CAS  Google Scholar 

  17. Soughayer JS, Wang Y, Li H, et al. Characterization of TAT-mediated transport of detachable kinase substrates. Biochemistry. 2004;43(26):8528–40.

    Article  PubMed  CAS  Google Scholar 

  18. Chalfie M, Tu Y, Euskirchen G, et al. Green fluorescent protein as a marker for gene expression. Science. 1994;263(5148):802–5.

    Article  PubMed  CAS  Google Scholar 

  19. Inouye S, Tsuji FI. Aequorea green fluorescent protein. Expression of the gene and fluorescence characteristics of the recombinant protein. FEBS Lett. 1994;341(2–3):277–80.

    Article  PubMed  CAS  Google Scholar 

  20. Tsien RY. The green fluorescent protein. Annu Rev Biochem. 1998;67:509–44.

    Article  PubMed  CAS  Google Scholar 

  21. Shen K, Meyer T. Dynamic control of CaMKII translocation and localization in hippocampal neurons by NMDA receptor stimulation. Science. 1999;284(5411):162–6.

    Article  PubMed  CAS  Google Scholar 

  22. Hirose K, Kadowaki S, Tanabe M, et al. Spatiotemporal dynamics of inositol 1,4,5-trisphosphate that underlies complex Ca2+ mobilization patterns. Science. 1999;284:1527–30.

    Article  PubMed  CAS  Google Scholar 

  23. Tanimura A, Nezu A, Morita T, et al. Interplay between calcium, diacylglycerol, and phosphorylation in the spatial and temporal regulation of PKCalpha-GFP. J Biol Chem. 2002;277: 29054–62.

    Article  PubMed  CAS  Google Scholar 

  24. Sawano A, Hama H, Saito N, et al. Multicolor imaging of Ca2+ and protein kinase C signals using novel epifluorescence microscopy. Biophys J. 2002;82:1076–85.

    Article  PubMed  CAS  Google Scholar 

  25. Pepperkok R, Squire A, Geley S, et al. Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy. Curr Biol. 1999;9:269–72.

    Article  PubMed  CAS  Google Scholar 

  26. Lackowicz JR. Principles of fluorescence spectroscopy. New York: Plenum Press. 1986.

    Google Scholar 

  27. Stryer L, Haugland RP. Energy transfer: a spectroscopic ruler. Proc Natl Acad Sci U S A. 1967;58:719–26.

    Article  PubMed  CAS  Google Scholar 

  28. Shaner NC, Campbell RE, Steinbach PA, et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol. 2004;22(12):1567–72.

    Article  PubMed  CAS  Google Scholar 

  29. Shaner NC, Steinbach PA, Tsien RY. A guide to choosing fluorescent proteins. Nat Methods. 2005;2(12):905–9.

    Article  PubMed  CAS  Google Scholar 

  30. Mochizuki N, Yamashita S, Kurokawa K, et al. Spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Nature. 2001;411:1065–8.

    Article  PubMed  CAS  Google Scholar 

  31. Miyawaki A, Llopis J, Heim R, et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature. 1997;388:882–7.

    Article  PubMed  CAS  Google Scholar 

  32. Zhang J, Allen MD. FRET-based biosensors for protein kinases: illuminating the kinome. Mol BioSyst. 2007;3:759–65.

    Article  PubMed  CAS  Google Scholar 

  33. Ni Q, Titov DV, Zhang J. Analyzing protein kinase dynamics in living cells with FRET reporters. Methods. 2006;40:279–86.

    Article  PubMed  CAS  Google Scholar 

  34. Zhang J, Ma Y, Taylor SS, et al. Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering. Proc Natl Acad Sci U S A. 2001;98:14997–5002.

    Article  PubMed  CAS  Google Scholar 

  35. Wang Y, Botvinick EL, Zhao Y, et al. Visualizing the mechanical activation of Src. Nature. 2005;434:1040–5.

    Article  PubMed  CAS  Google Scholar 

  36. Ting AY, Kain KH, Klemke RL, et al. Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells. Proc Natl Acad Sci U S A. 2001;98:15003–8.

    Article  PubMed  CAS  Google Scholar 

  37. Sato M, Ozawa T, Inukai K, et al. Fluorescent indicators for imaging protein phosphorylation in single living cells. Nat Biotechnol. 2002;20:287–94.

    Article  PubMed  CAS  Google Scholar 

  38. Violin JD, Zhang J, Tsien RY, et al. A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C. J Cell Biol. 2003;161:899–909.

    Article  PubMed  CAS  Google Scholar 

  39. Sasaki K, Sato M, Umezawa Y. Fluorescent indicators for Akt/protein kinase B and dynamics of Akt activity visualized in living cells. J Biol Chem. 2003;278:30945–51.

    Article  PubMed  CAS  Google Scholar 

  40. Zhang J, Hupfeld CJ, S.Taylor S, et al. Insulin disrupts \(\beta\)-adrenergic signalling to protein kinase A in adipocytes. Nature. 2005;437:569–73.

    Article  PubMed  CAS  Google Scholar 

  41. Kunkel MT, Ni Q, Tsien RY, et al. Spatio-temporal dynamics of protein kinase B/Akt signaling revealed by a genetically encoded fluorescent reporter. J Biol Chem. 2005;280:5581–7.

    Article  PubMed  CAS  Google Scholar 

  42. Johnson SA, You Z, Hunter T. Monitoring ATM kinase activity in living cells. DNA Repair (Amst). 2007;6:1277–84.

    Article  CAS  Google Scholar 

  43. Kunkel MT, Toker A, Tsien RY, et al. Calcium-dependent regulation of protein kinase D revealed by a genetically encoded kinase activity reporter. J Biol Chem. 2007;282:6733–42.

    Article  PubMed  CAS  Google Scholar 

  44. Sato M, Kawai Y, Umezawa Y. Genetically encoded fluorescent indicators to visualize protein phosphorylation by extracellular signal-regulated kinase in single living cells. Anal Chem. 2007;79:2570–5.

    Article  PubMed  CAS  Google Scholar 

  45. Calleja V, Ameer-Beg SM, Vojnovic B, et al. Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy. Biochem J. 2003;372:33–40.

    Article  PubMed  CAS  Google Scholar 

  46. Calleja V, Alcor D, Laguerre M, et al. Intramolecular and intermolecular interactions of protein kinase B define its activation in vivo. PLoS Biol. 2007;5(4):e95.

    Article  PubMed  Google Scholar 

  47. Takao K, Okamoto K, Nakagawa T, et al. Visualization of synaptic Ca2+/calmodulin-dependent protein kinase II activity in living neurons. J Neurosci. 2005;25:3107–12.

    Article  PubMed  CAS  Google Scholar 

  48. Fujioka A, Terai K, Itoh RE, et al. Dynamics of the Ras/ERK MAPK cascade as monitored by fluorescent probes. J Biol Chem. 2006;281(13):8917–26.

    Article  PubMed  CAS  Google Scholar 

  49. Neininger A, Thielemann H, Gaestel M. FRET-based detection of different conformations of MK2. EMBO Rep. 2001;2:703–8.

    Article  PubMed  CAS  Google Scholar 

  50. Nagai Y, Miyazaki M, Aoki R, et al. A fluorescent indicator for visualizing cAMP-induced phosphorylation in vivo. Nat Biotechnol. 2000;18:313–6.

    Article  PubMed  CAS  Google Scholar 

  51. Schleifenbaum A, Stier G, Gasch A, et al. Genetically encoded FRET probe for PKC activity based on pleckstrin. J Am Chem Soc. 2004;126(38):11786–7.

    Article  PubMed  CAS  Google Scholar 

  52. Green HM, Alberola-Ila J. Development of ERK activity sensor, an in vitro, FRET-based sensor of extracellular regulated kinase activity. BMC Chem Biol. 2005;5:1.

    Article  PubMed  Google Scholar 

  53. Gromova KV, Friedrich M, Noskov A, et al. Visualizing Smad1/4 signaling response to bone morphogenetic protein-4 activation by FRET biosensors. Biochimica et Biophysica Acta. 2007;1773:1759–73.

    Article  PubMed  CAS  Google Scholar 

  54. Chun W, Johnson GVW. Activation of glycogen synthase kinase 3\(\beta\) promotes the intermolecular association of tau. The use of fluorescence resonance energy transfer microscopy. J Biol Chem. 2007;282:23410–7.

    Article  PubMed  CAS  Google Scholar 

  55. Ruehr ML, Zakhary DR, Damron DS, et al. Cyclic AMP-dependent protein kinase binding to A-kinase anchoring proteins in living cells by fluorescence resonance energy transfer of green fluorescent protein fusion proteins. J Biol Chem. 1999;274:33092–6.

    Article  PubMed  CAS  Google Scholar 

  56. Heikal AA, Hess ST, Baird GS, et al. Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: coral red (dsRed) and yellow (Citrine). Proc Natl Acad Sci U S A. 2000;97: 11996–2001.

    Article  PubMed  CAS  Google Scholar 

  57. Ng T, Squire A, Hansra G, et al. Imaging protein kinase Calpha activation in cells. Science. 1999;283(5410):2085–9.

    Article  PubMed  CAS  Google Scholar 

  58. Verveer PJ, Wouters FS, Reynolds AR, et al. Quantitative imaging of lateral ErbB1 receptor signal propagation in the plasma membrane. Science. 2000;290:1567–70.

    Article  PubMed  CAS  Google Scholar 

  59. Wouters FS, Bastiaens PI. Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells. Curr Biol. 1999;9: 1127–30.

    Article  PubMed  CAS  Google Scholar 

  60. Hahn k, Toutchkine A. Live-cell fluorescent biosensors for activated signaling proteins. Curr Opin Cell Biol. 2002;14:167–72.

    Article  PubMed  CAS  Google Scholar 

  61. Kraynov VS, chamberlain C, Bokoch GM, et al. Localized Rac activation dynamics visualized in living cells. Science. 2000;290:333–7.

    Article  PubMed  CAS  Google Scholar 

  62. Chamberlain C, Hahn KM. Watching proteins in the wild: fluorescence methods to study protein dynamics in living cells. Traffic. 2000;1:755–62.

    Article  PubMed  CAS  Google Scholar 

  63. Cornish VW, Hahn KM, Schultz PG. Site-specific protein modification using a ketone handle. J Am Chem Soc. 1996;118: 8150–1.

    Article  CAS  Google Scholar 

  64. Wang L, Brock A, Herberich B, et al. Expanding the genetic code of Escherichia coli. Science. 2001;292:498–500.

    Article  PubMed  CAS  Google Scholar 

  65. Schwarze SR, Dowdy SF. In vivo protein transduction: intracellular delivery of biologically active proteins, compounds and DNA. Trends Pharmcol Sci. 2000;21:45–8.

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Institute of Biomaterials and Biomedical Engineering, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 164 College St., Room 407, M5S 3G9, Toronto, ON, Canada

    Weijia Wang & Julie Audet (Assistant Professor)

Authors
  1. Weijia Wang
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  2. Julie Audet
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Corresponding author

Correspondence to Julie Audet .

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Editors and Affiliations

  1. Memorial Sloan-Kettering Cancer Center, New York, NY, USA

    Vinagolu K. Rajasekhar MSc, MPhil, PhD

  2. Invitrogen Corporation, Frederick, MD, USA

    Mohan C. Vemuri Ph.D.

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC, a part of Springer Science+Business Media, LLC

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Wang, W., Audet, J. (2009). Single-Cell Approaches to Dissect Cellular Signaling Networks. In: Rajasekhar, V.K., Vemuri, M.C. (eds) Regulatory Networks in Stem Cells. Stem Cell Biology and Regenerative Medicine. Humana Press. https://doi.org/10.1007/978-1-60327-227-8_29

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