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Single-molecule methods for studying gene regulation in vivo

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Abstract

The recent emergence of new experimental tools employing sensitive fluorescence detection in vivo has made it possible to visualize various aspects of gene regulation at the single-molecule level in the native, intracellular context. In this review, we will first describe general considerations for in vivo, single-molecule fluorescence detection of DNA, mRNA, and protein molecules involved in gene regulation. We will then give an overview of the rapidly evolving suite of molecular tools available for observing gene regulation in vivo and discuss new insights they have brought into gene regulation.

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References

  1. Ambrose EJ (1956) A surface contact microscope for the study of cell movements. Nature 178:1194. doi:10.1038/1781194a0

    Article  PubMed  CAS  Google Scholar 

  2. Bakshi S, Siryaporn A, Goulian M, Weisshaar JC (2012) Superresolution imaging of ribosomes and RNA polymerase in live Escherichia coli cells. Mol Microbiol 85:21–38

    Article  PubMed  CAS  Google Scholar 

  3. Balázsi G, van Oudenaarden A, Collins JJ (2011) Cellular decision making and biological noise: from microbes to mammals. Cell 144:910–925

    Article  PubMed  Google Scholar 

  4. Bates D, Kleckner N (2005) Chromosome and replisome dynamics in E. coli: loss of sister cohesion triggers global chromosome movement and mediates chromosome segregation. Cell 121:899–911

    Article  PubMed  CAS  Google Scholar 

  5. Benke A, Manley S (2012) Live-cell dSTORM of cellular DNA based on direct DNA labeling. Chem Bio Chem 13:298–301

    Article  PubMed  CAS  Google Scholar 

  6. Benson RC, Meyer RA, Zaruba ME, McKhann GM (1979) Cellular autofluorescence—is it due to flavins? J Histochem Cytochem 27:44–48

    Article  PubMed  CAS  Google Scholar 

  7. Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645

    Article  PubMed  CAS  Google Scholar 

  8. Biteen JS, Thompson MA, Tselentis NK, Bowman GR, Shapiro L, Moerner WE (2008) Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP. Nature Methods 5:947–949

    Article  PubMed  CAS  Google Scholar 

  9. Blainey PC, Luo G, Kou SC, Mangel WF, Verdine GL, Bagchi B et al (2009) Nonspecifically bound proteins spin while diffusing along DNA. Nat Struct Mol Biol 16:1224–1229

    Article  PubMed  CAS  Google Scholar 

  10. Bumgarner SL, Neuert G, Voight BF, Symbor-Nagrabska A, Grisafi P, van Oudenaarden A et al (2012) Single-cell analysis reveals that noncoding RNAs contribute to clonal heterogeneity by modulating transcription factor recruitment. Molecular Cell 45:470–482

    Article  PubMed  CAS  Google Scholar 

  11. Cai L, Friedman N, Xie XS (2006) Stochastic protein expression in individual cells at the single molecule level. Nature 440:358–362

    Article  PubMed  CAS  Google Scholar 

  12. Choi PJ, Cai L, Frieda K, Xie XS (2008) A stochastic single-molecule event triggers phenotype switching of a bacterial cell. Science 322:442–446

    Article  PubMed  CAS  Google Scholar 

  13. Dickson RM, Cubitt AB, Tsien RY, Moerner WE (1997) On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature 388:355–358

    Article  PubMed  CAS  Google Scholar 

  14. Eldar A, Elowitz MB (2010) Functional roles for noise in genetic circuits. Nature 467:167–173

    Article  PubMed  CAS  Google Scholar 

  15. Elf J, Li G-W, Xie XS (2007) Probing transcription factor dynamics at the single-molecule level in a living cell. Science 316:1191–1194

    Article  PubMed  CAS  Google Scholar 

  16. Elowitz MB, Levine AJ, Siggia ED, Swain PS (2002) Stochastic gene expression in a single cell. Science 297:1183–1186

    Article  PubMed  CAS  Google Scholar 

  17. English BP, Hauryliuk V, Sanamrad A, Tankov S, Dekker NH, Elf J (2011) Single-molecule investigations of the stringent response machinery in living bacterial cells. PNAS 108:E365–E373

    Article  PubMed  CAS  Google Scholar 

  18. English BP, Sanamrad A, Tankov S, Hauryliuk V, Elf J (2010) Tracking of individual freely diffusing fluorescent protein molecules in the bacterial cytoplasm. arXiv:1003.2110

  19. Femino AM, Fay FS, Fogarty K, Singer RH (1998) Visualization of single RNA transcripts in situ. Science 280:585–590

    Article  PubMed  CAS  Google Scholar 

  20. Fernandez-Suarez M, Ting AY (2008) Fluorescent probes for super-resolution imaging in living cells. Nat Rev Mol Cell Biol 9:929–943

    Article  PubMed  CAS  Google Scholar 

  21. Flors C (2011) DNA and chromatin imaging with super-resolution fluorescence microscopy based on single-molecule localization. Biopolymers 95:290–297

    Article  PubMed  CAS  Google Scholar 

  22. Flors C, Ravarani CNJ, Dryden DTF (2009) Super-resolution imaging of DNA labelled with intercalating dyes. ChemPhysChem 10:2201–2204

    Article  PubMed  CAS  Google Scholar 

  23. Fusco D, Bertrand E, Singer RH (2004) Imaging of single mRNAs in the cytoplasm of living cells. Prog Mol Subcell Biol 35:135–150

    Article  PubMed  Google Scholar 

  24. Ghaemmaghami S, Huh WK, Bower K, Howson RW, Belle A, Dephoure N et al (2003) Global analysis of protein expression in yeast. Nature 425:737–741

    Article  PubMed  CAS  Google Scholar 

  25. Ghosh I, Hamilton AD, Regan L (2000) Antiparallel leucine zipper-directed protein reassembly: application to the green fluorescent protein. J Am Chem Soc 122:5658–5659

    Article  CAS  Google Scholar 

  26. Giepmans BN, Adams SR, Ellisman MH, Tsien RY (2006) The fluorescent toolbox for assessing protein location and function. Science 312:217–224

    Article  PubMed  CAS  Google Scholar 

  27. Golding I, Cox EC (2004) RNA dynamics in live Escherichia coli cells. PNAS 101:11310–11315

    Article  PubMed  CAS  Google Scholar 

  28. Golding I, Paulsson J, Zawilski SM, Cox EC (2005) Real-time kinetics of gene activity in individual bacteria. Cell 123:1025–1036

    Article  PubMed  CAS  Google Scholar 

  29. Guo W, Keckesova Z, Donaher JL, Shibue T, Tischler V, Reinhardt F et al (2012) Slug and Sox9 cooperatively determine the mammary stem cell state. Cell 148:1015–1028

    Article  PubMed  CAS  Google Scholar 

  30. Guptasarma P (1995) Does replication-induced transcription regulate synthesis of the myriad low copy number proteins of Escherichia coli? Bioessays 17:987–997

    Article  PubMed  CAS  Google Scholar 

  31. Hammar P, Leroy P, Mahmutovic A, Marklund EG, Berg OG, Elf J (2012) The lac repressor displays facilitated diffusion in living cells. Science 336:1595–1598

    Article  PubMed  CAS  Google Scholar 

  32. Hansen FG, Atlung T (2011) YGFP: a spectral variant of GFP. Biotechniques 50:411–412

    Article  PubMed  CAS  Google Scholar 

  33. Hensel Z, Feng H, Han B, Hatem C, Wang J, Xiao J (2012) Stochastic expression dynamics of a transcription factor revealed by single-molecule noise analysis. Nat Struct Mol Biol 19:797–802

    Article  PubMed  CAS  Google Scholar 

  34. Holland MJ (2002) Transcript abundance in yeast varies over six orders of magnitude. J Biol Chem 277:14363–14366

    Article  PubMed  CAS  Google Scholar 

  35. Huh D, Paulsson J (2011) Non-genetic heterogeneity from stochastic partitioning at cell division. Nat Genet 43:95–100

    Article  PubMed  CAS  Google Scholar 

  36. Itzkovitz S, Lyubimova A, Blat IC, Maynard M, van Es J, Lees J et al (2012) Single-molecule transcript counting of stem-cell markers in the mouse intestine. Nat Cell Biol 14:106–114

    Article  CAS  Google Scholar 

  37. Larson DR, Zenklusen D, Wu B, Chao JA, Singer RH (2011) Real-time observation of transcription initiation and elongation on an endogenous yeast gene. Science 332:475–478

    Article  PubMed  CAS  Google Scholar 

  38. Lau IF, Filipe SR, Soballe B, Okstad OA, Barre FX, Sherratt DJ (2003) Spatial and temporal organization of replicating Escherichia coli chromosomes. Mol Microbiol 49:731–743

    Article  PubMed  CAS  Google Scholar 

  39. Lee SF, Thompson MA, Schwartz MA, Shapiro L, Moerner WE (2011) Super-resolution imaging of the nucleoid-associated protein HU in Caulobacter crescentus. Biophys J 100:L31–L33

    Article  PubMed  CAS  Google Scholar 

  40. Li G-W, Berg OG, Elf J (2009) Effects of macromolecular crowding and DNA looping on gene regulation kinetics. Nat Phys 5:294–297

    Article  CAS  Google Scholar 

  41. Li G-W, Xie XS (2011) Central dogma at the single-molecule level in living cells. Nature 475:308–315

    Article  PubMed  CAS  Google Scholar 

  42. Lidstrom ME, Konopka MC (2010) The role of physiological heterogeneity in microbial population behavior. Nat Chem Biol 6:705–712

    Article  PubMed  CAS  Google Scholar 

  43. Little SC, Tkačik G, Kneeland TB, Wieschaus EF, Gregor T (2011) The formation of the bicoid morphogen gradient requires protein movement from anteriorly localized mRNA. PLoS Biol 9:e1000596

    Article  PubMed  CAS  Google Scholar 

  44. Llopis PM, Jackson AF, Sliusarenko O, Surovtsev I, Heinritz J, Emonet T et al (2010) Spatial organization of the flow of genetic information in bacteria. Nature 466:77–81

    Article  CAS  Google Scholar 

  45. Lubeck E, Cai L (2012) Single-cell systems biology by super-resolution imaging and combinatorial labeling. Nat Methods 9(7):743–748

    Article  PubMed  CAS  Google Scholar 

  46. McKinney SA, Murphy CS, Hazelwood KL, Davidson MW, Looger LL (2009) A bright and photostable photoconvertible fluorescent protein. Nat Methods 6:131–133

    Article  PubMed  CAS  Google Scholar 

  47. Middelkoop TC, Williams L, Yang P-T, Luchtenberg J, Betist MC, Ji N et al (2012) The thrombospondin repeat containing protein MIG-21 controls a left–right asymmetric Wnt signaling response in migrating C. elegans neuroblasts. Dev Biol 361:338–348

    Article  PubMed  CAS  Google Scholar 

  48. Mitarai N, Dodd IB, Crooks MT, Sneppen K (2008) The generation of promoter-mediated transcriptional noise in bacteria. PLoS Comput Biol 4:e1000109

    Article  PubMed  Google Scholar 

  49. Mor A, Suliman S, Ben-Yishay R, Yunger S, Brody Y, Shav-Tal Y (2010) Dynamics of single mRNP nucleocytoplasmic transport and export through the nuclear pore in living cells. Nature Cell Biology 12:543–552

    Article  PubMed  CAS  Google Scholar 

  50. Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, Miyawaki A (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol 20:87–90

    Article  PubMed  CAS  Google Scholar 

  51. Ostermayer FW, Allen RB, Dierschke EG (1971) Room-temperature cw operation of a GaAs1 − xPx diode-pumped YAG:Nd Laser. Appl Phys Lett 19:289–292

    Article  CAS  Google Scholar 

  52. Paige JS, Wu KY, Jaffrey SR (2011) RNA mimics of green fluorescent protein. Science 333:642–646

    Article  PubMed  CAS  Google Scholar 

  53. Pedraza JM, Paulsson J (2008) Effects of molecular memory and bursting on fluctuations in gene expression. Science 319:339–343

    Article  PubMed  CAS  Google Scholar 

  54. Pedraza JM, van Oudenaarden A (2005) Noise propagation in gene networks. Science 307:1965–1969

    Article  PubMed  CAS  Google Scholar 

  55. Pinaud F, Dahan M (2011) Targeting and imaging single biomolecules in living cells by complementation-activated light microscopy with split-fluorescent proteins. PNAS 108:E201–E210

    Article  PubMed  CAS  Google Scholar 

  56. Potrykus K, Cashel M (2008) (p)ppGpp Still Magical?*. Annu Rev Microbiol 62:35–51

    Article  PubMed  CAS  Google Scholar 

  57. Raj A, Peskin CS, Tranchina D, Vargas DY, Tyagi S (2006) Stochastic mRNA synthesis in mammalian cells. PLoS Biol 4:e309

    Article  PubMed  Google Scholar 

  58. Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S (2008) Imaging individual mRNA molecules using multiple singly labeled probes. Nature Methods 5:877–879

    Article  PubMed  CAS  Google Scholar 

  59. Raj A, van Oudenaarden A (2008) Nature, nurture, or chance: stochastic gene expression and its consequences. Cell 135:216–226

    Article  PubMed  CAS  Google Scholar 

  60. Raser JM, O’Shea EK (2004) Control of stochasticity in eukaryotic gene expression. Science 304:1811–1814

    Article  PubMed  CAS  Google Scholar 

  61. Rosenfeld N, Young JW, Alon U, Swain PS, Elowitz MB (2005) Gene regulation at the single-cell level. Science 307:1962–1965

    Article  PubMed  CAS  Google Scholar 

  62. Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3:793–795

    Article  PubMed  CAS  Google Scholar 

  63. Saffer AM, Kim DH, van Oudenaarden A, Horvitz HR (2011) The caenorhabditis elegans synthetic multivulva genes prevent ras pathway activation by tightly repressing global ectopic expression of lin-3 EGF. PLoS Genet 7:e1002418

    Article  PubMed  CAS  Google Scholar 

  64. Santangelo P, Nitin N, Bao G (2006) Nanostructured probes for RNA detection in living cells. Ann Biomed Eng 34:39–50

    Article  PubMed  Google Scholar 

  65. Schoen I, Ries J, Klotzsch E, Ewers H, Vogel V (2011) Binding-activated localization microscopy of DNA structures. Nano Lett 11:4008–4011

    Article  PubMed  CAS  Google Scholar 

  66. Schonhoft JD, Stivers JT (2012) Timing facilitated site transfer of an enzyme on DNA. Nat Chem Biol 8:205–210

    Article  PubMed  CAS  Google Scholar 

  67. Selvin PR, Ha T (2008) Single-molecule techniques: A laboratory manual, CSHL Press

  68. Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905–909

    Article  PubMed  CAS  Google Scholar 

  69. Silverman AP, Kool ET (2005) Quenched autoligation probes allow discrimination of live bacterial species by single nucleotide differences in rRNA. Nucl Acids Res 33:4978–4986

    Article  PubMed  CAS  Google Scholar 

  70. Snijder B, Pelkmans L (2011) Origins of regulated cell-to-cell variability. Nat Rev Mol Cell Biol 12:119–125

    Article  PubMed  CAS  Google Scholar 

  71. So L, Ghosh A, Zong C, Sepulveda LA, Segev R, Golding I (2011) General properties of transcriptional time series in Escherichia coli. Nat Genet 43:554–560

    Article  PubMed  CAS  Google Scholar 

  72. Stephen LZ-D (2012) E D Webb, Multicolour single molecule imaging on cells using a supercontinuum source. Biomedical Optics Express 3:400–406

    Article  Google Scholar 

  73. Straight AF, Belmont AS, Robinett CC, Murray AW (1996) GFP tagging of budding yeast chromosomes reveals that protein-protein interactions can mediate sister chromatid cohesion. Curr Biol 6:1599–1608

    Article  PubMed  CAS  Google Scholar 

  74. Tan RZ, van Oudenaarden A (2010) Transcript counting in single cells reveals dynamics of rDNA transcription. Mol Syst Biol 6:358

    Article  PubMed  Google Scholar 

  75. Taniguchi Y, Choi PJ, Li G-W, Chen H, Babu M, Hearn J et al (2010) Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells. Science 329:533–538

    Article  PubMed  CAS  Google Scholar 

  76. Testa I, Wurm CA, Medda R, Rothermel E, von Middendorf C, Fölling J et al (2010) Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength. Biophys J 99:2686–2694

    Article  PubMed  CAS  Google Scholar 

  77. Thattai M, van Oudenaarden A (2001) Intrinsic noise in gene regulatory networks. Proc Natl Acad Sci U S A 98:8614–8619

    Article  PubMed  CAS  Google Scholar 

  78. Trcek T, Larson DR, Moldón A, Query CC, Singer RH (2011) Single-molecule mRNA decay measurements reveal promoter-regulated mRNA stability in yeast. Cell 147:1484–1497

    Article  PubMed  CAS  Google Scholar 

  79. Umbarger MA, Toro E, Wright MA, Porreca GJ, Baù D, Hong S-H et al (2011) The three-dimensional architecture of a bacterial genome and its alteration by genetic perturbation. Molecular Cell 44:252–264

    Article  PubMed  CAS  Google Scholar 

  80. Valencia-Burton M, McCullough RM, Cantor CR, Broude NE (2007) RNA visualization in live bacterial cells using fluorescent protein complementation. Nature Methods 4:421–427

    PubMed  CAS  Google Scholar 

  81. van de Linde S, Löschberger A, Klein T, Heidbreder M, Wolter S, Heilemann M et al (2011) Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat Protoc 6:991–1009

    Article  PubMed  Google Scholar 

  82. van den Bogaard PTC, Tyagi S (2008) Using molecular beacons to study dispersal of mRNPs from the gene locus. In: Hancock R, Walker JM (eds) The Nucleus, Humana Press, pp 91–103

  83. Vargas DY, Raj A, Marras SAE, Kramer FR, Tyagi S (2005) Mechanism of mRNA transport in the nucleus. Proc Natl Acad Sci U S A 102:17008–17013

    Article  PubMed  CAS  Google Scholar 

  84. Vargas DY, Shah K, Batish M, Levandoski M, Sinha S, Marras SAE et al (2011) Single-molecule imaging of transcriptionally coupled and uncoupled splicing. Cell 147:1054–1065

    Article  PubMed  CAS  Google Scholar 

  85. Velculescu VE, Zhang L, Zhou W, Vogelstein J, Basrai MA, Bassett DE et al (1997) Characterization of the yeast transcriptome. Cell 88:243–251

    Article  PubMed  CAS  Google Scholar 

  86. Viollier PH, Thanbichler M, McGrath PT, West L, Meewan M, McAdams HH et al (2004) Rapid and sequential movement of individual chromosomal loci to specific subcellular locations during bacterial DNA replication. PNAS 101:9257–9262

    Article  PubMed  CAS  Google Scholar 

  87. Volfson D, Marciniak J, Blake WJ, Ostroff N, Tsimring LS, Hasty J (2006) Origins of extrinsic variability in eukaryotic gene expression. Nature 439:861–864

    Article  PubMed  CAS  Google Scholar 

  88. Wang W, Li G-W, Chen C, Xie XS, Zhuang X (2011) Chromosome organization by a nucleoid-associated protein in live bacteria. Science 333:1445–1449

    Article  PubMed  CAS  Google Scholar 

  89. Weber SC, Spakowitz AJ, Theriot JA (2012) Nonthermal ATP-dependent fluctuations contribute to the in vivo motion of chromosomal loci. PNAS 109:7338–7343

    Article  PubMed  CAS  Google Scholar 

  90. Wombacher R, Heidbreder M, van de Linde S, Sheetz MP, Heilemann M, Cornish VW et al (2010) Live-cell super-resolution imaging with trimethoprim conjugates. Nature Methods 7:717–719

    Article  PubMed  CAS  Google Scholar 

  91. Wu B, Piatkevich KD, Lionnet T, Singer RH, Verkhusha VV (2011) Modern fluorescent proteins and imaging technologies to study gene expression, nuclear localization, and dynamics. Curr Opin Cell Biol 23:310–317

    Article  PubMed  CAS  Google Scholar 

  92. Xie XS, Choi PJ, Li GW, Lee NK, Lia G (2008) Single-molecule approach to molecular biology in living bacterial cells. Annu Rev Biophys 37:417–444

    Article  PubMed  CAS  Google Scholar 

  93. Yu J, Xiao J, Ren X, Lao K, Xie XS (2006) Probing gene expression in live cells, one protein molecule at a time. Science 311:1600–1603

    Article  PubMed  CAS  Google Scholar 

  94. Zeng L, Skinner SO, Zong C, Sippy J, Feiss M, Golding I (2010) Decision making at a subcellular level determines the outcome of bacteriophage infection. Cell 141:682–691

    Article  PubMed  CAS  Google Scholar 

  95. Zenklusen D, Larson DR, Singer RH (2008) Single-RNA counting reveals alternative modes of gene expression in yeast. Nat Struct Mol Biol 15:1263–1271

    Article  PubMed  CAS  Google Scholar 

  96. Zhang M, Chang H, Zhang Y, Yu J, Wu L, Ji W et al (2012) Rational design of true monomeric and bright photoactivatable fluorescent proteins. Nat Methods 9:727–729

    Article  PubMed  CAS  Google Scholar 

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  1. Zach Hensel

    Present address: Okinawa Institute of Science and Technology, Okinawa, Japan

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  1. Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, 21211, USA

    Zach Hensel & Jie Xiao

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Hensel, Z., Xiao, J. Single-molecule methods for studying gene regulation in vivo. Pflugers Arch - Eur J Physiol 465, 383–395 (2013). https://doi.org/10.1007/s00424-013-1243-y

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