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In Vivo Tracking of Single Biomolecules: What Trajectories Tell Us About the Acting Forces

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Far-Field Optical Nanoscopy

Part of the book series: Springer Series on Fluorescence ((SS FLUOR,volume 14))

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Abstract

It would be the dream of many experimental scientists in cell biology to be able to follow the life of a protein molecule over time, to watch its encounters with other proteins, to record its conformational fluctuations, and from that data to understand its functionality. Indeed, technology is now at hand to detect the signal of a single molecule in a live cell context. In particular, researchers have been employing single molecule tracking to recover the forces that act on biomolecules: active transport can be discriminated from free or confined diffusion, and nanoscopic details within the molecular paths can be investigated. In the first part of this chapter, we provide an overview over typical diffusion models for biomolecular motion. Yet, experiment, data analysis, and interpretation are not that simple: trajectories are frequently too short, the data are too noisy, and only a vanishingly small fraction of proteins is actually visible. In the second part of this chapter, we therefore delineate how diffusion models can be implemented for data analysis. As a showcase, we discuss recent single molecule data obtained on Lck, an important kinase in T cell signaling.

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References

  1. Davis MM, Krogsgaard M, Huse M, Huppa J, Lillemeier BF, Li QJ (2007) T cells as a self-referential, sensory organ. Annu Rev Immunol 25:681–695

    CAS  Google Scholar 

  2. Boggon TJ, Eck MJ (2004) Structure and regulation of src family kinases. Oncogene 23(48):7918–7927

    CAS  Google Scholar 

  3. Kabouridis PS, Magee AI, Ley SC (1997) S-acylation of lck protein tyrosine kinase is essential for its signalling function in t lymphocytes. EMBO J 16(16):4983–4998

    CAS  Google Scholar 

  4. Zimmermann L, Paster W, Weghuber J, Eckerstorfer P, Stockinger H, Schütz GJ (2010) Direct observation and quantitative analysis of lck-exchange between plasma membrane and cytosol in living t cells. J Biol Chem 285(9):6063–6070

    CAS  Google Scholar 

  5. Veillette A, Bookman MA, Horak EM, Bolen JB (1988) The cd4 and cd8 t cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck. Cell 55(2):301–308

    CAS  Google Scholar 

  6. Shaw AS, Chalupny J, Whitney JA, Hammond C, Amrein KE, Kavathas P, Sefton BM, Rose JK (1990) Short related sequences in the cytoplasmic domains of cd4 and cd8 mediate binding to the amino-terminal domain of the p56lck tyrosine protein kinase. Mol Cell Biol 10(5):1853–1862

    CAS  Google Scholar 

  7. Turner JM, Brodsky MH, Irving BA, Levin SD, Perlmutter RM, Littman DR (1990) Interaction of the unique n-terminal region of tyrosine kinase p56lck with cytoplasmic domains of cd4 and cd8 is mediated by cysteine motifs. Cell 60(5):755–765

    CAS  Google Scholar 

  8. Filipp D, Leung BL, Zhang J, Veillette A, Julius M (2004) Enrichment of lck in lipid rafts regulates colocalized fyn activation and the initiation of proximal signals through tcr alpha beta. J Immunol 172(7):4266–4274

    CAS  Google Scholar 

  9. Kim PW, Sun ZY, Blacklow SC, Wagner G, Eck MJ (2003) A zinc clasp structure tethers lck to t cell coreceptors cd4 and cd8. Science 301(5640):1725–1728

    CAS  Google Scholar 

  10. Schwarzenbacher M, Kaltenbrunner M, Brameshuber M, Hesch C, Paster W, Weghuber J, Heise B, Sonnleitner A, Stockinger H, Schütz GJ (2008) Micropatterning for quantitative analysis of protein-protein interactions in living cells. Nat Methods 5(12):1053–1060

    CAS  Google Scholar 

  11. Li QJ, Dinner AR, Qi S, Irvine DJ, Huppa JB, Davis MM, Chakraborty AK (2004) Cd4 enhances t cell sensitivity to antigen by coordinating lck accumulation at the immunological synapse. Nat Immunol 5(8):791–799

    CAS  Google Scholar 

  12. Choudhuri K, Wiseman D, Brown MH, Gould K, van der Merwe PA (2005) T-cell receptor triggering is critically dependent on the dimensions of its peptide-mhc ligand. Nature 436(7050):578–582

    CAS  Google Scholar 

  13. Irles C, Symons A, Michel F, Bakker TR, van der Merwe PA, Acuto O (2003) Cd45 ectodomain controls interaction with gems and lck activity for optimal tcr signaling. Nat Immunol 4(2):189–197

    CAS  Google Scholar 

  14. Eck MJ, Atwell SK, Shoelson SE, Harrison SC (1994) Structure of the regulatory domains of the src-family tyrosine kinase lck. Nature 368(6473):764–769

    CAS  Google Scholar 

  15. Panchamoorthy G, Fukazawa T, Stolz L, Payne G, Reedquist K, Shoelson S, Songyang Z, Cantley L, Walsh C, Band H (1994) Physical and functional interactions between sh2 and sh3 domains of the src family protein tyrosine kinase p59fyn. Mol Cell Biol 14(9):6372–6385

    CAS  Google Scholar 

  16. Romir J, Lilie H, Egerer-Sieber C, Bauer F, Sticht H, Muller YA (2007) Crystal structure analysis and solution studies of human lck-sh3; zinc-induced homodimerization competes with the binding of proline-rich motifs. J Mol Biol 365(5):1417–1428

    CAS  Google Scholar 

  17. Lee-Fruman KK, Collins TL, Burakoff SJ (1996) Role of the lck src homology 2 and 3 domains in protein tyrosine phosphorylation. J Biol Chem 271(40):25003–25010

    CAS  Google Scholar 

  18. Schütz GJ, Kada G, Pastushenko VP, Schindler H (2000) Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy. EMBO J 19(5):892–901

    Google Scholar 

  19. Sako Y, Minoghchi S, Yanagida T (2000) Single-molecule imaging of egfr signalling on the surface of living cells. Nat Cell Biol 2(3):168–172

    CAS  Google Scholar 

  20. Lord SJ, Lee HL, Moerner WE (2010) Single-molecule spectroscopy and imaging of biomolecules in living cells. Anal Chem 82(6):2192–2203

    CAS  Google Scholar 

  21. Mortensen KI, Churchman LS, Spudich JA, Flyvbjerg H (2010) Optimized localization analysis for single-molecule tracking and super-resolution microscopy. Nat Methods 7(5):377–381

    CAS  Google Scholar 

  22. Stallinga S, Rieger B (2010) Accuracy of the gaussian point spread function model in 2d localization microscopy. Opt Express 18(24):24461–24476

    CAS  Google Scholar 

  23. Schmidt T, Schütz GJ, Baumgartner W, Gruber HJ, Schindler H (1996) Imaging of single molecule diffusion. Proc Natl Acad Sci USA 93(7):2926–2929

    CAS  Google Scholar 

  24. Yildiz A, Forkey JN, McKinney SA, Ha T, Goldman YE, Selvin PR (2003) Myosin v walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300(5628):2061–2065

    CAS  Google Scholar 

  25. Pertsinidis A, Zhang Y, Chu S (2010) Subnanometre single-molecule localization, registration and distance measurements. Nature 466(7306):647–651

    CAS  Google Scholar 

  26. Wieser S, Schütz GJ (2008) Tracking single molecules in the live cell plasma membrane-do's and don't's. Methods 46(2):131–140

    CAS  Google Scholar 

  27. Serge A, Bertaux N, Rigneault H, Marguet D (2008) Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes. Nat Methods 5(8):687–694

    CAS  Google Scholar 

  28. Jaqaman K, Loerke D, Mettlen M, Kuwata H, Grinstein S, Schmid SL, Danuser G (2008) Robust single-particle tracking in live-cell time-lapse sequences. Nat Methods 5(8):695–702

    CAS  Google Scholar 

  29. Schutz GJ, Axmann M, Freudenthaler S, Schindler H, Kandror K, Roder JC, Jeromin A (2004) Visualization of vesicle transport along and between distinct pathways in neurites of living cells. Microsc Res Tech 63(3):159–167

    Google Scholar 

  30. Mudrakola HV, Zhang K, Cui B (2009) Optically resolving individual microtubules in live axons. Structure 17(11):1433–1441

    CAS  Google Scholar 

  31. Rudnick J, Gaspari G (1987) The shapes of random walks. Science 237:384–389

    CAS  Google Scholar 

  32. Saxton MJ (1993) Lateral diffusion in an archipelago. Single-particle diffusion. Biophys J 64(6):1766–1780

    CAS  Google Scholar 

  33. Metzler R, Klafter J (2000) The random walk's guide to anomalous diffusion: a fractional dynamics approach. Phys Rep 339(1):1–77

    CAS  Google Scholar 

  34. Powles JG, Mallett MJD, Rickayzen G, Evans WAB (1992) Exact analytic solutions for diffusion impeded by an infinite array of partially permeable barriers. Proc R Soc Lond A 436(1897):391–403

    Google Scholar 

  35. Saxton MJ, Jacobson K (1997) Single-particle tracking: applications to membrane dynamics. Annu Rev Biophys Biomol Struct 26:373–399

    CAS  Google Scholar 

  36. Gambin Y, Lopez-Esparza R, Reffay M, Sierecki E, Gov NS, Genest M, Hodges RS, Urbach W (2006) Lateral mobility of proteins in liquid membranes revisited. Proc Natl Acad Sci USA 103(7):2098–2102

    CAS  Google Scholar 

  37. Saffman PG, Delbruck M (1975) Brownian motion in biological membranes. Proc Natl Acad Sci USA 72(8):3111–3113

    CAS  Google Scholar 

  38. Hughes BD, Pailthorpe BA, White LR, Sawyer WH (1982) Extraction of membrane microviscosity from translational and rotational diffusion coefficients. Biophys J 37(3):673–676

    CAS  Google Scholar 

  39. Hughes BD, Pailthorpe BA, White LR (1981) The translational and rotational drag on a cylinder moving in a membrane. J Fluid Mech 110:349–372

    CAS  Google Scholar 

  40. Petrov EP, Schwille P (2008) Translational diffusion in lipid membranes beyond the saffman-delbruck approximation. Biophys J 94(5):L41–L43

    CAS  Google Scholar 

  41. Kumar M, Mommer MS, Sourjik V (2010) Mobility of cytoplasmic, membrane, and DNA-binding proteins in escherichia coli. Biophys J 98(4):552–559

    CAS  Google Scholar 

  42. Guigas G, Weiss M (2006) Size-dependent diffusion of membrane inclusions. Biophys J 91(7):2393–2398

    CAS  Google Scholar 

  43. Falck E, Patra M, Karttunen M, Hyvonen MT, Vattulainen I (2005) Response to comment by almeida et al.: free area theories for lipid bilayers–predictive or not? Biophys J 89(1):745–752

    CAS  Google Scholar 

  44. Falck E, Patra M, Karttunen M, Hyvonen MT, Vattulainen I (2004) Lessons of slicing membranes: interplay of packing, free area, and lateral diffusion in phospholipid/cholesterol bilayers. Biophys J 87(2):1076–1091

    CAS  Google Scholar 

  45. Almeida PF, Vaz WL, Thompson TE (2005) Lipid diffusion, free area, and molecular dynamics simulations. Biophys J 88(6):4434–4438

    CAS  Google Scholar 

  46. Kusumi A, Sako Y, Yamamoto M (1993) Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells. Biophys J 65(5):2021–2040

    CAS  Google Scholar 

  47. Wieser S, Moertelmaier M, Fuertbauer E, Stockinger H, Schütz GJ (2007) (un)confined diffusion of cd59 in the plasma membrane determined by high-resolution single molecule microscopy. Biophys J 92(10):3719–3728

    CAS  Google Scholar 

  48. Jin S, Haggie PM, Verkman AS (2007) Single-particle tracking of membrane protein diffusion in a potential: simulation, detection, and application to confined diffusion of cftr cl- channels. Biophys J 93(3):1079–1088

    CAS  Google Scholar 

  49. Saxton MJ (1987) Lateral diffusion in an archipelago. The effect of mobile obstacles. Biophys J 52(6):989–997

    CAS  Google Scholar 

  50. Saxton MJ (1994) Anomalous diffusion due to obstacles: a monte carlo study. Biophys J 66(2 Pt 1):394–401

    CAS  Google Scholar 

  51. Saxton MJ (1993) Lateral diffusion in an archipelago. Dependence on tracer size. Biophys J 64(4):1053–1062

    CAS  Google Scholar 

  52. Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Doose S, Li JJ, Sundaresan G, Wu AM, Gambhir SS, Weiss S (2005) Quantum dots for live cells, in vivo imaging, and diagnostics. Science 307(5709):538–544

    CAS  Google Scholar 

  53. Pinaud F, Clarke S, Sittner A, Dahan M (2010) Probing cellular events, one quantum dot at a time. Nat Methods 7(4):275–285

    CAS  Google Scholar 

  54. Martin DS, Forstner MB, Kas JA (2002) Apparent subdiffusion inherent to single particle tracking. Biophys J 83(4):2109–2117

    CAS  Google Scholar 

  55. Goulian M, Simon SM (2000) Tracking single proteins within cells. Biophys J 79(4):2188–2198

    CAS  Google Scholar 

  56. Ritchie K, Shan XY, Kondo J, Iwasawa K, Fujiwara T, Kusumi A (2005) Detection of non-brownian diffusion in the cell membrane in single molecule tracking. Biophys J 88(3):2266–2277

    CAS  Google Scholar 

  57. Destainville N, Salome L (2006) Quantification and correction of systematic errors due to detector time-averaging in single-molecule tracking experiments. Biophys J 90(2):L17–L19

    CAS  Google Scholar 

  58. Adler J, Shevchuk AI, Novak P, Korchev YE, Parmryd I (2010) Plasma membrane topography and interpretation of single-particle tracks. Nat Methods 7(3):170–171

    CAS  Google Scholar 

  59. King MR (2004) Apparent 2-d diffusivity in a ruffled cell membrane. J Theor Biol 227(3):323–326

    CAS  Google Scholar 

  60. Reister E, Seifert U (2005) Lateral diffusion of a protein on a fluctuating membrane. Europhys Lett 71(5):859–865

    CAS  Google Scholar 

  61. Wieser S, Schütz GJ, Cooper ME, Stockinger H (2007) Single molecule diffusion analysis on cellular nanotubules: implications on plasma membrane structure below the diffraction limit. Appl Phys Lett 91(23):233901

    Google Scholar 

  62. Rustom A, Saffrich R, Markovic I, Walther P, Gerdes HH (2004) Nanotubular highways for intercellular organelle transport. Science 303(5660):1007–1010

    CAS  Google Scholar 

  63. Smith PR, Morrison IE, Wilson KM, Fernandez N, Cherry RJ (1999) Anomalous diffusion of major histocompatibility complex class i molecules on hela cells determined by single particle tracking. Biophys J 76(6):3331–3344

    CAS  Google Scholar 

  64. Horton MR, Hofling F, Radler JO, Franosch T (2010) Development of anomalous diffusion among crowding proteins. Soft Matter 6(12):2648–2656

    CAS  Google Scholar 

  65. Deverall MA, Gindl E, Sinner EK, Besir H, Ruehe J, Saxton MJ, Naumann CA (2005) Membrane lateral mobility obstructed by polymer-tethered lipids studied at the single molecule level. Biophys J 88(3):1875–1886

    CAS  Google Scholar 

  66. Weigel AV, Simon B, Tamkun MM, Krapf D (2011) Ergodic and nonergodic processes coexist in the plasma membrane as observed by single-molecule tracking. Proc Natl Acad Sci USA 108(16):6438–6443

    CAS  Google Scholar 

  67. Condamin S, Tejedor V, Voituriez R, Benichou O, Klafter J (2008) Probing microscopic origins of confined subdiffusion by first-passage observables. Proc Natl Acad Sci USA 105(15):5675–5680

    CAS  Google Scholar 

  68. Schmidt U, Weiss M (2011) Anomalous diffusion of oligomerized transmembrane proteins. J Chem Phys 134(16):165101

    Google Scholar 

  69. Nechyporuk-Zloy V, Dieterich P, Oberleithner H, Stock C, Schwab A (2008) Dynamics of single potassium channel proteins in the plasma membrane of migrating cells. Am J Physiol Cell Physiol 294(4):C1096–C1102

    CAS  Google Scholar 

  70. Tejedor V, Benichou O, Voituriez R, Jungmann R, Simmel F, Selhuber-Unkel C, Oddershede LB, Metzler R (2010) Quantitative analysis of single particle trajectories: mean maximal excursion method. Biophys J 98(7):1364–1372

    CAS  Google Scholar 

  71. Qian H, Sheetz MP, Elson EL (1991) Single particle tracking. Analysis of diffusion and flow in two-dimensional systems. Biophys J 60(4):910–921

    CAS  Google Scholar 

  72. Saxton MJ (1997) Single-particle tracking: the distribution of diffusion coefficients. Biophys J 72(4):1744–1753

    CAS  Google Scholar 

  73. Schütz GJ, Schindler H, Schmidt T (1997) Single-molecule microscopy on model membranes reveals anomalous diffusion. Biophys J 73(2):1073–1080

    Google Scholar 

  74. Lommerse PH, Blab GA, Cognet L, Harms GS, Snaar-Jagalska BE, Spaink HP, Schmidt T (2004) Single-molecule imaging of the h-ras membrane-anchor reveals domains in the cytoplasmic leaflet of the cell membrane. Biophys J 86(1):609–616

    CAS  Google Scholar 

  75. Pinaud F, Michalet X, Iyer G, Margeat E, Moore HP, Weiss S (2009) Dynamic partitioning of a glycosyl-phosphatidylinositol-anchored protein in glycosphingolipid-rich microdomains imaged by single-quantum dot tracking. Traffic 10(6):691–712

    CAS  Google Scholar 

  76. Semrau S, Schmidt T (2007) Particle image correlation spectroscopy (pics) retrieving nanometer-scale correlations from high-density single-molecule position data. Biophys J 92(2):613–621

    CAS  Google Scholar 

  77. Wieser S, Axmann M, Schütz GJ (2008) Versatile analysis of single-molecule tracking data by comprehensive testing against monte carlo simulations. Biophys J 95(12):5988–6001

    CAS  Google Scholar 

  78. Matsuoka S, Shibata T, Ueda M (2009) Statistical analysis of lateral diffusion and multistate kinetics in single-molecule imaging. Biophys J 97(4):1115–1124

    CAS  Google Scholar 

  79. Ying W, Huerta G, Steinberg S, Zuniga M (2009) Time series analysis of particle tracking data for molecular motion on the cell membrane. Bull Math Biol 71(8):1967–2024

    CAS  Google Scholar 

  80. Simson R, Sheets ED, Jacobson K (1995) Detection of temporary lateral confinement of membrane proteins using single-particle tracking analysis. Biophys J 69(3):989–993

    CAS  Google Scholar 

  81. Meier J, Vannier C, Serge A, Triller A, Choquet D (2001) Fast and reversible trapping of surface glycine receptors by gephyrin. Nat Neurosci 4(3):253–260

    CAS  Google Scholar 

  82. Meilhac N, Le Guyader L, Salome L, Destainville N (2006) Detection of confinement and jumps in single-molecule membrane trajectories. Phys Rev E 73(1):011915

    CAS  Google Scholar 

  83. Montiel D, Cang H, Yang H (2006) Quantitative characterization of changes in dynamical behavior for single-particle tracking studies. J Phys Chem B 110(40):19763–19770

    CAS  Google Scholar 

  84. Huet S, Karatekin E, Tran VS, Fanget I, Cribier S, Henry JP (2006) Analysis of transient behavior in complex trajectories: application to secretory vesicle dynamics. Biophys J 91(9):3542–3559

    CAS  Google Scholar 

  85. Manley S, Gillette JM, Patterson GH, Shroff H, Hess HF, Betzig E, Lippincott-Schwartz J (2008) High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat Methods 5(2):155–157

    CAS  Google Scholar 

  86. Jaqaman K, Kuwata H, Touret N, Collins R, Trimble William S, Danuser G, Grinstein S (2011) Cytoskeletal control of cd36 diffusion promotes its receptor and signaling function. Cell 146(4):593–606

    CAS  Google Scholar 

  87. Andrews NL, Lidke KA, Pfeiffer JR, Burns AR, Wilson BS, Oliver JM, Lidke DS (2008) Actin restricts fcepsilonri diffusion and facilitates antigen-induced receptor immobilization. Nat Cell Biol 10(8):955–963

    CAS  Google Scholar 

  88. Brameshuber M, Schutz GJ (2008) How the sum of its parts gets greater than the whole. Nat Methods 5(2):133–134

    CAS  Google Scholar 

  89. Lillemeier BF, Mortelmaier MA, Forstner MB, Huppa JB, Groves JT, Davis MM (2010) Tcr and lat are expressed on separate protein islands on t cell membranes and concatenate during activation. Nat Immunol 11(1):90–96

    CAS  Google Scholar 

  90. Howard J (2001) Mechanics of motor proteins and the cytoskeleton. Sinauer Assoc, Sunderland

    Google Scholar 

  91. Funatsu T, Harada Y, Tokunaga M, Saito K, Yanagida T (1995) Imaging of single fluorescent molecules and individual atp turnovers by single myosin molecules in aqueous-solution. Nature 374(6522):555–559

    CAS  Google Scholar 

  92. Schmidt T, Schütz GJ, Baumgartner W, Gruber HJ, Schindler H (1995) Characterization of photophysics and mobility of single molecules in a fluid lipid membrane. J Phys Chem 99:17662–17668

    CAS  Google Scholar 

  93. Sahl SJ, Leutenegger M, Hilbert M, Hell SW, Eggeling C (2010) Fast molecular tracking maps nanoscale dynamics of plasma membrane lipids. Proc Natl Acad Sci USA 107(15):6829–6834

    CAS  Google Scholar 

  94. Demtröder W (2003) Laser spectroscopy, 3rd edn. Springer, Berlin

    Google Scholar 

  95. Diaspro A, Chirico G, Usai C, Ramoino P, Dobrucki JW (2006) Photobleaching. In: Pawley JB (ed) Handbook of biological confocal microscopy, 3rd edn. Springer Science + Business Media, New York

    Google Scholar 

  96. Zondervan R, Kulzer F, Kol'chenko MA, Orrit M (2004) Photobleaching of rhodamine 6 g in poly(vinyl alcohol) at the ensemble and single-molecule levels. J Phys Chem A 108:1657–1665

    CAS  Google Scholar 

  97. Deschenes LA, Vanden Bout DA (2002) Single molecule photobleaching: increasing photon yield and survival time through suppression of two-step photolysis. Chem Phys Lett 365:387–395

    CAS  Google Scholar 

  98. Bernas T, Zarebski M, Dobrucki JW, Cook PR (2004) Minimizing photobleaching during confocal microscopy of fluorescent probes bound to chromatin: role of anoxia and photon flux. J Microsc 215(Pt 3):281–296

    CAS  Google Scholar 

  99. Dickson RM, Cubitt AB, Tsien RY, Moerner WE (1997) On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature 388(6640):355–358

    CAS  Google Scholar 

  100. Garcia-Parajo MF, Segers-Nolten GM, Veerman JA, Greve J, van Hulst NF (2000) Real-time light-driven dynamics of the fluorescence emission in single green fluorescent protein molecules. Proc Natl Acad Sci USA 97(13):7237–7242

    CAS  Google Scholar 

  101. Annibale P, Vanni S, Scarselli M, Rothlisberger U, Radenovic A (2011) Identification of clustering artifacts in photoactivated localization microscopy. Nat Methods 8:527–528

    CAS  Google Scholar 

  102. Heinze KG, Costantino S, De Koninck P, Wiseman PW (2009) Beyond photobleaching, laser illumination unbinds fluorescent proteins. J Phys Chem B 113(15):5225–5233

    CAS  Google Scholar 

  103. Douglass AD, Vale RD (2005) Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signaling molecules in t cells. Cell 121(6):937–950

    CAS  Google Scholar 

  104. Vrljic M, Nishimura SY, Brasselet S, Moerner WE, McConnell HM (2002) Translational diffusion of individual class ii mhc membrane proteins in cells. Biophys J 83(5):2681–2692

    CAS  Google Scholar 

  105. Drbal K, Moertelmaier M, Holzhauser C, Muhammad A, Fuertbauer E, Howorka S, Hinterberger M, Stockinger H, Schütz GJ (2007) Single-molecule microscopy reveals heterogeneous dynamics of lipid raft components upon tcr engagement. Int Immunol 19(5):675–684

    CAS  Google Scholar 

  106. Moertelmaier M, Brameshuber M, Linimeier M, Schütz GJ, Stockinger H (2005) Thinning out clusters while conserving stoichiometry of labeling. Appl Phys Lett 87:263903

    Google Scholar 

  107. Schmidt T, Schütz GJ, Gruber HJ, Schindler H (1996) Local stoichiometries determined by counting individual molecules. Anal Chem 68(24):4397–4401

    CAS  Google Scholar 

  108. Brameshuber M, Weghuber J, Ruprecht V, Gombos I, Horvath I, Vigh L, Eckerstorfer P, Kiss E, Stockinger H, Schutz GJ (2010) Imaging of mobile long-lived nanoplatforms in the live cell plasma membrane. J Biol Chem 285(53):41765–41771

    CAS  Google Scholar 

  109. Madl J, Weghuber J, Fritsch R, Derler I, Fahrner M, Frischauf I, Lackner B, Romanin C, Schutz GJ (2010) Resting state orai1 diffuses as homotetramer in the plasma membrane of live mammalian cells. J Biol Chem 285(52):41135–41142

    CAS  Google Scholar 

  110. Ruprecht V, Brameshuber M, Schütz GJ (2010) Two-color single molecule tracking combined with photobleaching for the detection of rare molecular interactions in fluid biomembranes. Soft Matter 6(3):568–581

    CAS  Google Scholar 

  111. Monks CR, Freiberg BA, Kupfer H, Sciaky N, Kupfer A (1998) Three-dimensional segregation of supramolecular activation clusters in t cells. Nature 395(6697):82–86

    CAS  Google Scholar 

  112. Grakoui A, Bromley SK, Sumen C, Davis MM, Shaw AS, Allen PM, Dustin ML (1999) The immunological synapse: a molecular machine controlling t cell activation. Science 285(5425):221–227

    CAS  Google Scholar 

  113. Bromley SK, Burack WR, Johnson KG, Somersalo K, Sims TN, Sumen C, Davis MM, Shaw AS, Allen PM, Dustin ML (2001) The immunological synapse. Annu Rev Immunol 19:375–396

    CAS  Google Scholar 

  114. Krogsgaard M, Davis MM (2005) How t cells 'see' antigen. Nat Immunol 6(3):239–245

    CAS  Google Scholar 

  115. Huppa JB, Axmann M, Mortelmaier MA, Lillemeier BF, Newell EW, Brameshuber M, Klein LO, Schütz GJ, Davis MM (2010) Tcr-peptide-mhc interactions in situ show accelerated kinetics and increased affinity. Nature 463(7283):963–967

    CAS  Google Scholar 

  116. Xiong Y, Kern P, Chang H, Reinherz E (2001) T cell receptor binding to a pmhcii ligand is kinetically distinct from and independent of cd4. J Biol Chem 276(8):5659–5667

    CAS  Google Scholar 

  117. Bunnell SC, Kapoor V, Trible RP, Zhang W, Samelson LE (2001) Dynamic actin polymerization drives t cell receptor-induced spreading: a role for the signal transduction adaptor lat. Immunity 14(3):315–329

    CAS  Google Scholar 

  118. Ike H, Kosugi A, Kato A, Iino R, Hirano H, Fujiwara T, Ritchie K, Kusumi A (2003) Mechanism of lck recruitment to the t-cell receptor cluster as studied by single-molecule-fluorescence video imaging. Chemphyschem 4(6):620–626

    Google Scholar 

  119. Campi G, Varma R, Dustin ML (2005) Actin and agonist mhc-peptide complex-dependent t cell receptor microclusters as scaffolds for signaling. J Exp Med 202(8):1031–1036

    CAS  Google Scholar 

  120. Saito T, Yokosuka T, Hashimoto-Tane A (2010) Dynamic regulation of t cell activation and co-stimulation through tcr-microclusters. FEBS Lett 584(24):4865–4871

    CAS  Google Scholar 

  121. Demond AL, Mossman KD, Starr T, Dustin ML, Groves JT (2008) T cell receptor microcluster transport through molecular mazes reveals mechanism of translocation. Biophys J 94:3286–3292

    CAS  Google Scholar 

  122. Morton WM, Ayscough KR, McLaughlin PJ (2000) Latrunculin alters the actin-monomer subunit interface to prevent polymerization. Nat Cell Biol 2(6):376–378

    CAS  Google Scholar 

  123. Wakatsuki T, Schwab B, Thompson NC, Elson EL (2001) Effects of cytochalasin d and latrunculin b on mechanical properties of cells. J Cell Sci 114(Pt 5):1025–1036

    CAS  Google Scholar 

  124. Lommerse PH, Vastenhoud K, Pirinen NJ, Magee AI, Spaink HP, Schmidt T (2006) Single-molecule diffusion reveals similar mobility for the lck, h-ras, and k-ras membrane anchors. Biophys J 91(3):1090–1097

    CAS  Google Scholar 

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Acknowledgments

This work was supported by the Austrian Science Fund (FWF project Y250-B03) and the GEN-AU project of the Austrian Federal Ministry for Science and Research.

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Authors and Affiliations

  1. Institute of Applied Physics, Vienna University of Technology, Wiedner Hauptstraße 8-10, A-1040, Wien, Austria

    Mario Brameshuber & Gerhard J. Schütz

  2. Biophysics Institute, Johannes Kepler University Linz, Altenbergerstr. 69, A-4040, Linz, Austria

    Mario Brameshuber & Gerhard J. Schütz

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  1. Mario Brameshuber
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  2. Gerhard J. Schütz
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Correspondence to Gerhard J. Schütz .

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  1. NanoBioSciences Institute of Physical and Theoretical Chemistry, TU Braunschweig, Braunschweig, Germany

    Philip Tinnefeld

  2. MRC Human Immunology Unit and Wolfson Imaging Centre Oxford, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom

    Christian Eggeling

  3. Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Stefan W. Hell

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Brameshuber, M., Schütz, G.J. (2012). In Vivo Tracking of Single Biomolecules: What Trajectories Tell Us About the Acting Forces. In: Tinnefeld, P., Eggeling, C., Hell, S. (eds) Far-Field Optical Nanoscopy. Springer Series on Fluorescence, vol 14. Springer, Berlin, Heidelberg. https://doi.org/10.1007/4243_2011_38

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