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TNF Receptor Membrane Dynamics Studied with Fluorescence Microscopy and Spectroscopy

  • Felix Neugart
  • Darius Widera
  • Barbara Kaltschmidt
  • Christian Kaltschmidt
  • Mike Heilemann
Chapter
Part of the Springer Series on Fluorescence book series (SS FLUOR, volume 13)

Abstract

Sensitive fluorescence techniques opened novel opportunities to study the function and interaction of proteins in living cells. Here, we review the contribution of fluorescence correlation spectroscopy (FCS) and single-molecule tracking to study the dynamics of TNF receptor 1 and 2 (TNFR1/2). Although these techniques greatly differ, both report a similar behavior of TNF receptor (TNFR) species in Hela cells under different experimental conditions. FCS as well as single-molecule tracking revealed an increase of the diffusion coefficient of TNFR1 after treating cells with methyl-cyclodextrin.

In addition, FCS studies of the activation of TNFR1 showed that ligand binding hardly affects its diffusion coefficient.

In contrast, unstimulated TNFR2 was observed to diffuse faster than TNFR1, whereas ligand stimulation of TNFR2 decreases the diffusion coefficient.

In conclusion, the results indicate that the two TNFRs compartmentalize in distinct domains of the plasma membrane most likely determined by the respective transmembrane domains and/or transmembrane domain near regions.

Keywords

FCS sptPALM TNFR1 TNFR2 

References

  1. 1.
    Hohlbein J, Gryte K, Heilemann M, Kapanidis AN (2010) Surfing on a new wave of single-molecule fluorescence methods. Phys Biol 7(3):031001. doi:S1478-3975(10)53547-7 [pii]  10.1088/1478-3975/7/3/031001 CrossRefGoogle Scholar
  2. 2.
    Hotamisligil GS, Murray DL, Choy LN, Spiegelman BM (1994) Tumor necrosis factor alpha inhibits signaling from the insulin receptor. Proc Natl Acad Sci USA 91(11):4854–4858CrossRefGoogle Scholar
  3. 3.
    Bright JJ (2007) Curcumin and autoimmune disease. Adv Exp Med Biol 595:425–451CrossRefGoogle Scholar
  4. 4.
    Pacifico F, Leonardi A (2006) Nf-kappab in solid tumors. Biochem Pharmacol 72(9):1142–1152CrossRefGoogle Scholar
  5. 5.
    Gray PW, Aggarwal BB, Benton CV, Bringman TS, Henzel WJ, Jarrett JA, Leung DW, Moffat B, Ng P, Svedersky LP et al (1984) Cloning and expression of cdna for human lymphotoxin, a lymphokine with tumour necrosis activity. Nature 312(5996):721–724CrossRefGoogle Scholar
  6. 6.
    Pennica D, Nedwin GE, Hayflick JS, Seeburg PH, Derynck R, Palladino MA, Kohr WJ, Aggarwal BB, Goeddel DV (1984) Human tumour necrosis factor: precursor structure, expression and homology to lymphotoxin. Nature 312(5996):724–729CrossRefGoogle Scholar
  7. 7.
    Aggarwal BB (2003) Signalling pathways of the tnf superfamily: a double-edged sword. Nat Rev Immunol 3(9):745–756. doi: 10.1038/nri1184 nri1184 [pii]CrossRefGoogle Scholar
  8. 8.
    Schmitz ML, Mattioli I, Buss H, Kracht M (2004) Nf-kappab: a multifaceted transcription factor regulated at several levels. Chembiochem 5(10):1348–1358CrossRefGoogle Scholar
  9. 9.
    Bonizzi G, Karin M (2004) The two nf-kappab activation pathways and their role in innate and adaptive immunity. Trends Immunol 25(6):280–288CrossRefGoogle Scholar
  10. 10.
    Wajant H, Pfizenmaier K, Scheurich P (2003) Tumor necrosis factor signaling. Cell Death Differ 10(1):45–65. doi: 10.1038/sj.cdd.4401189 4401189 [pii]CrossRefGoogle Scholar
  11. 11.
    Mathew SJ, Haubert D, Kronke M, Leptin M (2009) Looking beyond death: a morphogenetic role for the tnf signalling pathway. J Cell Sci 122(Pt 12):1939–1946. doi: 10.1242/jcs.044487122/12/1939 [pii]CrossRefGoogle Scholar
  12. 12.
    Legler DF, Micheau O, Doucey MA, Tschopp J, Bron C (2003) Recruitment of tnf receptor 1 to lipid rafts is essential for tnfalpha-mediated nf-kappab activation. Immunity 18(5):655–664CrossRefGoogle Scholar
  13. 13.
    Doan JE, Windmiller DA, Riches DW (2004) Differential regulation of tnf-r1 signaling: lipid raft dependency of p42mapk/erk2 activation, but not nf-kappab activation. J Immunol 172(12):7654–7660. doi: 172/12/7654 [pii]Google Scholar
  14. 14.
    Hunter I, Nixon GF (2006) Spatial compartmentalization of tumor necrosis factor (tnf) receptor 1-dependent signaling pathways in human airway smooth muscle cells. Lipid rafts are essential for tnf-alpha-mediated activation of rhoa but dispensable for the activation of the nf-kappab and mapk pathways. J Biol Chem 281(45):34705–34715. doi:M605738200 [pii]  10.1074/jbc.M605738200 CrossRefGoogle Scholar
  15. 15.
    van de Linde S, Wolter S, Heilemann M, Sauer M (2010) The effect of photoswitching kinetics and labeling densities on super-resolution fluorescence imaging. J Biotechnol 149(4):260–266. doi: 10.1016/j.jbiotec.2010.02.010 CrossRefGoogle Scholar
  16. 16.
    Eggeling C, Ringemann C, Medda R, Schwarzmann G, Sandhoff K, Polyakova S, Belov VN, Hein B, von Middendorff C, Schonle A, Hell SW (2009) Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457(7233):1159–1162. doi: 10.1038/nature07596 CrossRefGoogle Scholar
  17. 17.
    Sprague BL, McNally JG (2005) Frap analysis of binding: proper and fitting. Trends Cell Biol 15(2):84–91. doi: 10.1016/j.tcb.2004.12.001 CrossRefGoogle Scholar
  18. 18.
    Garcia-Saez AJ, Schwille P (2008) Fluorescence correlation spectroscopy for the study of membrane dynamics and protein/lipid interactions. Methods 46(2):116–122. doi: 10.1016/j.ymeth.2008.06.011 CrossRefGoogle Scholar
  19. 19.
    Anikovsky M, Wiltshire ZD, Weisshart K, Petersen NO (2011) Photon counting histogram analysis for two-dimensional systems. Chemphyschem: European J Chemical Physics Physical Chem 12(13):2439–2448. doi: 10.1002/cphc.201100414 CrossRefGoogle Scholar
  20. 20.
    Huang B, Perroud TD, Zare RN (2004) Photon counting histogram: One-photon excitation. Chemphyschem: European J Chemical Physics Physical Chem 5(10):1523–1531. doi: 10.1002/cphc.200400176 CrossRefGoogle Scholar
  21. 21.
    Bacia K, Kim SA, Schwille P (2006) Fluorescence cross-correlation spectroscopy in living cells. Nat Methods 3(2):83–89. doi: 10.1038/nmeth822 CrossRefGoogle Scholar
  22. 22.
    Hwang LC, Wohland T (2007) Recent advances in fluorescence cross-correlation spectroscopy. Cell Biochem Biophys 49(1):1–13CrossRefGoogle Scholar
  23. 23.
    Joo C, Balci H, Ishitsuka Y, Buranachai C, Ha T (2008) Advances in single-molecule fluorescence methods for molecular biology. Annu Rev Biochem 77:51–76. doi: 10.1146/annurev.biochem.77.070606.101543 CrossRefGoogle Scholar
  24. 24.
    Garcia-Saez AJ, Schwille P (2007) Single molecule techniques for the study of membrane proteins. Appl Microbiol Biotechnol 76(2):257–266. doi: 10.1007/s00253-007-1007-8 CrossRefGoogle Scholar
  25. 25.
    Gerken M, Krippner-Heidenreich A, Steinert S, Willi S, Neugart F, Zappe A, Wrachtrup J, Tietz C, Scheurich P (2010) Fluorescence correlation spectroscopy reveals topological segregation of the two tumor necrosis factor membrane receptors. Biochim Biophys Acta 1798(6):1081–1089. doi: 10.1016/j.bbamem.2010.02.021 CrossRefGoogle Scholar
  26. 26.
    Groc L, Lafourcade M, Heine M, Renner M, Racine V, Sibarita JB, Lounis B, Choquet D, Cognet L (2007) Surface trafficking of neurotransmitter receptor: comparison between single-molecule/quantum dot strategies. J Neurosci: Official J Soc Neurosci 27(46):12433–12437. doi: 10.1523/JNEUROSCI.3349-07.2007 CrossRefGoogle Scholar
  27. 27.
    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. doi: 10.1038/nmeth.1176 CrossRefGoogle Scholar
  28. 28.
    Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313(5793):1642–1645. doi: 10.1126/science.1127344 CrossRefGoogle Scholar
  29. 29.
    Wiedenmann J, Ivanchenko S, Oswald F, Schmitt F, Rocker C, Salih A, Spindler KD, Nienhaus GU (2004) Eosfp, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc Natl Acad Sci USA 101(45):15905–15910. doi: 10.1073/pnas.0403668101 CrossRefGoogle Scholar
  30. 30.
    Heidbreder M, Zander C, Malkusch S, Widera D, Kaltschmidt B, Kaltschmidt C, Nair D, Choquet D, Sibarita JB, Heilemann M (2012) Tnf-alpha influences the lateral dynamics of tnf receptor i in living cells. Biochim Biophys Acta. doi: 10.1016/j.bbamcr.2012.06.026
  31. 31.
    Ma X, Wang Q, Jiang Y, Xiao Z, Fang X, Chen YG (2007) Lateral diffusion of tgf-beta type i receptor studied by single-molecule imaging. Biochem Biophys Res Commun 356(1):67–71. doi:S0006-291X(07)00372-5 [pii]  10.1016/j.bbrc.2007.02.080 CrossRefGoogle Scholar
  32. 32.
    Franklin KJ (1948) Circulatory concepts. St Bartholomew’s Hospital J 52(1):2, passimGoogle Scholar
  33. 33.
    Giannone G, Hosy E, Levet F, Constals A, Schulze K, Sobolevsky AI, Rosconi MP, Gouaux E, Tampe R, Choquet D, Cognet L (2010) Dynamic superresolution imaging of endogenous proteins on living cells at ultra-high density. Biophys J 99(4):1303–1310. doi: 10.1016/j.bpj.2010.06.005 CrossRefGoogle Scholar
  34. 34.
    Neugart F, Zappe A, Buk DM, Ziegler I, Steinert S, Schumacher M, Schopf E, Bessey R, Wurster K, Tietz C, Borsch M, Wrachtrup J, Graeve L (2009) Detection of ligand-induced cntf receptor dimers in living cells by fluorescence cross correlation spectroscopy. Biochim Biophys Acta 1788(9):1890–1900. doi: 10.1016/j.bbamem.2009.05.013 CrossRefGoogle Scholar
  35. 35.
    Teramura Y, Ichinose J, Takagi H, Nishida K, Yanagida T, Sako Y (2006) Single-molecule analysis of epidermal growth factor binding on the surface of living cells. EMBO J 25(18):4215–4222. doi: 10.1038/sj.emboj.7601308 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  1. 1.Institute of Cell Biology and ImmunologyUniversity of StuttgartStuttgartGermany
  2. 2.Leica Microsystems CMS GmbHMannheimGermany
  3. 3.Cell BiologyUniversity of BielefeldBielefeldGermany
  4. 4.Molecular NeurobiologyUniversity of BielefeldBielefeldGermany
  5. 5.Department of Biotechnology & BiophysicsJulius-Maximilians-UniversitätWürzburgGermany

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