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Tracking the Activity and Position of Mitochondrial β-Barrel Proteins

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Transmembrane β-Barrel Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2778))

Abstract

Total interference reflection fluorescence (TIRF) microscopy of lipid bilayers is an effective technique for studying the lateral movement and ion channel activity of single integral membrane proteins. Here we describe how to integrate the mitochondrial outer membrane preprotein translocase TOM-CC and its β-barrel protein-conducting channel Tom40 into supported lipid bilayers to identify possible relationships between movement and channel activity. We propose that our approach can be readily applied to membrane protein channels where transient tethering to either membrane-proximal or intramembrane structures is accompanied by a change in channel permeation.

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References

  1. Sigworth FJ (1994) Voltage gating of ion channels. Q Rev Biophys 27:1–40

    Article  CAS  PubMed  Google Scholar 

  2. Delcour AH (2003) Solute uptake through general porins. Front Biosci 8:d1055–d1071

    Article  CAS  PubMed  Google Scholar 

  3. Benz R (2021) Historical perspective of pore-forming activity studies of voltage-dependent anion channel (Eukaryotic or Mitochondrial Porin) since its discovery in the 70th of the last century. Front Physiol 12:734226

    Article  PubMed  PubMed Central  Google Scholar 

  4. Dunlop J, Bowlby M, Peri R et al (2008) High-throughput electrophysiology: an emerging paradigm for ion-channel screening and physiology. Nat Rev Drug Discov 7:358–368

    Article  CAS  PubMed  Google Scholar 

  5. Liu C, Li T, Chen J (2019) Role of high-throughput electrophysiology in drug discovery. Curr Protoc Pharmacol 87:e69

    Article  PubMed  Google Scholar 

  6. Benz R (2006) Bacterial and eukaryotic porins: structure, function, mechanism. John Wiley & Sons, Hoboken

    Google Scholar 

  7. Zeth K, Thein M (2010) Porins in prokaryotes and eukaryotes: common themes and variations. Biochem J 431:13–22

    Article  CAS  PubMed  Google Scholar 

  8. Nestorovich EM, Danelon C, Winterhalter M et al (2002) Designed to penetrate: time-resolved interaction of single antibiotic molecules with bacterial pores. Proc Natl Acad Sci 99:9789–9794

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Pagès J-M, James CE, Winterhalter M (2008) The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria. Nat Rev Microbiol 6:893–903

    Article  PubMed  Google Scholar 

  10. Kasianowicz JJ, Brandin E, Branton D et al (1996) Characterization of individual polynucleotide molecules using a membrane channel. Proc Natl Acad Sci 93:13770–13773

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Branton D, Deamer DW, Marziali A et al (2008) The potential and challenges of nanopore sequencing. Nat Biotechnol 26:1146–1153

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Deamer D, Akeson M, Branton D (2016) Three decades of nanopore sequencing. Nat Biotechnol 34:518–524

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Restrepo-Pérez L, Joo C, Dekker C (2018) Paving the way to single-molecule protein sequencing. Nat Nanotechnol 13:786–796

    Article  PubMed  Google Scholar 

  14. Alfaro JA, Bohländer P, Dai M et al (2021) The emerging landscape of single-molecule protein sequencing technologies. Nat Methods 18:604–617

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Hu Z-L, Huo M-Z, Ying Y-L et al (2021) Biological nanopore approach for single-molecule protein sequencing. Angew Chem Int Ed Engl 60:14738–14749

    Article  CAS  PubMed  Google Scholar 

  16. Ensslen T, Sarthak K, Aksimentiev A et al (2022) Resolving isomeric posttranslational modifications using a biological nanopore as a sensor of molecular shape. J Am Chem Soc 144:16060–16068

    Article  CAS  PubMed  Google Scholar 

  17. Ahting U, Thieffry M, Engelhardt H et al (2001) Tom40, the pore-forming component of the protein-conducting TOM channel in the outer membrane of mitochondria. J Cell Biol 153:1151–1160

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Bausewein T, Mills DJ, Langer JD et al (2017) Cryo-EM structure of the TOM core complex from Neurospora crassa. Cell 170:693–700

    Article  CAS  PubMed  Google Scholar 

  19. Bausewein T, Naveed H, Liang J et al (2020) The structure of the TOM core complex in the mitochondrial outer membrane. Biol Chem 401:687–697

    Article  CAS  PubMed  Google Scholar 

  20. Ornelas P, Bausewein T, Martin J et al (2023) Two conformations of the Tom20 preprotein receptor in the TOM holo complex. Proc Natl Acad Sci 120:e2301447120

    Google Scholar 

  21. Wang S, Findeisen L, Leptihn S et al (2022) Spatiotemporal stop-and-go dynamics of the mitochondrial TOM core complex correlates with channel activity. Commun Biol 5:1–11

    Google Scholar 

  22. Ahting U, Thun C, Hegerl R et al (1999) The TOM core complex: the general protein import pore of the outer membrane of mitochondria. J Cell Biol 147:959–968

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Poynor M, Eckert R, Nussberger S (2008) Dynamics of the preprotein translocation channel of the outer membrane of mitochondria. Biophys J 95:1511–1522

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Romero-Ruiz M, Mahendran KR, Eckert R et al (2010) Interactions of mitochondrial presequence peptides with the mitochondrial outer membrane preprotein translocase TOM. Biophys J 99:774–781

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Gessmann D, Flinner N, Pfannstiel J et al (2011) Structural elements of the mitochondrial preprotein-conducting channel Tom40 dissolved by bioinformatics and mass spectrometry. Biochim Biophys Acta 1807:1647–1657

    Article  CAS  PubMed  Google Scholar 

  26. Mahendran KR, Romero-Ruiz M, Schlösinger A et al (2012) Protein translocation through Tom40: kinetics of peptide release. Biophys J 102:39–47

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Mahendran KR, Lamichhane U, Romero-Ruiz M et al (2013) Polypeptide translocation through the mitochondrial TOM channel: temperature-dependent rates at the single-molecule level. J Phys Chem Lett 4:78–82

    Article  CAS  PubMed  Google Scholar 

  28. Thompson JR, Heron AJ, Santoso Y et al (2007) Enhanced stability and fluidity in droplet on hydrogel bilayers for measuring membrane protein diffusion. Nano Lett 7:3875–3878

    Article  CAS  PubMed  Google Scholar 

  29. Bayley H, Cronin B, Heron A et al (2008) Droplet interface bilayers. Mol BioSyst 4:1191–1208

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Leptihn S, Castell OK, Cronin B et al (2013) Constructing droplet interface bilayers from the contact of aqueous droplets in oil. Nat Protoc 8:1048–1057

    Article  CAS  PubMed  Google Scholar 

  31. Huang S, Romero-Ruiz M, Castell OK et al (2015) High-throughput optical sensing of nucleic acids in a nanopore array. Nat Nanotechnol 10:986–991

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Manafirad A (2021) Single ion-channel analysis in droplet interface bilayer. In: Fahie MAV (ed) Nanopore technology: methods and protocols. Springer US, New York, pp 187–195

    Chapter  Google Scholar 

  33. Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji – an Open Source platform for biological image analysis. Nat Methods 9:676–68

    Google Scholar 

  34. Tinevez J-Y, Perry N, Schindelin J et al (2017) TrackMate: an open and extensible platform for single-particle tracking. Methods 115:80–90

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Kusumi A, Nakada C, Ritchie K et al (2005) Paradigm shift of the plasma membrane concept from the two-dimensional continuum fluid to the partitioned fluid: high-speed single-molecule tracking of membrane molecules. Annu Rev Biophys Biomol Struct 34:351–378

    Article  CAS  PubMed  Google Scholar 

  37. Kusumi A, Tsunoyama TA, Hirosawa KM et al (2014) Tracking single molecules at work in living cells. Nat Chem Biol 10:524–532

    Article  CAS  PubMed  Google Scholar 

  38. Schafer DA (2002) Coupling actin dynamics and membrane dynamics during endocytosis. Curr Opin Cell Biol 14:76–81

    Article  CAS  PubMed  Google Scholar 

  39. Rajendran L, Simons K (2005) Lipid rafts and membrane dynamics. J Cell Sci 118:1099–1102

    Article  CAS  PubMed  Google Scholar 

  40. Kusumi A, Shirai YM, Koyama-Honda I et al (2010) Hierarchical organization of the plasma membrane: investigations by single-molecule tracking vs. fluorescence correlation spectroscopy. FEBS Lett 584:1814–1823

    Article  CAS  PubMed  Google Scholar 

  41. Betaneli V, Schwille P (2013) Fluorescence correlation spectroscopy to examine protein–lipid interactions in membranes. In: Kleinschmidt JH (ed) Lipid-protein interactions: methods and protocols. Humana Press, Totowa, pp 253–278

    Chapter  Google Scholar 

  42. Schink KO, Tan K-W, Stenmark H (2016) Phosphoinositides in control of membrane dynamics. Annu Rev Cell Dev Biol 32:143–171

    Article  CAS  PubMed  Google Scholar 

  43. Engelman DM (2005) Membranes are more mosaic than fluid. Nature 438:578–580

    Article  CAS  PubMed  Google Scholar 

  44. Lingwood D, Ries J, Schwille P et al (2008) Plasma membranes are poised for activation of raft phase coalescence at physiological temperature. Proc Natl Acad Sci 105:10005–10010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Jacobson K, Liu P, Lagerholm BC (2019) The lateral organization and mobility of plasma membrane components. Cell 177:806–819

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Mokranjac D, Sichting M, Popov-Čeleketić D et al (2009) Role of Tim50 in the transfer of precursor proteins from the outer to the inner membrane of mitochondria. Mol Biol Cell 20:1400–1407

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Waegemann K, Popov-Čeleketić D, Neupert W et al (2015) Cooperation of TOM and TIM23 complexes during translocation of proteins into mitochondria. J Mol Biol 427:1075–1084

    Article  CAS  PubMed  Google Scholar 

  48. Gomkale R, Linden A, Neumann P et al (2021) Mapping protein interactions in the active TOM-TIM23 supercomplex. Nat Commun 12:5715

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Ershov D, Phan M-S, Pylvänäinen JW et al (2022) TrackMate 7: integrating state-of-the-art segmentation algorithms into tracking pipelines. Nat Methods 19:829–832

    Article  CAS  PubMed  Google Scholar 

  50. Jaqaman K, Loerke D, Mettlen M et al (2008) Robust single-particle tracking in live-cell time-lapse sequences. Nat Methods 5:695–702

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Lindsey H, Petersen NO, Chan SI (1979) Physicochemical characterization of 1,2-diphytanoyl-sn-glycero-3-phosphocholine in model membrane systems. Biochim Biophys Acta Biomembr 555:147–167

    Article  CAS  Google Scholar 

  52. Rosholm KR, Baker MAB, Ridone P et al (2017) Activation of the mechanosensitive ion channel MscL by mechanical stimulation of supported Droplet-Hydrogel bilayers. Sci Rep 7:45180

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Wang Y, Yan S, Zhang P et al (2018) Osmosis-driven motion-type modulation of biological nanopores for parallel optical nucleic acid sensing. ACS Appl Mater Interfaces 10:7788–7797

    Article  CAS  PubMed  Google Scholar 

  54. Heron AJ, Thompson JR, Cronin B et al (2009) Simultaneous measurement of ionic current and fluorescence from single protein pores. J Am Chem Soc 131:1652–1653

    Article  CAS  PubMed  Google Scholar 

  55. Thompson RE, Larson DR, Webb WW (2002) Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82:2775–2783

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Selvin PR, Lougheed T, Hoffman MT et al (2008) In vitro & in vivo FIONA and other acronyms for watching molecular motors walk. In: Selvin PR, Ha T (eds) Single-molecule techniques: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 37–71

    Google Scholar 

  57. Hirsch M, Wareham RJ, Martin-Fernandez ML et al (2013) A stochastic model for electron multiplication charge-coupled devices – from theory to practice. PLoS One 8:e53671

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

We thank Robin Ghosh for critically reviewing the manuscript. This work was supported in part by Laura Na Liu (2nd Physics Institute, University of Stuttgart) and by a grant from the Baden Württemberg Foundation (BiofMO-6 to SN).

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Authors and Affiliations

  1. Biophysics Department, Institute of Biomaterials and Biomolecular Systems, University of Stuttgart, Stuttgart, Germany

    Shuo Wang & Stephan Nussberger

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  1. Shuo Wang
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  2. Stephan Nussberger
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Correspondence to Stephan Nussberger .

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Editors and Affiliations

  1. Laboratoire de Microbiologie et Génétique Moléculaires, Centre de Biologie Intégrative, CNRS, Université de Toulouse, Toulouse, France

    Raffaele Ieva

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Wang, S., Nussberger, S. (2024). Tracking the Activity and Position of Mitochondrial β-Barrel Proteins. In: Ieva, R. (eds) Transmembrane β-Barrel Proteins. Methods in Molecular Biology, vol 2778. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3734-0_14

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  • DOI: https://doi.org/10.1007/978-1-0716-3734-0_14

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3733-3

  • Online ISBN: 978-1-0716-3734-0

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