AFM to Study Pore-Forming Proteins

  • Joseph D. Unsay
  • Ana J. García-SáezEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1886)


Atomic force microscopy (AFM) is a form of contact microscopy that uses a very sharp tip to scan the surface of a sample. It provides a 3D image of the surface structure and in the force mode it can also be used to test the mechanical properties of the sample. AFM has been successfully applied to study the molecular mechanism of pore-forming proteins on model membranes. It gives information about both the structural reorganization of the membrane surface and the changes in the force required for membrane piercing upon incubation with this special type of proteins. Here we describe robust protocols to investigate the effect of pore-forming proteins in supported lipid bilayers .

Key words

Atomic force microscopy Supported lipid bilayer Piercing force Pore-forming protein Bax 


  1. 1.
    Morris VJ, Kirby AR, Gunning AP (2010) Atomic force microscopy for biologists, 2nd edn. Imperial College Press, LondonGoogle Scholar
  2. 2.
    Hansma PK, Elings VB, Marti O, Bracker CE (1988) Scanning tunneling microscopy and atomic force microscopy: application to biology and technology. Science 242:209–216. Scholar
  3. 3.
    Goksu EI, Vanegas JM, Blanchette CD, Lin W-C, Longo ML (2009) AFM for structure and dynamics of biomembranes. BBA-Biomembranes 1788(1):254–266. Scholar
  4. 4.
    Muller DJ (2008) AFM: a nanotool in membrane biology. Biochemistry-US 47:7986–7998. Scholar
  5. 5.
    Fradin C, Satsoura D, Andrews DW (2009) Punching holes in membranes: how oligomeric pore-forming proteins and lipids cooperate to form aqueous channels in membranes, Handbook of modern biophysics, vol vol. 2. Humana Press, New York, pp 223–262. Scholar
  6. 6.
    Bischofberger M, Iacovache I, van der Goot FG (2012) Pathogenic pore-forming proteins: function and host response. Cell Host Microbe 12(3):266–275. Scholar
  7. 7.
    Iacovache I, Bischofberger M, van der Goot FG (2010) Structure and assembly of pore-forming proteins. Curr Opin Struct Biol 20(2):241–246. Scholar
  8. 8.
    Pipkin ME, Lieberman J (2007) Delivering the kiss of death: progress on understanding how perforin works. Curr Opin Immunol 19(3):301–308. Scholar
  9. 9.
    Bubeck D (2014) The making of a macromolecular machine: assembly of the membrane attack complex. Biochemistry-US 53(12):1908–1915. Scholar
  10. 10.
    Serna M, Giles JL, Morgan BP, Bubeck D (2016) Structural basis of complement membrane attack complex formation. Nat Commun 7:10587. Scholar
  11. 11.
    Cosentino K, Garcia-Saez AJ (2014) Mitochondrial alterations in apoptosis. Chem Phys Lipids 181:62–75. Scholar
  12. 12.
    Gross A, McDonnell JM, Korsmeyer SJ (1999) Bcl-2 family members and the mitochondria in apoptosis. Genes Dev 13:1899–1911CrossRefGoogle Scholar
  13. 13.
    Suen DF, Norris KL, Youle RJ (2008) Mitochondrial dynamics and apoptosis. Genes Dev 22(12):1577–1590. Scholar
  14. 14.
    Cosentino K, Ros U, Garcia-Saez AJ (2016) Assembling the puzzle: oligomerization of alpha-pore forming proteins in membranes. Biochim Biophys Acta 1858(3):457–466. Scholar
  15. 15.
    Ros U, Garcia-Saez AJ (2015) More than a pore: the interplay of pore-forming proteins and lipid membranes. J Membr Biol 248(3):545–561. Scholar
  16. 16.
    Huang HW (2000) Action of antimicrobial peptides: two-state model. Biochemistry-US 39:8347–8352CrossRefGoogle Scholar
  17. 17.
    Xu XP, Zhai D, Kim E, Swift M, Reed JC, Volkmann N, Hanein D (2013) Three-dimensional structure of Bax-mediated pores in membrane bilayers. Cell Death Dis 4:e683. Scholar
  18. 18.
    Parker MW, Feil SC (2005) Pore-forming protein toxins: from structure to function. Prog Biophys Mol Biol 88(1):91–142. Scholar
  19. 19.
    Sonnen AF, Plitzko JM, Gilbert RJ (2014) Incomplete pneumolysin oligomers form membrane pores. Open Biol 4:140044. Scholar
  20. 20.
    Metkar SS, Marchioretto M, Antonini V, Lunelli L, Wang B, Gilbert RJ, Anderluh G, Roth R, Pooga M, Pardo J, Heuser JE, Serra MD, Froelich CJ (2015) Perforin oligomers form arcs in cellular membranes: a locus for intracellular delivery of granzymes. Cell Death Differ 22(1):74–85. Scholar
  21. 21.
    Salvador-Gallego R, Mund M, Cosentino K, Schneider J, Unsay J, Schraermeyer U, Engelhardt J, Ries J, Garcia-Saez AJ (2016) Bax assembly into rings and arcs in apoptotic mitochondria is linked to membrane pores. EMBO J 35(4):389–401. Scholar
  22. 22.
    Castellana ET, Cremer PS (2006) Solid supported lipid bilayers: from biophysical studies to sensor design. Surf Sci Rep 61(10):429–444. Scholar
  23. 23.
    Frederix PLTM, Bosshart PD, Engel A (2009) Atomic force microscopy of biological membranes. Biophys J 96(2):329–338. Scholar
  24. 24.
    Mennicke U, Salditt T (2002) Preparation of solid-supported lipid bilayers by spin-coating. Langmuir 18:8172–8177. Scholar
  25. 25.
    Yilmaz N, Kobayashi T (2016) Assemblies of pore-forming toxins visualized by atomic force microscopy. Biochim Biophys Acta 1858(3):500–511. Scholar
  26. 26.
    Mou J, Yang J, Shao Z (1995) Atomic force microscopy of cholera toxin B-oligomers bound to bilayers of biologically relevant lipids. J Mol Biol 248:507–512CrossRefGoogle Scholar
  27. 27.
    Epand RF, Martinou J-C, Montessuit S, Epand RM, Yip CM (2002) Direct evidence of membrane pore formation by the apoptotic protein Bax. Biochem Biophys Res Commun 298:744–749CrossRefGoogle Scholar
  28. 28.
    Czajkowsky DM, Hotze EM, Shao Z, Tweten RK (2004) Vertical collapse of a cytolysin prepore moves its transmembrane beta-hairpins to the membrane. EMBO J 23:3206–3215. Scholar
  29. 29.
    Yilmaz N, Kobayashi T (2015) Visualization of lipid membrane reorganization induced by a pore-forming toxin using high-speed atomic force microscopy. ACS Nano 9:7960–7967CrossRefGoogle Scholar
  30. 30.
    Unsay J, Cosentino K, Garcia-Saez AJ (2015) Atomic force microscopy imaging and force spectroscopy of supported lipid bilayers. J Vis Exp 101:e52867. Scholar
  31. 31.
    Canale C, Jacono M, Diaspro A, Dante S (2010) Force spectroscopy as a tool to investigate the properties of supported lipid membranes. Microsc Res Tech 73(10):965–972. Scholar
  32. 32.
    Redondo-Morata L, Giannotti MI, Sanz F (2012) Stability of lipid bilayers as model membranes: atomic force microscopy and spectroscopy approach. In: Baró AM, Reifenberger RG (eds) Atomic force microscopy in liquid: biological applications, 1st edn. Wiley-VCH Verlag GmbH, Weinheim. Scholar
  33. 33.
    Butt H-J, Franz V (2002) Rupture of molecular thin films observed in atomic force microscopy I. Theory Phys Rev E 66:031601. Scholar
  34. 34.
    Unsay JD, Cosentino K, Sporbeck K, Garcia-Saez AJ (2017) Pro-apoptotic cBid and Bax exhibit distinct membrane remodeling activities: an AFM study. BBA-Biomembranes 1859:17–27. Scholar
  35. 35.
    Gräslund S, Nordlund P, Weigelt J, Bray J, Gileadi O, Knapp S, Oppermann U, Arrowsmith C, Hui R, Ming J, Dhe-Paganon S, Park H-W, Savchenko A, Yee A, Edwards A, Vincentelli R, Cambillau C, Kim R, Kim S-H, Rao Z, Shi Y, Terwilliger TC, Kim C-Y, Hung L-W, Waldo GS, Peleg Y, Albeck S, Unger T, Dym O, Prilusky J, Sussman JL, Stevens RC, Lesley SA, Wilson IA, Joachimiak A, Collart F, Dementieva I, Donnelly MI, Eschenfeldt WH, Kim Y, Stols L, Wu R, Zhou M, Burley SK, Emtage JS, Sauder JM, Thompson D, Bain K, Luz J, Gheyi T, Zhang F, Atwell S, Almo SC, Bonanno JB, Fiser A, Swaminathan S, Studier FW, Chance MR, Sali A, Acton TB, Xiao R, Zhao L, Ma LC, Hunt JF, Tong L, Cunningham K, Inouye M, Anderson S, Janjua H, Shastry R, Ho CK, Wang D, Wang H, Jiang M, Montelione GT, Stuart DI, Owens RJ, Daenke S, Schütz A, Heinemann U, Yokoyama S, Büssow K, Gunsalus KC (2008) Protein production and purification. Nat Methods 5(2):135–146. Scholar
  36. 36.
    Subburaj Y, Cosentino K, Axmann M, Pedrueza-Villalmanzo E, Hermann E, Bleicken S, Spatz J, Garcia-Saez AJ (2015) Bax monomers form dimer units in the membrane that further self-assemble into multiple oligomeric species. Nat Commun 6:8042. Scholar
  37. 37.
    Bleicken S, Jeschke G, Stegmueller C, Salvador-Gallego R, García-Sáez Ana J, Bordignon E (2014) Structural model of active Bax at the membrane. Mol Cell 56:496. Scholar
  38. 38.
    Desagher S, Osen-Sand A, Nichols A, Eskes R, Montessuit S, Lauper S, Maundrell K, Antonsson B, Martinou J-C (1999) Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J Cell Biol 144:891–901CrossRefGoogle Scholar
  39. 39.
    Subburaj Y (2014) Single particle tracking to characterize the mechanism of pore formation by pore-forming proteins. University of Tübingen, TübingenGoogle Scholar
  40. 40.
    Shamas-Din A, Binder S, Zhu W, Zaltsman Y, Campbell C, Gross A, Leber B, Andrews DW, Fradin C (2013) tBid undergoes multiple conformational changes at the membrane required for Bax activation. J Biol Chem 288(30):22111–22127. Scholar
  41. 41.
    Lovell JF, Billen LP, Bindner S, Shamas-Din A, Fradin C, Leber B, Andrews DW (2008) Membrane binding by tBid initiates an ordered series of events culminating in membrane permeabilization by Bax. Cell 135(6):1074–1084. Scholar
  42. 42.
    Burnham NA, Chen X, Hodges CS, Matei GA, Thoreson EJ, Roberts CJ, Davies MC, Tendler SJB (2002) Comparison of calibration methods for atomic-force microscopy. Nanotechnology 14:1–6CrossRefGoogle Scholar
  43. 43.
    Sader JE, Larson I, Mulvaney P, White LR (1995) Method for the calibration of atomic force microscope cantilevers. Rev Sci Instrum 66(7):3789. Scholar
  44. 44.
    Miller EJ, Trewby W, Payam AF, Piantanida L, Cafolla C, Voitchovsky K (2016) Sub-nanometer resolution imaging with amplitude-modulation atomic force microscopy in liquid. J Vis Exp:e54924.
  45. 45.
    Alessandrini A, Seeger Heiko M, Caramaschi T, Facci P (2012) Dynamic force spectroscopy on supported lipid bilayers: effect of temperature and sample preparation. Biophys J 103(1):38–47. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Interfaculty Institute of Biochemistry, University of TübingenTübingenGermany

Personalised recommendations