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. 2008 Oct 9;10(4):847–856. doi: 10.1111/j.1582-4934.2006.tb00529.x

Cellular secretion studied by force microscopy

D P Allison a,b,c,*, M J Doktycz a
PMCID: PMC3933080  PMID: 17125589

Abstract

Using the optical microscope, real adventures in cellular research began in earnest in the latter half of the nineteenth century. With the development of the electron microscope, ultramicroscopy, and improved cell staining techniques, significant advances were made in defining intracellular structures at the nanometer level. The invention of force microscopy, the atomic force microscope (AFM) in the mid 1980s, and the photonic force microscope (PFM) in the mid 1990s, finally provided the opportunity to study live cellular structure-function at the nanometer level. Working with the AFM, dynamic cellular and subcellular events at the molecular level were captured in the mid 1990s, and a new cellular structure ‘the porosome’ in the plasma membrane of all secretory cells has been defined, where specific docking and fusion of secretory vesicles occur. The molecular mechanism of fusion of the secretory vesicle membrane at the base of the porosome membrane in cells, and the regulated release of intravesicular contents through the porosome opening to the extracellular space, has been determined. These seminal discoveries provide for the first time a molecular mechanism of cell secretion, and the possibility to ameliorate secretory defects in disease states.

Keywords: cell secretion, porosome or fusion pore, atomic force microscopy, photonic force microscopy, membrane fusion, nanostructure

References

  • 1.Claude A. Fractionation of mammalian liver cells by differential centrifugation: I. Problems, methods, and preparation of extract. J Exp Med. 1946;84:51–9. [PubMed] [Google Scholar]
  • 2.Claude A. Fractionation of mammalian liver cells by differential centrifugation: II. Experimental procedures and results. J Exp Med. 1946;84:61–89. [PubMed] [Google Scholar]
  • 3.de Duve C. Tissue fractionation past and present. J Cell Biol. 1971;50:20–55. [PubMed] [Google Scholar]
  • 4.Palade GE. Nobel Lecture, Intracellular aspects of the process of protein synthesis. Science. 1975;189:347–58. doi: 10.1126/science.1096303. [DOI] [PubMed] [Google Scholar]
  • 5.Caro LG, Palade GE. Protein synthesis, storage, and discharge in the pancreatic exocrine cell: An autoradiographic study. J Cell Biol. 1964;20:473–95. doi: 10.1083/jcb.20.3.473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Palade GE. Hayashi T. Subcellular particles. New York: Ronald; 1959. pp. 64–83. [Google Scholar]
  • 7.de Duve C. 1963. p. 126. Lysosomes, Ciba Foundation Symposium; (Little Brown, Boston)
  • 8.Schneider SW, Sritharan KC, Geibel JP, Oberleithner H, Jena BP. Surface dynamics in living acinar cells imaged by atomic force microscopy: identification of plasma membrane structures involved in exocytosis. Proc Natl Acad Sci USA. 1997;94:316–21. doi: 10.1073/pnas.94.1.316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Jamieson JD. Current Topics in Membranes and Transport. New York and London: Academic Press; 1972. Transport and discharge of exportable proteins in pancreatic exocrine cells: in vitro studies; pp. 273–338. [Google Scholar]
  • 10.Chandler DE, Heuser JE. Arrest of membrane fusion in mast cells by quick-freezing. J Cell Biol. 1980;86:666–74. doi: 10.1083/jcb.86.2.666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Breckenridge LJ, Almers W. Currents through the fusion pore that forms during exocytosis of a secretory vesicle. Nature. 1987;328:814–7. doi: 10.1038/328814a0. [DOI] [PubMed] [Google Scholar]
  • 12.Breckenridge LJ, Almers W. Final steps in exocytosis observed in a cell with giant secretory granules. Proc Natl Acad Sci USA. 1987;84:1945–9. doi: 10.1073/pnas.84.7.1945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Heuser JE, Reese TS. Structural changes after transmitter release at the frog neuromuscular junction. J Cell Biol. 1981;88:564–80. doi: 10.1083/jcb.88.3.564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Alvarez de Toledo G, Fernandez JM. Cell Physiology of Blood. Rockefeller University Press; 1988. The events leading to secretory granule Fusion; pp. 334–44. [PubMed] [Google Scholar]
  • 15.Fernandez JM, Neher E, Gomperts BD. Capacitance measurements reveal stepwise fusion events in degranulat-ing mast cells. Nature. 1984;312:453–5. doi: 10.1038/312453a0. [DOI] [PubMed] [Google Scholar]
  • 16.Zimmerberg J, Curran M, Cohen FS, Brodwick M. Simultaneous electrical and optical measurements show that membrane fusion precedes secretory granule swelling during exocytosis of beige mouse mast cells. Proc Natl Acad Sci USA. 1987;84:1585–9. doi: 10.1073/pnas.84.6.1585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Alvarez de Toledo G, Fernandez-Chaocn R, Fernandez JM. Release of secretory products during transient vesicle fusion. Nature. 1993;363:554–8. doi: 10.1038/363554a0. [DOI] [PubMed] [Google Scholar]
  • 18.Monck JR, Oberhauser AF, Fernandez JM. The exocytotic fusion pore interface: a model of the site of neuro-transmitter release. Mol Memb Biol. 1995;12:151–6. doi: 10.3109/09687689509038511. [DOI] [PubMed] [Google Scholar]
  • 19.Cho SJ, Quinn AS, Stromer MH, Dash S, Cho J, Taatjes DJ, Jena BP. Structure and dynamics of the fusion pore in live cells. Cell Biol Int. 2002;26:35–42. doi: 10.1006/cbir.2001.0849. [DOI] [PubMed] [Google Scholar]
  • 20.Jena BP, Cho SJ, Jeremic A, Stromer MH Abu-Hamdah R, Structure and composition of the fusion pore. Biophys J. 2003;84:1337–43. doi: 10.1016/S0006-3495(03)74949-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Jeremic A, Kelly M, Cho SJ, Stromer MH, Jena BP. Reconstituted fusion pore. Biophys J. 2003;85:2522–7. doi: 10.1016/S0006-3495(03)74631-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Cho SJ, Wakade A, Pappas GD, Jena BP. New structure involved in transient membrane fusion and exocytosis. Ann New York Acad Sci. 2002;971:254–6. doi: 10.1111/j.1749-6632.2002.tb04471.x. [DOI] [PubMed] [Google Scholar]
  • 23.Cho SJ, Jeftinija K, Glavaski A, Jeftinija S, Jena BP, Anderson LL. Structure and dynamics of the fusion pore in live GH-secreting cells revealed using atomic force microscopy. Endocrinology. 2002;143:1144–8. doi: 10.1210/endo.143.3.8773. [DOI] [PubMed] [Google Scholar]
  • 24.Jena BP. Discovery of the porosome: revealing the molecular mechanism of secretion and membrane fusion in cells. J Cell Mol. Med. 2004;8:1–21. doi: 10.1111/j.1582-4934.2004.tb00255.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Cho WJ, Jeremic A, Rognlien KT, Zhvania MG, Lazrishvili I, Tamar B, Jena BP. Structure, isolation, composition and reconstitution of the neuronal fusion pore. Cell Biol Int. 2004;28:699–708. doi: 10.1016/j.cellbi.2004.07.004. [DOI] [PubMed] [Google Scholar]
  • 26.Cho SJ, Quinn AS, Stromer MH, Dash S, Cho J, Taatjes DJ, Jena BP. Structure and dynamics of the fusion pore in live cells. Cell Biol Int. 2002;26:35–42. doi: 10.1006/cbir.2001.0849. [DOI] [PubMed] [Google Scholar]
  • 27.Anderson LL. Discovery of the ’porosome’; the universal secretory machinery in cells. J Cell Mol Med. 2006;10:126–31. doi: 10.1111/j.1582-4934.2006.tb00294.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Jena BP. Cell secretion machinery: Studies using the AFM. Ultramicroscopy. 2006;106:663–9. doi: 10.1016/j.ultramic.2005.10.008. [DOI] [PubMed] [Google Scholar]
  • 29.Söller T, Whiteheart SW, Brunner M, Erdjument-Bromage H, Geromanos S, Tempst P, Rothman JE. SNAP receptors implicated in vesicle targeting and fusion. Nature. 1993;362:318–24. doi: 10.1038/362318a0. [DOI] [PubMed] [Google Scholar]
  • 30.Weber T, Zemelman BV, McNew JA, Westermann B, Gmachl M, Parlati F, Söllner TH, Rothman JE. SNAREpins: minimal machinery for membrane fusion. Cell. 1998;92:759–72. doi: 10.1016/s0092-8674(00)81404-x. [DOI] [PubMed] [Google Scholar]
  • 31.Cho SJ, Kelly M, Rognlien KT, Cho JA, Hörber JKH, Jena BP. SNAREs in opposing bilayers interact in a circular array to form conducting pores. Biophys J. 2002;83:2522–7. doi: 10.1016/s0006-3495(02)75263-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.MacDonald RC, MacDonald RI, Menco BP, Takeshita K, Subbarao NK, Hu LR. Small-volume extrusion apparatus for preparation of large unilamellar vesicles. Biochim Biophys Acta. 1991;1061:297–303. doi: 10.1016/0005-2736(91)90295-j. [DOI] [PubMed] [Google Scholar]
  • 33.Jeremic A, Kelly M, Cho JA, Cho SJ, Hörber JKH, Jena BP. Membrane fusion: what may transpire at the atomic level. Cell Biol Int. 2004;28:19–31. doi: 10.1016/j.cellbi.2003.11.004. [DOI] [PubMed] [Google Scholar]
  • 34.Cho WJ, Jeremic A, Jena BP. Size of supramolecular SNARE complex: membrane-directed self-assembly. J Am Chem Soc. 2005;127:10156–7. doi: 10.1021/ja052442m. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Israelachvili J. Intramolecular and Surface Forces. 2 ed. San Diego, CA: Academic Press; 1992. [Google Scholar]
  • 36.Ohki SJ. Effects of divalent cations, temperature, osmotic pressure gradient, and vesicle curvature on phos-phatidylserine vesicle fusion. J Memb Biol. 1984;77:265–75. doi: 10.1007/BF01870574. [DOI] [PubMed] [Google Scholar]
  • 37.Wilschut J, Duzgunes N, Papahadjopoulos D. Calcium/magnesium specificity in membrane fusion: kinetics of aggregation and fusion of phosphatidylserine vesicles and the role of bilayer curvature. Biochemistry. 1981;20:3126–33. doi: 10.1021/bi00514a022. [DOI] [PubMed] [Google Scholar]
  • 38.Leabu M. Membrane fusion in cells: molecular machinery and mechanisms. J Cell Mol Med. 2006;10:423–7. doi: 10.1111/j.1582-4934.2006.tb00409.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Jeong EH, Webster P, Khuong CQ, Sattar AKMA, Satchi M, Jena BP. The native membrane fusion machinery in cells. Cell Biol Int. 1998;22:657–70. doi: 10.1006/cbir.1999.0394. [DOI] [PubMed] [Google Scholar]
  • 40.Jeremic A, Quinn AS, Cho WJ, Taatjes DJ, Jena BP. Energy dependent disassembly of self-assembled SNARE complex: observation at nanometer resolution using atomic force microscopy. J Am Chem Soc. 2006;128:26–7. doi: 10.1021/ja056286v. [DOI] [PubMed] [Google Scholar]
  • 41.Kuznetsov SA, Langford GM, Weiss DG. Actin-dependent organelle movement in squid axoplasm. Nature. 1992;356:722–5. doi: 10.1038/356722a0. [DOI] [PubMed] [Google Scholar]
  • 42.Schroer TA, Sheetz MP. Functions of microtubule-based motors. Annu Rev Physiol. 1991;53:629–52. doi: 10.1146/annurev.ph.53.030191.003213. [DOI] [PubMed] [Google Scholar]
  • 43.Evans LL, Lee AJ, Bridgman PC, Mooseker MS. Vesicle-associated brain myosin-V can be activated to catalyze actin-based transport. J Cell Sci. 1998;111:2055–66. doi: 10.1242/jcs.111.14.2055. [DOI] [PubMed] [Google Scholar]
  • 44.Rudolf R, Kögel T, Kuznetsov SA, Salm T, Schlicker O, Hellwig A, Hammer IIIJA, Gerdes H-H. Myosin Va facilitates the distribution of secretory granules in the F-actin rich cortex of PC12 cells. J Cell Sci. 2003;116:1339–48. doi: 10.1242/jcs.00317. [DOI] [PubMed] [Google Scholar]
  • 45.Varadi A, Tsuboi T, Rutter GA. Myosin Va transports dense core secretory vesicles in pancreatic MIN6 â-Cells. Mol Biol Cell. 2005;16:2670–80. doi: 10.1091/mbc.E04-11-1001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Hirschberg K, Miller CM, Ellenberg J, Presley JF, Siggia ED, Phair RD, Lippincott-Schwartz J. Kinetic analysis of secretory protein traffic and characterization of Golgi to plasma membrane transport intermediates in living cells. J Cell Biol. 1998;143:1485–1503. doi: 10.1083/jcb.143.6.1485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Rudolf R, Salm T, Rustom A, Gerdes H-H. Dynamics of immature secretory granules: role of cytoskeletal elements during transport, cortical restriction, and F-actin-dependent tethering. Mol Biol Cell. 2001;12:1353–65. doi: 10.1091/mbc.12.5.1353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Manneville JB, Etienne-Manneville S, Skehel P, Carter T, Ogden D, Ferenczi M. Interaction of the actin cytoskeleton with microtubules regulates secretory organelle movement near the plasma membrane in human endothelial cells. J Cell Sci. 2003;116:3927–38. doi: 10.1242/jcs.00672. [DOI] [PubMed] [Google Scholar]
  • 49.Abu-Hamdah R, Cho WJ, Hörber JKH, Jena BP. Secretory vesicles in live cells are not free-floating but tethered to filamentous structures: a study using photonic force microscopy. Ultramicroscopy. 2006;106:670–3. doi: 10.1016/j.ultramic.2006.01.013. [DOI] [PubMed] [Google Scholar]
  • 50.Pralle A, Florin E-L, Stelzer EHK, Hörber JKH. Local viscosity probed by photonic force microscopy. Appl Phys A. 1998;66:S71. [Google Scholar]
  • 51.Jena BP. Fusion pore: structure and dynamics. J Endocrinol. 2003;176:169–74. doi: 10.1677/joe.0.1760169. [DOI] [PubMed] [Google Scholar]
  • 52.Jeftinija S. The story of cell secretion: events leading to the discovery of the ’porosome’ - the universal secretory machinery in cells. J Cell Mol Med. 2006;10:273–9. doi: 10.1111/j.1582-4934.2006.tb00398.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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