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Quantification of exocytosis kinetics by DIC image analysis of cortical lawns

  • Original Article
  • Published:
Journal of Chemical Biology

Abstract

Cortical lawns prepared from sea urchin eggs have offered a robust in vitro system for study of regulated exocytosis and membrane fusion events since their introduction by Vacquier almost 40 years ago (Vacquier in Dev Biol 43:62–74, 1975). Lawns have been imaged by phase contrast, darkfield, differential interference contrast, and electron microscopy. Quantification of exocytosis kinetics has been achieved primarily by light scattering assays. We present simple differential interference contrast image analysis procedures for quantifying the kinetics and extent of exocytosis in cortical lawns using an open vessel that allows rapid solvent equilibration and modification. These preparations maintain the architecture of the original cortices, allow for cytological and immunocytochemical analyses, and permit quantification of variation within and between lawns. When combined, these methods can shed light on factors controlling the rate of secretion in a spatially relevant cellular context. We additionally provide a subroutine for IGOR Pro® that converts raw data from line scans of cortical lawns into kinetic profiles of exocytosis. Rapid image acquisition reveals spatial variations in time of initiation of individual granule fusion events with the plasma membrane not previously reported.

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References

  1. Avery J, Ellis DJ, Lang T, Holroyd P, Riedel D, Henderson RM, Edwardson JM, Jahn R (2000) A cell-free system for regulated exocytosis in PC12 cells. J Cell Biol 148:317–324

    Article  CAS  Google Scholar 

  2. Blank PS, Cho MS, Vogel SS, Kaplan D, Kang A, Malley J, Zimmerberg J (1998) Submaximal responses in calcium-triggered exocytosis are explained by differences in the calcium sensitivity of individual secretory vesicles. J Gen Physiol 112:559–567

    Article  CAS  Google Scholar 

  3. Blank PS, Vogel SS, Malley JD, Zimmerberg J (2001) A kinetic analysis of calcium-triggered exocytosis. J Gen Physiol 118:145–156

    Article  CAS  Google Scholar 

  4. Chen X, Arac D, Wang T, Gilpin CJ, Zimmerberg J (2006) SNARE-mediated lipid mixing depends on the physical state of the vesicles. Biophys J 90:2062–2074

    Article  CAS  Google Scholar 

  5. Churchward M, Rogasevskaia T, Hofgen J, Bau J, Coorssen J (2005) Cholesterol facilitates the native mechanism of Ca2+-triggered membrane fusion. J Cell Sci 118:4833–4848

    Article  CAS  Google Scholar 

  6. Churchward MA, Rogasevskaia T, Brandman DM, Khosravani H, Nava P, Atkinson JK, Coorssen JR (2008) Specific lipids supply critical negative spontaneous curvature—an essential component of native Ca2+-triggered membrane fusion. Biophys J 94:3976–3986

    Article  CAS  Google Scholar 

  7. Coorssen JR, Blank PS, Tahara M, Zimmerberg J (1998) Biochemical and functional studies of cortical vesicle fusion: the SNARE complex and Ca2+ sensitivity. J Cell Biol 143:1845–1857

    Article  CAS  Google Scholar 

  8. Coorssen JR, Blank PS, Albertorio F, Bezrukov L, Kolosova I, Chen X, Backlund PS Jr, Zimmerberg J (2003) Regulated secretion: SNARE density, vesicle fusion, and calcium dependence. J Cell Sci 116:2087–2097

    Article  CAS  Google Scholar 

  9. Decker SJ, Kinsey WH (1983) Characterization of cortical secretory vesicles from the sea urchin egg. Dev Biol 96:37–45

    Article  CAS  Google Scholar 

  10. Detering NK, Decker GL, Schmell ED, Lennarz WJ (1977) Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs. J Cell Biol 75:899–914

    Article  CAS  Google Scholar 

  11. Kaplan D, Bungay P, Sullivan J, Zimmerberg J (1996) A rapid flow perfusion chamber for high-resolution microscopy. J Microsc 181:286–297

    Article  CAS  Google Scholar 

  12. Kinsey WH, Decker GL, Lennarz WJ (1980) Isolation and partial characterization of the plasma membrane of the sea urchin egg. J Cell Biol 87:248–254

    Article  CAS  Google Scholar 

  13. Kopf GS, Moy GW, Vacquier VD (1982) Isolation and characterization of sea urchin egg cortical granules. J Cell Biol 95:924–932

    Article  CAS  Google Scholar 

  14. Leguia M, Wessel GM (2007) The many faces of egg activation at fertilization. Signal Transduct 7:118–141

    Article  CAS  Google Scholar 

  15. Raveh A, Valitsky M, Shani L, Coorssen JR, Blank PS, Zimmerberg J, Rahamimoff R (2012) Observations of calcium dynamics in cortical secretory vesicles. Cell Calcium 52:217–225

    Article  CAS  Google Scholar 

  16. Rogasevskaia TP, Coorssen JR (2011) A new approach to the molecular analysis of docking, priming, and regulated membrane fusion. J Chem Biol 4:117–36

    Article  Google Scholar 

  17. Steinhardt R, Zucker R, Schatten G (1977) Intracellular calcium release at fertilization in the sea urchin egg. Dev Biol 58:185–196

    Article  CAS  Google Scholar 

  18. Sudhof TC (2004) The synaptic vesicle cycle. Annu Rev Neurosci 27:509–547

    Article  CAS  Google Scholar 

  19. Terasaki M (1995) Visualization of exocytosis during sea urchin egg fertilization using confocal microscopy. J Cell Sci 108:2293–2300

    CAS  Google Scholar 

  20. Terasaki M, Henson J, Begg D, Kaminer B, Sardet C (1991) Characterization of sea urchin egg endoplasmic reticulum in cortical preparations. Dev Biol 148:398–401

    Article  CAS  Google Scholar 

  21. Terasaki M, Reese TS (1992) Characterization of endoplasmic reticulum by co-localization of BiP and dicarbocyanine dyes. J Cell Sci 101:315–322

    CAS  Google Scholar 

  22. Terasaki M, Sardet C (1991) Demonstration of calcium uptake and release by sea urchin egg cortical endoplasmic reticulum. J Cell Biol 115:1031–1037

    Article  CAS  Google Scholar 

  23. Trikash IO, Kolchinskaya LI (2006) Fusion of synaptic vesicles and plasma membrane in the presence of synaptosomal soluble proteins. Neurochem Int 49:270–285

    Article  CAS  Google Scholar 

  24. Vacquier VD (1975) The isolation of intact cortical garanules from sea urchin eggs: calcium ions trigger granule discharge. Dev Biol 43:62–74

    Article  CAS  Google Scholar 

  25. Vogel SS, Blank PS, Zimmerberg J (1996) Poisson-distributed active fusion complexes underlie the control of the rate and extent of exocytosis by calcium. J Cell Biol 134:329–338

    Article  CAS  Google Scholar 

  26. Vogel SS, Zimmerberg J (1992) Proteins on exocytic vesicles mediate calcium-triggered fusion. Proc Natl Acad Sci 89:4749–4753

    Article  CAS  Google Scholar 

  27. Wojcik SM, Brose N (2007) Regulation of membrane fusion in synaptic excitation-secretion coupling: speed and accuracy matter. Neuron 55:11–24

    Article  CAS  Google Scholar 

  28. Wong JL, Wessel GM (2008) FRAP analysis of secretory granule lipids and proteins in the sea urchin egg. Methods Mol Biol 440:61–76

    Article  CAS  Google Scholar 

  29. Zimmerberg J, Sardet C, Epel D (1985) Exocytosis of sea urchin egg cortical vesicles in vitro is retarded by hyperosmotic sucrose: kinetics of fusion monitored by quantitative light-scattering microscopy. J Cell Biol 101:2398–2410

    Article  CAS  Google Scholar 

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Acknowledgments

We gratefully acknowledge Julie Fitzgerald and Fadi Hamati for technical assistance, Patrick Williamson for helpful discussions, and Ron Hebert for reaction vessel construction. Support was received from a SOMAS-URM grant through the Howard Hughes Medical Institute (grant no. 52005120) and the National Science Foundation (grant no. DUE-0930153) to J.G.T. and a Faculty Research Award of the Axel Schupf’57 Fund for Intellectual Life to D.P.

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Authors and Affiliations

  1. Department of Biology, Amherst College, Amherst, MA, 01002, USA

    Peter Kahng, Josef G. Trapani & Dominic Poccia

  2. Program in Neuroscience, Amherst College, Amherst, MA, 01002, USA

    James Mooney, Saumitra Thakur, Josef G. Trapani & Dominic Poccia

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  1. James Mooney
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  2. Saumitra Thakur
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  5. Dominic Poccia
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Correspondence to Dominic Poccia.

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Mooney, J., Thakur, S., Kahng, P. et al. Quantification of exocytosis kinetics by DIC image analysis of cortical lawns. J Chem Biol 7, 43–55 (2014). https://doi.org/10.1007/s12154-013-0104-7

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  • DOI: https://doi.org/10.1007/s12154-013-0104-7

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