Skip to main content
SpringerLink
Log in

Analysis of the Late Steps of Exocytosis: Biochemical and Total Internal Reflection Fluorescence Microscopy (TIRFM) Studies

  • Published:
Cellular and Molecular Neurobiology Aims and scope Submit manuscript

Abstract

1. Time with Julie in his laboratory at the NIH in the early 1970s is remembered. The experience led to a life-long interest in the regulation of catecholamine secretion. Here are summarized aspects of this work.

2. The relationship between ATP-dependent priming of exocytosis and the polyphosphoinositides is reviewed. In addition, studies are summarized in which total internal reflection fluorescent microscopy (TIRFM) was used to visualize secretory granule behavior before exocytosis and individual exocytotic events.

3. Quantitative optical analysis indicates that chromaffin granule motion is highly restricted but regulated. Granules can undergo significant motion in the 100 ms prior to fusion and interactions with the plasma membrane leading to fusion can occur within this time. The small motions may permit granules adjacent to the plasma membrane to repetitively sample microdomains of the plasma membrane, thereby increasing the probability of fruitful interactions that lead to fusion.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1.
Fig. 2.

Similar content being viewed by others

Abbreviations

PtdIns:

phosphatidyl inositol

TIRFM:

total internal reflection fluorescence microscopy.

REFERENCES

  • Ahnert-Hilger, G., Bader, M. F., Bhakdi, S., and Gratzl, M. (1989). Introduction of macromolecules into bovine adrenal medullary chromaffin cells and rat pheochromocytoma cells (PC12) by permeabilization with streptolysin O: inhibitory effect of tetanus toxin on catecholamine secretion. J. Neurochem. 52 :1751–1758.

    Article  PubMed  CAS  Google Scholar 

  • Allersma, M. W., Bittner, M. A., Axelrod, D., and Holz, R. W. (2006). Motion Matters: Secretory Granule Motion Adjacent to the Plasma Membrane and Exocytosis. Mol. Biol. Cell E05–E10.

  • Allersma, M. W., Wang, L., Axelrod, D., and Holz, R. W. (2004). Visualization of Regulated Exocytosis with a Granule-Membrane Probe using Total Internal Reflection Microscopy. Mol. Biol. Cell 15 :4658–4668.

    Google Scholar 

  • Axelrod, D. (1981). Cell-substrate contacts illuminated by total internal reflection fluorescence. J. Cell Biol. 89 :141–145.

    Article  PubMed  CAS  Google Scholar 

  • Axelrod, D. (2003). Total internal reflection fluorescence microscopy in cell biology. Methods Enzymol. 361 :1–33.

    Article  PubMed  CAS  Google Scholar 

  • Bai, J., Tucker, W. C., and Chapman, E. R. (2004). PIP2 increases the speed of response of synaptotagmin and steers its membrane-penetration activity toward the plasma membrane. Nat. Struct. Mol. Biol. 11 :36–44.

    Article  PubMed  CAS  Google Scholar 

  • Baker, P. F., and Knight, D. E. (1978). Calcium-dependent exocytosis in bovine adrenal medullary cells with leaky plasma membranes. Nature 276 :620–622.

    Article  PubMed  CAS  Google Scholar 

  • Bittner, M. A., and Holz, R. W. (1992a). A temperature-sensitive step in exocytosis. J. Biol. Chem. 267 :16226–16229.

    PubMed  CAS  Google Scholar 

  • Bittner, M. A., and Holz, R. W. (1992b). Kinetic analysis of secretion from permeabilized adrenal chromaffin cells reveals distinct components. J. Biol. Chem. 267 :16219–16225.

    PubMed  CAS  Google Scholar 

  • Bittner, M. A., and Holz, R. W. (2005). Phosphatidylinositol-4,5-bisphosphate:actin dynamics and the regulation of ATP-dependent and independent secretion. Mol. Pharmacol. 67 :1089–1098.

    Article  PubMed  CAS  Google Scholar 

  • Brooks, J. C., and Treml, S. (1983). Catecholamine secretion by chemically skinned cultured chromaffin cells. J. Neurochem. 40 :468–473.

    Article  PubMed  CAS  Google Scholar 

  • Buckley, J. T., Lefebvre, Y. A., and Hawthorne, J. N. (1971). Identification of an actively phosphorylated component of adrenal medulla chromaffin granules. Biochim. Biophys. Acta 239 :517–519.

    PubMed  CAS  Google Scholar 

  • Chung, S.-H., Song, W.-J., Kim, K., Bednarski, J. J., Chen, J., Prestwich, G. D., and Holz, R. W. (1998). The C2 domains of Rabphilin3a specifically bind PtdIns(4,5)P2-containing vesicles in a Ca2+-dependent manner: characteristics and possible physiological significance. J. Biol. Chem. 273 :10240–10248.

    Article  PubMed  CAS  Google Scholar 

  • Cremona, O., Di Paolo, G., Wenk, M. R., Luthi, A., Kim, W. T., Takei, K., Daniell, L., Nemoto, Y., Shears, S. B., Flavell, R. A., McCormick, D. A., and De Camilli, P. (1999). Essential role of phosphoinositide metabolism in synaptic vesicle recycling. Cell 99 :179–188.

    Article  PubMed  CAS  Google Scholar 

  • De Camilli, P., Emr, S. D., McPherson, P. S., and Novick, P. (1996). Phosphoinositides as regulators of membrane traffic. Science 271 :1533–1539.

    Article  PubMed  CAS  Google Scholar 

  • Dunn, L. A., and Holz, R. W. (1983). Catecholamine secretion from digitonin-treated adrenal medullary chromaffin cells. J. Biol. Chem. 258 :4989–4993.

    PubMed  CAS  Google Scholar 

  • Eberhard, D. A., Cooper, C. L., Low, M. G., and Holz, R. W. (1990). Evidence that the inositol phopholipids are necessary for exocytosis: loss of inositol phospholipids and inhibition of secretion in permeabilized cells caused by a bacterial phospholipase C and removal of ATP. Biochem. J. 268 :15–25.

    PubMed  CAS  Google Scholar 

  • Eichberg, J., and Dawson, R. M. C. (1965). Polyphosphoinositides in myelin. Biochem. J. 96 :644–650.

    PubMed  CAS  Google Scholar 

  • Griesinger, C. B., Richards, C. D., and Ashmore, J. F. (2005). Fast vesicle replenishment allows indefatigable signaling at the first auditory synapse. Nature 212–215.

  • Han, W., Ng, Y. K., Axelrod, D., and Levitan, E. S. (1999). Neuropeptide release by efficient recruitment of diffusing cytoplasmic secretory vesicles. Proc. Natl. Acad. Sci. USA 96 :14577–14582.

    Article  PubMed  CAS  Google Scholar 

  • Hay, J. C., Fisette, P. L., Jenkins, G. H., Fukami, K., Takenawa, T., Anderson, R. A., and Martin, T. F. J. (1995). ATP-dependent inositide phosphorylation required for Ca2+-activated secretion. Nature 374 :173–177.

    Article  PubMed  CAS  Google Scholar 

  • Hay, J. C., and Martin, T. F. J. (1992). Resolution of regulated secretion into sequential MgATP-dependent and calcium-dependent stages mediated by distinct cytosolic proteins. J. Cell Biol. 119 :139–151.

    Article  PubMed  CAS  Google Scholar 

  • Hay, J. C., and Martin, T. F. J. (1993). Phosphatidylinositol transfer protein required for ATP-dependent priming of Ca2+-activated secretion. Nature 366 :572–575.

    Article  PubMed  CAS  Google Scholar 

  • Heidelberger, R. (1998). Adenosine triphosphate and the late steps in calcium-dependent exocytosis at a ribbon synapse. J. Gen. Physiol. 111 :225–241.

    Article  PubMed  CAS  Google Scholar 

  • Hokin, L., and Hokin, M. R. (1964). The incorporation of 32P from triphosphate into polyphosphoinositides [γ-32P]adenosine and phsophatidic acid in erythrocyte membranes. Biochim. Biophys. Acta 84 :563–575.

    PubMed  CAS  Google Scholar 

  • Holz, R. W., Bittner, M. A., Peppers, S. C., Senter, R. A., and Eberhard, D. A. (1989). MgATP-independent and MgATP-dependent exocytosis. Evidence that MgATP primes adrenal chromaffin cells to undergo exocytosis. J. Biol. Chem. 264 :5412–5419.

    PubMed  CAS  Google Scholar 

  • Holz, R. W., Hlubek, M. D., Sorensen, S. D., Fisher, S. K., Balla, T., Ozaki, S., Prestwich, G. D., Stuenkel, E. L., and Bittner, M. A. (2000). A pleckstrin homology domain specific for PtdIns-4-5-P2 and fused to green fluorescent protein identifies plasma membrane PtdIns-4-5-P2 as being important in exocytosis. J Biol Chem. 275 :17878–17885.

    Article  PubMed  CAS  Google Scholar 

  • Husebye, E. S., and Flatmark, T. (1988). Phosphatidylinositol kinase of bovine adrenal chromaffin granules: Kinetic properties and inhibition by low concentrations of Ca2+. Biochim. Biophys. Acta 968 :261–265.

    Article  PubMed  CAS  Google Scholar 

  • Johns, L. M., Levitan, E. S., Shelden, E. S., Holz, R. W., and Axelrod, D. (2001). Restriction of secretory granule motion near the plasma membrane of chromaffin cells. J. Cell Biol. 153 :177–190.

    Article  PubMed  CAS  Google Scholar 

  • Khvotchev, M., and Sudhof, T. C. (1998). Newly synthesized phosphatidylinositol phosphates are required for synaptic norepinephrine but not glutamate or gamma-aminobutyric acid (GABA) release. J. Biol. Chem. 273 :21451–21454.

    Article  PubMed  CAS  Google Scholar 

  • Kurosawa, M., and Parker, C. (1986). A phosphatidylinositol kinase in rat mast cell granules. J. Immunol. 136 :616–622.

    PubMed  CAS  Google Scholar 

  • Loyet, K. M., Kowalchyk, J. A., Chaudhary, A., Chen, J., Prestwich, G. D., and Martin, T. F. J. (1998). Specific binding of phosphatidylinositol 4,5-bisphosphate to calcium-dependent activator protein for secretion (CAPS), a potential phosphoinositide effector protein for regulated exocytosis. J. Biol. Chem. 273 :8337–8343.

    Article  PubMed  CAS  Google Scholar 

  • Martin, T. F. (1997). Phosphoinositides as spatial regulators of membrane traffic. Curr. Opin. Neurobiol. 7 :331–338.

    Article  PubMed  CAS  Google Scholar 

  • Micheva, K. D., Buchanan, J., Holz, R. W., and Smith, S. J. (2003). Retrograde regulation of synaptic vesicle endocytosis and recycling. Nat. Neurosci. 6 :925–932.

    Article  PubMed  CAS  Google Scholar 

  • Micheva, K. D., Holz, R. W., and Smith, S. J. (2001). Regulation of presynaptic phosphatidylinositol 4,5-bisphosphate by neuronal activity. J. Cell Biol. 154 :355–368.

    Article  PubMed  CAS  Google Scholar 

  • Muller, T. W., and Kirshner, N. (1975). ATPase and phosphatidylinositol kinase activities of adrenal chromaffin vesicles. J. Neurochem. 24 :1155–1161.

    Article  PubMed  CAS  Google Scholar 

  • Nakata, T., and Hirokawa, N. (1992). Organization of cortical cytoskeleton of cultured chromaffin cells and involvement in secretion as revealed by quick-freeze, deep-etching, and double-label immunoelectron microscopy. J. Neurosci. 12 :2186–2197.

    PubMed  CAS  Google Scholar 

  • Ohara-Imaizumi, M., Nakamichi, Y., Tanaka, T., Ishida, H., and Nagamatsu, S. (2002). Imaging exocytosis of single insulin secretory granules with evanescent wave microscopy. Distinct behavior of granule motion in biphasic insulin release. J. Biol. Chem. 277 :3805–3808.

    Article  PubMed  CAS  Google Scholar 

  • Oheim, M., Loerke, D., Stuhmer, W., and Chow, R. H. (1998). The last few milliseconds in the life of a secretory granule. Docking, dynamics and fusion visualized by total internal reflection fluorescence microscopy (TIRFM). Eur. J. Biophys. 27 :83–98.

    Article  CAS  Google Scholar 

  • Parsons, T. D., Coorssen, J. R., Horstmann, H., and Almers, W. (1995). Docked granules, the exocytic burst, and the need for ATP hydrolysis in endocrine cells. Neuron 15 :1085–1096.

    Article  PubMed  CAS  Google Scholar 

  • Phillips, J. H. (1973). Phosphatidylinositol kinase. Biochem. J. 136 :579–587.

    PubMed  CAS  Google Scholar 

  • Schiavo, G., Gu, Q.-M., Prestwich, G. D., Sollner, T., and Rothman, J. E. (1996). Calcium-dependent switching of the specificity of phosphoinositide binding to synaptotagmin. Proc. Natl. Acad. Sci. USA 93 :13327–13332.

    Article  PubMed  CAS  Google Scholar 

  • Schu, P. V., Takegawa, K., Fry, M. J., Stack, J. H., Waterfield, M. D., and Emr, S. D. (1993). Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting. Science 260 :88–91.

    Article  PubMed  CAS  Google Scholar 

  • Sorensen, S. D., Linseman, D. A., McEwen, E. L., Heacock, A. M., and Fisher, S. K. (1999). A role for a wortmannin-sensitive phsophatidylinositol-4-kinase in the endocytosis of muscarinic cholinergic receptors. J. Pharmacol. Exp. Ther. 52 :827–836.

    Google Scholar 

  • Steyer, J. A., Horstman, H., and Almers, W. (1997). Transport, docking and exocytosis of single secretory granules in live chromaffin cells. Nature 388 :474–478.

    Article  PubMed  CAS  Google Scholar 

  • Voets, T. (2000). Dissection of three Ca2+-dependent steps leading to secretion in chromaffin cells from mouse adrenal slices. Neuron 28 :537–545.

    Article  PubMed  CAS  Google Scholar 

  • Whipps, D. E., Armston, A. E., Pryor, H. J., and Halestrap, A. P. (1987). Effects of glucagon and Ca2+ on the metabolism of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5- bisphosphate in isolated rat hepatocytes and plasma membranes. Biochem. J. 241 :835–845.

    PubMed  CAS  Google Scholar 

  • Wiedemann, C., Schafer, T., Burger, M. M., and Sihra, T. S. (1998). An essential role for a small synaptic vesicle-associated phosphatidylinositol 4-kinase in neurotransmitter release. J. Neurosci. 18 :5594–5602.

    PubMed  CAS  Google Scholar 

  • Wilson, S. P., and Kirshner, N. (1983). Calcium-evoked secretion from digitonin-permeabilized adrenal medullary chromaffin cells. J. Biol. Chem. 258 :4994–5000.

    PubMed  CAS  Google Scholar 

  • Wurmser, A. E., Gary, J. D., and Emr, S. D. (1999). Phosphoinositide 3-kinases and their FYVE domain-containing effectors as regulators of vacuolar/lysosomal membrane trafficking pathways. J. Biol. Chem. 274 :9129–9132.

    Article  PubMed  CAS  Google Scholar 

Download references

ACKNOWLEDGMENTS

This work was funded by grants from National Institutes of Health grant RO1-DK50127 (to RWH), R01-NS38129 (to Daniel Axelrod, University of Michigan) and a Michigan Economic Development Corporation and the Michigan Life Sciences Corridor Grant (to RWH).

Author information

Authors and Affiliations

  1. Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan, 48109-0632, USA

    Ronald W. Holz

Authors
  1. Ronald W. Holz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ronald W. Holz.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Holz, R.W. Analysis of the Late Steps of Exocytosis: Biochemical and Total Internal Reflection Fluorescence Microscopy (TIRFM) Studies. Cell Mol Neurobiol 26, 437–445 (2006). https://doi.org/10.1007/s10571-006-9049-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10571-006-9049-5

Key Words

Search

Navigation