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. 1995 Feb 2;128(4):589–598. doi: 10.1083/jcb.128.4.589

Actin filament disassembly is a sufficient final trigger for exocytosis in nonexcitable cells

PMCID: PMC2199902  PMID: 7860632

Abstract

Although the actin cytoskeleton has been implicated in vesicle trafficking, docking and fusion, its site of action and relation to the Ca(2+)-mediated activation of the docking and fusion machinery have not been elucidated. In this study, we examined the role of actin filaments in regulated exocytosis by introducing highly specific actin monomer- binding proteins, the beta-thymosins or a gelsolin fragment, into streptolysin O-permeabilized pancreatic acinar cells. These proteins had stimulatory and inhibitory effects. Low concentrations elicited rapid and robust exocytosis with a profile comparable to the initial phase of regulated exocytosis, but without raising [Ca2+], and even when [Ca2+] was clamped at low levels by EGTA. No additional cofactors were required. Direct visualization and quantitation of actin filaments showed that beta-thymosin, like agonists, induced actin depolymerization at the apical membrane where exocytosis occurs. Blocking actin depolymerization by phalloidin or neutralizing beta- thymosin by complexing with exogenous actin prevented exocytosis. These findings show that the cortical actin network acts as a dominant negative clamp which blocks constitutive exocytosis. In addition, actin filaments also have a positive role. High concentrations of the actin depolymerizing proteins inhibited all phases of exocytosis. The inhibition overrides stimulation by agonists and all downstream effectors tested, suggesting that exocytosis cannot occur without a minimal actin cytoskeletal structure.

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Selected References

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  1. Bader M. F., Garcia A. G., Ciesielski-Treska J., Thierse D., Aunis D. Contractile proteins in chromaffin cells. Prog Brain Res. 1983;58:21–29. doi: 10.1016/S0079-6123(08)60003-5. [DOI] [PubMed] [Google Scholar]
  2. Bauduin H., Stock C., Vincent D., Grenier J. F. Microfilamentous system and secretion of enzyme in the exocrine pancreas. Effect of cytochalasin B. J Cell Biol. 1975 Jul;66(1):165–181. doi: 10.1083/jcb.66.1.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bengtsson T., Dahlgren C., Stendahl O., Andersson T. Actin assembly and regulation of neutrophil function: effects of cytochalasin B and tetracaine on chemotactic peptide-induced O2- production and degranulation. J Leukoc Biol. 1991 Mar;49(3):236–244. doi: 10.1002/jlb.49.3.236. [DOI] [PubMed] [Google Scholar]
  4. Bennett M. K., Scheller R. H. The molecular machinery for secretion is conserved from yeast to neurons. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2559–2563. doi: 10.1073/pnas.90.7.2559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Brady S. T., Lasek R. J., Allen R. D., Yin H. L., Stossel T. P. Gelsolin inhibition of fast axonal transport indicates a requirement for actin microfilaments. Nature. 1984 Jul 5;310(5972):56–58. doi: 10.1038/310056a0. [DOI] [PubMed] [Google Scholar]
  7. Cano M. L., Cassimeris L., Joyce M., Zigmond S. H. Characterization of tetramethylrhodaminyl-phalloidin binding to cellular F-actin. Cell Motil Cytoskeleton. 1992;21(2):147–158. doi: 10.1002/cm.970210208. [DOI] [PubMed] [Google Scholar]
  8. Cassimeris L., McNeill H., Zigmond S. H. Chemoattractant-stimulated polymorphonuclear leukocytes contain two populations of actin filaments that differ in their spatial distributions and relative stabilities. J Cell Biol. 1990 Apr;110(4):1067–1075. doi: 10.1083/jcb.110.4.1067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cassimeris L., Safer D., Nachmias V. T., Zigmond S. H. Thymosin beta 4 sequesters the majority of G-actin in resting human polymorphonuclear leukocytes. J Cell Biol. 1992 Dec;119(5):1261–1270. doi: 10.1083/jcb.119.5.1261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fath K. R., Burgess D. R. Golgi-derived vesicles from developing epithelial cells bind actin filaments and possess myosin-I as a cytoplasmically oriented peripheral membrane protein. J Cell Biol. 1993 Jan;120(1):117–127. doi: 10.1083/jcb.120.1.117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Feany M. B., Buckley K. M. The synaptic vesicle protein synaptotagmin promotes formation of filopodia in fibroblasts. Nature. 1993 Aug 5;364(6437):537–540. doi: 10.1038/364537a0. [DOI] [PubMed] [Google Scholar]
  12. Fechheimer M., Zigmond S. H. Focusing on unpolymerized actin. J Cell Biol. 1993 Oct;123(1):1–5. doi: 10.1083/jcb.123.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gardner J. D., Jensen R. T. Receptors and cell activation associated with pancreatic enzyme secretion. Annu Rev Physiol. 1986;48:103–117. doi: 10.1146/annurev.ph.48.030186.000535. [DOI] [PubMed] [Google Scholar]
  14. Goldschmidt-Clermont P. J., Machesky L. M., Doberstein S. K., Pollard T. D. Mechanism of the interaction of human platelet profilin with actin. J Cell Biol. 1991 Jun;113(5):1081–1089. doi: 10.1083/jcb.113.5.1081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Howard T. H., Oresajo C. O. The kinetics of chemotactic peptide-induced change in F-actin content, F-actin distribution, and the shape of neutrophils. J Cell Biol. 1985 Sep;101(3):1078–1085. doi: 10.1083/jcb.101.3.1078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jena B. P., Padfield P. J., Ingebritsen T. S., Jamieson J. D. Protein tyrosine phosphatase stimulates Ca(2+)-dependent amylase secretion from pancreatic acini. J Biol Chem. 1991 Sep 25;266(27):17744–17746. [PubMed] [Google Scholar]
  17. Kitagawa M., Williams J. A., De Lisle R. C. Amylase release from streptolysin O-permeabilized pancreatic acini. Am J Physiol. 1990 Aug;259(2 Pt 1):G157–G164. doi: 10.1152/ajpgi.1990.259.2.G157. [DOI] [PubMed] [Google Scholar]
  18. Kitagawa M., Williams J. A., De Lisle R. C. Interactions of intracellular mediators of amylase secretion in permeabilized pancreatic acini. Biochim Biophys Acta. 1991 Jan 23;1073(1):129–135. doi: 10.1016/0304-4165(91)90192-j. [DOI] [PubMed] [Google Scholar]
  19. Koffer A., Tatham P. E., Gomperts B. D. Changes in the state of actin during the exocytotic reaction of permeabilized rat mast cells. J Cell Biol. 1990 Sep;111(3):919–927. doi: 10.1083/jcb.111.3.919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kuznetsov S. A., Langford G. M., Weiss D. G. Actin-dependent organelle movement in squid axoplasm. Nature. 1992 Apr 23;356(6371):722–725. doi: 10.1038/356722a0. [DOI] [PubMed] [Google Scholar]
  21. Kwiatkowski D. J., Janmey P. A., Mole J. E., Yin H. L. Isolation and properties of two actin-binding domains in gelsolin. J Biol Chem. 1985 Dec 5;260(28):15232–15238. [PubMed] [Google Scholar]
  22. Kwiatkowski D. J., Stossel T. P., Orkin S. H., Mole J. E., Colten H. R., Yin H. L. Plasma and cytoplasmic gelsolins are encoded by a single gene and contain a duplicated actin-binding domain. Nature. 1986 Oct 2;323(6087):455–458. doi: 10.1038/323455a0. [DOI] [PubMed] [Google Scholar]
  23. Lelkes P. I., Friedman J. E., Rosenheck K., Oplatka A. Destabilization of actin filaments as a requirement for the secretion of catecholamines from permeabilized chromaffin cells. FEBS Lett. 1986 Nov 24;208(2):357–363. doi: 10.1016/0014-5793(86)81049-3. [DOI] [PubMed] [Google Scholar]
  24. Martin T. F. Identification of proteins required for Ca(2+)-activated secretion. Ann N Y Acad Sci. 1994 Mar 9;710:328–332. doi: 10.1111/j.1749-6632.1994.tb26639.x. [DOI] [PubMed] [Google Scholar]
  25. Matter K., Dreyer F., Aktories K. Actin involvement in exocytosis from PC12 cells: studies on the influence of botulinum C2 toxin on stimulated noradrenaline release. J Neurochem. 1989 Feb;52(2):370–376. doi: 10.1111/j.1471-4159.1989.tb09131.x. [DOI] [PubMed] [Google Scholar]
  26. McLaughlin P. J., Gooch J. T., Mannherz H. G., Weeds A. G. Structure of gelsolin segment 1-actin complex and the mechanism of filament severing. Nature. 1993 Aug 19;364(6439):685–692. doi: 10.1038/364685a0. [DOI] [PubMed] [Google Scholar]
  27. Minta A., Kao J. P., Tsien R. Y. Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J Biol Chem. 1989 May 15;264(14):8171–8178. [PubMed] [Google Scholar]
  28. Muallem S. The ins and outs of Ca2+ in exocrine cells. Adv Second Messenger Phosphoprotein Res. 1992;26:351–368. [PubMed] [Google Scholar]
  29. Nachmias V. T. Small actin-binding proteins: the beta-thymosin family. Curr Opin Cell Biol. 1993 Feb;5(1):56–62. doi: 10.1016/s0955-0674(05)80008-0. [DOI] [PubMed] [Google Scholar]
  30. Norman J. C., Price L. S., Ridley A. J., Hall A., Koffer A. Actin filament organization in activated mast cells is regulated by heterotrimeric and small GTP-binding proteins. J Cell Biol. 1994 Aug;126(4):1005–1015. doi: 10.1083/jcb.126.4.1005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nüsse O., Lindau M. The dynamics of exocytosis in human neutrophils. J Cell Biol. 1988 Dec;107(6 Pt 1):2117–2123. doi: 10.1083/jcb.107.6.2117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. O'Konski M. S., Pandol S. J. Cholecystokinin JMV-180 and caerulein effects on the pancreatic acinar cell cytoskeleton. Pancreas. 1993 Sep;8(5):638–646. doi: 10.1097/00006676-199309000-00018. [DOI] [PubMed] [Google Scholar]
  33. O'Konski M. S., Pandol S. J. Effects of caerulein on the apical cytoskeleton of the pancreatic acinar cell. J Clin Invest. 1990 Nov;86(5):1649–1657. doi: 10.1172/JCI114887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Padfield P. J., Balch W. E., Jamieson J. D. A synthetic peptide of the rab3a effector domain stimulates amylase release from permeabilized pancreatic acini. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1656–1660. doi: 10.1073/pnas.89.5.1656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pandol S. J., Schoeffield M. S., Sachs G., Muallem S. Role of free cytosolic calcium in secretagogue-stimulated amylase release from dispersed acini from guinea pig pancreas. J Biol Chem. 1985 Aug 25;260(18):10081–10086. [PubMed] [Google Scholar]
  36. Popov S. V., Poo M. M. Synaptotagmin: a calcium-sensitive inhibitor of exocytosis? Cell. 1993 Jul 2;73(7):1247–1249. doi: 10.1016/0092-8674(93)90352-q. [DOI] [PubMed] [Google Scholar]
  37. Sanders M. C., Goldstein A. L., Wang Y. L. Thymosin beta 4 (Fx peptide) is a potent regulator of actin polymerization in living cells. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4678–4682. doi: 10.1073/pnas.89.10.4678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sontag J. M., Aunis D., Bader M. F. Peripheral actin filaments control calcium-mediated catecholamine release from streptolysin-O-permeabilized chromaffin cells. Eur J Cell Biol. 1988 Jun;46(2):316–326. [PubMed] [Google Scholar]
  39. Southwick F. S., Young C. L. The actin released from profilin--actin complexes is insufficient to account for the increase in F-actin in chemoattractant-stimulated polymorphonuclear leukocytes. J Cell Biol. 1990 Jun;110(6):1965–1973. doi: 10.1083/jcb.110.6.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Söllner T., Bennett M. K., Whiteheart S. W., Scheller R. H., Rothman J. E. A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. Cell. 1993 Nov 5;75(3):409–418. doi: 10.1016/0092-8674(93)90376-2. [DOI] [PubMed] [Google Scholar]
  41. Trifaró J. M., Rodríguez del Castillo A., Vitale M. L. Dynamic changes in chromaffin cell cytoskeleton as prelude to exocytosis. Mol Neurobiol. 1992 Winter;6(4):339–358. doi: 10.1007/BF02757940. [DOI] [PubMed] [Google Scholar]
  42. Vitale M. L., Rodríguez Del Castillo A., Tchakarov L., Trifaró J. M. Cortical filamentous actin disassembly and scinderin redistribution during chromaffin cell stimulation precede exocytosis, a phenomenon not exhibited by gelsolin. J Cell Biol. 1991 Jun;113(5):1057–1067. doi: 10.1083/jcb.113.5.1057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Walent J. H., Porter B. W., Martin T. F. A novel 145 kd brain cytosolic protein reconstitutes Ca(2+)-regulated secretion in permeable neuroendocrine cells. Cell. 1992 Sep 4;70(5):765–775. doi: 10.1016/0092-8674(92)90310-9. [DOI] [PubMed] [Google Scholar]
  44. Way M., Gooch J., Pope B., Weeds A. G. Expression of human plasma gelsolin in Escherichia coli and dissection of actin binding sites by segmental deletion mutagenesis. J Cell Biol. 1989 Aug;109(2):593–605. doi: 10.1083/jcb.109.2.593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Weber A., Nachmias V. T., Pennise C. R., Pring M., Safer D. Interaction of thymosin beta 4 with muscle and platelet actin: implications for actin sequestration in resting platelets. Biochemistry. 1992 Jul 14;31(27):6179–6185. doi: 10.1021/bi00142a002. [DOI] [PubMed] [Google Scholar]
  46. Whitters E. A., Cleves A. E., McGee T. P., Skinner H. B., Bankaitis V. A. SAC1p is an integral membrane protein that influences the cellular requirement for phospholipid transfer protein function and inositol in yeast. J Cell Biol. 1993 Jul;122(1):79–94. doi: 10.1083/jcb.122.1.79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Williams J. A., Blevins G. T., Jr Cholecystokinin and regulation of pancreatic acinar cell function. Physiol Rev. 1993 Oct;73(4):701–723. doi: 10.1152/physrev.1993.73.4.701. [DOI] [PubMed] [Google Scholar]
  48. Yin H. L. Gelsolin: calcium- and polyphosphoinositide-regulated actin-modulating protein. Bioessays. 1987 Oct;7(4):176–179. doi: 10.1002/bies.950070409. [DOI] [PubMed] [Google Scholar]
  49. Yu F. X., Johnston P. A., Südhof T. C., Yin H. L. gCap39, a calcium ion- and polyphosphoinositide-regulated actin capping protein. Science. 1990 Dec 7;250(4986):1413–1415. doi: 10.1126/science.2255912. [DOI] [PubMed] [Google Scholar]
  50. Yu F. X., Lin S. C., Morrison-Bogorad M., Atkinson M. A., Yin H. L. Thymosin beta 10 and thymosin beta 4 are both actin monomer sequestering proteins. J Biol Chem. 1993 Jan 5;268(1):502–509. [PubMed] [Google Scholar]
  51. Yu F. X., Lin S. C., Morrison-Bogorad M., Yin H. L. Effects of thymosin beta 4 and thymosin beta 10 on actin structures in living cells. Cell Motil Cytoskeleton. 1994;27(1):13–25. doi: 10.1002/cm.970270103. [DOI] [PubMed] [Google Scholar]
  52. Yu F. X., Sun H. Q., Janmey P. A., Yin H. L. Identification of a polyphosphoinositide-binding sequence in an actin monomer-binding domain of gelsolin. J Biol Chem. 1992 Jul 25;267(21):14616–14621. [PubMed] [Google Scholar]
  53. Zarbock J., Oschkinat H., Hannappel E., Kalbacher H., Voelter W., Holak T. A. Solution conformation of thymosin beta 4: a nuclear magnetic resonance and simulated annealing study. Biochemistry. 1990 Aug 28;29(34):7814–7821. doi: 10.1021/bi00486a006. [DOI] [PubMed] [Google Scholar]
  54. Zhang B. X., Zhao H., Muallem S. Ca(2+)-dependent kinase and phosphatase control inositol 1,4,5-trisphosphate-mediated Ca2+ release. Modification by agonist stimulation. J Biol Chem. 1993 May 25;268(15):10997–11001. [PubMed] [Google Scholar]

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