Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1992 Apr 2;117(2):357–367. doi: 10.1083/jcb.117.2.357

Astrocyte process growth induction by actin breakdown

PMCID: PMC2289411  PMID: 1313815

Abstract

cAMP analogues such as dibutyryl cAMP (dBcAMP) have been shown to induce the formation of processes in cultured primary astrocytes. We observe that the processes form by elongation as well as the previously reported retraction of cytoplasm around cytoskeletal elements. The most prominent cytoskeletal change that occurs in response to dBcAMP is a rearrangement of actin filaments characterized by a loss of cortical F- actin staining and the appearance of actin filament staining at the tips of the processes. If cortical actin filaments are disrupted with dihydrocytochalasin B, processes form that are similar to those induced by dBcAMP suggesting that the disruption of the cortical actin network is the pivotal step in process formation. Reorganization of the actin filament network in response to cAMP is accompanied by a decrease in phosphate incorporation into the regulatory light chain of myosin (MLC). Two selective inhibitors of MLC kinase (MLCK), ML-9 and KT5926, as well as a calmodulin antagonist (W7), which would also inhibit MLCK activation, all induce astrocytic process growth implicating MLCK as a control point in process initiation. We also found that dBcAMP and ML-9 both cause a decrease in the phosphate content of actin depolymerizing factor, suggesting that this protein and myosin light chain are the effectors of actin cytoskeleton reorganization and process growth.

Full Text

The Full Text of this article is available as a PDF (1.3 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bamburg J. R., Bray D. Distribution and cellular localization of actin depolymerizing factor. J Cell Biol. 1987 Dec;105(6 Pt 1):2817–2825. doi: 10.1083/jcb.105.6.2817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bamburg J. R., Minamide L. S., Morgan T. E., Hayden S. M., Giuliano K. A., Koffer A. Purification and characterization of low-molecular-weight actin-depolymerizing proteins from brain and cultured cells. Methods Enzymol. 1991;196:125–140. doi: 10.1016/0076-6879(91)96014-i. [DOI] [PubMed] [Google Scholar]
  3. Bray D., Heath J., Moss D. The membrane-associated 'cortex' of animal cells: its structure and mechanical properties. J Cell Sci Suppl. 1986;4:71–88. doi: 10.1242/jcs.1986.supplement_4.5. [DOI] [PubMed] [Google Scholar]
  4. Burmeister D. W., Rivas R. J., Goldberg D. J. Substrate-bound factors stimulate engorgement of growth cone lamellipodia during neurite elongation. Cell Motil Cytoskeleton. 1991;19(4):255–268. doi: 10.1002/cm.970190404. [DOI] [PubMed] [Google Scholar]
  5. Conti M. A., Adelstein R. S. Phosphorylation by cyclic adenosine 3':5'-monophosphate-dependent protein kinase regulates myosin light chain kinase. Fed Proc. 1980 Apr;39(5):1569–1573. [PubMed] [Google Scholar]
  6. Cornell-Bell A. H., Finkbeiner S. M., Cooper M. S., Smith S. J. Glutamate induces calcium waves in cultured astrocytes: long-range glial signaling. Science. 1990 Jan 26;247(4941):470–473. doi: 10.1126/science.1967852. [DOI] [PubMed] [Google Scholar]
  7. Croop J., Holtzer H. Response of myogenic and fibrogenic cells to cytochalasin B and to colcemid. I. Light microscope observations. J Cell Biol. 1975 May;65(2):271–285. doi: 10.1083/jcb.65.2.271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Forscher P., Smith S. J. Actions of cytochalasins on the organization of actin filaments and microtubules in a neuronal growth cone. J Cell Biol. 1988 Oct;107(4):1505–1516. doi: 10.1083/jcb.107.4.1505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Giuliano K. A., Khatib F. A., Hayden S. M., Daoud E. W., Adams M. E., Amorese D. A., Bernstein B. W., Bamburg J. R. Properties of purified actin depolymerizing factor from chick brain. Biochemistry. 1988 Dec 13;27(25):8931–8938. doi: 10.1021/bi00425a009. [DOI] [PubMed] [Google Scholar]
  10. Goldberg D. J., Burmeister D. W. Looking into growth cones. Trends Neurosci. 1989 Dec;12(12):503–506. doi: 10.1016/0166-2236(89)90110-0. [DOI] [PubMed] [Google Scholar]
  11. Goldman J. E., Abramson B. Cyclic AMP-induced shape changes of astrocytes are accompanied by rapid depolymerization of actin. Brain Res. 1990 Oct 1;528(2):189–196. doi: 10.1016/0006-8993(90)91657-3. [DOI] [PubMed] [Google Scholar]
  12. Goldman J. E., Chiu F. C. Dibutyryl cyclic AMP causes intermediate filament accumulation and actin reorganization in astrocytes. Brain Res. 1984 Jul 23;306(1-2):85–95. doi: 10.1016/0006-8993(84)90358-5. [DOI] [PubMed] [Google Scholar]
  13. Harris A. K., Stopak D., Wild P. Fibroblast traction as a mechanism for collagen morphogenesis. Nature. 1981 Mar 19;290(5803):249–251. doi: 10.1038/290249a0. [DOI] [PubMed] [Google Scholar]
  14. Harris A. K., Wild P., Stopak D. Silicone rubber substrata: a new wrinkle in the study of cell locomotion. Science. 1980 Apr 11;208(4440):177–179. doi: 10.1126/science.6987736. [DOI] [PubMed] [Google Scholar]
  15. Hatten M. E. Neuronal regulation of astroglial morphology and proliferation in vitro. J Cell Biol. 1985 Feb;100(2):384–396. doi: 10.1083/jcb.100.2.384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kimelberg H. K., Norenberg M. D. Astrocytes. Sci Am. 1989 Apr;260(4):66-72, 74, 76. doi: 10.1038/scientificamerican0489-66. [DOI] [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Lamb N. J., Fernandez A., Conti M. A., Adelstein R., Glass D. B., Welch W. J., Feramisco J. R. Regulation of actin microfilament integrity in living nonmuscle cells by the cAMP-dependent protein kinase and the myosin light chain kinase. J Cell Biol. 1988 Jun;106(6):1955–1971. doi: 10.1083/jcb.106.6.1955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lim R., Mitsunobu K., Li W. K. Maturation-stimulating effect of brain extract and dibutyryl cyclic AMP on dissociated embryonic brain cells in culture. Exp Eye Res. 1973 Apr;79(1):243–246. [PubMed] [Google Scholar]
  20. Luby-Phelps K., Castle P. E., Taylor D. L., Lanni F. Hindered diffusion of inert tracer particles in the cytoplasm of mouse 3T3 cells. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4910–4913. doi: 10.1073/pnas.84.14.4910. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Luby-Phelps K., Taylor D. L., Lanni F. Probing the structure of cytoplasm. J Cell Biol. 1986 Jun;102(6):2015–2022. doi: 10.1083/jcb.102.6.2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Luby-Phelps K., Taylor D. L. Subcellular compartmentalization by local differentiation of cytoplasmic structure. Cell Motil Cytoskeleton. 1988;10(1-2):28–37. doi: 10.1002/cm.970100107. [DOI] [PubMed] [Google Scholar]
  23. Ludowyke R. I., Peleg I., Beaven M. A., Adelstein R. S. Antigen-induced secretion of histamine and the phosphorylation of myosin by protein kinase C in rat basophilic leukemia cells. J Biol Chem. 1989 Jul 25;264(21):12492–12501. [PubMed] [Google Scholar]
  24. Madreperla S. A., Adler R. Opposing microtubule- and actin-dependent forces in the development and maintenance of structural polarity in retinal photoreceptors. Dev Biol. 1989 Jan;131(1):149–160. doi: 10.1016/s0012-1606(89)80046-6. [DOI] [PubMed] [Google Scholar]
  25. Mason C. A., Edmondson J. C., Hatten M. E. The extending astroglial process: development of glial cell shape, the growing tip, and interactions with neurons. J Neurosci. 1988 Sep;8(9):3124–3134. doi: 10.1523/JNEUROSCI.08-09-03124.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. McCarthy K. D., Prime J., Harmon T., Pollenz R. Receptor-mediated phosphorylation of astroglial intermediate filament proteins in cultured astroglia. J Neurochem. 1985 Mar;44(3):723–730. doi: 10.1111/j.1471-4159.1985.tb12875.x. [DOI] [PubMed] [Google Scholar]
  27. McCarthy K. D., de Vellis J. Alpah-adrenergic receptor modulation of beta-adrenergic, adenosine and prostaglandin E1 increased adenosine 3':5'-cyclic monophosphate levels in primary cultures of glia. J Cyclic Nucleotide Res. 1978 Feb;4(1):15–26. [PubMed] [Google Scholar]
  28. Menko A. S., Toyama Y., Boettiger D., Holtzer H. Altered cell spreading in cytochalasin B: a possible role for intermediate filaments. Mol Cell Biol. 1983 Jan;3(1):113–125. doi: 10.1128/mcb.3.1.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Moriyama K., Nishida E., Yonezawa N., Sakai H., Matsumoto S., Iida K., Yahara I. Destrin, a mammalian actin-depolymerizing protein, is closely related to cofilin. Cloning and expression of porcine brain destrin cDNA. J Biol Chem. 1990 Apr 5;265(10):5768–5773. [PubMed] [Google Scholar]
  30. Mrwa U., Hartshorne D. J. Phosphorylation of smooth muscle myosin and myosin light chains. Fed Proc. 1980 Apr;39(5):1564–1568. [PubMed] [Google Scholar]
  31. Murphy S., Pearce B. Functional receptors for neurotransmitters on astroglial cells. Neuroscience. 1987 Aug;22(2):381–394. doi: 10.1016/0306-4522(87)90342-3. [DOI] [PubMed] [Google Scholar]
  32. Nakanishi S., Yamada K., Iwahashi K., Kuroda K., Kase H. KT5926, a potent and selective inhibitor of myosin light chain kinase. Mol Pharmacol. 1990 Apr;37(4):482–488. [PubMed] [Google Scholar]
  33. Nishida E., Maekawa S., Muneyuki E., Sakai H. Action of a 19K protein from porcine brain on actin polymerization: a new functional class of actin-binding proteins. J Biochem. 1984 Feb;95(2):387–398. doi: 10.1093/oxfordjournals.jbchem.a134619. [DOI] [PubMed] [Google Scholar]
  34. Nishikawa M., Hidaka H., Adelstein R. S. Phosphorylation of smooth muscle heavy meromyosin by calcium-activated, phospholipid-dependent protein kinase. The effect on actin-activated MgATPase activity. J Biol Chem. 1983 Dec 10;258(23):14069–14072. [PubMed] [Google Scholar]
  35. Pollenz R. S., McCarthy K. D. Analysis of cyclic AMP-dependent changes in intermediate filament protein phosphorylation and cell morphology in cultured astroglia. J Neurochem. 1986 Jul;47(1):9–17. doi: 10.1111/j.1471-4159.1986.tb02824.x. [DOI] [PubMed] [Google Scholar]
  36. Saitoh M., Naka M., Hidaka H. The modulatory role of myosin light chain phosphorylation in human platelet activation. Biochem Biophys Res Commun. 1986 Oct 15;140(1):280–287. doi: 10.1016/0006-291x(86)91087-9. [DOI] [PubMed] [Google Scholar]
  37. Shain W., Forman D. S., Madelian V., Turner J. N. Morphology of astroglial cells is controlled by beta-adrenergic receptors. J Cell Biol. 1987 Nov;105(5):2307–2314. doi: 10.1083/jcb.105.5.2307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sherry J. M., Górecka A., Aksoy M. O., Dabrowska R., Hartshorne D. J. Roles of calcium and phosphorylation in the regulation of the activity of gizzard myosin. Biochemistry. 1978 Oct 17;17(21):4411–4418. doi: 10.1021/bi00614a009. [DOI] [PubMed] [Google Scholar]
  39. Stossel T. P. Contribution of actin to the structure of the cytoplasmic matrix. J Cell Biol. 1984 Jul;99(1 Pt 2):15s–21s. doi: 10.1083/jcb.99.1.15s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Trimmer P. A., Reier P. J., Oh T. H., Eng L. F. An ultrastructural and immunocytochemical study of astrocytic differentiation in vitro: changes in the composition and distribution of the cellular cytoskeleton. J Neuroimmunol. 1982 Jun;2(3-4):235–260. doi: 10.1016/0165-5728(82)90058-3. [DOI] [PubMed] [Google Scholar]
  42. Walz W. Role of glial cells in the regulation of the brain ion microenvironment. Prog Neurobiol. 1989;33(4):309–333. doi: 10.1016/0301-0082(89)90005-1. [DOI] [PubMed] [Google Scholar]
  43. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES