Abstract
Low concentrations of calcium and magnesium ions have been shown to influence microtubule assembly in vitro. To test whether these cations also have an effect on microtubules in vivo, specimens of Actinosphaerium eichhorni were exposed to different concentrations of Ca++ and Mg++ and the divalent cation ionophore A23187. Experimental degradation and reformation of axopodia were studied by light and electron microscopy. In the presence of Ca++ and the ionophore axopodia gradually shorten, the rate of shortening depending on the concentrations of Ca++ and the ionophore used. Retraction of axopodia was observed with a concentration of Ca++ as low as 0.01 mM. After transfer to a Ca++-free solution containing EGTA, axopodia re-extend; the initial length is reached after about 2 h. Likewise, reformation of axopodia of cold-treated organisms is observed only in solutions of EGTA or Mg++, whereas it is completely inhibited in a Ca++ solution. Electron microscope studies demonstrate degradation of the axonemal microtubular array in organisms treated with Ca++ and A23187. No alteration was observed in organisms treated with Mg++ or EGTA plus ionophore. The results suggest that, in the presence of the ionophore, formation of axonemal microtubules can be regulated by varying the Ca++ concentration in the medium. Since A23187 tends to equilibrate the concentrations of divalent cations between external medium and cell interior, it is likely that microtubule formation invivo is influenced by micromolar concentrations of Ca++. These concentrations are low enough to be of physiological significance for a role in the regulation of microtubule assembly in vivo.
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- Baker P. F., Hodgkin A. L., Ridgway E. B. Depolarization and calcium entry in squid giant axons. J Physiol. 1971 Nov;218(3):709–755. doi: 10.1113/jphysiol.1971.sp009641. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Borisy G. G., Marcum J. M., Olmsted J. B., Murphy D. B., Johnson K. A. Purification of tubulin and associated high molecular weight proteins from porcine brain and characterization of microtubule assembly in vitro. Ann N Y Acad Sci. 1975 Jun 30;253:107–132. doi: 10.1111/j.1749-6632.1975.tb19196.x. [DOI] [PubMed] [Google Scholar]
- Case G. D., Vanderkooi J. M., Scarpa A. Physical properties of biological membranes determined by the fluorescence of the calcium ionophore A23187. Arch Biochem Biophys. 1974 May;162(1):174–185. doi: 10.1016/0003-9861(74)90116-7. [DOI] [PubMed] [Google Scholar]
- Diamond I., Goldberg A. L. Uptake and release of 45Ca by brain microsomes, synaptosomes and synaptic vesicles. J Neurochem. 1971 Aug;18(8):1419–1431. doi: 10.1111/j.1471-4159.1971.tb00005.x. [DOI] [PubMed] [Google Scholar]
- Ebashi S., Endo M., Otsuki I. Control of muscle contraction. Q Rev Biophys. 1969 Nov;2(4):351–384. doi: 10.1017/s0033583500001190. [DOI] [PubMed] [Google Scholar]
- Edds K. T. Motility in Echinosphaerium nucleofilum. II. Cytoplasmic contractility and its molecular basis. J Cell Biol. 1975 Jul;66(1):156–164. doi: 10.1083/jcb.66.1.156. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Foreman J. C., Mongar J. L., Gomperts B. D. Calcium ionophores and movement of calcium ions following the physiological stimulus to a secretory process. Nature. 1973 Oct 5;245(5423):249–251. doi: 10.1038/245249a0. [DOI] [PubMed] [Google Scholar]
- Gallin J. I., Rosenthal A. S. The regulatory role of divalent cations in human granulocyte chemotaxis. Evidence for an association between calcium exchanges and microtubule assembly. J Cell Biol. 1974 Sep;62(3):594–609. doi: 10.1083/jcb.62.3.594. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gambetti P., Erulkar S. E., Somlyo A. P., Gonatas N. K. Calcium-containing structures in vertebrate glial cells. Ultrastructural and microprobe analysis. J Cell Biol. 1975 Feb;64(2):322–330. doi: 10.1083/jcb.64.2.322. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gaskin F., Cantor C. R., Shelanski M. L. Biochemical studies on the in vitro assembly and disassembly of microtubules. Ann N Y Acad Sci. 1975 Jun 30;253:133–146. doi: 10.1111/j.1749-6632.1975.tb19197.x. [DOI] [PubMed] [Google Scholar]
- Haga T., Abe T., Kurokawa M. Polymerization and depolymerization of microtubules in vitro as studied by flow birefringence. FEBS Lett. 1974 Mar 1;39(3):291–295. doi: 10.1016/0014-5793(74)80133-x. [DOI] [PubMed] [Google Scholar]
- Hales C. N., Luzio J. P., Chandler J. A., Herman L. Localization of calcium in the smooth endoplasmic reticulum of rat isolated fat cells. J Cell Sci. 1974 Jun;15(1):1–15. doi: 10.1242/jcs.15.1.1. [DOI] [PubMed] [Google Scholar]
- Heumann H. G. Calciumakkumulierende Strukturen in einem glatten Wirbellosenmuskel. Protoplasma. 1969;67(1):111–115. doi: 10.1007/BF01256771. [DOI] [PubMed] [Google Scholar]
- Inoué S., Borisy G. G., Kiehart D. P. Growth and lability of Chaetopterus oocyte mitotic spindles isolated in the presence of porcine brain tubulin. J Cell Biol. 1974 Jul;62(1):175–184. doi: 10.1083/jcb.62.1.175. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kagayama M., Douglas W. W. Electron microscope evidence of calcium-induced exocytosis in mast cells treated with 48-80 or the ionophores A-23187 and X-537A. J Cell Biol. 1974 Aug;62(2):519–526. doi: 10.1083/jcb.62.2.519. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kinoshita S., Yazaki I. The behaviour and localization of intracellular relaxing system during cleavage in the sea urchin egg. Exp Cell Res. 1967 Sep;47(3):449–458. doi: 10.1016/0014-4827(67)90003-1. [DOI] [PubMed] [Google Scholar]
- Lee Y. C., Samson F. E., Jr, Houston L. L., Himes R. H. The in vitro polymerization of tubulin from beef brain. J Neurobiol. 1974;5(4):317–330. doi: 10.1002/neu.480050404. [DOI] [PubMed] [Google Scholar]
- Levy J. V., Cohen J. A., Inesi G. Contractile effects of a calcium ionophore. Nature. 1973 Apr 13;242(5398):461–463. doi: 10.1038/242461a0. [DOI] [PubMed] [Google Scholar]
- Ockleford C. D., Tucker J. B. Growth, breakdown, repair, and rapid contraction of microtubular axopodia in the heliozoan Actinophrys sol. J Ultrastruct Res. 1973 Sep;44(5):369–387. doi: 10.1016/s0022-5320(73)90005-1. [DOI] [PubMed] [Google Scholar]
- Olmsted J. B., Borisy G. G. Ionic and nucleotide requirements for microtubule polymerization in vitro. Biochemistry. 1975 Jul;14(13):2996–3005. doi: 10.1021/bi00684a032. [DOI] [PubMed] [Google Scholar]
- Petzelt C. Ca 2+ -activated APTase during the cell cycle of the sea urchin Strongylocentrotus purpuratus. Exp Cell Res. 1972 Feb;70(2):333–339. doi: 10.1016/0014-4827(72)90144-9. [DOI] [PubMed] [Google Scholar]
- Petzelt C., von Ledebur-Villiger M. Ca2+-stimulated ATPase during the early development of parthenogenetically activated eggs of the sea urchin Paracentrotus lividus. Exp Cell Res. 1973 Sep;81(1):87–94. doi: 10.1016/0014-4827(73)90114-6. [DOI] [PubMed] [Google Scholar]
- Pressman B. C. Properties of ionophores with broad range cation selectivity. Fed Proc. 1973 Jun;32(6):1698–1703. [PubMed] [Google Scholar]
- REYNOLDS E. S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol. 1963 Apr;17:208–212. doi: 10.1083/jcb.17.1.208. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rebhun L. I., Jemiolo D., Ivy N., Mellon M., Nath J. Regulation of the in vivo mitotic apparatus by glycols and metabolic inhibitors. Ann N Y Acad Sci. 1975 Jun 30;253:362–377. doi: 10.1111/j.1749-6632.1975.tb19214.x. [DOI] [PubMed] [Google Scholar]
- Rebhun L. I., Rosenbaum J., Lefebvre P., Smith G. Reversible restoration of the birefringence of cold-treated, isolated mitotic apparatus of surf clam eggs with chick brain tubulin. Nature. 1974 May 10;249(453):113–115. doi: 10.1038/249113a0. [DOI] [PubMed] [Google Scholar]
- Reed P. W., Lardy H. A. A23187: a divalent cation ionophore. J Biol Chem. 1972 Nov 10;247(21):6970–6977. [PubMed] [Google Scholar]
- Roth L. E., Pihlaja D. J., Shigenaka Y. Microtubules in the heliozoan axopodium. I. The gradion hypothesis of allosterism in structural proteins. J Ultrastruct Res. 1970 Jan;30(1):7–37. doi: 10.1016/s0022-5320(70)90062-6. [DOI] [PubMed] [Google Scholar]
- Roth L. E., Shigenaka Y. Microtubules in the heliozoan axopodium. II. Rapid degradation by cupric and nickelous ions. J Ultrastruct Res. 1970 May;31(3):356–374. doi: 10.1016/s0022-5320(70)90138-3. [DOI] [PubMed] [Google Scholar]
- Schatzmann H. J. Dependence on calcium concentration and stoichiometry of the calcium pump in human red cells. J Physiol. 1973 Dec;235(2):551–569. doi: 10.1113/jphysiol.1973.sp010403. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schlaepfer W. W., Bunge R. P. Effects of calcium ion concentration on the degeneration of amputated axons in tissue culture. J Cell Biol. 1973 Nov;59(2 Pt 1):456–470. doi: 10.1083/jcb.59.2.456. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schliwa M. Cytoarchitecture of surface layer cells of the teleost epidermis. J Ultrastruct Res. 1975 Sep;52(3):377–386. doi: 10.1016/s0022-5320(75)80076-1. [DOI] [PubMed] [Google Scholar]
- Schroeder T. E., Strickland D. L. Ionophore A23187, calcium and contractility in frog eggs. Exp Cell Res. 1974 Jan;83(1):139–142. doi: 10.1016/0014-4827(74)90696-x. [DOI] [PubMed] [Google Scholar]
- Shelanski M. L., Gaskin F., Cantor C. R. Microtubule assembly in the absence of added nucleotides. Proc Natl Acad Sci U S A. 1973 Mar;70(3):765–768. doi: 10.1073/pnas.70.3.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shigenaka Y., Roth L. E., Pihlaja D. J. Microtubules in the heliozoan axopodium. 3. Degradation and reformation after dilute urea treatment. J Cell Sci. 1971 Jan;8(1):127–151. doi: 10.1242/jcs.8.1.127. [DOI] [PubMed] [Google Scholar]
- Steinhardt R. A., Epel D. Activation of sea-urchin eggs by a calcium ionophore. Proc Natl Acad Sci U S A. 1974 May;71(5):1915–1919. doi: 10.1073/pnas.71.5.1915. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tilney L. G., Byers B. Studies on the microtubules in heliozoa. V. Factors controlling the organization of microtubules in the Axonemal pattern in Echinosphaerium (Actinosphaerium) nucleofilum. J Cell Biol. 1969 Oct;43(1):148–165. doi: 10.1083/jcb.43.1.148. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tilney L. G., Porter K. R. Studies on microtubules in Heliozoa. I. The fine structure of Actinosphaerium nucleofilum (Barrett), with particular reference to the axial rod structure. Protoplasma. 1965;60(4):317–344. doi: 10.1007/BF01247886. [DOI] [PubMed] [Google Scholar]
- Tilney L. G., Porter K. R. Studies on the microtubules in heliozoa. II. The effect of low temperature on these structures in the formation and maintenance of the axopodia. J Cell Biol. 1967 Jul;34(1):327–343. doi: 10.1083/jcb.34.1.327. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wong D. T., Wilkinson J. R., Hamill R. L., Horng J. S. Effects of antibiotic ionophore, A23187, on oxidative phosphorylation and calcium transport of liver mitochondria. Arch Biochem Biophys. 1973 Jun;156(2):578–585. doi: 10.1016/0003-9861(73)90308-1. [DOI] [PubMed] [Google Scholar]