Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Nov 1;109(5):2379–2394. doi: 10.1083/jcb.109.5.2379

A squid dynein isoform promotes axoplasmic vesicle translocation

PMCID: PMC2115859  PMID: 2478567

Abstract

Axoplasmic vesicles that translocate on isolated microtubules in an ATP- dependent manner have an associated ATP-binding polypeptide with a previously estimated relative molecular mass of 292 kD (Gilbert, S. P., and R. D. Sloboda. 1986. J. Cell Biol. 103:947-956). Here, data are presented showing that this polypeptide (designated H1) and another high molecular mass polypeptide (H2) can be isolated in association with axoplasmic vesicles or optic lobe microtubules. The H1 and H2 polypeptides dissociate from microtubules in the presence of MgATP and can be further purified by gel filtration chromatography. The peak fraction thus obtained demonstrates MgATPase activity and promotes the translocation of salt-extracted vesicles (mean = 0.87 microns/s) and latex beads (mean = 0.92 microns/s) along isolated microtubules. The H1 polypeptide binds [alpha 32P]8-azidoATP and is thermosoluble, but the H2 polypeptide does not share these characteristics. In immunofluorescence experiments with dissociated squid axoplasm, affinity-purified H1 antibodies yield a punctate pattern that corresponds to vesicle-like particles, and these antibodies inhibit the bidirectional movement of axoplasmic vesicles. H2 is cleaved by UV irradiation in the presence of MgATP and vanadate to yield vanadate- induced peptides of 240 and 195 kD, yet H1 does not cleave under identical conditions. These experiments also demonstrate that the actual relative molecular mass of the H1 and H2 polypeptides is approximately 435 kD. On sucrose density gradients, H1 and H2 sediment at 19-20 S, and negatively stained samples reveal particles comprised of two globular heads with stems that contact each other and extend to a common base. The results demonstrate that the complex purified is a vesicle-associated ATPase whose characteristics indicate that it is a squid isoform of dynein. Furthermore, the data suggest that this vesicle-associated dynein promotes membranous organelle motility during fast axoplasmic transport.

Full Text

The Full Text of this article is available as a PDF (4.4 MB).

Selected References

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

  1. Allen R. D., Allen N. S. Video-enhanced microscopy with a computer frame memory. J Microsc. 1983 Jan;129(Pt 1):3–17. doi: 10.1111/j.1365-2818.1983.tb04157.x. [DOI] [PubMed] [Google Scholar]
  2. Allen R. D., Weiss D. G., Hayden J. H., Brown D. T., Fujiwake H., Simpson M. Gliding movement of and bidirectional transport along single native microtubules from squid axoplasm: evidence for an active role of microtubules in cytoplasmic transport. J Cell Biol. 1985 May;100(5):1736–1752. doi: 10.1083/jcb.100.5.1736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bell C. W., Fraser C., Sale W. S., Tang W. J., Gibbons I. R. Preparation and purification of dynein. Methods Cell Biol. 1982;24:373–397. doi: 10.1016/s0091-679x(08)60666-4. [DOI] [PubMed] [Google Scholar]
  4. Blake M. S., Johnston K. H., Russell-Jones G. J., Gotschlich E. C. A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem. 1984 Jan;136(1):175–179. doi: 10.1016/0003-2697(84)90320-8. [DOI] [PubMed] [Google Scholar]
  5. Borisy G. G., Olmsted J. B., Marcum J. M., Allen C. Microtubule assembly in vitro. Fed Proc. 1974 Feb;33(2):167–174. [PubMed] [Google Scholar]
  6. 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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  7. Brady S. T. A novel brain ATPase with properties expected for the fast axonal transport motor. Nature. 1985 Sep 5;317(6032):73–75. doi: 10.1038/317073a0. [DOI] [PubMed] [Google Scholar]
  8. Burton P. R., Paige J. L. Polarity of axoplasmic microtubules in the olfactory nerve of the frog. Proc Natl Acad Sci U S A. 1981 May;78(5):3269–3273. doi: 10.1073/pnas.78.5.3269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cohen A. I. An ultrastructural analysis of the photoreceptors of the squid and their synaptic connections. 3. Photoreceptor terminations in the optic lobes. J Comp Neurol. 1973 Feb 1;147(3):399–426. doi: 10.1002/cne.901470306. [DOI] [PubMed] [Google Scholar]
  10. Dentler W. L., Granett S., Rosenbaum J. L. Ultrastructural localization of the high molecular weight proteins associated with in vitro-assembled brain microtubules. J Cell Biol. 1975 Apr;65(1):237–241. doi: 10.1083/jcb.65.1.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Do C. V., Sears E. B., Gilbert S. P., Sloboda R. D. Vesikin, a vesicle associated ATPase from squid axoplasm and optic lobe, has characteristics in common with vertebrate brain MAP 1 and MAP 2. Cell Motil Cytoskeleton. 1988;10(1-2):246–254. doi: 10.1002/cm.970100129. [DOI] [PubMed] [Google Scholar]
  12. Euteneuer U., Koonce M. P., Pfister K. K., Schliwa M. An ATPase with properties expected for the organelle motor of the giant amoeba, Reticulomyxa. Nature. 1988 Mar 10;332(6160):176–178. doi: 10.1038/332176a0. [DOI] [PubMed] [Google Scholar]
  13. Evans J. A., Gibbons I. R. Activation of dynein 1 adenosine triphosphatase by organic solvents and by Triton X-100. J Biol Chem. 1986 Oct 25;261(30):14044–14048. [PubMed] [Google Scholar]
  14. Fairbanks G., Steck T. L., Wallach D. F. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971 Jun 22;10(13):2606–2617. doi: 10.1021/bi00789a030. [DOI] [PubMed] [Google Scholar]
  15. Fellous A., Francon J., Lennon A. M., Nunez J. Microtubule assembly in vitro. Purification of assembly-promoting factors. Eur J Biochem. 1977 Aug 15;78(1):167–174. doi: 10.1111/j.1432-1033.1977.tb11726.x. [DOI] [PubMed] [Google Scholar]
  16. Foltz K. R., Asai D. J. Ionic strength-dependent isoforms of sea urchin egg dynein. J Biol Chem. 1988 Feb 25;263(6):2878–2883. [PubMed] [Google Scholar]
  17. Gibbons I. R., Fronk E. A latent adenosine triphosphatase form of dynein 1 from sea urchin sperm flagella. J Biol Chem. 1979 Jan 10;254(1):187–196. [PubMed] [Google Scholar]
  18. Gibbons I. R., Lee-Eiford A., Mocz G., Phillipson C. A., Tang W. J., Gibbons B. H. Photosensitized cleavage of dynein heavy chains. Cleavage at the "V1 site" by irradiation at 365 nm in the presence of ATP and vanadate. J Biol Chem. 1987 Feb 25;262(6):2780–2786. [PubMed] [Google Scholar]
  19. Gilbert S. P., Allen R. D., Sloboda R. D. Translocation of vesicles from squid axoplasm on flagellar microtubules. Nature. 1985 May 16;315(6016):245–248. doi: 10.1038/315245a0. [DOI] [PubMed] [Google Scholar]
  20. Gilbert S. P., Sloboda R. D. Bidirectional transport of fluorescently labeled vesicles introduced into extruded axoplasm of squid Loligo pealei. J Cell Biol. 1984 Aug;99(2):445–452. doi: 10.1083/jcb.99.2.445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gilbert S. P., Sloboda R. D. Identification of a MAP 2-like ATP-binding protein associated with axoplasmic vesicles that translocate on isolated microtubules. J Cell Biol. 1986 Sep;103(3):947–956. doi: 10.1083/jcb.103.3.947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Goodenough U., Heuser J. Structural comparison of purified dynein proteins with in situ dynein arms. J Mol Biol. 1984 Dec 25;180(4):1083–1118. doi: 10.1016/0022-2836(84)90272-9. [DOI] [PubMed] [Google Scholar]
  23. Grafstein B., Forman D. S. Intracellular transport in neurons. Physiol Rev. 1980 Oct;60(4):1167–1283. doi: 10.1152/physrev.1980.60.4.1167. [DOI] [PubMed] [Google Scholar]
  24. Haghighat N., Cohen R. S., Pappas G. D. Fine structure of squid (Loligo pealei) optic lobe synapses. Neuroscience. 1984 Oct;13(2):527–546. doi: 10.1016/0306-4522(84)90246-x. [DOI] [PubMed] [Google Scholar]
  25. Hastie A. T., Marchese-Ragona S. P., Johnson K. A., Wall J. S. Structure and mass of mammalian respiratory ciliary outer arm 19S dynein. Cell Motil Cytoskeleton. 1988;11(3):157–166. doi: 10.1002/cm.970110303. [DOI] [PubMed] [Google Scholar]
  26. Heidemann S. R., Landers J. M., Hamborg M. A. Polarity orientation of axonal microtubules. J Cell Biol. 1981 Dec;91(3 Pt 1):661–665. doi: 10.1083/jcb.91.3.661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Herzog W., Weber K. Fractionation of brain microtubule-associated proteins. Isolation of two different proteins which stimulate tubulin polymerization in vitro. Eur J Biochem. 1978 Dec 1;92(1):1–8. doi: 10.1111/j.1432-1033.1978.tb12716.x. [DOI] [PubMed] [Google Scholar]
  28. Hirokawa N. 270K microtubule-associated protein cross-reacting with anti-MAP2 IgG in the crayfish peripheral nerve axon. J Cell Biol. 1986 Jul;103(1):33–39. doi: 10.1083/jcb.103.1.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hisanaga S., Hirokawa N. Substructure of sea urchin egg cytoplasmic dynein. J Mol Biol. 1987 Jun 20;195(4):919–927. doi: 10.1016/0022-2836(87)90495-5. [DOI] [PubMed] [Google Scholar]
  30. Hollenbeck P. J., Chapman K. A novel microtubule-associated protein from mammalian nerve shows ATP-sensitive binding to microtubules. J Cell Biol. 1986 Oct;103(4):1539–1545. doi: 10.1083/jcb.103.4.1539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Johnson G. D., Nogueira Araujo G. M. A simple method of reducing the fading of immunofluorescence during microscopy. J Immunol Methods. 1981;43(3):349–350. doi: 10.1016/0022-1759(81)90183-6. [DOI] [PubMed] [Google Scholar]
  32. Johnson K. A. The pathway of ATP hydrolysis by dynein. Kinetics of a presteady state phosphate burst. J Biol Chem. 1983 Nov 25;258(22):13825–13832. [PubMed] [Google Scholar]
  33. Johnson K. A., Wall J. S. Structure and molecular weight of the dynein ATPase. J Cell Biol. 1983 Mar;96(3):669–678. doi: 10.1083/jcb.96.3.669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kim H., Binder L. I., Rosenbaum J. L. The periodic association of MAP2 with brain microtubules in vitro. J Cell Biol. 1979 Feb;80(2):266–276. doi: 10.1083/jcb.80.2.266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. King S. M., Witman G. B. Structure of the alpha and beta heavy chains of the outer arm dynein from Chlamydomonas flagella. Masses of chains and sites of ultraviolet-induced vanadate-dependent cleavage. J Biol Chem. 1987 Dec 25;262(36):17596–17604. [PubMed] [Google Scholar]
  36. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  37. 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]
  38. Lasek R. J., Brady S. T. Attachment of transported vesicles to microtubules in axoplasm is facilitated by AMP-PNP. Nature. 1985 Aug 15;316(6029):645–647. doi: 10.1038/316645a0. [DOI] [PubMed] [Google Scholar]
  39. Lee-Eiford A., Ow R. A., Gibbons I. R. Specific cleavage of dynein heavy chains by ultraviolet irradiation in the presence of ATP and vanadate. J Biol Chem. 1986 Feb 15;261(5):2337–2342. [PubMed] [Google Scholar]
  40. Lye R. J., Porter M. E., Scholey J. M., McIntosh J. R. Identification of a microtubule-based cytoplasmic motor in the nematode C. elegans. Cell. 1987 Oct 23;51(2):309–318. doi: 10.1016/0092-8674(87)90157-7. [DOI] [PubMed] [Google Scholar]
  41. MARTIN R. G., AMES B. N. A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J Biol Chem. 1961 May;236:1372–1379. [PubMed] [Google Scholar]
  42. Marchese-Ragona S. P., Gagnon C., White D., Isles M. B., Johnson K. A. Structure and mass analysis of 12S and 19S dynein obtained from bull sperm flagella. Cell Motil Cytoskeleton. 1987;8(4):368–374. doi: 10.1002/cm.970080409. [DOI] [PubMed] [Google Scholar]
  43. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  44. Murphy D. B., Borisy G. G. Association of high-molecular-weight proteins with microtubules and their role in microtubule assembly in vitro. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2696–2700. doi: 10.1073/pnas.72.7.2696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Neely M. D., Boekelheide K. Sertoli cell processes have axoplasmic features: an ordered microtubule distribution and an abundant high molecular weight microtubule-associated protein (cytoplasmic dynein). J Cell Biol. 1988 Nov;107(5):1767–1776. doi: 10.1083/jcb.107.5.1767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Paschal B. M., Shpetner H. S., Vallee R. B. MAP 1C is a microtubule-activated ATPase which translocates microtubules in vitro and has dynein-like properties. J Cell Biol. 1987 Sep;105(3):1273–1282. doi: 10.1083/jcb.105.3.1273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Paschal B. M., Vallee R. B. Retrograde transport by the microtubule-associated protein MAP 1C. Nature. 1987 Nov 12;330(6144):181–183. doi: 10.1038/330181a0. [DOI] [PubMed] [Google Scholar]
  48. Pfister K. K., Haley B. E., Witman G. B. Labeling of Chlamydomonas 18 S dynein polypeptides by 8-azidoadenosine 5'-triphosphate, a photoaffinity analog of ATP. J Biol Chem. 1985 Oct 15;260(23):12844–12850. [PubMed] [Google Scholar]
  49. Pfister K. K., Witman G. B. Subfractionation of Chlamydomonas 18 S dynein into two unique subunits containing ATPase activity. J Biol Chem. 1984 Oct 10;259(19):12072–12080. [PubMed] [Google Scholar]
  50. Potter R. L., Haley B. E. Photoaffinity labeling of nucleotide binding sites with 8-azidopurine analogs: techniques and applications. Methods Enzymol. 1983;91:613–633. doi: 10.1016/s0076-6879(83)91054-6. [DOI] [PubMed] [Google Scholar]
  51. Pratt M. M., Hisanaga S., Begg D. A. An improved purification method for cytoplasmic dynein. J Cell Biochem. 1984;26(1):19–33. doi: 10.1002/jcb.240260103. [DOI] [PubMed] [Google Scholar]
  52. Pratt M. M. Homology of egg and flagellar dynein. Comparison of ATP-binding sites and primary structure. J Biol Chem. 1986 Jan 15;261(2):956–964. [PubMed] [Google Scholar]
  53. Rebhun L. I. Polarized intracellular particle transport: saltatory movements and cytoplasmic streaming. Int Rev Cytol. 1972;32:93–137. doi: 10.1016/s0074-7696(08)60339-3. [DOI] [PubMed] [Google Scholar]
  54. Sale W. S., Goodenough U. W., Heuser J. E. The substructure of isolated and in situ outer dynein arms of sea urchin sperm flagella. J Cell Biol. 1985 Oct;101(4):1400–1412. doi: 10.1083/jcb.101.4.1400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Schacterle G. R., Pollack R. L. A simplified method for the quantitative assay of small amounts of protein in biologic material. Anal Biochem. 1973 Feb;51(2):654–655. doi: 10.1016/0003-2697(73)90523-x. [DOI] [PubMed] [Google Scholar]
  56. Scherson T., Kreis T. E., Schlessinger J., Littauer U. Z., Borisy G. G., Geiger B. Dynamic interactions of fluorescently labeled microtubule-associated proteins in living cells. J Cell Biol. 1984 Aug;99(2):425–434. doi: 10.1083/jcb.99.2.425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Schliwa M. Mechanisms of intracellular organelle transport. Cell Muscle Motil. 1984;5:1-82,403-6. doi: 10.1007/978-1-4684-4592-3_1. [DOI] [PubMed] [Google Scholar]
  58. Schnapp B. J., Vale R. D., Sheetz M. P., Reese T. S. Single microtubules from squid axoplasm support bidirectional movement of organelles. Cell. 1985 Feb;40(2):455–462. doi: 10.1016/0092-8674(85)90160-6. [DOI] [PubMed] [Google Scholar]
  59. Scholey J. M., Neighbors B., McIntosh J. R., Salmon E. D. Isolation of microtubules and a dynein-like MgATPase from unfertilized sea urchin eggs. J Biol Chem. 1984 May 25;259(10):6516–6525. [PubMed] [Google Scholar]
  60. Scholey J. M., Porter M. E., Grissom P. M., McIntosh J. R. Identification of kinesin in sea urchin eggs, and evidence for its localization in the mitotic spindle. Nature. 1985 Dec 5;318(6045):483–486. doi: 10.1038/318483a0. [DOI] [PubMed] [Google Scholar]
  61. 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]
  62. Sloboda R. D., Rosenbaum J. L. Decoration and stabilization of intact, smooth-walled microtubules with microtubule-associated proteins. Biochemistry. 1979 Jan 9;18(1):48–55. doi: 10.1021/bi00568a008. [DOI] [PubMed] [Google Scholar]
  63. Sloboda R. D., Rudolph S. A., Rosenbaum J. L., Greengard P. Cyclic AMP-dependent endogenous phosphorylation of a microtubule-associated protein. Proc Natl Acad Sci U S A. 1975 Jan;72(1):177–181. doi: 10.1073/pnas.72.1.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Tang W. J., Bell C. W., Sale W. S., Gibbons I. R. Structure of the dynein-1 outer arm in sea urchin sperm flagella. I. Analysis by separation of subunits. J Biol Chem. 1982 Jan 10;257(1):508–515. [PubMed] [Google Scholar]
  65. 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]
  66. Vale R. D. Intracellular transport using microtubule-based motors. Annu Rev Cell Biol. 1987;3:347–378. doi: 10.1146/annurev.cb.03.110187.002023. [DOI] [PubMed] [Google Scholar]
  67. Vale R. D., Reese T. S., Sheetz M. P. Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell. 1985 Aug;42(1):39–50. doi: 10.1016/s0092-8674(85)80099-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Vale R. D., Schnapp B. J., Mitchison T., Steuer E., Reese T. S., Sheetz M. P. Different axoplasmic proteins generate movement in opposite directions along microtubules in vitro. Cell. 1985 Dec;43(3 Pt 2):623–632. doi: 10.1016/0092-8674(85)90234-x. [DOI] [PubMed] [Google Scholar]
  69. Vale R. D., Schnapp B. J., Reese T. S., Sheetz M. P. Movement of organelles along filaments dissociated from the axoplasm of the squid giant axon. Cell. 1985 Feb;40(2):449–454. doi: 10.1016/0092-8674(85)90159-x. [DOI] [PubMed] [Google Scholar]
  70. Valentine R. C., Shapiro B. M., Stadtman E. R. Regulation of glutamine synthetase. XII. Electron microscopy of the enzyme from Escherichia coli. Biochemistry. 1968 Jun;7(6):2143–2152. doi: 10.1021/bi00846a017. [DOI] [PubMed] [Google Scholar]
  71. Vallee R. B., Wall J. S., Paschal B. M., Shpetner H. S. Microtubule-associated protein 1C from brain is a two-headed cytosolic dynein. Nature. 1988 Apr 7;332(6164):561–563. doi: 10.1038/332561a0. [DOI] [PubMed] [Google Scholar]
  72. Vandenbunder B., Borisy G. G. Decoration of microtubules by fluorescently labeled microtubule-associated protein 2 (MAP2) does not interfere with their spatial organization and progress through mitosis in living fibroblasts. Cell Motil Cytoskeleton. 1986;6(6):570–579. doi: 10.1002/cm.970060605. [DOI] [PubMed] [Google Scholar]
  73. Warren R. H. Axonal microtubules of crayfish and spiny lobster nerve cords are decorated with a heat-stable protein of high molecular weight. J Cell Sci. 1984 Oct;71:1–15. doi: 10.1242/jcs.71.1.1. [DOI] [PubMed] [Google Scholar]

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

RESOURCES