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. 1996 Aug;7(8):1167–1180. doi: 10.1091/mbc.7.8.1167

Functionally distinct isoforms of dynactin are expressed in human neurons.

M K Tokito 1, D S Howland 1, V M Lee 1, E L Holzbaur 1
PMCID: PMC275970  PMID: 8856662

Abstract

P150Glued is the largest subunit of dynactin, which binds to cytoplasmic dynein and activates vesicle transport along microtubules. We have isolated human cDNAs encoding p150Glued as well as a 135-kDa isoform; these isoforms are expressed in human brain by alternative mRNA splicing of the human DCTN1 gene. The p135 isoform lacks the consensus microtubule-binding motif shared by members of the p150Glued/Glued/CLIP-170/BIK1 family of microtubule-associated proteins and, therefore, is predicted not to bind directly to microtubules. We used transient transfection assays and in vitro microtubule-binding assays to demonstrate that the p150 isoform binds to microtubules, but the p135 isoform does not. However, both isoforms bind to cytoplasmic dynein, and both partition similarly into cytosolic and membrane cellular fractions. Sequential immunoprecipitations with an isoform-specific antibody for p150 followed by a pan-isoform antibody revealed that, in brain, these polypeptides assemble to form distinct complexes, each of which sediments at approximately 20 S. On the basis of these observations, we hypothesize that there is a conserved neuronal function for a distinct form of the dynactin complex that cannot bind directly to cellular microtubules.

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Selected References

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

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Andrews P. W., Damjanov I., Simon D., Banting G. S., Carlin C., Dracopoli N. C., Føgh J. Pluripotent embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2. Differentiation in vivo and in vitro. Lab Invest. 1984 Feb;50(2):147–162. [PubMed] [Google Scholar]
  3. Andrews P. W. Retinoic acid induces neuronal differentiation of a cloned human embryonal carcinoma cell line in vitro. Dev Biol. 1984 Jun;103(2):285–293. doi: 10.1016/0012-1606(84)90316-6. [DOI] [PubMed] [Google Scholar]
  4. Berlin V., Styles C. A., Fink G. R. BIK1, a protein required for microtubule function during mating and mitosis in Saccharomyces cerevisiae, colocalizes with tubulin. J Cell Biol. 1990 Dec;111(6 Pt 1):2573–2586. doi: 10.1083/jcb.111.6.2573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Clark S. W., Meyer D. I. ACT3: a putative centractin homologue in S. cerevisiae is required for proper orientation of the mitotic spindle. J Cell Biol. 1994 Oct;127(1):129–138. doi: 10.1083/jcb.127.1.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clark S. W., Meyer D. I. Centractin is an actin homologue associated with the centrosome. Nature. 1992 Sep 17;359(6392):246–250. doi: 10.1038/359246a0. [DOI] [PubMed] [Google Scholar]
  7. Collins C. A., Vallee R. B. Preparation of microtubules from rat liver and testis: cytoplasmic dynein is a major microtubule associated protein. Cell Motil Cytoskeleton. 1989;14(4):491–500. doi: 10.1002/cm.970140407. [DOI] [PubMed] [Google Scholar]
  8. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dillman J. F., 3rd, Dabney L. P., Pfister K. K. Cytoplasmic dynein is associated with slow axonal transport. Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):141–144. doi: 10.1073/pnas.93.1.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Echeverri C. J., Paschal B. M., Vaughan K. T., Vallee R. B. Molecular characterization of the 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis. J Cell Biol. 1996 Feb;132(4):617–633. doi: 10.1083/jcb.132.4.617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Eshel D., Urrestarazu L. A., Vissers S., Jauniaux J. C., van Vliet-Reedijk J. C., Planta R. J., Gibbons I. R. Cytoplasmic dynein is required for normal nuclear segregation in yeast. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11172–11176. doi: 10.1073/pnas.90.23.11172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Garen S. H., Kankel D. R. Golgi and genetic mosaic analyses of visual system mutants in Drosophila melanogaster. Dev Biol. 1983 Apr;96(2):445–466. doi: 10.1016/0012-1606(83)90182-3. [DOI] [PubMed] [Google Scholar]
  13. Gill S. R., Schroer T. A., Szilak I., Steuer E. R., Sheetz M. P., Cleveland D. W. Dynactin, a conserved, ubiquitously expressed component of an activator of vesicle motility mediated by cytoplasmic dynein. J Cell Biol. 1991 Dec;115(6):1639–1650. doi: 10.1083/jcb.115.6.1639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Harte P. J., Kankel D. R. Genetic analysis of mutations at the Glued locus and interacting loci in Drosophila melanogaster. Genetics. 1982 Jul-Aug;101(3-4):477–501. doi: 10.1093/genetics/101.3-4.477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hendry I. A., Stöckel K., Thoenen H., Iversen L. L. The retrograde axonal transport of nerve growth factor. Brain Res. 1974 Mar 15;68(1):103–121. doi: 10.1016/0006-8993(74)90536-8. [DOI] [PubMed] [Google Scholar]
  16. Hirokawa N., Sato-Yoshitake R., Yoshida T., Kawashima T. Brain dynein (MAP1C) localizes on both anterogradely and retrogradely transported membranous organelles in vivo. J Cell Biol. 1990 Sep;111(3):1027–1037. doi: 10.1083/jcb.111.3.1027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Holzbaur E. L., Hammarback J. A., Paschal B. M., Kravit N. G., Pfister K. K., Vallee R. B. Homology of a 150K cytoplasmic dynein-associated polypeptide with the Drosophila gene Glued. Nature. 1991 Jun 13;351(6327):579–583. doi: 10.1038/351579a0. [DOI] [PubMed] [Google Scholar]
  18. Holzbaur E. L., Tokito M. K. Localization of the DCTN1 gene encoding p150Glued to human chromosome 2p13 by fluorescence in situ hybridization. Genomics. 1996 Feb 1;31(3):398–399. doi: 10.1006/geno.1996.0068. [DOI] [PubMed] [Google Scholar]
  19. Holzbaur E. L., Vallee R. B. DYNEINS: molecular structure and cellular function. Annu Rev Cell Biol. 1994;10:339–372. doi: 10.1146/annurev.cb.10.110194.002011. [DOI] [PubMed] [Google Scholar]
  20. Karki S., Holzbaur E. L. Affinity chromatography demonstrates a direct binding between cytoplasmic dynein and the dynactin complex. J Biol Chem. 1995 Dec 1;270(48):28806–28811. doi: 10.1074/jbc.270.48.28806. [DOI] [PubMed] [Google Scholar]
  21. Kristensson K., Olsson Y. Retrograde axonal transport of protein. Brain Res. 1971 Jun 18;29(2):363–365. doi: 10.1016/0006-8993(71)90044-8. [DOI] [PubMed] [Google Scholar]
  22. Lee V. M., Andrews P. W. Differentiation of NTERA-2 clonal human embryonal carcinoma cells into neurons involves the induction of all three neurofilament proteins. J Neurosci. 1986 Feb;6(2):514–521. doi: 10.1523/JNEUROSCI.06-02-00514.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lees-Miller J. P., Helfman D. M., Schroer T. A. A vertebrate actin-related protein is a component of a multisubunit complex involved in microtubule-based vesicle motility. Nature. 1992 Sep 17;359(6392):244–246. doi: 10.1038/359244a0. [DOI] [PubMed] [Google Scholar]
  24. Li Y. Y., Yeh E., Hays T., Bloom K. Disruption of mitotic spindle orientation in a yeast dynein mutant. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10096–10100. doi: 10.1073/pnas.90.21.10096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. McGrail M., Gepner J., Silvanovich A., Ludmann S., Serr M., Hays T. S. Regulation of cytoplasmic dynein function in vivo by the Drosophila Glued complex. J Cell Biol. 1995 Oct;131(2):411–425. doi: 10.1083/jcb.131.2.411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Melloni R. H., Jr, Tokito M. K., Holzbaur E. L. Expression of the p150Glued component of the dynactin complex in developing and adult rat brain. J Comp Neurol. 1995 Jun 19;357(1):15–24. doi: 10.1002/cne.903570103. [DOI] [PubMed] [Google Scholar]
  27. Meyerowitz E. M., Kankel D. R. A genetic analysis of visual system development in Drosophilia melanogaster. Dev Biol. 1978 Jan;62(1):112–142. doi: 10.1016/0012-1606(78)90096-9. [DOI] [PubMed] [Google Scholar]
  28. Muhua L., Karpova T. S., Cooper J. A. A yeast actin-related protein homologous to that in vertebrate dynactin complex is important for spindle orientation and nuclear migration. Cell. 1994 Aug 26;78(4):669–679. doi: 10.1016/0092-8674(94)90531-2. [DOI] [PubMed] [Google Scholar]
  29. Obar R. A., Holzbaur E. L. Immunoselection and characterization of cDNA clones. Methods Cell Biol. 1993;37:361–405. doi: 10.1016/s0091-679x(08)60258-7. [DOI] [PubMed] [Google Scholar]
  30. Paschal B. M., Holzbaur E. L., Pfister K. K., Clark S., Meyer D. I., Vallee R. B. Characterization of a 50-kDa polypeptide in cytoplasmic dynein preparations reveals a complex with p150GLUED and a novel actin. J Biol Chem. 1993 Jul 15;268(20):15318–15323. [PubMed] [Google Scholar]
  31. 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]
  32. Pierre P., Scheel J., Rickard J. E., Kreis T. E. CLIP-170 links endocytic vesicles to microtubules. Cell. 1992 Sep 18;70(6):887–900. doi: 10.1016/0092-8674(92)90240-d. [DOI] [PubMed] [Google Scholar]
  33. Plamann M., Minke P. F., Tinsley J. H., Bruno K. S. Cytoplasmic dynein and actin-related protein Arp1 are required for normal nuclear distribution in filamentous fungi. J Cell Biol. 1994 Oct;127(1):139–149. doi: 10.1083/jcb.127.1.139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pleasure S. J., Lee V. M. NTera 2 cells: a human cell line which displays characteristics expected of a human committed neuronal progenitor cell. J Neurosci Res. 1993 Aug 15;35(6):585–602. doi: 10.1002/jnr.490350603. [DOI] [PubMed] [Google Scholar]
  35. Pleasure S. J., Page C., Lee V. M. Pure, postmitotic, polarized human neurons derived from NTera 2 cells provide a system for expressing exogenous proteins in terminally differentiated neurons. J Neurosci. 1992 May;12(5):1802–1815. doi: 10.1523/JNEUROSCI.12-05-01802.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Plough H H, Ives P T. Induction of Mutations by High Temperature in Drosophila. Genetics. 1935 Jan;20(1):42–69. doi: 10.1093/genetics/20.1.42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rickard J. E., Kreis T. E. Binding of pp170 to microtubules is regulated by phosphorylation. J Biol Chem. 1991 Sep 15;266(26):17597–17605. [PubMed] [Google Scholar]
  38. Schafer D. A., Gill S. R., Cooper J. A., Heuser J. E., Schroer T. A. Ultrastructural analysis of the dynactin complex: an actin-related protein is a component of a filament that resembles F-actin. J Cell Biol. 1994 Jul;126(2):403–412. doi: 10.1083/jcb.126.2.403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Steuer E. R., Wordeman L., Schroer T. A., Sheetz M. P. Localization of cytoplasmic dynein to mitotic spindles and kinetochores. Nature. 1990 May 17;345(6272):266–268. doi: 10.1038/345266a0. [DOI] [PubMed] [Google Scholar]
  40. Swaroop A., Sun J. W., Paco-Larson M. L., Garen A. Molecular organization and expression of the genetic locus glued in Drosophila melanogaster. Mol Cell Biol. 1986 Mar;6(3):833–841. doi: 10.1128/mcb.6.3.833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Swaroop A., Swaroop M., Garen A. Sequence analysis of the complete cDNA and encoded polypeptide for the Glued gene of Drosophila melanogaster. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6501–6505. doi: 10.1073/pnas.84.18.6501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sweeney H. L., Holzbaur E. L. Mutational analysis of motor proteins. Annu Rev Physiol. 1996;58:751–792. doi: 10.1146/annurev.ph.58.030196.003535. [DOI] [PubMed] [Google Scholar]
  43. Tinsley J. H., Minke P. F., Bruno K. S., Plamann M. p150Glued, the largest subunit of the dynactin complex, is nonessential in Neurospora but required for nuclear distribution. Mol Biol Cell. 1996 May;7(5):731–742. doi: 10.1091/mbc.7.5.731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Vaisberg E. A., Koonce M. P., McIntosh J. R. Cytoplasmic dynein plays a role in mammalian mitotic spindle formation. J Cell Biol. 1993 Nov;123(4):849–858. doi: 10.1083/jcb.123.4.849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Vallee R. B. A taxol-dependent procedure for the isolation of microtubules and microtubule-associated proteins (MAPs). J Cell Biol. 1982 Feb;92(2):435–442. doi: 10.1083/jcb.92.2.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Vaughan K. T., Vallee R. B. Cytoplasmic dynein binds dynactin through a direct interaction between the intermediate chains and p150Glued. J Cell Biol. 1995 Dec;131(6 Pt 1):1507–1516. doi: 10.1083/jcb.131.6.1507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Waterman-Storer C. M., Holzbaur E. L. The product of the Drosophila gene, Glued, is the functional homologue of the p150Glued component of the vertebrate dynactin complex. J Biol Chem. 1996 Jan 12;271(2):1153–1159. doi: 10.1074/jbc.271.2.1153. [DOI] [PubMed] [Google Scholar]
  48. Waterman-Storer C. M., Karki S., Holzbaur E. L. The p150Glued component of the dynactin complex binds to both microtubules and the actin-related protein centractin (Arp-1). Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1634–1638. doi: 10.1073/pnas.92.5.1634. [DOI] [PMC free article] [PubMed] [Google Scholar]

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