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. 1992 May 1;117(3):595–606. doi: 10.1083/jcb.117.3.595

Suppression of kinesin expression in cultured hippocampal neurons using antisense oligonucleotides

PMCID: PMC2289440  PMID: 1533397

Abstract

Kinesin, a microtubule-based force-generating molecule, is thought to translocate organelles along microtubules. To examine the function of kinesin in neurons, we sought to suppress kinesin heavy chain (KHC) expression in cultured hippocampal neurons using antisense oligonucleotides and study the phenotype of these KHC "null" cells. Two different antisense oligonucleotides complementary to the KHC sequence reduced the protein levels of the heavy chain by greater than 95% within 24 h after application and produced identical phenotypes. After inhibition of KHC expression for 24 or 48 h, neurons extended an array of neurites often with one neurite longer than the others; however, the length of all these neurites was significantly reduced. Inhibition of KHC expression also altered the distribution of GAP-43 and synapsin I, two proteins thought to be transported in association with membranous organelles. These proteins, which are normally localized at the tips of growing neurites, were confined to the cell body in antisense-treated cells. Treatment of the cells with the corresponding sense oligonucleotides affected neither the distribution of GAP-43 and synapsin I, nor the length of neurites. A full recovery of neurite length occurred after removal of the antisense oligonucleotides from the medium. These data indicate that KHC plays a role in the anterograde translocation of vesicles containing GAP-43 and synapsin I. A deficiency in vesicle delivery may also explain the inhibition of neurite outgrowth. Despite the inhibition of KHC and the failure of GAP- 43 and synapsin I to move out of the cell body, hippocampal neurons can extend processes and acquire as asymmetric morphology.

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Selected References

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  1. Baas P. W., Black M. M., Banker G. A. Changes in microtubule polarity orientation during the development of hippocampal neurons in culture. J Cell Biol. 1989 Dec;109(6 Pt 1):3085–3094. doi: 10.1083/jcb.109.6.3085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baas P. W., Deitch J. S., Black M. M., Banker G. A. Polarity orientation of microtubules in hippocampal neurons: uniformity in the axon and nonuniformity in the dendrite. Proc Natl Acad Sci U S A. 1988 Nov;85(21):8335–8339. doi: 10.1073/pnas.85.21.8335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baetge E. E., Hammang J. P. Neurite outgrowth in PC12 cells deficient in GAP-43. Neuron. 1991 Jan;6(1):21–30. doi: 10.1016/0896-6273(91)90118-j. [DOI] [PubMed] [Google Scholar]
  4. Baitinger C., Willard M. Axonal transport of synapsin I-like proteins in rabbit retinal ganglion cells. J Neurosci. 1987 Nov;7(11):3723–3735. doi: 10.1523/JNEUROSCI.07-11-03723.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bartlett W. P., Banker G. A. An electron microscopic study of the development of axons and dendrites by hippocampal neurons in culture. I. Cells which develop without intercellular contacts. J Neurosci. 1984 Aug;4(8):1944–1953. doi: 10.1523/JNEUROSCI.04-08-01944.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Benowitz L. I., Shashoua V. E., Yoon M. G. Specific changes in rapidly transported proteins during regeneration of the goldfish optic nerve. J Neurosci. 1981 Mar;1(3):300–307. doi: 10.1523/JNEUROSCI.01-03-00300.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bloom G. S., Wagner M. C., Pfister K. K., Brady S. T. Native structure and physical properties of bovine brain kinesin and identification of the ATP-binding subunit polypeptide. Biochemistry. 1988 May 3;27(9):3409–3416. doi: 10.1021/bi00409a043. [DOI] [PubMed] [Google Scholar]
  8. Bottenstein J. E., Sato G. H. Growth of a rat neuroblastoma cell line in serum-free supplemented medium. Proc Natl Acad Sci U S A. 1979 Jan;76(1):514–517. doi: 10.1073/pnas.76.1.514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Brady S. T. Molecular motors in the nervous system. Neuron. 1991 Oct;7(4):521–533. doi: 10.1016/0896-6273(91)90365-7. [DOI] [PubMed] [Google Scholar]
  10. Brady S. T., Pfister K. K., Bloom G. S. A monoclonal antibody against kinesin inhibits both anterograde and retrograde fast axonal transport in squid axoplasm. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1061–1065. doi: 10.1073/pnas.87.3.1061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Caceres A., Banker G., Steward O., Binder L., Payne M. MAP2 is localized to the dendrites of hippocampal neurons which develop in culture. Brain Res. 1984 Apr;315(2):314–318. doi: 10.1016/0165-3806(84)90167-6. [DOI] [PubMed] [Google Scholar]
  12. De Camilli P., Cameron R., Greengard P. Synapsin I (protein I), a nerve terminal-specific phosphoprotein. I. Its general distribution in synapses of the central and peripheral nervous system demonstrated by immunofluorescence in frozen and plastic sections. J Cell Biol. 1983 May;96(5):1337–1354. doi: 10.1083/jcb.96.5.1337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Dotti C. G., Sullivan C. A., Banker G. A. The establishment of polarity by hippocampal neurons in culture. J Neurosci. 1988 Apr;8(4):1454–1468. doi: 10.1523/JNEUROSCI.08-04-01454.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Endow S. A., Hatsumi M. A multimember kinesin gene family in Drosophila. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4424–4427. doi: 10.1073/pnas.88.10.4424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ferreira A., Busciglio J., Cáceres A. Microtubule formation and neurite growth in cerebellar macroneurons which develop in vitro: evidence for the involvement of the microtubule-associated proteins, MAP-1a, HMW-MAP2 and Tau. Brain Res Dev Brain Res. 1989 Oct 1;49(2):215–228. doi: 10.1016/0165-3806(89)90023-0. [DOI] [PubMed] [Google Scholar]
  17. Fletcher T. L., Cameron P., De Camilli P., Banker G. The distribution of synapsin I and synaptophysin in hippocampal neurons developing in culture. J Neurosci. 1991 Jun;11(6):1617–1626. doi: 10.1523/JNEUROSCI.11-06-01617.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gispen W. H., Leunissen J. L., Oestreicher A. B., Verkleij A. J., Zwiers H. Presynaptic localization of B-50 phosphoprotein: the (ACTH)-sensitive protein kinase substrate involved in rat brain polyphosphoinositide metabolism. Brain Res. 1985 Mar 4;328(2):381–385. doi: 10.1016/0006-8993(85)91054-6. [DOI] [PubMed] [Google Scholar]
  19. Goslin K., Banker G. Experimental observations on the development of polarity by hippocampal neurons in culture. J Cell Biol. 1989 Apr;108(4):1507–1516. doi: 10.1083/jcb.108.4.1507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Goslin K., Banker G. Rapid changes in the distribution of GAP-43 correlate with the expression of neuronal polarity during normal development and under experimental conditions. J Cell Biol. 1990 Apr;110(4):1319–1331. doi: 10.1083/jcb.110.4.1319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hall D. H., Hedgecock E. M. Kinesin-related gene unc-104 is required for axonal transport of synaptic vesicles in C. elegans. Cell. 1991 May 31;65(5):837–847. doi: 10.1016/0092-8674(91)90391-b. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Hirokawa N., Sato-Yoshitake R., Kobayashi N., Pfister K. K., Bloom G. S., Brady S. T. Kinesin associates with anterogradely transported membranous organelles in vivo. J Cell Biol. 1991 Jul;114(2):295–302. doi: 10.1083/jcb.114.2.295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hollenbeck P. J., Swanson J. A. Radial extension of macrophage tubular lysosomes supported by kinesin. Nature. 1990 Aug 30;346(6287):864–866. doi: 10.1038/346864a0. [DOI] [PubMed] [Google Scholar]
  25. Hollenbeck P. J. The distribution, abundance and subcellular localization of kinesin. J Cell Biol. 1989 Jun;108(6):2335–2342. doi: 10.1083/jcb.108.6.2335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hotani H., Miyamoto H. Dynamic features of microtubules as visualized by dark-field microscopy. Adv Biophys. 1990;26:135–156. doi: 10.1016/0065-227x(90)90010-q. [DOI] [PubMed] [Google Scholar]
  27. Houliston E., Elinson R. P. Evidence for the involvement of microtubules, ER, and kinesin in the cortical rotation of fertilized frog eggs. J Cell Biol. 1991 Sep;114(5):1017–1028. doi: 10.1083/jcb.114.5.1017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ingold A. L., Cohn S. A., Scholey J. M. Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains. J Cell Biol. 1988 Dec;107(6 Pt 2):2657–2667. doi: 10.1083/jcb.107.6.2657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Jahn R., Schiebler W., Greengard P. A quantitative dot-immunobinding assay for proteins using nitrocellulose membrane filters. Proc Natl Acad Sci U S A. 1984 Mar;81(6):1684–1687. doi: 10.1073/pnas.81.6.1684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Knops J., Kosik K. S., Lee G., Pardee J. D., Cohen-Gould L., McConlogue L. Overexpression of tau in a nonneuronal cell induces long cellular processes. J Cell Biol. 1991 Aug;114(4):725–733. doi: 10.1083/jcb.114.4.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kosik K. S., Finch E. A. MAP2 and tau segregate into dendritic and axonal domains after the elaboration of morphologically distinct neurites: an immunocytochemical study of cultured rat cerebrum. J Neurosci. 1987 Oct;7(10):3142–3153. doi: 10.1523/JNEUROSCI.07-10-03142.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kosik K. S., Orecchio L. D., Schnapp B., Inouye H., Neve R. L. The primary structure and analysis of the squid kinesin heavy chain. J Biol Chem. 1990 Feb 25;265(6):3278–3283. [PubMed] [Google Scholar]
  33. Kucik D. F., Elson E. L., Sheetz M. P. Forward transport of glycoproteins on leading lamellipodia in locomoting cells. Nature. 1989 Jul 27;340(6231):315–317. doi: 10.1038/340315a0. [DOI] [PubMed] [Google Scholar]
  34. Kuznetsov S. A., Vaisberg E. A., Shanina N. A., Magretova N. N., Chernyak V. Y., Gelfand V. I. The quaternary structure of bovine brain kinesin. EMBO J. 1988 Feb;7(2):353–356. doi: 10.1002/j.1460-2075.1988.tb02820.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Otsuka A. J., Jeyaprakash A., García-Añoveros J., Tang L. Z., Fisk G., Hartshorne T., Franco R., Born T. The C. elegans unc-104 gene encodes a putative kinesin heavy chain-like protein. Neuron. 1991 Jan;6(1):113–122. doi: 10.1016/0896-6273(91)90126-k. [DOI] [PubMed] [Google Scholar]
  37. Pfenninger K. H., Maylié-Pfenninger M. F. Lectin labeling of sprouting neurons. II. Relative movement and appearance of glycoconjugates during plasmalemmal expansion. J Cell Biol. 1981 Jun;89(3):547–559. doi: 10.1083/jcb.89.3.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Pfister K. K., Wagner M. C., Stenoien D. L., Brady S. T., Bloom G. S. Monoclonal antibodies to kinesin heavy and light chains stain vesicle-like structures, but not microtubules, in cultured cells. J Cell Biol. 1989 Apr;108(4):1453–1463. doi: 10.1083/jcb.108.4.1453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Porter M. E., Scholey J. M., Stemple D. L., Vigers G. P., Vale R. D., Sheetz M. P., McIntosh J. R. Characterization of the microtubule movement produced by sea urchin egg kinesin. J Biol Chem. 1987 Feb 25;262(6):2794–2802. [PubMed] [Google Scholar]
  40. Rodionov V. I., Gyoeva F. K., Gelfand V. I. Kinesin is responsible for centrifugal movement of pigment granules in melanophores. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4956–4960. doi: 10.1073/pnas.88.11.4956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Saxton W. M., Hicks J., Goldstein L. S., Raff E. C. Kinesin heavy chain is essential for viability and neuromuscular functions in Drosophila, but mutants show no defects in mitosis. Cell. 1991 Mar 22;64(6):1093–1102. doi: 10.1016/0092-8674(91)90264-y. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Shea T. B., Perrone-Bizzozero N. I., Beermann M. L., Benowitz L. I. Phospholipid-mediated delivery of anti-GAP-43 antibodies into neuroblastoma cells prevents neuritogenesis. J Neurosci. 1991 Jun;11(6):1685–1690. doi: 10.1523/JNEUROSCI.11-06-01685.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Skene J. H., Virág I. Posttranslational membrane attachment and dynamic fatty acylation of a neuronal growth cone protein, GAP-43. J Cell Biol. 1989 Feb;108(2):613–624. doi: 10.1083/jcb.108.2.613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Skene J. H., Willard M. Changes in axonally transported proteins during axon regeneration in toad retinal ganglion cells. J Cell Biol. 1981 Apr;89(1):86–95. doi: 10.1083/jcb.89.1.86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Small R. K., Blank M., Ghez R., Pfenninger K. H. Components of the plasma membrane of growing axons. II. Diffusion of membrane protein complexes. J Cell Biol. 1984 Apr;98(4):1434–1443. doi: 10.1083/jcb.98.4.1434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. Tanaka E. M., Kirschner M. W. Microtubule behavior in the growth cones of living neurons during axon elongation. J Cell Biol. 1991 Oct;115(2):345–363. doi: 10.1083/jcb.115.2.345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Urrutia R., McNiven M. A., Albanesi J. P., Murphy D. B., Kachar B. Purified kinesin promotes vesicle motility and induces active sliding between microtubules in vitro. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6701–6705. doi: 10.1073/pnas.88.15.6701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. 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]
  52. Valtorta F., Villa A., Jahn R., De Camilli P., Greengard P., Ceccarelli B. Localization of synapsin I at the frog neuromuscular junction. Neuroscience. 1988 Feb;24(2):593–603. doi: 10.1016/0306-4522(88)90353-3. [DOI] [PubMed] [Google Scholar]
  53. Wright B. D., Henson J. H., Wedaman K. P., Willy P. J., Morand J. N., Scholey J. M. Subcellular localization and sequence of sea urchin kinesin heavy chain: evidence for its association with membranes in the mitotic apparatus and interphase cytoplasm. J Cell Biol. 1991 May;113(4):817–833. doi: 10.1083/jcb.113.4.817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Yang J. T., Laymon R. A., Goldstein L. S. A three-domain structure of kinesin heavy chain revealed by DNA sequence and microtubule binding analyses. Cell. 1989 Mar 10;56(5):879–889. doi: 10.1016/0092-8674(89)90692-2. [DOI] [PubMed] [Google Scholar]
  55. Yankner B. A., Benowitz L. I., Villa-Komaroff L., Neve R. L. Transfection of PC12 cells with the human GAP-43 gene: effects on neurite outgrowth and regeneration. Brain Res Mol Brain Res. 1990 Jan;7(1):39–44. doi: 10.1016/0169-328x(90)90071-k. [DOI] [PubMed] [Google Scholar]

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