Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Jun 1;108(6):2335–2342. doi: 10.1083/jcb.108.6.2335

The distribution, abundance and subcellular localization of kinesin

PMCID: PMC2115587  PMID: 2525563

Abstract

An antiserum which binds kinesin specifically on Western blots was used to determine the distribution and abundance of chicken kinesin by correlated immunoblotting and immunolocalization. Quantitative immunoblotting showed that the abundance of kinesin varied widely in different cell and tissue types, from 0.039% of total protein in epidermal fibroblasts to 0.309% in sympathetic neurons; of the types examined, only red blood cells lacked detectable kinesin. The molar ratio of tubulin/kinesin varied over a narrower range. To analyze the intracellular distribution of kinesin, cultured fibroblasts were fractionated by sequential extraction with saponin-, Triton X-100-, and SDS-containing buffer. Quantitative blotting of the resulting cell fractions indicated that 68% of fibroblast kinesin is in soluble form, 32% is membrane- or organelle-associated, and none is detectable in cytoskeletal fractions. To visualize this distribution, cells treated by the same extraction protocol were immunofluorescently stained with antikinesin and antitubulin. Without extraction, kinesin staining was located throughout cultured neurons and fibroblasts. However, when fibroblasts were extracted with saponin or Brij 58 before fixation, subsequent staining revealed that the remaining kinesin fraction was colocalized with interphase microtubules, but not with mitotic spindles. Prefixation extraction with Triton abolished antikinesin staining. These data suggest that kinesin may play a role in tubovesicular movement but provide no evidence for a role in mitosis.

Full Text

The Full Text of this article is available as a PDF (1.9 MB).

Selected References

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

  1. Amos L. A. Kinesin from pig brain studied by electron microscopy. J Cell Sci. 1987 Feb;87(Pt 1):105–111. doi: 10.1242/jcs.87.1.105. [DOI] [PubMed] [Google Scholar]
  2. Ansorge S., Bohley P., Kirschke H., Langner J., Wiederanders B., Hanson H. Metabolism of insulin and glucagon. Glutathione-insulin transhydrogenase from microsomes of rat liver. Eur J Biochem. 1973 Jan 3;32(1):27–35. doi: 10.1111/j.1432-1033.1973.tb02574.x. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Beutler E., West C., Blume K. G. The removal of leukocytes and platelets from whole blood. J Lab Clin Med. 1976 Aug;88(2):328–333. [PubMed] [Google Scholar]
  5. 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]
  6. Blose S. H., Meltzer D. I., Feramisco J. R. 10-nm filaments are induced to collapse in living cells microinjected with monoclonal and polyclonal antibodies against tubulin. J Cell Biol. 1984 Mar;98(3):847–858. doi: 10.1083/jcb.98.3.847. [DOI] [PMC free article] [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. Bramhall S., Noack N., Wu M., Loewenberg J. R. A simple colorimetric method for determination of protein. Anal Biochem. 1969 Oct 1;31(1):146–148. doi: 10.1016/0003-2697(69)90251-6. [DOI] [PubMed] [Google Scholar]
  9. Breitling F., Little M. Carboxy-terminal regions on the surface of tubulin and microtubules. Epitope locations of YOL1/34, DM1A and DM1B. J Mol Biol. 1986 May 20;189(2):367–370. doi: 10.1016/0022-2836(86)90517-6. [DOI] [PubMed] [Google Scholar]
  10. Brown S., Levinson W., Spudich J. A. Cytoskeletal elements of chick embryo fibroblasts revealed by detergent extraction. J Supramol Struct. 1976;5(2):119–130. doi: 10.1002/jss.400050203. [DOI] [PubMed] [Google Scholar]
  11. Burridge K., Bray D. Purification and structural analysis of myosins from brain and other non-muscle tissues. J Mol Biol. 1975 Nov 25;99(1):1–14. doi: 10.1016/s0022-2836(75)80154-9. [DOI] [PubMed] [Google Scholar]
  12. Cande W. Z., McDonald K., Meeusen R. L. A permeabilized cell model for studying cell division: a comparison of anaphase chromosome movement and cleavage furrow constriction in lysed PtK1 cells. J Cell Biol. 1981 Mar;88(3):618–629. doi: 10.1083/jcb.88.3.618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Caron J. M., Jones A. L., Kirschner M. W. Autoregulation of tubulin synthesis in hepatocytes and fibroblasts. J Cell Biol. 1985 Nov;101(5 Pt 1):1763–1772. doi: 10.1083/jcb.101.5.1763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Citi S., Kendrick-Jones J. Regulation of non-muscle myosin structure and function. Bioessays. 1987 Oct;7(4):155–159. doi: 10.1002/bies.950070404. [DOI] [PubMed] [Google Scholar]
  15. Clark T. G., Rosenbaum J. L. Pigment particle translocation in detergent-permeabilized melanophores of Fundulus heteroclitus. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4655–4659. doi: 10.1073/pnas.79.15.4655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Cohn S. A., Ingold A. L., Scholey J. M. Correlation between the ATPase and microtubule translocating activities of sea urchin egg kinesin. Nature. 1987 Jul 9;328(6126):160–163. doi: 10.1038/328160a0. [DOI] [PubMed] [Google Scholar]
  17. Craig R., Smith R., Kendrick-Jones J. Light-chain phosphorylation controls the conformation of vertebrate non-muscle and smooth muscle myosin molecules. 1983 Mar 31-Apr 6Nature. 302(5907):436–439. doi: 10.1038/302436a0. [DOI] [PubMed] [Google Scholar]
  18. Crimaudo C., Hortsch M., Gausepohl H., Meyer D. I. Human ribophorins I and II: the primary structure and membrane topology of two highly conserved rough endoplasmic reticulum-specific glycoproteins. EMBO J. 1987 Jan;6(1):75–82. doi: 10.1002/j.1460-2075.1987.tb04721.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Dabora S. L., Sheetz M. P. The microtubule-dependent formation of a tubulovesicular network with characteristics of the ER from cultured cell extracts. Cell. 1988 Jul 1;54(1):27–35. doi: 10.1016/0092-8674(88)90176-6. [DOI] [PubMed] [Google Scholar]
  20. Dawson D. B., Varandani P. T. Characterization and application of monoclonal antibodies directed to separate epitopes of glutathione-insulin transhydrogenase. Biochim Biophys Acta. 1987 Mar 19;923(3):389–400. doi: 10.1016/0304-4165(87)90047-x. [DOI] [PubMed] [Google Scholar]
  21. Forman D. S., Brown K. J., Livengood D. R. Fast axonal transport in permeabilized lobster giant axons is inhibited by vanadate. J Neurosci. 1983 Jun;3(6):1279–1288. doi: 10.1523/JNEUROSCI.03-06-01279.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Forman D. S. Vanadate inhibits saltatory organelle movement in a permeabilized cell model. Exp Cell Res. 1982 Sep;141(1):139–147. doi: 10.1016/0014-4827(82)90076-3. [DOI] [PubMed] [Google Scholar]
  23. Gelles J., Schnapp B. J., Sheetz M. P. Tracking kinesin-driven movements with nanometre-scale precision. Nature. 1988 Feb 4;331(6155):450–453. doi: 10.1038/331450a0. [DOI] [PubMed] [Google Scholar]
  24. Hafner M., Petzelt C. Inhibition of mitosis by an antibody to the mitotic calcium transport system. Nature. 1987 Nov 19;330(6145):264–266. doi: 10.1038/330264a0. [DOI] [PubMed] [Google Scholar]
  25. Harris P. The role of membranes in the ogranization of the mitotic apparatus. Exp Cell Res. 1975 Sep;94(2):409–425. doi: 10.1016/0014-4827(75)90507-8. [DOI] [PubMed] [Google Scholar]
  26. Hollenbeck P. J., Bray D., Adams R. J. Effects of the uncoupling agents FCCP and CCCP on the saltatory movements of cytoplasmic organelles. Cell Biol Int Rep. 1985 Feb;9(2):193–199. doi: 10.1016/0309-1651(85)90094-3. [DOI] [PubMed] [Google Scholar]
  27. Hollenbeck P. J., Bray D. Rapidly transported organelles containing membrane and cytoskeletal components: their relation to axonal growth. J Cell Biol. 1987 Dec;105(6 Pt 1):2827–2835. doi: 10.1083/jcb.105.6.2827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Hortsch M., Meyer D. I. Immunochemical analysis of rough and smooth microsomes from rat liver. Segregation of docking protein in rough membranes. Eur J Biochem. 1985 Aug 1;150(3):559–564. doi: 10.1111/j.1432-1033.1985.tb09057.x. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Kilmartin J. V., Wright B., Milstein C. Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line. J Cell Biol. 1982 Jun;93(3):576–582. doi: 10.1083/jcb.93.3.576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kreibich G., Freienstein C. M., Pereyra B. N., Ulrich B. L., Sabatini D. D. Proteins of rough microsomal membranes related to ribosome binding. II. Cross-linking of bound ribosomes to specific membrane proteins exposed at the binding sites. J Cell Biol. 1978 May;77(2):488–506. doi: 10.1083/jcb.77.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kuznetsov S. A., Gelfand V. I. Bovine brain kinesin is a microtubule-activated ATPase. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8530–8534. doi: 10.1073/pnas.83.22.8530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kyhse-Andersen J. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods. 1984 Dec;10(3-4):203–209. doi: 10.1016/0165-022x(84)90040-x. [DOI] [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. 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]
  37. Lee C., Chen L. B. Dynamic behavior of endoplasmic reticulum in living cells. Cell. 1988 Jul 1;54(1):37–46. doi: 10.1016/0092-8674(88)90177-8. [DOI] [PubMed] [Google Scholar]
  38. Leslie R. J., Hird R. B., Wilson L., McIntosh J. R., Scholey J. M. Kinesin is associated with a nonmicrotubule component of sea urchin mitotic spindles. Proc Natl Acad Sci U S A. 1987 May;84(9):2771–2775. doi: 10.1073/pnas.84.9.2771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Marcantonio E. E., Amar-Costesec A., Kreibich G. Segregation of the polypeptide translocation apparatus to regions of the endoplasmic reticulum containing ribophorins and ribosomes. II. Rat liver microsomal subfractions contain equimolar amounts of ribophorins and ribosomes. J Cell Biol. 1984 Dec;99(6):2254–2259. doi: 10.1083/jcb.99.6.2254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Matlin K. S. Ammonium chloride slows transport of the influenza virus hemagglutinin but does not cause mis-sorting in a polarized epithelial cell line. J Biol Chem. 1986 Nov 15;261(32):15172–15178. [PubMed] [Google Scholar]
  41. Matlin K. S., Simons K. Reduced temperature prevents transfer of a membrane glycoprotein to the cell surface but does not prevent terminal glycosylation. Cell. 1983 Aug;34(1):233–243. doi: 10.1016/0092-8674(83)90154-x. [DOI] [PubMed] [Google Scholar]
  42. Miller R. H., Lasek R. J., Katz M. J. Preferred microtubules for vesicle transport in lobster axons. Science. 1987 Jan 9;235(4785):220–222. doi: 10.1126/science.2432661. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Neighbors B. W., Williams R. C., Jr, McIntosh J. R. Localization of kinesin in cultured cells. J Cell Biol. 1988 Apr;106(4):1193–1204. doi: 10.1083/jcb.106.4.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Penningroth S. M., Rose P. M., Peterson D. D. Evidence that the 116 kDa component of kinesin binds and hydrolyzes ATP. FEBS Lett. 1987 Sep 28;222(1):204–210. doi: 10.1016/0014-5793(87)80220-x. [DOI] [PubMed] [Google Scholar]
  46. Petzelt C., Hafner M. Visualization of the Ca-transport system of the mitotic apparatus of sea urchin eggs with a monoclonal antibody. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1719–1722. doi: 10.1073/pnas.83.6.1719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Pollard T. D. Cytoplasmic contractile proteins. J Cell Biol. 1981 Dec;91(3 Pt 2):156s–165s. doi: 10.1083/jcb.91.3.156s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. 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]
  50. 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]
  51. Terasaki M., Chen L. B., Fujiwara K. Microtubules and the endoplasmic reticulum are highly interdependent structures. J Cell Biol. 1986 Oct;103(4):1557–1568. doi: 10.1083/jcb.103.4.1557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Vale R. D., Hotani H. Formation of membrane networks in vitro by kinesin-driven microtubule movement. J Cell Biol. 1988 Dec;107(6 Pt 1):2233–2241. doi: 10.1083/jcb.107.6.2233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. 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]
  54. 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]
  55. 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]
  56. Vale R. D., Schnapp B. J., Reese T. S., Sheetz M. P. Organelle, bead, and microtubule translocations promoted by soluble factors from the squid giant axon. Cell. 1985 Mar;40(3):559–569. doi: 10.1016/0092-8674(85)90204-1. [DOI] [PubMed] [Google Scholar]
  57. Varandani P. T. Insulin degradation. V. Unmasking of glutathione-insulin transhydrogenase in rat liver microsomal membrane. Biochim Biophys Acta. 1973 May 28;304(3):642–659. doi: 10.1016/0304-4165(73)90210-9. [DOI] [PubMed] [Google Scholar]
  58. Wessel D., Flügge U. I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem. 1984 Apr;138(1):141–143. doi: 10.1016/0003-2697(84)90782-6. [DOI] [PubMed] [Google Scholar]

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

RESOURCES