Abstract
Biochemical, pharmacological and immunocytochemical studies have implicated the microtubule-activated ATPase, kinesin, in the movement of membrane bounded organelles in fast axonal transport. In vitro studies suggested that kinesin moves organelles preferentially in the anterograde direction, but data about the function and precise localization of kinesin in the living axon were lacking. The current study was undertaken to establish whether kinesin associates with anterograde or retrograde moving organelles in vivo. Peripheral nerves were ligated to produce accumulations of organelles moving in defined directions. Regions proximal (anterograde) and distal (retrograde) to the ligation were analyzed for kinesin localization by immunofluorescence, and by immunogold electron microscopy using ultracryomicrotomy. Substantial amounts of kinesin were associated with anterograde moving organelles on the proximal side, while significantly less kinesin was detected distally. Statistical analyses indicated that kinesin was mostly associated with membrane-bounded organelles. These observations indicate that axonal kinesin is primarily associated with anterograde moving organelles in vivo.
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- 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]
- 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]
- 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]
- 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]
- Fahim M. A., Lasek R. J., Brady S. T., Hodge A. J. AVEC-DIC and electron microscopic analyses of axonally transported particles in cold-blocked squid giant axons. J Neurocytol. 1985 Oct;14(5):689–704. doi: 10.1007/BF01170822. [DOI] [PubMed] [Google Scholar]
- 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]
- Hirokawa N., Bloom G. S., Vallee R. B. Cytoskeletal architecture and immunocytochemical localization of microtubule-associated proteins in regions of axons associated with rapid axonal transport: the beta,beta'-iminodipropionitrile-intoxicated axon as a model system. J Cell Biol. 1985 Jul;101(1):227–239. doi: 10.1083/jcb.101.1.227. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hirokawa N. Cross-linker system between neurofilaments, microtubules, and membranous organelles in frog axons revealed by the quick-freeze, deep-etching method. J Cell Biol. 1982 Jul;94(1):129–142. doi: 10.1083/jcb.94.1.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hirokawa N., Glicksman M. A., Willard M. B. Organization of mammalian neurofilament polypeptides within the neuronal cytoskeleton. J Cell Biol. 1984 Apr;98(4):1523–1536. doi: 10.1083/jcb.98.4.1523. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hirokawa N., Pfister K. K., Yorifuji H., Wagner M. C., Brady S. T., Bloom G. S. Submolecular domains of bovine brain kinesin identified by electron microscopy and monoclonal antibody decoration. Cell. 1989 Mar 10;56(5):867–878. doi: 10.1016/0092-8674(89)90691-0. [DOI] [PubMed] [Google Scholar]
- 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]
- Hirokawa N., Sobue K., Kanda K., Harada A., Yorifuji H. The cytoskeletal architecture of the presynaptic terminal and molecular structure of synapsin 1. J Cell Biol. 1989 Jan;108(1):111–126. doi: 10.1083/jcb.108.1.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hisanaga S., Murofushi H., Okuhara K., Sato R., Masuda Y., Sakai H., Hirokawa N. The molecular structure of adrenal medulla kinesin. Cell Motil Cytoskeleton. 1989;12(4):264–272. doi: 10.1002/cm.970120407. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Leopold P. L., Snyder R., Bloom G. S., Brady S. T. Nucleotide specificity for the bidirectional transport of membrane-bounded organelles in isolated axoplasm. Cell Motil Cytoskeleton. 1990;15(4):210–219. doi: 10.1002/cm.970150404. [DOI] [PubMed] [Google Scholar]
- 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]
- Miller R. H., Lasek R. J. Cross-bridges mediate anterograde and retrograde vesicle transport along microtubules in squid axoplasm. J Cell Biol. 1985 Dec;101(6):2181–2193. doi: 10.1083/jcb.101.6.2181. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Pfister K. K., Wagner M. C., Bloom G. S., Brady S. T. Modification of the microtubule-binding and ATPase activities of kinesin by N-ethylmaleimide (NEM) suggests a role for sulfhydryls in fast axonal transport. Biochemistry. 1989 Nov 14;28(23):9006–9012. doi: 10.1021/bi00449a008. [DOI] [PubMed] [Google Scholar]
- 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]
- Schnapp B. J., Reese T. S. Dynein is the motor for retrograde axonal transport of organelles. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1548–1552. doi: 10.1073/pnas.86.5.1548. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Scholey J. M., Heuser J., Yang J. T., Goldstein L. S. Identification of globular mechanochemical heads of kinesin. Nature. 1989 Mar 23;338(6213):355–357. doi: 10.1038/338355a0. [DOI] [PubMed] [Google Scholar]
- Schroer T. A., Steuer E. R., Sheetz M. P. Cytoplasmic dynein is a minus end-directed motor for membranous organelles. Cell. 1989 Mar 24;56(6):937–946. doi: 10.1016/0092-8674(89)90627-2. [DOI] [PubMed] [Google Scholar]
- Smith D. S. On the significance of cross-bridges between microtubules and synaptic vesicles. Philos Trans R Soc Lond B Biol Sci. 1971 Jun 17;261(839):395–405. doi: 10.1098/rstb.1971.0074. [DOI] [PubMed] [Google Scholar]
- Smith R. S. Studies on the mechanism of the reversal of rapid organelle transport in myelinated axons of Xenopus laevis. Cell Motil Cytoskeleton. 1988;10(1-2):296–308. doi: 10.1002/cm.970100134. [DOI] [PubMed] [Google Scholar]
- Smith R. S. The short term accumulation of axonally transported organelles in the region of localized lesions of single myelinated axons. J Neurocytol. 1980 Feb;9(1):39–65. doi: 10.1007/BF01205226. [DOI] [PubMed] [Google Scholar]
- Tokuyasu K. T. Immunochemistry on ultrathin frozen sections. Histochem J. 1980 Jul;12(4):381–403. doi: 10.1007/BF01011956. [DOI] [PubMed] [Google Scholar]
- 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]
- Tsukita S., Ishikawa H. The movement of membranous organelles in axons. Electron microscopic identification of anterogradely and retrogradely transported organelles. J Cell Biol. 1980 Mar;84(3):513–530. doi: 10.1083/jcb.84.3.513. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Walker R. A., Salmon E. D., Endow S. A. The Drosophila claret segregation protein is a minus-end directed motor molecule. Nature. 1990 Oct 25;347(6295):780–782. doi: 10.1038/347780a0. [DOI] [PubMed] [Google Scholar]