Abstract
In a previous study using PC-12 cells (Lim, S. S., P. J. Sammak, and G. G. Borisy, 1989. J. Cell Biol. 109:253-263), we presented evidence that the microtubule component of the neuronal cytoskeleton is differentially dynamic but stationary. However, neurites of PC-12 cells grow slowly, hindering a stringent test of slow axonal transport mechanisms under conditions where growth was substantial. We therefore extended our studies to primary cultures of dorsal root ganglion cells where the rate of neurite outgrowth is rapid. Cells were microinjected with X-rhodamine-labeled tubulin 7-16 h after plating. After a further incubation for 6-18 h, the cells were photobleached with an argon ion laser. Using a cooled charged couple device and video microscopy, the cells were monitored for growth of the neurite and movement and recovery of fluorescence in the bleached zone. As for PC-12 cells, all bleached zones in the neurite recovered their fluorescence, indicating that incorporation of tubulin occurred along the neurite. Despite increases in neurite length of up to 70 microns, and periods of observation of up to 5 h, no movement of bleached zones was observed. We conclude that neurite elongation cannot be accounted for by the transport of a microtubule network assembled only at the cell body. Rather, microtubules turn over all along the length of the neurite and neurite elongation occurs by net assembly at the tip.
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Selected References
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- Bamburg J. R., Bray D., Chapman K. Assembly of microtubules at the tip of growing axons. Nature. 1986 Jun 19;321(6072):788–790. doi: 10.1038/321788a0. [DOI] [PubMed] [Google Scholar]
- Black M. M., Keyser P., Sobel E. Interval between the synthesis and assembly of cytoskeletal proteins in cultured neurons. J Neurosci. 1986 Apr;6(4):1004–1012. doi: 10.1523/JNEUROSCI.06-04-01004.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Black M. M., Lasek R. J. Slow components of axonal transport: two cytoskeletal networks. J Cell Biol. 1980 Aug;86(2):616–623. doi: 10.1083/jcb.86.2.616. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bray D. Branching patterns of individual sympathetic neurons in culture. J Cell Biol. 1973 Mar;56(3):702–712. doi: 10.1083/jcb.56.3.702. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gorbsky G. J., Borisy G. G. Microtubules of the kinetochore fiber turn over in metaphase but not in anaphase. J Cell Biol. 1989 Aug;109(2):653–662. doi: 10.1083/jcb.109.2.653. [DOI] [PMC free article] [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]
- 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]
- Hollenbeck P. J. The transport and assembly of the axonal cytoskeleton. J Cell Biol. 1989 Feb;108(2):223–227. doi: 10.1083/jcb.108.2.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Keith C. H. Slow transport of tubulin in the neurites of differentiated PC12 cells. Science. 1987 Jan 16;235(4786):337–339. doi: 10.1126/science.2432662. [DOI] [PubMed] [Google Scholar]
- Lasek R. J. Polymer sliding in axons. J Cell Sci Suppl. 1986;5:161–179. doi: 10.1242/jcs.1986.supplement_5.10. [DOI] [PubMed] [Google Scholar]
- Letourneau P. C. Analysis of microtubule number and length in cytoskeletons of cultured chick sensory neurons. J Neurosci. 1982 Jun;2(6):806–814. doi: 10.1523/JNEUROSCI.02-06-00806.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Letourneau P. C. Possible roles for cell-to-substratum adhesion in neuronal morphogenesis. Dev Biol. 1975 May;44(1):77–91. doi: 10.1016/0012-1606(75)90378-4. [DOI] [PubMed] [Google Scholar]
- Lim S. S., Sammak P. J., Borisy G. G. Progressive and spatially differentiated stability of microtubules in developing neuronal cells. J Cell Biol. 1989 Jul;109(1):253–263. doi: 10.1083/jcb.109.1.253. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mitchison T., Kirschner M. Dynamic instability of microtubule growth. Nature. 1984 Nov 15;312(5991):237–242. doi: 10.1038/312237a0. [DOI] [PubMed] [Google Scholar]
- Okabe S., Hirokawa N. Microtubule dynamics in nerve cells: analysis using microinjection of biotinylated tubulin into PC12 cells. J Cell Biol. 1988 Aug;107(2):651–664. doi: 10.1083/jcb.107.2.651. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Okabe S., Hirokawa N. Turnover of fluorescently labelled tubulin and actin in the axon. Nature. 1990 Feb 1;343(6257):479–482. doi: 10.1038/343479a0. [DOI] [PubMed] [Google Scholar]
- Robson S. J., Burgoyne R. D. Differential localisation of tyrosinated, detyrosinated, and acetylated alpha-tubulins in neurites and growth cones of dorsal root ganglion neurons. Cell Motil Cytoskeleton. 1989;12(4):273–282. doi: 10.1002/cm.970120408. [DOI] [PubMed] [Google Scholar]
- Sammak P. J., Borisy G. G. Detection of single fluorescent microtubules and methods for determining their dynamics in living cells. Cell Motil Cytoskeleton. 1988;10(1-2):237–245. doi: 10.1002/cm.970100128. [DOI] [PubMed] [Google Scholar]
- Sammak P. J., Borisy G. G. Direct observation of microtubule dynamics in living cells. Nature. 1988 Apr 21;332(6166):724–726. doi: 10.1038/332724a0. [DOI] [PubMed] [Google Scholar]
- Sammak P. J., Gorbsky G. J., Borisy G. G. Microtubule dynamics in vivo: a test of mechanisms of turnover. J Cell Biol. 1987 Mar;104(3):395–405. doi: 10.1083/jcb.104.3.395. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Saxton W. M., Stemple D. L., Leslie R. J., Salmon E. D., Zavortink M., McIntosh J. R. Tubulin dynamics in cultured mammalian cells. J Cell Biol. 1984 Dec;99(6):2175–2186. doi: 10.1083/jcb.99.6.2175. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schulze E., Kirschner M. Microtubule dynamics in interphase cells. J Cell Biol. 1986 Mar;102(3):1020–1031. doi: 10.1083/jcb.102.3.1020. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Soltys B. J., Borisy G. G. Polymerization of tubulin in vivo: direct evidence for assembly onto microtubule ends and from centrosomes. J Cell Biol. 1985 May;100(5):1682–1689. doi: 10.1083/jcb.100.5.1682. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vigers G. P., Coue M., McIntosh J. R. Fluorescent microtubules break up under illumination. J Cell Biol. 1988 Sep;107(3):1011–1024. doi: 10.1083/jcb.107.3.1011. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weisenberg R. C., Flynn J., Gao B. C., Awodi S., Skee F., Goodman S. R., Riederer B. M. Microtubule gelation-contraction: essential components and relation to slow axonal transport. Science. 1987 Nov 20;238(4830):1119–1122. doi: 10.1126/science.2446388. [DOI] [PubMed] [Google Scholar]