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. 1993 Mar 1;120(5):1177–1186. doi: 10.1083/jcb.120.5.1177

Do photobleached fluorescent microtubules move?: re-evaluation of fluorescence laser photobleaching both in vitro and in growing Xenopus axon

PMCID: PMC2119730  PMID: 7679673

Abstract

We previously documented differences in the behavior of microtubules in growing axons of two types of neurons, adult mouse sensory neurons and Xenopus embryonal spinal cord neurons. Namely, the bulk of microtubules was stationary in mouse sensory neurons both by the method of photoactivation of caged-fluorescein-labeled tubulin and photobleaching of fluorescein-labeled tubulin, but the bulk of microtubules did translocate anterogradely by the method of photoactivation. Although these results indicated that the stationary nature of photobleached microtubules in mouse neurons is not an artifact derived from the high levels of energy required for the procedure, it has not yet been settled whether the photobleaching method can detect the movement of microtubules properly. Here we report photobleaching experiments on growing axons of Xenopus embryonal neurons. Anterograde movement of photobleached microtubules was observed at a frequency and translocation rate similar to the values determined by the method of photoactivation. Our results suggest that, under appropriate conditions, the photobleaching method is able to reveal the behavior of microtubules as accurately as the photoactivation method.

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Selected References

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  1. Axelrod D., Koppel D. E., Schlessinger J., Elson E., Webb W. W. Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J. 1976 Sep;16(9):1055–1069. doi: 10.1016/S0006-3495(76)85755-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Gorbsky G. J., Sammak P. J., Borisy G. G. Microtubule dynamics and chromosome motion visualized in living anaphase cells. J Cell Biol. 1988 Apr;106(4):1185–1192. doi: 10.1083/jcb.106.4.1185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Herman B. Resonance energy transfer microscopy. Methods Cell Biol. 1989;30:219–243. doi: 10.1016/s0091-679x(08)60981-4. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Kellogg D. R., Mitchison T. J., Alberts B. M. Behaviour of microtubules and actin filaments in living Drosophila embryos. Development. 1988 Aug;103(4):675–686. doi: 10.1242/dev.103.4.675. [DOI] [PubMed] [Google Scholar]
  8. Kishino A., Yanagida T. Force measurements by micromanipulation of a single actin filament by glass needles. Nature. 1988 Jul 7;334(6177):74–76. doi: 10.1038/334074a0. [DOI] [PubMed] [Google Scholar]
  9. Kreis T. E., Geiger B., Schlessinger J. Mobility of microinjected rhodamine actin within living chicken gizzard cells determined by fluorescence photobleaching recovery. Cell. 1982 Jul;29(3):835–845. doi: 10.1016/0092-8674(82)90445-7. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Lim S. S., Edson K. J., Letourneau P. C., Borisy G. G. A test of microtubule translocation during neurite elongation. J Cell Biol. 1990 Jul;111(1):123–130. doi: 10.1083/jcb.111.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Mitchison T. J. Polewards microtubule flux in the mitotic spindle: evidence from photoactivation of fluorescence. J Cell Biol. 1989 Aug;109(2):637–652. doi: 10.1083/jcb.109.2.637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Okabe S., Hirokawa N. Actin dynamics in growth cones. J Neurosci. 1991 Jul;11(7):1918–1929. doi: 10.1523/JNEUROSCI.11-07-01918.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Okabe S., Hirokawa N. Axonal transport. Curr Opin Cell Biol. 1989 Feb;1(1):91–97. doi: 10.1016/s0955-0674(89)80043-2. [DOI] [PubMed] [Google Scholar]
  15. Okabe S., Hirokawa N. Differential behavior of photoactivated microtubules in growing axons of mouse and frog neurons. J Cell Biol. 1992 Apr;117(1):105–120. doi: 10.1083/jcb.117.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Reinsch S. S., Mitchison T. J., Kirschner M. Microtubule polymer assembly and transport during axonal elongation. J Cell Biol. 1991 Oct;115(2):365–379. doi: 10.1083/jcb.115.2.365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. 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]
  22. 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]
  23. Schulze E., Kirschner M. New features of microtubule behaviour observed in vivo. Nature. 1988 Jul 28;334(6180):356–359. doi: 10.1038/334356a0. [DOI] [PubMed] [Google Scholar]
  24. Simon J. R., Gough A., Urbanik E., Wang F., Lanni F., Ware B. R., Taylor D. L. Analysis of rhodamine and fluorescein-labeled F-actin diffusion in vitro by fluorescence photobleaching recovery. Biophys J. 1988 Nov;54(5):801–815. doi: 10.1016/S0006-3495(88)83018-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Theriot J. A., Mitchison T. J. Actin microfilament dynamics in locomoting cells. Nature. 1991 Jul 11;352(6331):126–131. doi: 10.1038/352126a0. [DOI] [PubMed] [Google Scholar]
  27. 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]

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