Home LiteratureArticle Details
PMID: 2646309 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Microtubule dynamics investigated by microinjection of Paramecium axonemal tubulin: lack of nucleation but proximal assembly of microtubules at the kinetochore during prometaphase.

The Journal of cell biology ·Vol. 108 ·No. 3 ·1989-03-00 ·Pages 939-53

Geuens G, Hill AM, Levilliers N, Adoutte A, DeBrabander M

Abstract

Microtubule (MT) dynamics in PtK2 cells have been investigated using in vivo injection of unmodified Paramecium ciliary tubulin and time-lapse fixation. The sites of incorporation of the axonemal tubulin were localized using a specific antibody which does not react with vertebrate cytoplasmic tubulin (Adoutte, A., M. Claisse, R. Maunoury, and J. Beisson. 1985. J. Mol. Evol. 22:220-229), followed by immunogold labeling, Nanovid microscopy, and ultrastructural observation of the same cells. We confirm data from microinjection of labeled tubulins in other cell types (Soltys, B. J., and G. G. Borisy. 1985. J. Cell Biol. 100:1682-1689; Mitchison, T., L. Evans, E. Schulze, and M. Kirschner. 1986. Cell. 45:515-527; Schulze, E., and M. Kirschner. 1986. J. Cell Biol. 102:1020-1031). In agreement with the dynamic instability model (Mitchison, T., and M. Kirschner. 1984. Nature (Lond.). 312:237-242), during interphase, fast (2.6 microns/min) distal growth of MTs occurs, together with new centrosomal nucleation. Most of the cytoplasmic MT complex is replaced within 15-30 min. During mitosis, astral MTs display the same pattern of renewal, but the turnover of the MT system is much faster (approximately 6 min). We have concentrated on the construction of the kinetochore fibers during prometaphase and observe that (a) incorporation of tubulin in the vicinity of the kinetochores is not seen during prophase and early prometaphase as long as the kinetochores are not yet connected to a pole by MTs; (b) proximal time-dependent incorporation occurs only into preexisting kinetochore MTs emanating from centrosomes. Consequently, in undisturbed prometaphase cells, the kinetochores probably do not act as independent nucleation sites. This confirms a model in which, at prometaphase, fast probing centrosomal MTs are grabbed by the kinetochores, where tubulin incorporation then takes place.

MeSH Terms
Animals Cell Line Centromere/metabolism,ultrastructure Chromosomes/metabolism Fluorescent Antibody Technique Immunohistochemistry Interphase Metaphase Microinjections Microscopy, Electron Microtubules/metabolism,ultrastructure Paramecium Prophase Spindle Apparatus/ultrastructure Tubulin/metabolism
Chemicals
Tubulin
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Geuens G
Department of Cellular Biology and Pathology, Janssen Research Foundation, Beerse, Belgium.
Hill A M
Levilliers N
Adoutte A
DeBrabander M
References (54)
54 references, click to expand
  1. Microtubule dynamics in interphase cells.
    J Cell Biol. 1986 Mar;102(3):1020-31 PMID: 3512576
  2. An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy.
    J Cell Biol. 1987 Jul;105(1):41-8 PMID: 3112165
  3. Microtubule dynamics during the cell cycle: the effects of taxol and nocodazole on the microtubule system of Pt K2 cells at different stages of the mitotic cycle.
    Int Rev Cytol. 1986;101:215-74 PMID: 2870994
  4. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
    Nature. 1970 Aug 15;227(5259):680-5 PMID: 5432063
  5. Structural polarity and directional growth of microtubules of Chlamydomonas flagella.
    J Mol Biol. 1974 Dec 5;90(2):381-402 PMID: 4476803
  6. Assembly of chick brain tubulin onto flagellar microtubules from Chlamydomonas and sea urchin sperm.
    Proc Natl Acad Sci U S A. 1975 Mar;72(3):1122-6 PMID: 1055370
  7. Immunofluorescence of mitotic spindles by using monospecific antibody against bovine brain tubulin.
    Science. 1975 Mar 14;187(4180):948-50 PMID: 1096300
  8. Light and electron microscopy of rat kangaroo cells in mitosis. III. Patterns of chromosome behavior during prometaphase.
    Chromosoma. 1976 Mar 10;54(4):363-85 PMID: 1253643
  9. Tubulin-like protein from Aspergillus nidulans.
    Biochem Biophys Res Commun. 1976 Mar 22;69(2):285-90 PMID: 773373
  10. Identification of alpha and beta tubulin in yeast.
    Biochem Biophys Res Commun. 1976 Jun 7;70(3):704-8 PMID: 779780
  11. In vitro polymerization of flagellar and ciliary outer fiber tubulin into microtubules.
    J Biochem. 1976 Jul;80(1):153-65 PMID: 184079
  12. Induction of microtubule protein synthesis in Chlamydomonas reinhardi during flagellar regeneration.
    Cell. 1976 Sep;9(1):15-27 PMID: 975238
  13. Characterization and in vitro polymerization of Tetrahymena tubulin.
    J Biochem. 1978 Apr;83(4):1065-75 PMID: 659381
  14. Microtubule reassembly in vitro of Strongylocentrotus purpuratus sperm tail outer doublet tubulin.
    J Mol Biol. 1978 May 25;121(3):393-410 PMID: 671544
  15. Radioimmunoassay for tubulin: a quantitative comparison of the tubulin content of different established tissue culture cells and tissues.
    Cell. 1978 Aug;14(4):795-804 PMID: 688394
  16. The in vitro assembly of flagellar outer doublet tubulin.
    J Cell Biol. 1978 Nov;79(2 Pt 1):500-15 PMID: 569158
  17. Characterization of the in vitro reassembly of tubulin derived from stable Strongylocentrotus purpuratus outer doublet microtubules.
    Biochemistry. 1979 Mar 6;18(5):905-11 PMID: 33707
  18. Programmed appearance of translatable flagellar tubulin mRNA during cell differentiation in Naegleria.
    Cell. 1979 Aug;17(4):867-78 PMID: 487433
  19. 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):4350-4 PMID: 388439
  20. Biochemical studies of the excitable membrane of Paramecium tetraurelia. III. Proteins of cilia and ciliary membranes.
    J Cell Biol. 1980 Mar;84(3):717-38 PMID: 7358796
  21. Origin of kinetochore microtubules in Chinese hamster ovary cells.
    Chromosoma. 1980;81(3):483-505 PMID: 7449572
  22. Direct visualization of fluorescein-labeled microtubules in vitro and in microinjected fibroblasts.
    J Cell Biol. 1981 Jan;88(1):234-40 PMID: 7193677
  23. Reassembly of flagellar B (alpha beta) tubulin into singlet microtubules: consequences for cytoplasmic microtubule structure and assembly.
    J Cell Biol. 1981 May;89(2):323-37 PMID: 7251656
  24. Structural polarity of kinetochore microtubules in PtK1 cells.
    J Cell Biol. 1981 May;89(2):338-45 PMID: 7251657
  25. Decoration of spindle microtubules with Dynein: evidence for uniform polarity.
    J Cell Biol. 1981 May;89(2):373-8 PMID: 6454693
  26. Multiple forms of tubulin in the cytoskeletal and flagellar microtubules of Polytomella.
    J Cell Biol. 1981 Nov;91(2 Pt 1):352-60 PMID: 7309786
  27. A dot-immunobinding assay for monoclonal and other antibodies.
    Anal Biochem. 1982 Jan 1;119(1):142-7 PMID: 7072935
  28. Improved system for capillary microinjection into living cells.
    Exp Cell Res. 1982 Jul;140(1):31-7 PMID: 6286332
  29. Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line.
    J Cell Biol. 1982 Jun;93(3):576-82 PMID: 6811596
  30. A model for the microtubule organizing activity of the centrosomes and kinetochores in mammalian cells.
    Cell Biol Int Rep. 1982 Oct;6(10):901-15 PMID: 7139723
  31. Nucleated assembly of mitotic microtubules in living PTK2 cells after release from nocodazole treatment.
    Cell Motil. 1981;1(4):469-83 PMID: 7348606
  32. Rethinking mitosis.
    Cell. 1982 Jul;29(3):729-44 PMID: 6217897
  33. The formation, structure, and composition of the mammalian kinetochore and kinetochore fiber.
    Int Rev Cytol. 1982;79:1-58 PMID: 6185450
  34. Tubulin evolution: an electrophoretic and immunological analysis.
    Orig Life. 1984 Mar;13(3-4):177-82 PMID: 6203079
  35. Spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching.
    J Cell Biol. 1984 Dec;99(6):2165-74 PMID: 6501418
  36. Tubulin dynamics in cultured mammalian cells.
    J Cell Biol. 1984 Dec;99(6):2175-86 PMID: 6501419
  37. Microtubule assembly nucleated by isolated centrosomes.
    Nature. 1984 Nov 15-21;312(5991):232-7 PMID: 6504137
  38. Dynamic instability of microtubule growth.
    Nature. 1984 Nov 15-21;312(5991):237-42 PMID: 6504138
  39. Polymerization of tubulin in vivo: direct evidence for assembly onto microtubule ends and from centrosomes.
    J Cell Biol. 1985 May;100(5):1682-9 PMID: 3886672
  40. Properties of the kinetochore in vitro. I. Microtubule nucleation and tubulin binding.
    J Cell Biol. 1985 Sep;101(3):755-65 PMID: 4030893
  41. Properties of the kinetochore in vitro. II. Microtubule capture and ATP-dependent translocation.
    J Cell Biol. 1985 Sep;101(3):766-77 PMID: 4030894
  42. Tubulin evolution: ciliate-specific epitopes are conserved in the ciliary tubulin of Metazoa.
    J Mol Evol. 1985;22(3):220-9 PMID: 2416941
  43. A chicken-yeast chimeric beta-tubulin protein is incorporated into mouse microtubules in vivo.
    Cell. 1986 Feb 14;44(3):461-8 PMID: 3753663
  44. Direct observation of steady-state microtubule dynamics.
    J Cell Biol. 1986 Mar;102(3):1007-19 PMID: 3005332
  45. Sites of microtubule assembly and disassembly in the mitotic spindle.
    Cell. 1986 May 23;45(4):515-27 PMID: 3708686
  46. The use of submicroscopic gold particles combined with video contrast enhancement as a simple molecular probe for the living cell.
    Cell Motil Cytoskeleton. 1986;6(2):105-13 PMID: 3011284
  47. Visualization of the dynamic instability of individual microtubules by dark-field microscopy.
    Nature. 1986 Jun 5-11;321(6070):605-7 PMID: 3713844
  48. Evolutionary aspects of tubulin structure.
    Ann N Y Acad Sci. 1986;466:8-12 PMID: 3460454
  49. Chromosomes move poleward in anaphase along stationary microtubules that coordinately disassemble from their kinetochore ends.
    J Cell Biol. 1987 Jan;104(1):9-18 PMID: 3793763
  50. Direct observation of microtubule dynamics in living cells.
    Nature. 1988 Apr 21;332(6166):724-6 PMID: 3357537
  51. Protein measurement with the Folin phenol reagent.
    J Biol Chem. 1951 Nov;193(1):265-75 PMID: 14907713
  52. Cell motility by labile association of molecules. The nature of mitotic spindle fibers and their role in chromosome movement.
    J Gen Physiol. 1967 Jul;50(6):Suppl:259-92 PMID: 6058222
  53. Microtubule dynamics in vivo: a test of mechanisms of turnover.
    J Cell Biol. 1987 Mar;104(3):395-405 PMID: 3546333
  54. Analysis of the treadmilling model during metaphase of mitosis using fluorescence redistribution after photobleaching.
    J Cell Biol. 1986 Mar;102(3):1032-8 PMID: 3949871
Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.108.3.939
Published
1989-03-00
Pages
939-53
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2115398
Subset
IM
External Links
PubMed source DOI: 10.1083/jcb.108.3.939 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: [email protected]

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2