Advertisement

Cell and Tissue Biology

, Volume 1, Issue 3, pp 225–234 | Cite as

Free and centrosome-attached microtubules: Quantitative analysis and modeling of two-component system

  • K. M. Smurova
  • I. B. Alieva
  • I. A. Vorobjev
Article

Abstract

In cultivated in vitro interphase animal cells, microtubules form a network whose density is highest in the central cell area, in the region of centrosome, and decreases towards the cell periphery. Since identification of individual microtubules in the central cell area is significantly difficult and more often is impossible, there are several approaches to studying microtubules in the internal cell cytoplasm. These approaches are based on a decrease of microtubule density—both real, due to their partial depolymerization (by the action of cold temperatures or cytostatics), or apparent, due to a decrease of cell thickness (by photobleaching of preexisting microtubules and analysis of newly formed ones). In the present work, we propose a method based on the determination of optical density which allows evaluation of the state of the cytoplasmic microtubule system as a whole. The method consists of a comparison of the dependences describing changes of the microtubule optical density from the cell center to the periphery in controls and in experiments. Analysis of living cells by the proposed method has shown that the character of curves describing the decrease of optical density from the cell center to its periphery is different for various cell types; the dependence can be described both as an exponential regression (the CHO cell line) and as a linear regression (the NIH-3T3 and REF cell lines). Our previous studies have allowed the suggestion that the character of the dependence is determined by the ratio of free and centrosome-attached microtubules and by the position of their ends in the cell cytoplasm. To test this hypothesis, we considered model systems with all microtubules assumed to be in a straight orientation and divergent radially from the centrosome, but with different arrangements of plus-and minus-ends. In the model system, in which all the microtubule minus-ends are attached to the centrosome while the plus-ends are at different distances from it, the microtubule density is described by the exponential (f(x) = ae bx ). Introduction of free microtubules into the system leads to a change of the character of this dependence, and the system in which the concentration of free microtubules with minus ends located at different distances from the cytoplasm is 5 times higher than that of the centrosome-attached microtubules is described by the linear regression equation (f(x) = k * x + b), which corresponds to the experimentally obtained dependences for 3T3 and REF cells. Thus, we believe that even in cells with a radial microtubule system, free microtubules may constitute the majority.

Kew words

centrosome centrosome-attached microtubules free microtubules distribution of microtubules in the cell videomicroscopy 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, JD., The Cytoskeleton, Molecular Biology of the Cell, New York: Garland, 2002.Google Scholar
  2. Alieva, I.B. and Vorobjev, I.A., Interphase Microtubules in Cultured Cells: Long or Short? Membr. Cell Biol., 2000, vol.14, no. 1, pp. 57–67.PubMedGoogle Scholar
  3. Alieva, I.B. and Vorobjev, I.A., Centrosome Behavior under the Action of Uncoupler and the Effect of Disruption of Cytoskeleton Elements on the Uncoupler-induced Alterations of the Centrosome, Struct. Biol., 1994, vol. 113, pp. 217–224.CrossRefGoogle Scholar
  4. Badley, R.A., Woods, A., Carruthers, L., and Rees, D.A., Cytoskeleton Changes in Fibroblast Adhesion and Detachment, J Cell Sci., 1980, vol. 43, pp. 379–390.PubMedGoogle Scholar
  5. Borisy, G.G. and Rodionov, V.I., Lessons from the Melanophore, FASEB, 1999, vol. 13,suppl. 2, pp. S221–S224.Google Scholar
  6. Brinkley, B.R., Fuller, E.M., and Highfield, D.P., Cytoplasmic Microtubules in Normal and Transformed Cells in Culture: Analysis by Tubulin Antibody Immunofluorescence, Proc. Natl. Acad. Sci. USA, 1975, vol. 72, no. 12, pp. 4981–4985.PubMedCrossRefGoogle Scholar
  7. Brinkley, B.R., Wible, L.J., Asch, B.B., Medina, D., Mace, M.M., Beall, P.T., and Cailleau, R.M., The Microtubule Cytoskeleton in Normal and Transformed Cells in vitro, Results Probl. Cell Differ., 1980, vol. 11, pp. 132–138.PubMedGoogle Scholar
  8. Chernobelskaya, O.A., Alieva, I.B., and Vorobjev, I.A., Dynamics of Recovery of Microtubules in the Cell: A Fast Growth from Centrosome and A Slow Recovery of Free Microtubules, Tsitologiya, 2004, vol. 46, vol. 6, pp. 531–544.Google Scholar
  9. Cytrynbaum, E.N., Rodionov, V., and Mogilner, A., Computational Model of Dynein-dependent Self-organization of Microtubule Asters, J. Cell Sci., 2004, vol. 117, no. 8, pp. 1381–1397.PubMedCrossRefGoogle Scholar
  10. Danowski, B.A., Microtubule Dynamics in Serum-starved and Serum-stimulated Swiss 3T3 Mouse Fibroblasts: Implications for the Relationship between Serum-induced Contractility and Microtubules, Cell. Motil. Cytoskel., 1998, vol. 40, no. 1, pp. 1–12.CrossRefGoogle Scholar
  11. De Brabander, M., Geuens, G., Nuydens, R., Willebrords, R., Aerts, F., and De Mey, J., 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, vol. 101, pp. 215–274.PubMedGoogle Scholar
  12. De Harven, E., Electron Microscope Study of Human Leukemic Cells in Tissue Culture, Bibl. Haematol., 1968, vol. 30, pp. 300–302.PubMedGoogle Scholar
  13. Dhamodharan, R. and Wadsworth, P., Modulation of Microtubule Dynamic Instability in vivo by Brain Microtubule Associated Proteins, J. Cell Sci., 1995, vol. 108, no. 4, pp. 1679–1689.PubMedGoogle Scholar
  14. Gudima, G.O., Vorobjev, I.A., and Chentsov, Yu.S., Centriolar Location during Blood Cell Spreading and Motion in vitro: an Ultrastructural Analysis, J. Cell Sci., 1988, vol. 89, no. 2, pp. 225–241.PubMedGoogle Scholar
  15. Keating, T.J., Peloquin, J.G., Rodionov, V.I., Momcilovic, D., and Borisy, G.G., Microtubule Release from the Centrosome, Proc. Natl. Acad. Sci. USA, 1997, vol. 94, no. 10, pp. 5078–5083.PubMedCrossRefGoogle Scholar
  16. Keryer, G., Ris, H., and Borisy, G.G., Centriole Distribution during Tripolar Mitosis in Chinese Hamster Ovary Cells, J. Cell Biol., 1984, vol. 98, no. 6, pp. 2222–2229.PubMedCrossRefGoogle Scholar
  17. Komarova, Y.A., Vorobjev, I.A., and Borisy, G.G., Life Cycle of MTs: Persistent Growth in the Cell Interior; Asymmetric Transition Frequencies and Effects of Cell Boundary, J. Cell Sci., 2002, vol. 115, no. 17, pp. 3517–3539.Google Scholar
  18. Ledbetter, M.C. and Porter, K.R., A Microtubule in Plant Cells: Fine Structure, J. Cell Biol., 1963, vol. 19, pp. 239–258.CrossRefGoogle Scholar
  19. Malikov, V., Cytrynbaum, E.N., Kashina, A., Mogilner, A., and Rodionov, V., Centering of a Radial Microtubule Array by Translocation along Microtubules Spontaneously Nucleated in the Cytoplasm, Nat. Cell Biol., 2005, vol. 7, no. 12, pp. 1213–1218.PubMedCrossRefGoogle Scholar
  20. Maly, I.V. and Borisy, G.G., Self-organization of Treadmilling Microtubules into a Polar Array Trends Cell Biol., 2002, vol. 12, no. 10, pp. 462–465.PubMedCrossRefGoogle Scholar
  21. Mikhailov, A.V. and Gundersen, G.G., Centripetal Transport of Microtubules in Motile Cells, Cell Motil. Cytoskel., 1995, vol. 32, pp. 173–186.CrossRefGoogle Scholar
  22. Mimori-Kiyosue, Y., Grigoriev, I., Lansbergen, G., Sasaki, H., Matsui, C., Severin, F., Galjart, N., Grosveld, F., Vorobiev, I., Tsukita, S., and Akhmanova, A., CLASP1 and CLASP2 Bind to EB1 and Regulate Microtubule Plus End Dynamics at the Cell Cortex, J Cell Biol., 2005, vol. 68, no. 1, pp. 141–153.Google Scholar
  23. Mogensen, M., Microtubule Organizing Centers in Polarized Epithelial Cells, Centrosome in Development and Disease, WILEY-VCH: Weinheim, 2004.Google Scholar
  24. Moudjou, M., Bordes, N., Paintrand, M., and Bomens, M., Gamma-Tubulin in Mammalian Cells: The Centrosomal and The Cytosolic Forms, J. Cell Sci., 1996, vol. 109, no. 4, pp. 875–887.PubMedGoogle Scholar
  25. Osborn, M., Born, T., Koitsch, H.J., and Weber, K., Stereo Immunofluorescence Microscopy: I. Three-dimensional Arrangement of Microfilaments, Microtubules and Tonofilaments, Cell, 1978, vol. 14, no. 3, pp. 477–488.PubMedCrossRefGoogle Scholar
  26. Osborn, M. and Weber, K., The Display of Microtubules in Transformed cells, Cell, 1977, vol. 12, no. 3, pp. 561–571.PubMedCrossRefGoogle Scholar
  27. Porter, K.R., Cytoplasmic Microtubules and Their Function, Ciba Found. Symp., 1966, vol. 8, pp. 308–356.Google Scholar
  28. Rodionov, V.I. and Borisy, G.G., Self-centering Activity of Cytoplasm, Nature, 1997a, vol. 386, no. 6621, pp. 170–173.PubMedCrossRefGoogle Scholar
  29. Rodionov, V.I. and Borisy, G.G., Microtubule Treadmilling in vivo, Science, 1997b, vol. 275, no. 5297, pp. 215–218.PubMedCrossRefGoogle Scholar
  30. Rodionov, V., Nadezhdina, E., and Borisy, G., Centrosomal Control of Microtubule Dynamics, Proc. Natl. Acad. Sci. USA, 1999, vol. 96, no. 1, pp. 115–120.PubMedCrossRefGoogle Scholar
  31. Rusan, N.M., Fagerstrom, C.J., Yvon, A.M., and Wadsworth, P., Cell Cycle-dependent Changes in Microtubule Dynamics in Living Cells Expressing Green Fluorescent Protein-alpha Tubulin, Mol. Biol. Cell, 2001, vol. 12, no. 4, pp. 971–980.PubMedGoogle Scholar
  32. Schliwa, M., Microtubular Apparatus of Melanophores. Three-dimensional Organization, J. Cell Biol., 1978, vol. 76, no. 3, pp. 605–614.PubMedCrossRefGoogle Scholar
  33. Schliwa, M., Euteneuer, U., Herzog, W., and Weber, K., Evidence for Rapid Structural and Functional Changes of the Melanophore Microtubule-organizing Center upon Pigment Movements, J. Cell Biol., 1979, vol. 83, no. 3, pp. 623–632.PubMedCrossRefGoogle Scholar
  34. Shelden, E. and Wadsworth, P., Observation and Quantification of Individual Microtubule Behavior in vivo: Microtubule Dynamics are Cell Type Specific, J. Cell Biol., 1993, vol. 120, pp. 935–945.PubMedCrossRefGoogle Scholar
  35. Smurova, K.M., Alieva, I.B., and Vorobjev, I.A., Dynamics of Recovery of Cytoplasmic Microtubules after Their Destruction with Nocodazol in Cells of the Vero Culture, Biolog. Membr., 2002, vol. 19, no. 6, pp. 472–482.Google Scholar
  36. Smurova, K.M., Biryukova, A.A., Garcia, J., Vorobjev, I.A., Alieva, I.B., and Verin, A.D., Reorganization of Microtubular System in Pulmonary Endothelial Cells in Response to Action of Thrombin, Tsitologiya, 2004, vol. 46, no. 8, pp. 695–703.Google Scholar
  37. Stubblefield, E. and Brinkley, B.R., Cilia Formation in Chinese Hamster Fibroblasts in vitro as a Response to Colcemid Treatment, J. Cell Biol., 1966, vol. 30, no. 3, pp. 645–652.PubMedCrossRefGoogle Scholar
  38. Svitkina, T.M. and Borisy, G.G., Correlative Light and Electron Microscopy of the Cytoskeleton of Cultured Cells, Methods Enzymol., 1998, vol. 298, pp. 570–592.PubMedCrossRefGoogle Scholar
  39. Vidair, C.A., Doxsey, S.J., and Dewey, W.C., Heat Shock Alters Centrosome Organization Leading to Mitotic Dysfunction and Cell Death, J. Cell Physiol., 1993, vol. 154, no. 3, pp. 443–455.PubMedCrossRefGoogle Scholar
  40. Vorobjev, I.A., Alieva, I.B., Grigoriev, I.S., and Borisy, G.G., Microtubule Dynamics in Living Cells: Direct Analysis in the Internal Cytoplasm, Cell Biol. Int., 2003, vol. 27, no. 3, pp. 293–294.PubMedCrossRefGoogle Scholar
  41. Vorobjev, I., Malikov, V., and Rodionov, V., Self-organization of a Radial Microtubule Array by Dynein-dependent Nucleation of Microtubules, Proc. Natl. Acad. Sci. USA, 2001, vol. 98, no. 18, pp. 10 160–10 165.CrossRefGoogle Scholar
  42. Vorobjev, I.A., Rodionov, V.I., Maly, I.V., and Borisy, G.G., Contribution of Plus and Minus End Pathways to Microtubule Turnover J. Cell Sci., 1999, vol. 112, no. 14, pp. 2277–2289.PubMedGoogle Scholar
  43. Vorobjev I.A., Svitkina T.M., and Borisy, G.G., Cytoplasmic Assembly of Microtubules in Cultured Cells, J. Cell Sci., 1997, vol. 110, no. 21, pp. 2635–2645.PubMedGoogle Scholar
  44. Wadsworth, P., Regional Regulation of Microtubule Dynamics in Polarized, Motile Cells, Cell Motil. Cytoskel., 1999, vol. 42, no. 1, pp. 48–59.CrossRefGoogle Scholar
  45. Waterman-Storer, C.M. and Salmon, E.D., Microtubule Dynamics: Treadmilling Comes Around Again, Curr. Biol., 1997, vol. 7, R369–R372.PubMedCrossRefGoogle Scholar
  46. Weber, K., Pollack, R., and Bibring, T., Antibody against Tubulin: the Specific Visualization of Cytoplasmic Microtubules in Tissue Culture Cells, Proc. Natl. Acad. Sci. USA, 1975, vol. 72, no. 2, pp. 459–463.PubMedCrossRefGoogle Scholar
  47. Yvon, A.M., Wadsworth, P., and Jordan, M.A., Taxol Suppresses Dynamics of Individual Microtubules in Living Human Tumor Cells, Mol. Biol. Cell, 1999, vol. 10, no. 4, pp. 947–959.PubMedGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2007

Authors and Affiliations

  • K. M. Smurova
    • 1
  • I. B. Alieva
    • 1
  • I. A. Vorobjev
    • 1
    • 2
  1. 1.Belozersky Institute of Physicochemical BiologyMoscow State UniversityMoscowRussia
  2. 2.Department of Cell Biology and HistologyMoscow State UniversityMoscowRussia

Personalised recommendations