Journal of Cell Science, Vol 107, Issue 4 881-890, Copyright © 1994 by Company of Biologists

JOURNAL ARTICLES

Quantitative determination of the proportion of microtubule polymer present during the mitosis-interphase transition

Y Zhai and GG Borisy
Laboratory of Molecular Biology, University of Wisconsin-Madison 53706.

We have developed a new method for determining levels of tubulin polymer, based on quantitative fluorescence detection of x-rhodamine tubulin microinjected into living cells and we have applied this method to analysis of the mitosis-interphase transition. LLC-PK cells in interphase and mitosis were microinjected, then cooled and rewarmed to drive tubulin incorporation. Total tubulin fluorescence in individual, living cells was quantified using a cooled, scientific grade CCD image sensor. Cells were then washed and lysed into a microtubule-stabilizing buffer to extract the soluble pool. Total tubulin polymer fluorescence was determined for the extracted cells in the same way as for living cells. Fluorescence images were corrected by flat-fielding and background subtraction. The ratio of extracted cell fluorescence/living cell fluorescence for individual cells, was taken as the proportion of tubulin as polymer. Cells in M-phase, G1 and random interphase were analyzed. G1 cells had almost the same proportion as random interphase cells. Mitotic cells gave a value of 90 +/- 5% of G1 cells at 37 degrees C. Within M-phase, levels of tubulin as polymer in metaphase and early anaphase were not significantly different. In contrast to the general expectation of microtubule depolymerization at anaphase onset, these results indicate that as cells exit mitosis, the overall proportion of tubulin as polymer does not change dramatically even though the mitotic spindle disassembles. We conclude that the mitosis-interphase transition is accompanied by a redistribution of tubulin at an essentially constant polymer level. Therefore, a global shift to depolymerization conditions is not the driving force for anaphase chromosome movement.

This article has been cited by other articles:

				Y. S. Aratyn, T. E. Schaus, E. W. Taylor, and G. G. Borisy Intrinsic Dynamic Behavior of Fascin in Filopodia Mol. Biol. Cell, October 1, 2007; 18(10): 3928 - 3940. [Abstract] [Full Text] [PDF]

				J. M. Bunker, K. Kamath, L. Wilson, M. A. Jordan, and S. C. Feinstein FTDP-17 Mutations Compromise the Ability of Tau to Regulate Microtubule Dynamics in Cells J. Biol. Chem., April 28, 2006; 281(17): 11856 - 11863. [Abstract] [Full Text] [PDF]

				H. Guo, C. Xu, C. Liu, E. Qu, M. Yuan, Z. Li, B. Cheng, and D. Zhang Mechanism and Dynamics of Breakage of Fluorescent Microtubules Biophys. J., March 15, 2006; 90(6): 2093 - 2098. [Abstract] [Full Text] [PDF]

				M. A. Jordan, K. Kamath, T. Manna, T. Okouneva, H. P. Miller, C. Davis, B. A. Littlefield, and L. Wilson The primary antimitotic mechanism of action of the synthetic halichondrin E7389 is suppression of microtubule growth Mol. Cancer Ther., July 1, 2005; 4(7): 1086 - 1095. [Abstract] [Full Text] [PDF]

				T. J. Mitchison, P. Maddox, J. Gaetz, A. Groen, M. Shirasu, A. Desai, E. D. Salmon, and T. M. Kapoor Roles of Polymerization Dynamics, Opposed Motors, and a Tensile Element in Governing the Length of Xenopus Extract Meiotic Spindles Mol. Biol. Cell, June 1, 2005; 16(6): 3064 - 3076. [Abstract] [Full Text] [PDF]

				P. Fanara, S. Turner, R. Busch, S. Killion, M. Awada, H. Turner, A. Mahsut, K. L. LaPrade, J. M. Stark, and M. K. Hellerstein In Vivo Measurement of Microtubule Dynamics Using Stable Isotope Labeling with Heavy Water: EFFECT OF TAXANES J. Biol. Chem., November 26, 2004; 279(48): 49940 - 49947. [Abstract] [Full Text] [PDF]

				J. M. Bunker, L. Wilson, M. A. Jordan, and S. C. Feinstein Modulation of Microtubule Dynamics by Tau in Living Cells: Implications for Development and Neurodegeneration Mol. Biol. Cell, June 1, 2004; 15(6): 2720 - 2728. [Abstract] [Full Text] [PDF]

				T. Wittmann, G. M. Bokoch, and C. M. Waterman-Storer Regulation of Microtubule Destabilizing Activity of Op18/Stathmin Downstream of Rac1 J. Biol. Chem., February 13, 2004; 279(7): 6196 - 6203. [Abstract] [Full Text] [PDF]

				P. Holmfeldt, G. Brattsand, and M. Gullberg Interphase and monoastral-mitotic phenotypes of overexpressed MAP4 are modulated by free tubulin concentrations J. Cell Sci., September 15, 2003; 116(18): 3701 - 3711. [Abstract] [Full Text] [PDF]

				T. Wittmann, G. M. Bokoch, and C. M. Waterman-Storer Regulation of leading edge microtubule and actin dynamics downstream of Rac1 J. Cell Biol., June 9, 2003; 161(5): 845 - 851. [Abstract] [Full Text] [PDF]

				K. I. Swenson, K. E. Winkler, and A. R. Means A New Identity for MLK3 as an NIMA-related, Cell Cycle-regulated Kinase That Is Localized near Centrosomes and Influences Microtubule Organization Mol. Biol. Cell, January 1, 2003; 14(1): 156 - 172. [Abstract] [Full Text]

				M. Caplow and L. Fee Dissociation of the Tubulin Dimer Is Extremely Slow, Thermodynamically Very Unfavorable, and Reversible in the Absence of an Energy Source Mol. Biol. Cell, June 1, 2002; 13(6): 2120 - 2131. [Abstract] [Full Text] [PDF]

				I. Vorobjev, V. Malikov, and V. Rodionov Self-organization of a radial microtubule array by dynein-dependent nucleation of microtubules PNAS, August 10, 2001; (2001) 181354198. [Abstract] [Full Text] [PDF]

				A. Khodjakov and C. L. Rieder The Sudden Recruitment of {gamma}-Tubulin to the Centrosome at the Onset of Mitosis and its Dynamic Exchange Throughout the Cell Cycle, Do Not Require Microtubules J. Cell Biol., August 9, 1999; 146(3): 585 - 596. [Abstract] [Full Text] [PDF]

				V. Rodionov, E. Nadezhdina, and G. Borisy Centrosomal control of microtubule dynamics PNAS, January 5, 1999; 96(1): 115 - 120. [Abstract] [Full Text] [PDF]

				F. J. McNally and S. Thomas Katanin Is Responsible for the M-Phase Microtubule-severing Activity in Xenopus Eggs Mol. Biol. Cell, July 1, 1998; 9(7): 1847 - 1861. [Abstract] [Full Text]

				S. Illenberger, Q. Zheng-Fischhöfer, U. Preuss, K. Stamer, K. Baumann, B. Trinczek, J. Biernat, R. Godemann, E.-M. Mandelkow, and E. Mandelkow The Endogenous and Cell Cycle-dependent Phosphorylation of tau Protein in Living Cells: Implications for Alzheimer's Disease Mol. Biol. Cell, June 1, 1998; 9(6): 1495 - 1512. [Abstract] [Full Text]

				A. Verkhovsky, T. Svitkina, and G. Borisy Polarity sorting of actin filaments in cytochalasin-treated fibroblasts J. Cell Sci., January 8, 1997; 110(15): 1693 - 1704. [Abstract] [PDF]

				H. Nguyen, S Chari, D Gruber, C. Lue, S. Chapin, and J. Bulinski Overexpression of full- or partial-length MAP4 stabilizes microtubules and alters cell growth J. Cell Sci., January 1, 1997; 110(2): 281 - 294. [Abstract] [PDF]

				E Brisch, M. Daggett, and K. Suprenant Cell cycle-dependent phosphorylation of the 77 kDa echinoderm microtubule-associated protein (EMAP) in vivo and association with the p34cdc2 kinase J. Cell Sci., January 12, 1996; 109(12): 2885 - 2893. [Abstract] [PDF]

				R Dhamodharan and P Wadsworth Modulation of microtubule dynamic instability in vivo by brain microtubule associated proteins J. Cell Sci., January 4, 1995; 108(4): 1679 - 1689. [Abstract] [PDF]

				N. Kourmouli, G. Dialynas, C. Petraki, A. Pyrpasopoulou, P. B. Singh, S. D. Georgatos, and P. A. Theodoropoulos Binding of Heterochromatin Protein 1 to the Nuclear Envelope Is Regulated by a Soluble Form of Tubulin J. Biol. Chem., April 13, 2001; 276(16): 13007 - 13014. [Abstract] [Full Text] [PDF]

				B. Eichenmuller, P. Everley, J. Palange, D. Lepley, and K. A. Suprenant The Human EMAP-like Protein-70 (ELP70) Is a Microtubule Destabilizer That Localizes to the Mitotic Apparatus J. Biol. Chem., January 4, 2002; 277(2): 1301 - 1309. [Abstract] [Full Text] [PDF]

				I. Vorobjev, V. Malikov, and V. Rodionov Self-organization of a radial microtubule array by dynein-dependent nucleation of microtubules PNAS, August 28, 2001; 98(18): 10160 - 10165. [Abstract] [Full Text] [PDF]