Live-cell Analysis of Mitotic Spindle Formation in Taxol-treated Cells

Overview

Journal Cell Motil Cytoskeleton

Specialty Cell Biology

Date 2008 May 16

PMID 18481305

Citations 35

Authors

Jessica E Hornick

Jason R Bader

Emily K Tribble

Kayleigh Trimble

J Scott Breunig

Elizabeth S Halpin

Kevin T Vaughan

Edward H Hinchcliffe

Affiliations

Soon will be listed here.

Abstract

Taxol functions to suppress the dynamic behavior of individual microtubules, and induces multipolar mitotic spindles. However, little is known about the mechanisms by which taxol disrupts normal bipolar spindle assembly in vivo. Using live imaging of GFP-alpha tubulin expressing cells, we examined spindle assembly after taxol treatment. We find that as taxol-treated cells enter mitosis, there is a dramatic re-distribution of the microtubule network from the centrosomes to the cell cortex. As they align there, the cortical microtubules recruit NuMA to their embedded ends, followed by the kinesin motor HSET. These cortical microtubules then bud off to form cytasters, which fuse into multipolar spindles. Cytoplasmic dynein and dynactin do not re-localize to cortical microtubules, and disruption of dynein/dynactin interactions by over-expression of p50 "dynamitin" does not prevent cytaster formation. Taxol added well before spindle poles begin to form induces multipolarity, but taxol added after nascent spindle poles are visible-but before NEB is complete-results in bipolar spindles. Our results suggest that taxol prevents rapid transport of key components, such as NuMA, to the nascent spindle poles. The net result is loss of mitotic spindle pole cohesion, microtubule re-distribution, and cytaster formation.

Citing Articles

Protease-Activated Receptor 2 (PAR2) Expressed in Sensory Neurons Contributes to Signs of Pain and Neuropathy in Paclitaxel Treated Mice.

Kume M, Ahmad A, DeFea K, Vagner J, Dussor G, Boitano S J Pain. 2023; 24(11):1980-1993.

PMID: 37315729 PMC: 10615692. DOI: 10.1016/j.jpain.2023.06.006.

Substances of Natural Origin in Medicine: Plants vs. Cancer.

Gielecinska A, Kciuk M, Mujwar S, Celik I, Kolat D, Kaluzinska-Kolat Z Cells. 2023; 12(7).

PMID: 37048059 PMC: 10092955. DOI: 10.3390/cells12070986.

Early Pharmacological Profiling of Antiproliferative Compounds by Live Cell Imaging.

Puerta A, Gonzalez-Bakker A, Santos G, Padron J Molecules. 2022; 27(16).

PMID: 36014500 PMC: 9415461. DOI: 10.3390/molecules27165261.

Preparation and Cytotoxic Evaluation of PGV-1 Derivative, CCA-1.1, as a New Curcumin Analog with Improved-Physicochemical and Pharmacological Properties.

Utomo R, Wulandari F, Novitasari D, Lestari B, Asmah Susidarti R, Jenie R Adv Pharm Bull. 2022; 12(3):603-612.

PMID: 35935043 PMC: 9348534. DOI: 10.34172/apb.2022.063.

Dual role of deubiquitinating enzyme USP19 regulates mitotic progression and tumorigenesis by stabilizing survivin.

Chandrasekaran A, Tyagi A, Poondla N, Sarodaya N, Karapurkar J, Kaushal K Mol Ther. 2022; 30(11):3414-3429.

PMID: 35918893 PMC: 9637645. DOI: 10.1016/j.ymthe.2022.07.019.

References

Compton D, Cleveland D . NuMA, a nuclear protein involved in mitosis and nuclear reformation. Curr Opin Cell Biol. 1994; 6(3):343-6. DOI: 10.1016/0955-0674(94)90024-8. View

Vaughan K . Surfing, regulating and capturing: are all microtubule-tip-tracking proteins created equal?. Trends Cell Biol. 2004; 14(9):491-6. DOI: 10.1016/j.tcb.2004.07.011. View

Amin-Hanjani S, Wadsworth P . Inhibition of spindle elongation by taxol. Cell Motil Cytoskeleton. 1991; 20(2):136-44. DOI: 10.1002/cm.970200206. View

Merdes A, Heald R, Samejima K, Earnshaw W, Cleveland D . Formation of spindle poles by dynein/dynactin-dependent transport of NuMA. J Cell Biol. 2000; 149(4):851-62. PMC: 2174573. DOI: 10.1083/jcb.149.4.851. View

Yvon A, Wadsworth P, Jordan M . Taxol suppresses dynamics of individual microtubules in living human tumor cells. Mol Biol Cell. 1999; 10(4):947-59. PMC: 25218. DOI: 10.1091/mbc.10.4.947. View

Balczon R, Bao L, Zimmer W, Brown K, Zinkowski R, Brinkley B . Dissociation of centrosome replication events from cycles of DNA synthesis and mitotic division in hydroxyurea-arrested Chinese hamster ovary cells. J Cell Biol. 1995; 130(1):105-15. PMC: 2120504. DOI: 10.1083/jcb.130.1.105. View

Saxton W, Stemple D, Leslie R, Salmon E, Zavortink M, McIntosh J . Tubulin dynamics in cultured mammalian cells. J Cell Biol. 1984; 99(6):2175-86. PMC: 2113582. DOI: 10.1083/jcb.99.6.2175. View

Moore J . The Ran-GTPase and cell-cycle control. Bioessays. 2001; 23(1):77-85. DOI: 10.1002/1521-1878(200101)23:1<77::AID-BIES1010>3.0.CO;2-E. View

Moritz M, Braunfeld M, Sedat J, Alberts B, Agard D . Microtubule nucleation by gamma-tubulin-containing rings in the centrosome. Nature. 1995; 378(6557):638-40. DOI: 10.1038/378638a0. View

10.

Wilde A, Zheng Y . Stimulation of microtubule aster formation and spindle assembly by the small GTPase Ran. Science. 1999; 284(5418):1359-62. DOI: 10.1126/science.284.5418.1359. View

11.

Snyder J, Mullins J . Analysis of spindle microtubule organization in untreated and taxol-treated PtK1 cells. Cell Biol Int. 1993; 17(12):1075-84. DOI: 10.1006/cbir.1993.1040. View

12.

De Brabander M, Geuens G, Nuydens R, Willebrords R, de Mey J . Taxol induces the assembly of free microtubules in living cells and blocks the organizing capacity of the centrosomes and kinetochores. Proc Natl Acad Sci U S A. 1981; 78(9):5608-612. PMC: 348802. DOI: 10.1073/pnas.78.9.5608. View

13.

Gaglio T, Dionne M, Compton D . Mitotic spindle poles are organized by structural and motor proteins in addition to centrosomes. J Cell Biol. 1997; 138(5):1055-66. PMC: 2136753. DOI: 10.1083/jcb.138.5.1055. View

14.

Piehl M, Cassimeris L . Organization and dynamics of growing microtubule plus ends during early mitosis. Mol Biol Cell. 2003; 14(3):916-25. PMC: 151569. DOI: 10.1091/mbc.e02-09-0607. View

15.

Jordan M, Wilson L . The use and action of drugs in analyzing mitosis. Methods Cell Biol. 1999; 61:267-95. DOI: 10.1016/s0091-679x(08)61986-x. View

16.

Hinchcliffe E . Using long-term time-lapse imaging of mammalian cell cycle progression for laboratory instruction and analysis. Cell Biol Educ. 2005; 4(4):284-90. PMC: 1305890. DOI: 10.1187/cbe.05-02-0064. View

17.

Echeverri C, Paschal B, Vaughan K, Vallee R . Molecular characterization of the 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis. J Cell Biol. 1996; 132(4):617-33. PMC: 2199864. DOI: 10.1083/jcb.132.4.617. View

18.

Schiff P, Horwitz S . Taxol stabilizes microtubules in mouse fibroblast cells. Proc Natl Acad Sci U S A. 1980; 77(3):1561-5. PMC: 348536. DOI: 10.1073/pnas.77.3.1561. View

19.

Rusan N, Tulu U, Fagerstrom C, Wadsworth P . Reorganization of the microtubule array in prophase/prometaphase requires cytoplasmic dynein-dependent microtubule transport. J Cell Biol. 2002; 158(6):997-1003. PMC: 2173209. DOI: 10.1083/jcb.200204109. View

20.

Cassimeris L, Pryer N, Salmon E . Real-time observations of microtubule dynamic instability in living cells. J Cell Biol. 1988; 107(6 Pt 1):2223-31. PMC: 2115680. DOI: 10.1083/jcb.107.6.2223. View