Dynamic reorganization of Eg5 in the mammalian spindle throughout mitosis requires dynein and TPX2

Gable, Alyssa; Qiu, Minhua; Titus, Janel; Balchand, Sai Keshavan; Ferenz, Nick P.; Ma, Nan; Collins, Elizabeth; Fagerstrom, Carey J.; Ross, Jennifer L.; Yang, Ge; Wadsworth, Patricia

doi:10.1091/mbc.e11-09-0820

Cited by 50 publications

(87 citation statements)

References 60 publications

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“…Cell extracts were made from LLC-Pk1 cells stably expressing LAP-tagged Eg5 from a bacterial artificial chromosome (12). To prepare the extract, a confluent 100-mm diameter cell culture dish was washed twice with 5 ml of room temperature PBS.…”

Section: Methodsmentioning

confidence: 99%

“…Functional Eg5 from Mammalian Cell Extracts-We prepared cytoplasmic extracts (19) from a mammalian cell line stably expressing full-length, localization, and affinity purification-tagged Eg5 (hereafter referred to as Eg5-EGFP) expressed from a bacterial artificial chromosome under control of the native promoter (12,20) depending on the extract (Fig. 3A).…”

Section: Tpx2mentioning

confidence: 99%

“…Because the cells expressing Eg5-EGFP also express endogenous Eg5, the motile motors could be composed of between one and four EGFP molecules. In this cell line, the Eg5-EGFP is not resistant to the siRNA designed to deplete endogenous Eg5 (12). Therefore, to estimate the number of labeled Eg5 motors in the motile puncta, we depleted endogenous Eg5 from parental cells cotransfected with siRNA-resistant Eg5-mEmerald and prepared cell extracts.…”

Section: Tpx2mentioning

confidence: 99%

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TPX2 Inhibits Eg5 by Interactions with Both Motor and Microtubule

Balchand

Mann

Titus

et al. 2015

Journal of Biological Chemistry

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Background: TPX2 is a mitotic microtubule-associated protein that regulates the kinesin, Eg5. Results: Full-length TPX2 is a more potent inhibitor of Eg5 velocity than truncated TPX2; differential regulation by TPX2 was not observed for monomeric Eg5. Conclusion: TPX2 inhibits Eg5 as a roadblock and by direct interaction with Eg5. Significance: TPX2 may contribute to the spatial and temporal regulation of the mitotic motor Eg5.

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Section: Methodsmentioning

confidence: 99%

Section: Tpx2mentioning

confidence: 99%

Section: Tpx2mentioning

confidence: 99%

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TPX2 Inhibits Eg5 by Interactions with Both Motor and Microtubule

Balchand

Mann

Titus

et al. 2015

Journal of Biological Chemistry

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“…In that report, a decrease in tubulin subunit arrival and departure events at the microtubule tip was shown to correspond with a decrease in the variability of microtubule growth, owing to a reduction in the total number of on-off events (Skellam, 1946;Oosawa, 1970). Thus, to evaluate microtubule growth rate variability in the presence and absence of TPX2, time-lapse movies of growing Alexa-488-labeled microtubules were collected using total internal reflection fluorescence (TIRF) microscopy, and then the microtubule tip position was evaluated with sub-pixel resolution by fitting the microtubule tip to a Gaussian decay function (as previously described by Demchouk et al, 2011) (TPX2 purification described by Ma et al, 2011;Gable et al, 2012) (Fig. 1A).…”

Section: Tpx2 Slows Tubulin Subunit Off-rates During Microtubule Assementioning

confidence: 99%

Suppression of microtubule assembly kinetics by the mitotic protein TPX2

Reid

Schuster

Mann

et al. 2016

Journal of Cell Science

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TPX2 is a widely conserved microtubule-associated protein that is required for mitotic spindle formation and function. Previous studies have demonstrated that TPX2 is required for the nucleation of microtubules around chromosomes; however, the molecular mechanism by which TPX2 promotes microtubule nucleation remains a mystery. In this study, we found that TPX2 acts to suppress tubulin subunit off-rates during microtubule assembly and disassembly, thus allowing for the support of unprecedentedly slow rates of plus-end microtubule growth, and also leading to a dramatically reduced microtubule shortening rate. These changes in microtubule dynamics can be explained in computational simulations by a moderate increase in tubulin-tubulin bond strength upon TPX2 association with the microtubule lattice, which in turn acts to reduce the departure rate of tubulin subunits from the microtubule ends. Thus, the direct suppression of tubulin subunit off-rates by TPX2 during microtubule growth and shortening could provide a molecular mechanism to explain the nucleation of new microtubules in the presence of TPX2.

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“…During metaphase, kinesin-5 localizes along spindle microtubules and at spindle poles. The motor relocalizes to the spindle midzone in anaphase (Gable et al, 2012). Kinesin-5 has been reported to associate directly with NuMa ( Nu clear M itotic A pparatus protein).…”

Section: Regulation Of Kinesin-5 By Binding Partnersmentioning

confidence: 99%

Mechanism and regulation of kinesin‐5, an essential motor for the mitotic spindle

Waitzman

Rice

2013

Biology of the Cell

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Mitotic cell division is the most fundamental task of all living cells. Cells have intricate and tightly regulated machinery to ensure that mitosis occurs with appropriate frequency and high fidelity. A core element of this machinery is the kinesin-5 motor protein, which plays essential roles in spindle formation and maintenance. In this review, we discuss how the structural and mechanical properties of kinesin-5 motors uniquely suit them to their mitotic role. We describe some of the small molecule inhibitors and regulatory proteins that act on kinesin-5, and discuss how these regulators may influence the process of cell division. Finally, we touch on some more recently described functions of kinesin-5 motors in non-dividing cells. Throughout, we highlight a number of open questions that impede our understanding of both this motor's function and the potential utility of kinesin-5 inhibitors.

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Dynamic reorganization of Eg5 in the mammalian spindle throughout mitosis requires dynein and TPX2

Cited by 50 publications

References 60 publications

TPX2 Inhibits Eg5 by Interactions with Both Motor and Microtubule

TPX2 Inhibits Eg5 by Interactions with Both Motor and Microtubule

Suppression of microtubule assembly kinetics by the mitotic protein TPX2

Mechanism and regulation of kinesin‐5, an essential motor for the mitotic spindle

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