Prophase microtubule arrays undergo flux-like behaviour in mammalian cells

In higher eukaryotic cells, microtubules within metaphase and anaphase spindles undergo poleward flux, the slow, poleward movement of tubulin subunits through the spindle microtubule lattice. Although a number of studies have documented this phenomenon across a wide range of model systems, the possibility of poleward flux before nuclear envelope breakdown (NEB) has not been examined. Using a mammalian cell line expressing photoactivatable green fluorescent protein (GFP)-tubulin, we observe microtubule motion, both toward and away from centrosomes, at a wide range of rates (0.5–4.5 μm/min) in prophase cells. Rapid microtubule motion in both directions is dynein dependent. In contrast, slow microtubule motion, which occurs at rates consistent with metaphase flux, is insensitive to inhibition of dynein but sensitive to perturbation of Eg5 and Kif2a, two proteins with previously documented roles in flux. Our results demonstrate that microtubules in prophase cells are unexpectedly dynamic and that a subpopulation of these microtubules shows motion that is consistent with flux. We propose that the marked reduction in rate and directionality of microtubule motion from prophase to metaphase results from changes in microtubule organization during spindle formation

Mitotic functions of kinesin-5

In all eukaryotic cells, molecular motor proteins play essential roles in spindle assembly and function. The homotetrameric kinesin-5 motors in particular generate outward forces that establish and maintain spindle bipolarity and contribute to microtubule flux. Cell cycle dependent phosphorylation of kinesin-5 motors regulates their localization to the mitotic spindle. Analysis of live cells further shows that kinesin-5 motors are highly dynamic in the spindle. Understanding the interactions of kinesin-5 motors with microtubules and other spindle proteins is likely to broaden the documented roles of kinesin-5 motors during cell division

Molecular requirements for kinetochore-associated microtubule formation in mammalian cells

SummaryIn centrosome-containing cells, microtubules nucleated at centrosomes are thought to play a major role in spindle assembly [1]. In addition, microtubule formation at kinetochores has also been observed [2–5], most recently under physiological conditions in live cells [6]. The relative contributions of microtubule formation at kinetochores and centrosomes to spindle assembly, and their molecular requirements, remain incompletely understood. Using mammalian cells released from nocodazole-induced disassembly, we observed microtubule formation at centrosomes and at Bub1-positive sites on chromosomes. Kinetochore-associated microtubules rapidly coalesced into pole-like structures in a dynein-dependent manner. Microinjection of excess importin-β or depletion of the Ran-dependent spindle assembly factor, TPX2, blocked kinetochore-associated microtubule formation, enhanced centrosome-associated microtubule formation, but did not prevent chromosome capture by centrosomal microtubules. Depletion of the chromosome passenger protein, survivin, reduced microtubule formation at kinetochores in an MCAK-dependent manner. Microtubule formation in cells depleted of Bub1 or Nuf2 was indistinguishable from that in controls. Our data demonstrate that microtubule assembly at centrosomes and kinetochores is kinetically distinct and differentially regulated. The presence of microtubules at kinetochores provides a mechanism to reconcile the time required for spindle assembly in vivo with that observed in computer simulations of search and capture

Imaging protein dynamics in live mitotic cells

To ensure that genetic material is accurately segregated during mitosis, eukaryotic cells assemble a mitotic spindle, a dynamic structure composed of microtubules and associated regulatory, structural and motor proteins. Although much has been learned in the past decades from direct observations of live cells expressing fluorescently tagged spindle proteins, a complete understanding of spindle assembly requires a detailed analysis of the dynamic behavior of component parts. Proteins tagged with conventional fluorophores, however, make such an analysis difficult because all of the molecules are uniformly fluorescent. To alleviate this problem, we have tagged proteins with a photoactivatable variant of GFP (PA-GFP), thereby allowing one to follow the behavior of a subset of tagged molecules in the cell. Here, we describe methods to tag and express proteins with PA-GFP, locally photoactivate the recombinant protein and record the dynamic behavior of the photoactivated molecules in live cells. We provide examples of photoactivable proteins in mammalian and yeast cells to illustrate the power of this approach to examine the dynamics of spindle formation and function in diverse cells

Dynein Antagonizes Eg5 by Crosslinking and Sliding Antiparallel Microtubules

SummaryMitotic spindle assembly requires the combined activity of various molecular motor proteins, including Eg5 [1] and dynein [2]. Together, these motors generate antagonistic forces during mammalian bipolar spindle assembly [3]; what remains unknown, however, is how these motors are functionally coordinated such that antagonism is possible. Given that Eg5 generates an outward force by crosslinking and sliding apart antiparallel microtubules (MTs) [4–6], we explored the possibility that dynein generates an inward force by likewise sliding antiparallel MTs. We reasoned that antiparallel overlap, and therefore the magnitude of a dynein-mediated force, would be inversely proportional to the initial distance between centrosomes. To capitalize on this relationship, we utilized a nocodazole washout assay to mimic spindle assembly. We found that Eg5 inhibition led to either monopolar or bipolar spindle formation, depending on whether centrosomes were initially separated by less than or greater than 5.5 μm, respectively. Mathematical modeling predicted this same spindle bistability in the absence of functional Eg5 and required dynein acting on antiparallel MTs to do so. Our results suggest that dynein functionally coordinates with Eg5 by crosslinking and sliding antiparallel MTs, a novel role for dynein within the framework of spindle assembly

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

The kinesin Eg5 moves toward minus ends of astral microtubules in early mitosis, switching to plus-end motion in anaphase. Dynein is required for minus-end motion; depletion of TPX2 results in a switch to plus-end motion. On midzone microtubules, Eg5 moves in both directions. Our results explain the redistribution of Eg5 throughout mitosis

The sequential activation of the mitotic microtubule assembly pathways favors bipolar spindle formation

Centrosome maturation is the process by which the duplicated centrosomes recruit pericentriolar components and increase their microtubule nucleation activity before mitosis. The role of this process in cells entering mitosis has been mostly related to the separation of the duplicated centrosomes and thereby to the assembly of a bipolar spindle. However, spindles can form without centrosomes. In fact, all cells, whether they have centrosomes or not, rely on chromatin-driven microtubule assembly to form a spindle. To test whether the sequential activation of these microtubule assembly pathways, defined by centrosome maturation and nuclear envelope breakdown, plays any role in spindle assembly, we combined experiments in tissue culture cells and Xenopus laevis egg extracts with a mathematical model. We found that interfering with the sequential activation of the microtubule assembly pathways compromises bipolar spindle assembly in tissue culture cells but not in X. laevis egg extracts. Our data suggest a novel function for centrosome maturation that determines the contribution of the chromosomal microtubule assembly pathway and favors bipolar spindle formation in most animal cells in which tubulin is in limiting amounts.T.C. was supported by Formación de Personal Investigador (FPI) Fellowship BES-2010-031355. This work was supported by Spanish ministry grants BFU2009-10202 and BFU2012-37163. We acknowledge support of the Spanish Ministry of Economy and Competitiveness, Centro de Excelencia Severo Ochoa 2013-2017, SEV-2012-0208