Self-organization of stabilized microtubules by both spindle and midzone mechanisms in Xenopus egg cytosol

© The Author(s), 2013. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Molecular Biology of the Cell 24 (2013): 1559-1573, doi:10.1091/mbc.E12-12-0850.Previous study of self-organization of Taxol-stabilized microtubules into asters in Xenopus meiotic extracts revealed motor-dependent organizational mechanisms in the spindle. We revisit this approach using clarified cytosol with glycogen added back to supply energy and reducing equivalents. We added probes for NUMA and Aurora B to reveal microtubule polarity. Taxol and dimethyl sulfoxide promote rapid polymerization of microtubules that slowly self-organize into assemblies with a characteristic morphology consisting of paired lines or open circles of parallel bundles. Minus ends align in NUMA-containing foci on the outside, and plus ends in Aurora B–containing foci on the inside. Assemblies have a well-defined width that depends on initial assembly conditions, but microtubules within them have a broad length distribution. Electron microscopy shows that plus-end foci are coated with electron-dense material and resemble similar foci in monopolar midzones in cells. Functional tests show that two key spindle assembly factors, dynein and kinesin-5, act during assembly as they do in spindles, whereas two key midzone assembly factors, Aurora B and Kif4, act as they do in midzones. These data reveal the richness of self-organizing mechanisms that operate on microtubules after they polymerize in meiotic cytoplasm and provide a biochemically tractable system for investigating plus-end organization in midzones.Our work was funded primarily by National Institutes of Health Grant GM23928

Spindle-to-cortex communication in cleaving, polyspermic Xenopus eggs

© The Author(s), 2015. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Molecular Biology of the Cell 26 (2015): 3628-3640, doi:10.1091/mbc.E15-04-0233.Mitotic spindles specify cleavage planes in early embryos by communicating their position and orientation to the cell cortex using microtubule asters that grow out from the spindle poles during anaphase. Chromatin also plays a poorly understood role. Polyspermic fertilization provides a natural experiment in which aster pairs from the same spindle (sister asters) have chromatin between them, whereas asters pairs from different spindles (nonsisters) do not. In frogs, only sister aster pairs induce furrows. We found that only sister asters recruited two conserved furrow-inducing signaling complexes, chromosome passenger complex (CPC) and Centralspindlin, to a plane between them. This explains why only sister pairs induce furrows. We then investigated factors that influenced CPC recruitment to microtubule bundles in intact eggs and a cytokinesis extract system. We found that microtubule stabilization, optimal starting distance between asters, and proximity to chromatin all favored CPC recruitment. We propose a model in which proximity to chromatin biases initial CPC recruitment to microtubule bundles between asters from the same spindle. Next a positive feedback between CPC recruitment and microtubule stabilization promotes lateral growth of a plane of CPC-positive microtubule bundles out to the cortex to position the furrow.This work was supported by National Institutes of Health Grant GM39565 (T.J.M.) and MBL fellowships from the Evans Foundation, MBL Associates, and the Colwin Fund (T.J.M. and C.M.F.)

XRHAMM Functions in Ran-Dependent Microtubule Nucleation and Pole Formation during Anastral Spindle Assembly

Background: The regulated assembly of microtubules is essential for bipolar spindle formation. Depending on cell type, microtubules nucleate through two different pathways: centrosome-driven or chromatin-driven. The chromatin-driven pathway dominates in cells lacking centrosomes.Results: Human RHAMM (receptor for hyaluronic-acid-mediated motility) was originally implicated in hyaluronic-acid-induced motility but has since been shown to associate with centrosomes and play a role in astral spindle pole integrity in mitotic systems. We have identified the Xenopus ortholog of human RHAMM as a microtubule-associated protein that plays a role in focusing spindle poles and is essential for efficient microtubule nucleation during spindle assembly without centrosomes. XRHAMM associates both with γ-TuRC, a complex required for microtubule nucleation and with TPX2, a protein required for microtubule nucleation and spindle pole organization.Conclusions: XRHAMM facilitates Ran-dependent, chromatin-driven nucleation in a process that may require coordinate activation of TPX2 and γ-TuRC

Spindle assembly in the absence of a RanGTP gradient requires localized CPC activity

Author Posting. © The Author(s), 2009. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Current Biology 19 (2009): 1210-1215, doi:10.1016/j.cub.2009.05.061.During animal cell division, a gradient of GTP-bound Ran is generated around mitotic chromatin. It is generally accepted that this RanGTP gradient is essential for organizing the spindle since it locally activates critical spindle assembly factors. Here, we show in Xenopus egg extract, where the gradient is best characterized, that spindles can assemble in the absence of a RanGTP gradient. Gradient-free spindle assembly occurred around sperm nuclei but not around chromatin-coated beads and required the chromosomal passenger complex (CPC). Artificial enrichment of CPC activity within hybrid bead arrays containing both immobilized chromatin and the CPC supported local microtubule assembly even in the absence of a RanGTP gradient. We conclude that RanGTP and the CPC constitute the two major molecular signals that spatially promote microtubule polymerization around chromatin. Furthermore, we hypothesize that the two signals mainly originate from discreet physical sites on the chromosomes to localize microtubule assembly around chromatin: a RanGTP signal from any chromatin, and a CPC-dependent signal predominantly generated from centromeric chromatin.This work was supported by the American Cancer Society (grant PF0711401 to T.J. Maresca), the National Cancer Institute (grant CA078048-09 to T.J. Mitchison) and the National Institutes of Health (grant F32GM080049 to J.C. Gatlin and grant GM24364 to E.D. Salmon)

A new method reveals microtubule minus ends throughout the meiotic spindle

Anastral meiotic spindles are thought to be organized differently from astral mitotic spindles, but the field lacks the basic structural information required to describe and model them, including the location of microtubule-nucleating sites and minus ends. We measured the distributions of oriented microtubules in metaphase anastral spindles in Xenopus laevis extracts by fluorescence speckle microscopy and cross-correlation analysis. We localized plus ends by tubulin incorporation and combined this with the orientation data to infer the localization of minus ends. We found that minus ends are localized throughout the spindle, sparsely at the equator and at higher concentrations near the poles. Based on these data, we propose a model for maintenance of the metaphase steady-state that depends on continuous nucleation of microtubules near chromatin, followed by sorting and outward transport of stabilized minus ends, and, eventually, their loss near poles

The kinesin Eg5 drives poleward microtubule flux in Xenopus laevis egg extract spindles

Although mitotic and meiotic spindles maintain a steady-state length during metaphase, their antiparallel microtubules slide toward spindle poles at a constant rate. This “poleward flux” of microtubules occurs in many organisms and may provide part of the force for chromosome segregation. We use quantitative image analysis to examine the role of the kinesin Eg5 in poleward flux in metaphase Xenopus laevis egg extract spindles. Pharmacological inhibition of Eg5 results in a dose–responsive slowing of flux, and biochemical depletion of Eg5 significantly decreases the flux rate. Our results suggest that ensembles of nonprocessive Eg5 motors drive flux in metaphase Xenopus extract spindles

Growth, interaction, and positioning of microtubule asters in extremely large vertebrate embryo cells

Ray Rappaport spent many years studying microtubule asters, and how they induce cleavage furrows. Here, we review recent progress on aster structure and dynamics in zygotes and early blastomeres of Xenopus laevis and Zebrafish, where cells are extremely large. Mitotic and interphase asters differ markedly in size, and only interphase asters span the cell. Growth of interphase asters occurs by a mechanism that allows microtubule density at the aster periphery to remain approximately constant as radius increases. We discuss models for aster growth, and favor a branching nucleation process. Neighboring asters that grow into each other interact to block further growth at the shared boundary. We compare the morphology of interaction zones formed between pairs of asters that grow out from the poles of the same mitotic spindle (sister asters) and between pairs not related by mitosis (non-sister asters) that meet following polyspermic fertilization. We argue growing asters recognize each other by interaction between antiparallel microtubules at the mutual boundary, and discuss models for molecular organization of interaction zones. Finally, we discuss models for how asters, and the centrosomes within them, are positioned by dynein-mediated pulling forces so as to generate stereotyped cleavage patterns. Studying these problems in extremely large cells is starting to reveal how general principles of cell organization scale with cell size. V C 2012 Wiley Periodicals, Inc Key Words: aster, embryo, microtubule, cleavage, centrosome Introduction M icrotubule asters-radial arrays of microtubules radiating from centrosomes-play a central organizing role in early embryos. Ray Rappaport was fascinated by the question of how asters, in particular pairs of asters, induce cleavage furrows. One of his most celebrated discoveries The amphibian Xenopus laevis and the fish Danio rerio (Zebrafish) are easy to rear in the laboratory, and offer complementary technical advantages. Xenopus eggs cleave completely and are easy to fertilize with one or multiple sperm and to microinject. They are opaque, which precludes live imaging of internal events, but fixed embryos can be cleared for immunofluorescence imaging by immersion in a high refractive index medium REVIEW ARTICLE n 738 live imaging Xenopus and Zebrafish zygotes and early blastomeres are extremely large cells, with zygotes $1200 lm and$600 lm in diameter, respectively. They are also unusually fast compared to somatic cells, in the sense that the cell cycle takes 20-30 min to complete at room temperature (the first cell cycles are longer). These sizes and speeds represent physical extremes compared to typical somatic cells, which may require special adaptations of conserved cell organizing mechanisms, and/or reveal underappreciated intrinsic capabilities of those mechanisms. One well-studied example is adaptation of replication origins for very fast genome duplication Aster Growth in Large Cells The question of how microtubule asters grow in extremely large embryo cells has received little attention, but we believe that answering it will reveal principles of size scaling and unexpected molecular mechanisms. Figures 1 and 2 illustrate aster morphology and growth during the first and second cell cycle in frog and fish embryos. Inspection of these and similar images n 740 Mitchison et al. CYTOSKELETON Possibly consistent with this model, prometaphase asters in tissue culture cells capture and orient non-centrosomal microtubules using dynein An important question is whether the unusual aster growth mechanisms illustrated in Aster size in frog and fish embryos is temporally controlled by the cell cycle, with important implications for growth mechanisms and embryo organization. Aster radius at the poles of the first metaphase spindle is $30-40 lm in Xenopus [Figs. 1A and 1B, Wühr et al., 2008] and similar in Zebrafish [Fig. 2, 4 min, Wühr et al., 2010]. In both cases, this is much smaller than the zygote radius. Asters grow dramatically at anaphase onset, presumably due to decreased activity of Cdk1 (Cdc2.Cyclin B) kinase. In mitosis, Cdk1 acts on a complex network of microtubule interacting proteins to promote catastrophes (growing to shrinking transitions) and limit length CYTOSKELETON Growth, Interaction, and Positioning of Microtubule Asters 741 n presumably limited by the length distribution of microtubules in this bounded regime. Cdk1 levels drop shortly after fertilization, and at anaphase onset [reviewed in Cell cycle regulation of aster size has important implications for spatial organization of the early embryo. In early frog or fish blastomeres metaphase spindles are centrally located, and their short astral microtubules do not reach the cortex Aster-Aster Interactions What happens when two neighboring asters grow to touch each other? This question was of great interest to Rappaport, since cleavage furrows are typically induced where and when microtubules growing from aster pairs meet at the cortex. Asters grow into each other in early embryos under different circumstances. Two asters grow out from the poles of each mitotic spindle at anaphase, and meet each other at the midplane of the cell The most characteristic consequence of aster-aster interaction in interphase frog and fish embryos, seen for both n 742 Mitchison et al. The microtubule distribution in metaphase (A) appears radial, and microtubule density decreases rapidly with radius. In late anaphase (B), it appears more bundled and bushy, and microtubule density decreases less with radius. The dark zone in the center in B is presumably caused by a steric block to antibody penetration. A similar block is present at the center of the anaphase midzone and telophase midbody in somatic cells, and may also be present at the center of the aster-aster interaction zone in frog and fish embryos. CYTOSKELETON Growth, Interaction, and Positioning of Microtubule Asters 743 n sister ( Despite common features, there are reasons to suspect that not all aster-aster interaction zones are the same. Most notably, furrows are induced where the interaction zones between sister asters reach the cortex after 1st mitosis, and not where interaction zones between non-sisters reach the cortex, in the frogs Rana fusca and X. laevis, [Fig. 6C, Brachet, 1910; n 744 Mitchison et al. CYTOSKELETON the interaction between non-sister asters from two different spindles efficiently induced furrows if they were sufficiently close together. Non-sister asters also generated furrows at their interaction zone in tissue culture cells What molecules are likely to mediate aster-aster interactions in early frog and fish embryos? To our knowledge, no molecule has been specifically localized to aster-aster interaction zones in frog or fish embryos, but one logical set of candidates are molecules that organize cytokinesis midzone complexes in smaller cells [reviewed in Glotzer, 2005; Midzones are organized by three conserved protein modules or complexes Aster and Centrosome Positioning How asters, and the centrosomes at their centers, position themselves within embryos was also of great interest to Rappaport. Aster movement in large embryo cells is driven mainly by cytoplasmic dynein pulling on microtubules Hamaguchi and Hiramoto [1986] postulated that the sperm centrosome centers in the zygote due to length-dependent pulling forces on astral microtubules, combined with limitation of microtubule length by interaction with the cortex. Their model was based on elegant experiments CYTOSKELETON Growth, Interaction, and Positioning of Microtubule Asters 745 n where local inactivation of colcemid with UV light was used to artificially control microtubule length distribution in echinoderm embryos. Recent mathematical models support the concept that asters center by pulling forces that increase with microtubule length We hypothesized that Hiramoto's basic idea, which is cartooned in Perhaps the most intriguing unexplained aspect of aster and centrosome movement is orthogonal orientation of successive cleavage planes in embryos with an orthoradial cleavage pattern n 746 Mitchison et al. CYTOSKELETON because the yolk-free cytoplasm is laid down as a sheet in the oocyte. Successive cleavage planes are approximately orthogonal in two dimensions. Although cleavage plane geometry is stereotyped in frog eggs, it is not rigidly prespecified. Changing the shape of the egg, or the distribution of yolk within it 11 , spread between passivated coverslips and imaged by widefield fluorescence microscopy with a Â10 objective. Large asters grew with a bushy morphology at their peripheries (e.g., 32 min [Â3]). When asters grew to touch each they generated interaction zones with locally low microtubule density (e.g., arrows at 42 min, shown at higher mag. in 42 min [Â3]). These interaction zones blocked aster expansion and were stable for tens of minutes (compare 32, 52 min). When two artificial centrosomes were initially close together, they tended to initiate a single aster, and later split apart within that aster (e.g., the pair indicated by arrowheads at 2, 22, and 53 min). This splitting was reminiscent of centrosome separation within telophase asters in embryos Cell-Free Reconstitution of Interphase Aster Growth and Interaction It will be difficult to elucidate the molecular and biophysical mechanisms involved in aster dynamics using whole, living embryos as the only experimental system, especially in Xenopus where the egg is opaque. The related problem of meiosis-II spindle assembly in Xenopus eggs was tackled using cell-free extracts that accurately recapitulated the assembly process and greatly facilitated imaging and perturbation experiments Questions and Directions In closing, we will highlight key questions from each section of this review where we need to uncover new molecular and biophysical mechanisms. (i) Aster growth: what is the mechanism for keeping microtubule density constant as aster radius expands? If microtubules nucleate away from the centrosome as we suspect, what is the mechanism? (ii) Aster-aster interaction: how do growing asters recognize each other when they touch, and how does this recognition lead to inhibition of aster growth? To what extent are interaction zones between sister and non-sister asters similar at the molecular level, and why do only the former induce furrows in frog zygotes at 1st mitosis? (iii) Aster positioning: can we find further experimental validation for the Hiramoto model for aster centering? How do centrosomes split apart within growing asters, and what determines the axis on which they separate? Answering these questions will surely require interdisciplinary approaches that combine imaging, biochemistry, genetics, physical perturbation, force measurement, and computational modeling. Different biological systems have complementary advantages for these approaches, and we expect that Xenopus egg extract will prove particularly versatile. Vertebrate embryos with extremely large cells, where aster dynamics operate at a physical extreme, will help elucidate not only general principles of physical organization of cells, but also how these principles scale with cell size

Dynamic phosphoregulation of the cortical actin cytoskeleton and endocytic machinery revealed by real-time chemical genetic analysis

We used chemical genetics to control the activity of budding yeast Prk1p, which is a protein kinase that is related to mammalian GAK and AAK1, and which targets several actin regulatory proteins implicated in endocytosis. In vivo Prk1p inhibition blocked pheromone receptor endocytosis, and caused cortical actin patches to rapidly aggregate into large clumps that contained Abp1p, Sla2p, Pan1p, Sla1p, and Ent1p. Clump formation depended on Arp2p, suggesting that this phenotype might result from unregulated Arp2/3-stimulated actin assembly. Electron microscopy/immunoelectron microscopy analysis and tracking of the endocytic membrane marker FM4-64 revealed vesicles of likely endocytic origin within the actin clumps. Upon inhibitor washout, the actin clumps rapidly disassembled, and properly polarized actin patches reappeared. Our results suggest that actin clumps result from blockage at a normally transient step during which actin assembly is stimulated by endocytic proteins. Thus, we revealed tight phosphoregulation of an intrinsically dynamic, actin patch–related process, and propose that Prk1p negatively regulates the actin assembly–stimulating activity of endocytic proteins

Functional overlap of microtubule assembly factors in chromatin-promoted spindle assembly

Author Posting. © American Society for Cell Biology, 2009. This article is posted here by permission of American Society for Cell Biology for personal use, not for redistribution. The definitive version was published in Molecular Biology of the Cell 20 (2009): 2766-2773, doi:10.1091/mbc.E09-01-0043.Distinct pathways from centrosomes and chromatin are thought to contribute in parallel to microtubule nucleation and stabilization during animal cell mitotic spindle assembly, but their full mechanisms are not known. We investigated the function of three proposed nucleation/stabilization factors, TPX2, {gamma}-tubulin and XMAP215, in chromatin-promoted assembly of anastral spindles in Xenopus laevis egg extract. In addition to conventional depletion-add back experiments, we tested whether factors could substitute for each other, indicative of functional redundancy. All three factors were required for microtubule polymerization and bipolar spindle assembly around chromatin beads. Depletion of TPX2 was partially rescued by the addition of excess XMAP215 or EB1, or inhibiting MCAK (a Kinesin-13). Depletion of either {gamma}-tubulin or XMAP215 was partially rescued by adding back XMAP215, but not by adding any of the other factors. These data reveal functional redundancy between specific assembly factors in the chromatin pathway, suggesting individual proteins or pathways commonly viewed to be essential may not have entirely unique functions.This work was supported by the American Cancer Society (grant PF0711401 to T. J. Maresca), the National Cancer Institute (grant CA078048-09 to T. J. Mitchison) and the National Institutes of Health (grant F32GM080049 to J. C. Gatlin and grant GM24364 to E. D. Salmon)