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 1200lmand600 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
