TPX 2 levels modulate meiotic spindle size and architecture in Xenopus egg extracts

The function of the spindle to accurately segregate chromosomes during cell division is universal among eukaryotes. A common feature of metaphase spindles is their bipolar structure, with microtubule (MT) minus ends pointing toward the poles and MT plus ends toward the center, with a subset of them connecting to chromosomes at the kinetochores. However, wide variation in spindle assembly, size, and morphology is observed among different cell types, presumably to optimize spindle function (Goshima et al., 2005; Helmke et al., 2013). For example, in cultured somatic cells centrosomes serve as the dominant MTnucleating sites at each spindle pole that direct spindle formation and also generate astral MTs that function in spindle positioning, whereas meiotic spindles of eggs and oocytes frequently lack centrosomes and astral MT arrays and assemble by selforganization of MTs stabilized by chromatin. It is now accepted that spindles form through a combination of mechanisms, but how a particular spindle architecture is established and contributes to spindle function is poorly understood. Xenopus provides a valuable system to study a variety of spindle types in vitro because spindles formed in egg and embryo extracts recapitulate morphologies observed in vivo. The ellipsoidal, 35-μm-long Xenopus laevis meiotic spindle has been studied most extensively and is thought to be built from a tiled array of dynamic, overlapping MTs generated by a gradient of RanGTP around chromatin and organized by motor proteins (Yang et al., 2008; Loughlin et al., 2010; Needleman et al., 2010; Brugués et al., 2012). Meiotic spindles assembled in egg extracts of the smaller Xenopus tropicalis frog possess a similar anastral appearance but are significantly shorter at 22 μm (Brown et al., 2007). In vitro spindle size scaling can also be seen by comparing mitotic spindles assembled in extracts from X. laevis embryos containing four large cells at stage 3 to extracts prepared from 4,000 small cells at stage 8 (Wilbur and Heald, 2013). In both interspecies and developmental spindle scaling, modulation of the activity of factors that destabilize MTs contributed to differences in spindle lengths (Loughlin et al., 2011; Wilbur and Heald, 2013). Also apparent among these spindles were differences in morphology and the apparent role of centrosomes and kinetochores in spindle assembly and organization. Although centrosomes were present in both mitotic spindle types, connections between centrosome-nucleated MTs and chromosomes were more prominent in the smaller stage 8 spindles, which unlike the larger stage 3 spindles were not disrupted by RanGTP inhibition (Wilbur and Heald, 2013). The spindle segregates chromosomes in dividing eukaryotic cells, and its assembly pathway and morphology vary across organisms and cell types. We investigated mechanisms underlying differences between meiotic spindles formed in egg extracts of two frog species. Small Xenopus tropicalis spindles resisted inhibition of two factors essential for assembly of the larger Xenopus laevis spindles: RanGTP, which functions in chromatindriven spindle assembly, and the kinesin-5 motor Eg5, which drives antiparallel microtubule (MT) sliding. This suggested a role for the MT-associated protein TPX2 (targeting factor for Xenopus kinesin-like protein 2), which is regulated by Ran and binds Eg5. Indeed, TPX2 was threefold more abundant in X. tropicalis extracts, and elevated TPX2 levels in X. laevis extracts reduced spindle length and sensitivity to Ran and Eg5 inhibition. Higher TPX2 levels recruited Eg5 to the poles, where MT density increased. We propose that TPX2 levels modulate spindle architecture through Eg5, partitioning MTs between a tiled, antiparallel array that promotes spindle expansion and a cross-linked, parallel architecture that concentrates MTs at spindle poles. TPX2 levels modulate meiotic spindle size and architecture in Xenopus egg extracts