Interspecies Comparisons of Xenopus Egg Extracts Reveal Mechanisms for Modulating Meiotic Spindle Size and Architecture

The function of the spindle to faithfully segregate chromosomes is universal among eukaryotes. A common feature of metaphase spindles is their self-organizing, bipolar structure, with microtubules organized into polar arrays by the activity of motors and other proteins. However, wide variation in spindle assembly, size and morphology is observed among different cell types, presumably to optimize spindle function. While many of the hundreds of conserved spindle assembly factors have been identified, how these proteins interact to establish a particular spindle architecture is poorly understood./nXenopus provides a valuable system to study a variety of spindle types in vitro, since 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 microtubules. Meiotic spindles assembled in egg extracts of the smaller Xenopus tropicalis frog possess a similar anastral appearance but are significantly shorter at ~22 μm. Previously, these two extracts were used to identify proteins which regulate spindle length, and a microtubule-severing protein p60 katanin was shown to be differentially phospho-regulated between these two systems. Activity differences in p60 katanin were not sufficient to explain the size differences between X. tropicalis and X. laevis, and this study identifies TPX2 as an additional length regulator that also contributes to other features of spindle architecture./nIn addition to evaluating spindle length, we probed the involvement of positional microtubule nucleation, motor organization, and microtubule distribution as contributors to architectural features. Spindles formed in X. laevis extracts have been previously shown to rely heavily on both microtubule nucleation near the chromatin governed by the protein RanGTP as well as organization of antiparallel arrays by the kinesin-5 motor Eg5, resulting in a structure with a tiled array of microtubules in almost constant density pole-to-pole. Intriguingly, X. tropicalis extract spindles do not require either process, as indicated by resistance to inhibition of these pathways. Furthermore, X. tropicalis microtubule density is significantly reduced in the spindle midzone./nWe focused on the role of the microtubule-associated factor TPX2 in mediating these differences, since it has been shown to interact both with Ran and Eg5. Levels of TPX2 were measured to be approximately 3-fold higher in X. tropicalis. Addition of excess TPX2 to X. laevis extract recapitulated differences seen in the architectures of the two spindles: rendering spindles less sensitive to RanGTP and Eg5 inhibition and as well as reducing spindle length. These spindles also showed increased recruitment of Eg5 to the spindle poles, where microtubule density increased. We propose that TPX2 levels modulate spindle architecture by partitioning microtubules between a tiled antiparallel array that promotes spindle expansion, and a compact, parallel cross-linked architecture that concentrates MTs at spindle poles./nAdditionally, TPX2 has been previously shown in the literature to have microtubule-nucleating activity. Examining the two Xenopus species orthologs of this protein, we determined that X. tropicalis had much higher nucleation capacity, resulting in spindle astral microtubules not normally seen in the Xenopus egg extract spindle as well as increased non-spindle associated microtubule formation. Comparing primary sequence of these proteins, we discovered a short c-terminal 7 amino acid stretch absent in the X. tropicalis protein. Removing this sequence from X. laevis TPX2 greatly enhanced microtubule nucleation and altered spindle morphology. Using these TPX2 nucleation mutants in spindle assembly, we were able to ask what the effect of altering microtubule nucleation was on spindle length. Surprisingly, changing microtubule nucleation did not affect spindle length but did correspond to altered amounts of astral microtubules at the pole./nIn summary, we show here how TPX2 is involved in many features of spindle architecture: establishing MT distribution and length through its interaction with Eg5 and polar architecture through its nucleation activity. TPX2 is not well-conserved through evolution, and many species retain only certain domains of the protein, and therefore only some facets of its activities. As a major contributor to spindle architecture, it is an attractive hypothesis that species tailor their spindle morphology through evolutionary retention or rejection of certain TPX2-mediated architectural features.

Advisor: Heald, Rebecca