Optimized expansion microscopy reveals species-specific spindle microtubule organization in <i>Xenopus</i> egg extracts

The spindle is key to cell division, ensuring accurate chromosome segregation. Although its assembly and function are well studied, the mechanisms regulating spindle architecture remain elusive. Here, we investigate spindle organization differences between <i>Xenopus laevis</i> and <i>tropicalis</i>, leveraging expansion microscopy (ExM) to overcome conventional imaging limitations. We optimized an ExM protocol tailored for <i>Xenopus</i> egg extract spindles, refining fixation, denaturation, and gelation to achieve higher resolution while preserving spindle integrity. Our protocol enables preexpansion immunofluorescence and is seamlessly compatible with both species. To quantitatively compare microtubule organization in expanded spindles between the two species, we developed an analysis pipeline that is able to characterize microtubule bundles throughout spindles. We show that <i>X. laevis</i> spindles exhibit overall a broader range of bundle sizes, while <i>X. tropicalis</i> spindles contain mostly smaller bundles. Although both species show larger bundles near the spindle center, <i>X. tropicalis</i> spindles otherwise consist of very small bundles, whereas <i>X. laevis</i> spindles contain more medium-sized bundles. Altogether, our work reveals species-specific spindle architectures and suggests their adaptation to the different spindle size and chromatin amount. By enhancing resolution and minimizing artifacts, our ExM approach provides new insights into spindle morphology and a robust tool for further studying these large cellular assemblies.