The spindle is a key structure in cell division as it orchestrates the accurate segregation of genetic material. While its assembly and function are well-studied, the mechanisms regulating spindle architecture remain elusive. In this study, we focus on the differences in spindle organization betweenXenopus laevisandXenopus tropicalis, leveraging expansion microscopy (ExM) to overcome the limitations of conventional imaging techniques. We optimized an ExM protocol tailored forXenopusegg extract spindles, improving upon fixation, denaturation and gelation methods to achieve higher resolution imaging of spindles. Our protocol preserves spindle integrity and allows effective pre-expansion immunofluorescence. This method enabled detailed analysis of the differences in microtubule organization between the two species.X. laevisspindles overall exhibit a broader range of bundle sizes, whileX. tropicalisspindles are more limited to smaller bundles. Moreover, while both species favor larger bundle sizes near and at the spindle center,X. tropicalisspindles otherwise prefer very small bundles, andX. laevisspindles medium-sized bundles. By enhancing resolution and minimizing distortions and fixation artifacts, our optimized ExM approach offers new insights into spindle morphology and provides a robust tool for studying the structural intricacies of these large cellular assemblies. This work advances our understanding of spindle architecture and opens up new avenues for exploring spindle-related questions.SIGNIFICANCE STATEMENTCorrect spindle morphology is key to its function; however, traditional microscopy methods limit our view of spindle architecture. This study addresses the gap in resolving detailed spindle microtubule organization by using advanced imaging.The research utilizes Expansion Microscopy (ExM) to reveal previously unobservable details of spindle morphology in egg extracts of twoXenopusspecies (X. laevisandX. tropicalis). This approach provides unprecedented clarity on microtubule arrangement and variations in spindle architecture.This work establishes a new protocol for high-resolution imaging of spindle structures, offering insights into how spindle architecture is adapted in differently-sized spindles to ensure proper function.
