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bioRxiv · 10.1101/2024.09.11.612005

Optimized expansion microscopy reveals species-specific spindle microtubule organization in Xenopus egg extracts

Guilloux, G.·Kitaoka, M.·Mocaer, K.·Heichette, C.·Duchesne, L.·Heald, R.·Pecot, T.·Gibeaux, R.
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Abstract

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 investigate the differences in spindle organization between Xenopus laevis and Xenopus tropicalis, leveraging expansion microscopy (ExM) to overcome the limitations of conventional imaging techniques. We optimized an ExM protocol tailored for Xenopus egg 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. laevis spindles overall exhibited a broader range of bundle sizes, while X. tropicalis spindles contained mostly smaller bundles. Moreover, while both species exhibited larger bundle sizes near and at the spindle center, X. tropicalis spindles otherwise consisted of very small bundles, and X. laevis spindles 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 underlying mechanisms. SIGNIFICANCE STATEMENTO_LICorrect 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. C_LIO_LIThe research utilizes Expansion Microscopy (ExM) to reveal previously unobservable details of spindle morphology in egg extracts of two Xenopus species (X. laevis and X. tropicalis). This approach provides unprecedented clarity on microtubule arrangement and variations in spindle architecture. C_LIO_LIThis 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. C_LI

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Guilloux, G., Kitaoka, M., Mocaer, K., Heichette, C., Duchesne, L., Heald, R., Pecot, T., Gibeaux, R.. 2024-09-11. Optimized expansion microscopy reveals species-specific spindle microtubule organization in Xenopus egg extracts. https://doi.org/10.1101/2024.09.11.612005

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