
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.09.11.612005
preprint
1
Article
Optimized expansion microscopy reveals species-specific spindle microtubule organization in Xenopus egg extracts
Guilloux Gabriel http://orcid.org/0000-0003-4674-3478

Kitaoka Maiko http://orcid.org/0000-0002-1240-7903

Mocaer Karel http://orcid.org/0000-0002-9706-6457

Heichette Claire
Duchesne Laurence http://orcid.org/0000-0003-1985-6266

Heald Rebecca http://orcid.org/0000-0001-6671-6528

Pécot Thierry http://orcid.org/0000-0003-0772-9753

Gibeaux Romain http://orcid.org/0000-0001-5081-1985

11 9 2024
2024.09.11.612005https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
http://biorxiv.org/lookup/doi/10.1101/2024.09.11.612005
nihpp-2024.09.11.612005.pdf
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 focus on 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 exhibit a broader range of bundle sizes, while X. tropicalis spindles are more limited to smaller bundles. Moreover, while both species favor larger bundle sizes near and at the spindle center, X. tropicalis spindles otherwise prefer 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 spindle-related questions.

SIGNIFICANCE STATEMENT

Correct 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 two Xenopus species ( X. laevis and X. 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.
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