Data supporting "Expanding Our View of Drosophila Centrioles"

Drosophila melanogaster serves as a powerful model system for investigating the fundamental principles of centrosome and centriole biology. However, a significant and persistent challenge in the field has been the relatively small size of most Drosophila centrioles, which, at approximately 200 nm in length and width, reside below the diffraction limit of conventional light microscopy. While advanced super-resolution techniques have provided valuable insights, they often require specialized equipment and can be difficult to implement in tissues. Expansion Microscopy (ExM), a method that achieves super-resolution images by physically expanding the biological sample itself, offers a robust, cost-effective, and accessible alternative. Despite its potential, the application of ExM to Drosophila centrioles has been sparse. We have provided a detailed and optimized Ultrastructure Expansion Microscopy (U-ExM) protocol for both cultured S2 cells and fly tissues. Our protocol, which is adapted from several other protocols, has uncovered many new insights into centriole biology. First, we demonstrate that U-ExM dramatically improves antibody-based detection of endogenous centriolar proteins, which allowed us to reliably visualize key inner centriole proteins such as Sas6, Ana1, and Ana2. Second, we reveal new insights into pro-centriole biology in both cells and tissues. In S2 cells, we were able to document centriole overduplication in the form of the classic "rosettes" In developing spermatids, U-ExM uncovered an unexpected lateral movement of the pro-centriole-like (PCL) structure. Finally, the nanoscale resolution afforded by U-ExM enabled us to refine existing molecular models. In spermatids, we spatially segregated the inner nuclear membrane protein Spag4 and the cytoplasmic protein Yuri at the centriole invagination site, while in S2 cells, we visualized the distal tip protein Cep97 as a distinct ring, providing high-confidence localization data that clarifies its role in capping the growing centriole microtubules. Overall, U-ExM proves to be a transformative and broadly applicable tool for the Drosophila research community. While our step-by-step protocol was used to gain insight into centriole architecture, duplication, and dynamics, it is broadly applicable to all studies in fly models and should be considered a very powerful method of discovery.