Visualizing Drosophila centrioles by expansion microscopy

A significant challenge in studying the biology of the Drosophila centriole is its small size. Advanced super-resolution techniques have provided valuable insights but require specialized equipment and can be difficult to implement in tissues. Expansion microscopy (ExM) offers an accessible alternative, yet its application in Drosophila centriole research has been sparse. We provide an ExM protocol for cultured S2 cells and fly tissues that reveals new insights into procentriole biology. In S2 cells we document overduplication in the form of the classic ‘rosettes’, while in spermatids we uncover an unexpected movement of the procentriole-like structure (PCL). ExM has also refined existing molecular models. In S2 cells we document the distal tip protein Cep97 as a ring, which clarifies its role in capping the growing centriole. In spermatids, we spatially segregate the inner nuclear membrane protein Spag4 and the cytoplasmic protein Yuri, leading to the new hypothesis that they play independent roles at the centriole–nucleus contact site. Finally, we show that our ExM protocol is a hypothesis generator and discovery tool applicable beyond Drosophila centrioles by imaging synaptonemal complexes in the Plodia interpunctella moth.