Cytoskeleton club - Romain Gibeaux

Romain Gibeaux -  "Microtubules, from Tubulin to Spindles" Team - IGDR, Rennes

Title : Xenopus spermatid microtubules contain an internal helix

Abstract: The production of mature and functional sperm requires proper microtubule organization. In addition to their roles in protein transport and chromosome segregation, microtubules form cytoskeletal structures essential for sperm morphogenesis. During sperm differentiation, known as spermiogenesis, the round spermatid undergoes dramatic morphological changes, in particular nuclear reshaping, which involves a transient microtubule-based structure called the manchette. While well described in mammals, the existence of a manchette in other vertebrates like Xenopus, in which extreme nuclear reshaping leads to its characteristic corkscrew-shaped sperm head, remains unclear. To characterize microtubules of Xenopus spermatids, we first optimized spermatid isolation. Immunofluorescence of the microtubules surrounding the nucleus together with molecular and structural analyses revealed the presence of a manchette-like structure in Xenopus spermatids. To explore their architecture in intact spermatids, we employed cryo-FIB-SEM followed by electron tomography of lamellae. Unexpectedly, we observed a repeating density within the microtubule lumen. Subtomogram averaging uncovered the helical nature of this structure. We next extracted spermatid microtubules, applied a single-particle analysis strategy to reach near-atomic resolution, and identified the two main proteins composing this internal helix. Furthermore, RNA-FISH revealed their spermatid-specific expression; mass spectrometry proteomics of isolated manchettes confirmed their presence in this structure; and immunofluorescence localized them within spermatid manchette microtubules. Through this study, we discovered the existence of a manchette-like structure conserved in Xenopus species, and the presence of an internal helix in these microtubules, which represents the first observation of an internal structure in manchette microtubules of any species. These findings furthermore highlight a potentially important new mechanism for regulating manchette microtubules to ensure the fidelity of spermiogenesis and the integrity of sperm function.