Nanoscale imaging resolves canonical topology and intracellular dynamics of SUN5/SPAG4L during mammalian spermiogenesis

SUN5 is a testis-specific SUN domain protein essential for connecting the sperm tail to the nucleus. However, until now, its precise localization, intracellular dynamics, and membrane topology during spermiogenesis have remained controversial. To address these discrepancies, we applied ultrastructure expansion microscopy (U-ExM) to systematically track SUN5 redistribution throughout spermiogenesis. This approach enabled a detailed reconstruction of SUN5 localization across developmental stages and revealed previously undescribed enrichment at the perinuclear ring (PNR) and the microtubule manchette, suggesting secondary functions at the PNR or a potential role in intra-manchette transport (IMT). Complementary immunogold labelling using the Tokuyasu method, together with biochemical assays, demonstrated that SUN5 adopts a membrane localization and topology consistent with that of classical SUN domain proteins. Quantitative measurements of the nuclear envelope architecture at the head-to-tail coupling apparatus (HTCA) further enabled us to present a refined structural model of SUN5 positioning at the head-tail junction. Overall, our findings resolve previous discrepancies in the field and provide a coherent framework for understanding SUN5 organization and its role in mammalian spermiogenesis. Summary StatementIn the presented study, we analyzed the dynamic redistribution of SUN5 during mammalian spermiogenesis and resolved its topology in developing spermatids to gain insights concerning the proteins molecular function in head-tail coupling.