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

Abstract SUN-domain proteins are core components of LINC complexes that connect the cytoskeleton to the nucleus across the nuclear envelope. SUN5 is a testis-specific SUN-domain protein essential for anchoring the sperm tail to the nucleus, but its functional organization remained controversial, with previous reports suggesting that SUN5 represents an exception to the canonical topology of SUN-domain proteins. Here, we analyzed SUN5 organization during mammalian spermiogenesis using ultrastructure expansion microscopy (U-ExM), Tokuyasu immunogold labeling, and complementary biochemical assays. We show that SUN5 adopts a canonical SUN-domain protein topology at the head-to-tail junction and present a refined model of SUN5 positioning within the nuclear envelope. Stage-resolved U-ExM further reveals SUN5 redistribution including enrichment at the microtubule manchette and the perinuclear ring (PNR), suggesting additional roles in manchette-associated processes. Together, these findings clarify SUN5 topology and localization and provide a framework for understanding its function during mammalian spermiogenesis.