Septins regulate cytokinesis and multicellular development in the closest living relatives of animals

Septins are cytoskeletal proteins that regulate cytokinesis in fungi and animals, yet their functions in choanoflagellates—the closest living relatives of animals—have remained unknown. Salpingoeca rosetta , a choanoflagellate that switches between unicellular and multicellular forms, encodes four septins that are closely related to animal and fungal septins. Disruption of three of these septins altered cell size and impaired multicellular rosette development, with two of these three also required for normal rosette integrity. Characterization of Sros_septA , which showed the strongest phenotype, revealed a role in cytokinesis: mutant cells underwent furrow ingression but frequently failed to complete cell division, resulting in enlarged, multinucleated cells. Cell size and rates of cytokinesis failure and multinucleation were elevated in uninduced Sros_septA mutant cells and increased further upon rosette induction, revealing a heightened requirement for Sros_septA in cytokinesis during multicellular development in S. rosetta . Consistent with these cytokinesis defects, endogenously tagged Sros _SeptA dynamically redistributed from the basal pole in interphase cells to the cleavage furrow and nascent intercellular bridge during cell division. These findings identify roles for septins in cytokinesis and multicellular development in S. rosetta and offer a framework for exploring the evolution of cytokinetic regulation alongside the emergence of multicellularity in the animal stem lineage.