Reconstituting centriole biogenesis on an engineered cellular platform uncovers organelle assembly principles

Summary Centriole copy number is tightly regulated, with one procentriole emanating from a torus surrounding each pre-existing centriole. Which proteins are sufficient to generate a full-fledged organelle is unclear. We address this question by engineering a high valency low copy number phase-separated droplet platform to concentrate proteins in an ectopic cellular location. We establish that droplet targeting of the torus protein Cep63 suffices to initiate procentriole assembly. Ectopic procentrioles mature when a limiting interaction involving STIL is alleviated or when pre-existing centrioles are lacking. Ectopic procentrioles disengage from the droplet during mitosis in a PLK1-dependent manner, organize supernumerary spindle poles, and seed procentriole formation at the next cell cycle, demonstrating that synthetic centrioles have been reconstituted. Moreover, we uncover that procentriole number scales with platform surface area. Since the torus surface area is set by pre-existing centriole dimensions, we propose that this constitutes a closed circuit mechanism dictating organelle number homeostasis. Highlights □ A high valency low copy number droplet platform engineered for cellular reconstitution □ Targeting the torus protein Cep63 to the droplet leads to naked cartwheel assembly □ Eliminating competition from resident centrioles enables synthetic centriole reconstitution □ Synthetic centriole number scales linearly with reconstituted torus surface area