Confinement size determines the architecture of Ran-induced microtubule networks

The organization of microtubules (MTs) within cells is critical for its internal organization during interphase and mitosis. During mitotic spindle assembly, MTs are made and organized around chromosomes in a process regulated by RanGTP. The role of RanGTP has been explored in Xenopus egg extracts, which are not limited by a cell membrane. Here, we investigated whether cell-sized confinements affect the assembly of RanGTP-induced MT networks in Xenopus egg extracts. We used microfluidics to encapsulate extract within monodisperse extract-in-oil droplets. Importantly, we find that the architecture of Ran-induced MT networks depends on the droplet diameter and the Ran concentration, and differs from structures formed in bulk extract. Our results highlight that both MT nucleation and physical confinement play critical roles in determining the spatial organization of the MT cytoskeleton. STATEMENT OF SIGNIFICANCEDuring cell division, chromosomes are segregated by the mitotic spindle, whose framework consists of up to hundreds of thousands of microtubules (MTs). Spindle MTs are generated via several pathways, one of which is regulated by RanGTP. Yet, how Ran-induced MTs self-organize within cell-sized confinement remains unclear. This work reports unexpected architectures of Ran-induced MT networks confined in cell-sized droplets, which depend on the droplet diameter and the RanGTP concentration. Thus, MT nucleation and confinement together give rise to specific MT network architectures, which are otherwise not observed in unconfined assays. The findings provide a simple strategy to engineer the architectures of MT networks and could have direct implications in nucleation-controlled soft material processing.