A Minimal System to Establish Microtubule-Based Cell Polarity In Vitro

From yeast to fibroblasts, many cell types have a defined polarity that allows them to directionally move, grow or divide. This polarity is typically defined by a polarized distribution of proteins at the cell cortex. We are interested in the emergence of polarity in systems where microtubules are directly involved in its establishment and maintenance by delivering polarity markers to the plasma membrane.We developed two in vitro systems that allow for microtubule-based delivery of proteins to bio-mimetic cortices. These systems, consisting of elongated micro-fabricated chambers or emulsion droplets, allow for dynamic microtubules to self-assemble and organize in response to interactions with the chamber or droplet boundaries.In micro-chambers, single events of microtubule-based delivery to a wall are imaged and quantified. Our experiments show that clustering of proteins at microtubule tips (fission yeast's mal3, kinesin tea2, and tip1) enhances prolonged docking of proteins to the wall receptors as opposed to non-clustering proteins (EB analog protein mal3). Moreover, pre-docked clusters at the wall can capture growing microtubule tips enhancing repeated deliveries at the same spot. These observations are very similar to the observation of clustering of polarity markers in living fission yeast cells.With elongated emulsion droplets the global emergence of polarity in a closed system can be assessed under conditions where proteins can additionally diffuse within the lipid boundaries. With this minimal system we aim to establish the minimal mechanism by which microtubules can establish and maintain cell polarity in living cells. In parallel, we performed experiments in living yeast cells, which suggest that a simple artificial protein that combines membrane affinity with microtubule tip affinity is in principle enough to establish (but not maintain) polarity.