The PhyB-PIF light-inducible dimerization system is used to achieve rapid, reversible, and titratable control of protein localization in budding yeast. This system can be used to dynamically activate or inactivate proteins of interest. It is used here to probe where and when Clb2 activity is required for nuclear fission and spindle stabilization.

Jost and Waters review best practices for validation of quantitative microscopy methods and strategies to avoid unconscious bias in imaging experiments.

We recently developed a technique for rapidly and reversibly inhibiting protein function through light-inducible sequestration of proteins away from their normal sites of action. Here, we adapt this method for inducible inactivation of Bem1, a scaffold protein involved in budding yeast polarity. We find that acute inhibition of Bem1 produces profound defects in cell polarization and cell viability that are not observed in bem1Δ. By disrupting Bem1 activity at specific points in the cell cycle, we demonstrate that Bem1 is essential for the establishment of polarity and bud emergence but is dispensable for the growth of an emerged bud. By taking advantage of the reversibility of Bem1 inactivation, we show that pole size scales with cell size, and that this scaling is dependent on the actin cytoskeleton. Our experiments reveal how rapid reversible inactivation of protein function complements traditional genetic approaches. This strategy should be widely applicable to other biological contexts.