Designing a rigorous microscopy experiment: Validating methods and avoiding bias.

Images generated by a microscope are never a perfect representation of the biological specimen. Microscopes and specimen preparation methods are prone to error and can impart images with unintended attributes that might be misconstrued as belonging to the biological specimen. In addition, our brains are wired to quickly interpret what we see, and with an unconscious bias toward that which makes the most sense to us based on our current understanding. Unaddressed errors in microscopy images combined with the bias we bring to visual interpretation of images can lead to false conclusions and irreproducible imaging data. Here we review important aspects of designing a rigorous light microscopy experiment: validation of methods used to prepare samples and of imaging system performance, identification and correction of errors, and strategies for avoiding bias in the acquisition and analysis of images.

Probing Yeast Polarity with Acute, Reversible, Optogenetic Inhibition of Protein Function.

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.

A light-inducible organelle-targeting system for dynamically activating and inactivating signaling in budding yeast.

Protein localization plays a central role in cell biology. Although powerful tools exist to assay the spatial and temporal dynamics of proteins in living cells, our ability to control these dynamics has been much more limited. We previously used the phytochrome B- phytochrome-interacting factor light-gated dimerization system to recruit proteins to the plasma membrane, enabling us to control the activation of intracellular signals in mammalian cells. Here we extend this approach to achieve rapid, reversible, and titratable control of protein localization for eight different organelles/positions in budding yeast. By tagging genes at the endogenous locus, we can recruit proteins to or away from their normal sites of action. This system provides a general strategy for dynamically activating or inactivating proteins of interest by controlling their localization and therefore their availability to binding partners and substrates, as we demonstrate for galactose signaling. More importantly, the temporal and spatial precision of the system make it possible to identify when and where a given protein's activity is necessary for function, as we demonstrate for the mitotic cyclin Clb2 in nuclear fission and spindle stabilization. Our light-inducible organelle-targeting system represents a powerful approach for achieving a better understanding of complex biological systems.