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Light can be controlled with high spatial and temporal accuracy. Therefore, optogenetics is an attractive experimental approach to modulate intracellular cytoskeleton dynamics at much faster timescales than by genetic modification. For example, in mammalian cells, microtubules (MTs) grow tens of micrometers per minute and many intracellular MT functions are mediated by a complex of +TIP proteins that dynamically associate with growing MT plus ends. EB1 is a central component of this +TIP protein network, and we recently developed a photo-inactivated π-EB1 by inserting a blue light-sensitive LOV2/Zdk1 module between the EB1 MT-binding domain and the +TIP adaptor domain. Blue light-induced π-EB1 photodissociation results in disassembly of the +TIP complex and strongly attenuates MT growth in mammalian cells.In this chapter, we discuss theoretical and practical aspects of how to perform high-resolution live-cell microscopy in combination with π-EB1 photodissociation. However, these techniques are broadly applicable to other LOV2-based and likely other blue light-sensitive optogenetics. In addition to being a tool to investigate +TIP functions acutely and with subcellular resolution, because of its dramatic and rapid change in intracellular localization, π-EB1 can serve as a powerful tool to test and characterize optogenetic illumination setups. We describe protocols on how to achieve micrometer-scale intracellular control of π-EB1 activity using patterned illumination, and we introduce a do-it-yourself LED cube design compatible with transmitted light microscopy in multiwell plates.

Genetically encoded live-cell reporters measure signaling pathway activity at the cellular level with high temporal resolution, often revealing a high degree of cell-to-cell heterogeneity. By using multiple spectrally distinct reporters within the same cell, signal transmission from one node to another within a signaling pathway can be analyzed to quantify factors such as signaling efficiency and delay. With other reporter configurations, correlation between different signaling pathways can be quantified. Such analyses can be used to establish the mechanisms and consequences of cell-to-cell heterogeneity and can inform new models of the functional properties of signaling pathways. In this unit, we describe an approach for designing and executing live-cell multiplexed reporter experiments. We also describe approaches for analyzing the resulting time-course data to quantify correlations and trends between the measured parameters at the single-cell level. © 2018 by John Wiley & Sons, Inc.