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. Author manuscript; available in PMC: 2021 Oct 1.
Published in final edited form as: Curr Opin Cell Biol. 2020 May 1;66:1–10. doi: 10.1016/j.ceb.2020.03.003

Figure 3. Example of a spatiotemporal optogenetics experiment with subcellular resolution.

Figure 3.

Shown is a human tissue culture cell in which a photosensitive LOV2/Zdk1 protein-protein interaction module was inserted into the endogenous EB1 gene by CRISPR/Cas9 genome editing, which replaces the wild-type EB1 protein with the light sensitive π-EB1 variant in one step. The images show an overlay of the mCherry-tagged π-EB1 C-terminal half on growing microtubule ends from short time-lapse sequences (8 images every 5 seconds) in alternating green and magenta before, during and after localized 470 nm blue light exposure. The light-exposed region is indicated by the blue overlay in the middle panel. Note the gradient of π-EB1 dissociation near the edge of the light-exposed region that results from diffusion of photoactivated molecules. Sharper boundaries can be achieved with LOV2 domain variants with faster dark recovery rates [45].