Wittmann (Austria)

This protocol describes procedures for generating cell lines in which the MT plus end tracking protein EB1 is replaced by a photo-inactivated variant \(π-EB1), which we generated by inserting a light sensitive protein interaction module between functional domains of EB1.π-EB1 photo-dissociation allows control of microtubule dynamics with high spatial and temporal accuracy and local investigation of microtubule functions in complex cell behaviors.Here we describe two protocols we have used to functionally replace EB1 with π-EB1 in a cancer cell line: \(I) an shRNA-based protocol in which endogenous EB1 is depleted in cells stably expressing π-EB1, and \(II) a CRISPR/Cas9-based protocol in which expression from the endogenous EB1 and EB3 loci is deleted, and subsequently replaced by the photo-inactivated variant.These protocols serve as guides for generating other π-EB1 cell lines.In addition, this domain-splitting strategy for replacing speci c protein activities with photo-inactivated variants could be adapted to other multi-domain proteins.