Fig. 4.

Manipulation of PIs by the blue-light-induced CRY2-CIBN heterodimerization system. A. Schematic drawing showing the principle underlying blue-light-induced PI(4,5)P₂ dephosphorylation. CIBN is targeted to the plasma membrane while CRY2 is fused to a 5-phosphatase (5-ptase) domain and carries an mCherry-tag (mCh) for microscopic detection. mCh-CRY2-5-ptase is cytosolic under dark conditions but binds CIBN, and thus is recruited at the target membrane, upon blue-light illumination. This translocation results in dephosphorylation of PI(4,5)P₂ and this reaction can be detected as loss of iRFP-PH-PLC_δ1 [a PI(4,5)P₂ binding module] from the plasma membrane. B. Confocal microscopy images of a HeLa cell showing loss of iRFP-PH-PLC_δ1 from the plasma membrane upon blue-light-induced recruitment of mCh-CRY2-5-ptase to this cellular compartment. Pictures were taken 10 s before, 10 s after and 15 min after a 200 ms pulse of blue-light illumination [112]. C. Schematic drawing showing the principle underlying blue-light-induced PI(3,4,5)P₃ synthesis via the recruitment of the endogenous p110α subunit of type I PI 3-kinase to a CRY2 fusion of the iSH2-region of its regulatory subunit p85 [101,112]. PI(3,4,5)P₃ is detected by the RFP-tagged PH-domain from Akt. D. Confocal microscopy images of a migrating RAW264.7 macrophage expressing the fusion proteins shown in C. Continuous focal illumination (blue circle) results in local PI(3,4,5)P₃ synthesis and reverses the direction of cell movement occurring before illumination by inducing local ruffling (arrows). E. Illustrations showing the changes in cell shape and position induced by local PI(3,4,5)P₃ synthesis.