Optogenetic Control of Cofilin Dynamics

Antibodies were from the following sources: Cofilin (D3F9) XP® Rabbit mAb (Cell Signaling #5175), β-Actin (8H10D10) Mouse mAb (Cell Signaling #3700), Tks5 (Santa Cruz Biotechnology; sc-30122), Cortactin (Abcam; ab33333). The cDNA of the LOV2 domain from Avena sativa (oat) Phototropin1 (L404-L547) was used to generate photo-sensitive constructs. Three variants of LOV2 were used: wild-type, dark state mutant (C450A, L514K, G528A, L531E, and N538E), and lit state mutant (I510E/I539E) (Supplementary Table 2). ^{24 (link)} The cDNA of full-length rat cofilin was used for all constructs. The Z affibodies that selectively bind dark state LOV2 have been described elsewhere. ^{24 (link)} For transient expression in mammalian cells, constructs were cloned into pmCherry-C1 (Clontech Laboratories, Inc.). Cells were transfected with Lipofectamine 2000 (Life Technologies) using the manufacturer’s protocol 24 h before imaging. For imaging of living cells, cells were co-transfected with mCherry Z-lock cofilin and a membrane-anchored yPet (KRas C-terminus) to visualize the cell edge. ⁵¹