Cloning of Optogenetic Constructs

Cloning of I-BAR–Cry2–mCh, Cry2–mCh–I-BAR, I-BAR–Cry2–mCh–WH2, and Cry2–mCh–MTSS1 constructs was conducted using a previously described cloning scheme (34 (link)). Briefly, the I-BAR domain from MTSS1, the WH2 domain from MTSS1, and MTSS1 were PCR amplified from human MTSS1 complementary DNA obtained from the Arizona State University DNA repository (DNASU ID: HsCD00746054). N-terminal I-BAR genes were cloned into Cry2PHR–mCherry (Addgene; #26866) using NheI and XhoI restriction sites (23 (link)). C-terminal I-BAR, WH2, and MTSS1 genes were cloned into Cry2PHR–mCherry using BsrgI and NotI restriction sites. The ezrin–GFP expression construct was a gift from Stephen Shaw (plasmid pHJ421; Addgene; #20680 (35 (link))). The actin expression construct (pCAG-mGFP-actin; Addgene; #21948) was a gift from Ryohei Yasuda. CIBN–GFP–CAAX (pCIBN(deltaNLS)-pmGFP; Addgene; #26867) was a gift from Chandra Tucker. Midi prep quantities of DNA of each construct were created from Escherichia coli and collected for cell transfection.