Domain arrangement combinations of BcLOV4, mCherry, and proteins-of-interest (with a flexible (GGGS)2 linker between each pair) were assembled by Gibson cloning using NEB HiFi DNA Assembly Master Mix (E2621) into the pcDNA3.1 mammalian expression vector under the CMV promoter. BcLOV4 and mCherry were amplified from their mammalian codon-optimized reported fusion (Addgene plasmid 114595).10 (link) Wildtype Cdc42 GTPase was amplified from CLPIT Cry2PHR-mCherry-Cdc42, a kind gift from Dr. Lukasz Bugaj, with the ‘CAAX’ removed to prevent prenylation. The DH domain of Tiam1 was identified using the PROSITE ExPASy database and amplified from pMXs3-TIAM1 (Addgene plasmid 86143). Other effectors were amplified from previously reported plasmids, all available on Addgene: Rac1 (plasmid 135396),7 Intersectin1-DHPH (plasmid 174509),6 (link) RhoA (plasmid 164472), ARHGEF11-DH (plasmid 164473).31 Genetic constructs were transformed into competent E. coli (New England Biolabs, C2984H), and sequence-verified by Sanger sequencing. For filopodia quantification, the miRFP703-tagged LifeAct plasmid was acquired from Addgene (plasmid 79993). Plasmids for opto-Tiam1 and opto-Cdc42 (plasmids 174508 and 174509) and plasmids for POI screening of BcLOV4 fusions (plasmids 174511 and 174512) will be available through Addgene.
Protocol detail
Optogenetic control of Rho GTPases
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Corresponding Organization : University of Pennsylvania
Other organizations : Waseda University, Center for Systems Biology, Harvard University, McGill University, CUNY Advanced Science Research Center, City University of New York, The Graduate Center, CUNY, City College of New York
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Variable analysis
independent variables
- Domain arrangement combinations of BcLOV4, mCherry, and proteins-of-interest (with a flexible (GGGS)2 linker between each pair)
- Filopodia quantification
- Wildtype Cdc42 GTPase (with the 'CAAX' removed to prevent prenylation)
- DH domain of Tiam1
- Intersectin1-DHPH
- ARHGEF11-DH
- MiRFP703-tagged LifeAct plasmid
- Not explicitly mentioned
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