Construction of fluorescent protein fusions

EGFP-C1-TGAT was a kind gift from Susanne Schmidt [21]. mCherry-C1-TGAT (https://www.addgene.org/84334/) was obtained by cutting the EGFP-C1-TGAT vector with XhoI and AgeI and replacing the EGFP for mCherry, cut from mCherry-C1 with the same enzymes. Restriction sites and oligonucleotide overhangs are marked in bold in primer sequences.

To obtain mCherry-C1-TGAT^Δ15, we performed PCR with EGFP-C1-TGAT as template, by amplifying with forward 5’-GCGCGATCACATGGTCCTG-3′ and reverse 5′- TTTGGTACCTCAGGCTACGATTTTCCCGTC-3′. TGAT^Δ15 was ligated into mCherry-C1 by cutting the vector and PCR product with KpnI and HindIII.

To obtain EGFP-C1-TGAT^C242S, we performed a mutagenesis PCR with EGFP-C1-TGAT as template, by amplifying with forward 5’-GCGCGATCACATGGTCCTG-3′ and reverse 5’-CTCTTTGAACCGATGGCTCAGGGCTACGATTTTCC-3′. mCherry-C1-TGAT^C242S was obtained by cutting the EGFP-C1-TGAT^C242S vector with HindIII and AgeI and replacing the EGFP for mCherry, cut from mCherry-C1 with the same enzymes.

To obtain mCherry-C1-TGAT^C253S and mCherry-C1-TGAT^{C242S, C253S} (https://www.addgene.org/84336/), we performed a PCR with EGFP-C1-TGAT and EGFP-C1-TGAT^C242S as template, respectively, by amplifying with forward 5’-AGGTCTATATAAGCAGAGC-3′ and reverse 5’-TTGGTACCTCAAAACCAACTAATTTCACGAAAAGTCTCTTTG-3′. TGAT^C253S and TGAT^{C242S, C253S} were ligated into mCherry-C1 by cutting the vector and PCR products with Acc651 and HindIII.

CFP and YFP color variants were obtained by cutting mCherry-C1-TGAT, mCherry-C1-TGAT^Δ15, mCherry-C1-TGAT^C242S, mCherry-C1-TGAT^C253S and mCherry-C1-TGAT^{C242S, C253S} with AgeI and HindIII and replacing the mCherry with mTurquoise1 or mVenus, cut from mTurquoise1-C1 and mVenus-C1 with the same enzymes.

For the rapamycin experiments, mCherry-FKBP12-C1-TGAT^Δ15 was created by cutting mCherry-C1-TGAT^Δ15 with AgeI and HindIII and replacing mCherry with FKBP12-mCherry, cut from FKBP12-mCherry-C1 [26] with the same enzymes.

A membrane targeting sequence (derived from amino acid residue 1–10 of Lck; MGCVCSSNPE) was constructed by annealing [27] two oligonucleotide linkers, 5′-ctagccaccatgggctgcgtgtgcagcagcaaccccgagcta-3′ and 5′-ccggtagctcggggttgctgctgcacacgcagcccatggtgg-3′, with sticky overhangs and inserting it into an mVenus-C1 plasmid cut with NheI and AgeI, resulting in Lck-mVenus (https://www.addgene.org/84337/). Lck-mTurquoise2 (https://www.addgene.org/98822/) was obtained by exchanging mVenus for mTurquoise2 in the Lck-mVenus plasmid by cutting with AgeI and BsrGI.

The Lck-FRB-ECFP(W66A) was a kind gift from M. Putyrski [28]. FRB-ECFP(W66A)-Giantin, ECFP(W66A)-FRB-MoA, mVenus-MKL2 and the DORA RhoA sensors were previously described [26]. In order to obtain mTurquoise2-CAAX(RhoA), two oligonucleotides were annealed as previously described [27]. Annealing forward 5’-GTACaagctgcaagctagacgtgggaagaaaaaatctgggtgccttgtcttgtgaG-3′ and reverse 5′- GATCctcacaagacaaggcacccagattttttcttcccacgtctagcttgcagCTT-3′ oligonucleotides yielded the coding sequence for the last 15 amino acids of the C-terminus of RhoA (LQARRGKKKSGCLVL*) with overhangs (in capitals) on both sides, compatible with BsrGI and BamHI restriction sites. The double stranded linker was ligated into a C1-mTurquoise2 vector cut with BsrGI and BamHI, resulting in mTurquoise2-CAAX(RhoA). FRB-ECFP-CAAX(RhoA) was obtained by ligating the CAAX(RhoA) fragment, cut from mTurquoise2-CAAX(RhoA), into FRB-ECFP(W66A)-Giantin cut with the same enzymes. mRFP-RhoGDI was a kind gift from Martin A. Schwartz [29]. Plasmids constructed in this study will be made available through Addgene: http://www.addgene.org/Dorus_Gadella/.