Biosensor construction and fluorometry

The previously published single-chain FRET-biosensor for Rac1 (25) was modified to improve the dynamic range of response and the expression stability in living cells. Briefly, the original Rac1 biosensor contained a monomeric Cerulean1 (mCer1) as the FRET donor, two tandem p21 binding domains (PBD) of p21 activated kinase 1 (PAK1, amino acid residues 70-149) to achieve autoinhibitory regulation, separated by a structurally optimized linker (GSGGPPGSGGSG), monomeric Venus (mVen) as FRET acceptor, and a full-length wild-type (WT) Rac1 (25). The second PBD contained H83D, H86D point mutations to render it unable to bind to active GTPase. To further optimize the stability and response of this system, the FRET acceptor mVen was replaced by monomeric circularly permutated (cp) versions cp49, cp157, cp173, cp195 or cp229Ven (32), PCR amplified using the primer pairs: 5'-cataagaatgcggccgcaatgaccggcaagctgcccg-3' and 5'-gggggaattccttggtgcagatcaacttcagggtc-3' (cp49); 5'-gcgtaccatggatgcagaagaacggcatcaaggc-3' and 5'-ggccgaattccttcttgtcggcggtgatatagac-3' (cp157); 5'-cataagaatgcggccgcaatggacggcggcgtgcagc-3' and 5'-ggaattccttctcgatgttgtggcggatcttgaagtttgc-3' (cp173); 5'-cataagaatgcggccgcaatgctgcccgacaaccactacc-3' and 5' -ccggaattccttcagcacggggccgtcgccgatg-3' (cp195); and 5'-cataagaatgcggccgcaatgatcactctcggcatggacgag-3' and 5'-ggaattccttcccggcggcggtcacgaactc-3' (cp229). The PCR-amplified fragments were subcloned into the biosensor backbone using the NotI and EcoRI restriction sites (Supplemental Fig. 1A). In order to achieve the required expression stability needed for the production of stable-inducible cell lines, the codon usages of the mCer1 and the second PBD were synonymously modified (33) in Rac1 (25) and Cdc42 (24) biosensors (Supplemental Fig. 1B, C). Furthermore, the final version of the optimized Rac1 biosensor construct also contained additional linker optimization applied to monomeric cp229Ven (mcp229Ven), by replacing the first 16 amino acids with the flexible, structureless linker that is resistant to protease cleavage (34) Briefly, mcp229Ven was PCR amplified using a primer pair 5'-gatatatatgcggccgcaatgggcagcaccagcggcagcggcaaaccgggcagcggcgaaggcagcatggtgagcaagggcgaggagctg-3' (optimized linker sequence underlined (34)) and 5'-taaataaataaagaattccccggcggcggtcacgaactccag-3', and subcloned into the biosensor backbone using NotI and EcoRI (Supplemental Fig. 1A) restriction sites. This version of the biosensor did not yield different fluorometric responses from the original mcp229Ven version of the sensor without this modification (data not shown).

To create the Rac2 biosensor, Rac1 was replaced by full-length WT Rac2 in the mVen and mcp229Ven versions of the Rac1 sensor. WT Rac2 was PCR amplified using the primer pair 5'-gtatatatatatatgaattcatgcaggccatcaagtgtgtg-3' and 5'-ccaattaattaattaactcgagctagaggaggctgcaggcgcg-3', and was subcloned into the biosensor backbone using the EcoRI and XhoI restriction sites (Supplemental Fig. 1A). The synonymous modifications (Supplemental Fig. 1B, C) were applied to the mCer1 and the second PBD as in Rac1 biosensor. To optimize the mcp229Ven version of the Rac2 biosensor, the linker was also optimized by replacing the first 16 amino acids within the mcp229Ven to confer protease resistance and stability, as in the optimized Rac1 sensor.

The biosensor cDNA cassettes were subcloned into pTriEX-4 (Novagen) for transient expression. Characterization of biosensor response was performed in HEK293T cells by overexpressing WT or mutant versions of the biosensor, with or without the appropriate upstream regulators as previously described (35). Briefly, HEK293T cells were plated overnight at 1.2×10⁶ cells/well of 6-well plates coated with poly-L-lysine, and transfected the next day using Lipofectamine2000 (Invitrogen) following the manufacturer's protocols. Biosensors were co-transfected at ratios of 1:3 (Rac1) and 1:4 (Rac2) with GDI or GAP, or 1:0.5 – 10 for co-transfection with GEFs (+/− GDI) as indicated. Adherent cells were washed in PBS and fixed using 3.7% formaldehyde 48 h following the transfection, and fluorescence emission spectra was measured by spectrofluorometer using a plate reader (Horiba-Jobin-Yvon Fluorolog-3MF2 with MicroMax plate reader). The spectra were obtained by exciting with 433 nm light, with emission scanned from 450 – 600 nm. The fluorescence reading of cells with empty vector (pCDNA3.1) was used to measure light scatter and autofluorescence, which were subtracted from the data. The resulting spectra were normalized to the peak mCer1 emission intensity at 474 nm to generate the final ratiometric spectra.

The second generation tetracycline-inducible system was used to produce stable-inducible cell lines. Tet-OFF tetracycline Trans-Activator (tTA) was restriction digested out of a pRetroX-advanced tet-OFF backbone (Clontech) using the BamHI/EcoRI sites flanking the tTA gene cassette. This fragment was then ligated into the new pQEXIN retroviral vector at its multiple cloning sites. The pQEXIN vector was produced by modifying the pQCXIN (Clontech) retroviral vector, originally containing the CMV promoter, a multiple cloning site, an internal ribosomal entry site, and a neomycin resistance gene cassette. The CMV promoter was digested out using the BglII/NotI sites flanking the promoter, and a PCR-amplified EF1α promoter containing BamHI/NotI sites was ligated into the backbone (amplified using the primer pair: 5’-gtcgacattattgactagatggatccgcgtgaggctccggtgcccgtcagtg-3’ and 5’-gctagcgcatatgcttaattgcggccgctatattcctcacgacacctgaaatggaag-3’). This produced the pQEXIN backbone system, resistant to gene silencing in hematopoietic/stem cells.

The expression cassettes for the optimized Rac1, Rac2 and Cdc42 biosensors were placed into pRetro-X inducible retroviral backbone system (Clontech), modified to contain the Gateway recombination cassette (Invitrogen) and an antibiotic resistance for Zeocin (pRetro-X-Zeo-DEST). Briefly, Rac1, Rac2 and Cdc42 biosensor cassettes were subcloned into the Gateway pENTR-4 entry vector using the NcoI/XhoI restriction sites. The Gateway recombination reaction was performed following the manufacturer's protocols (Invitrogen), producing pRetro-X-Zeo-Rac1, pRetro-X-Zeo-Rac2 and pRetro-X-Zeo-Cdc42.