Journal: Cell chemical biology
Article Title: A chemogenetic platform for controlling plasma membrane signaling and synthetic signal oscillation.
doi: 10.1016/j.chembiol.2022.06.005
Figure Lengend Snippet: Figure 7. Chemogenetic control of Rac activation and lamellipodia formation (A) Schematic illustration of the experimental setup. A synthetic, mDcTMP-responsive guanine nucleotide exchange factor for Rac was constructed by fusing the DH-PH domain of Tiam1 (Inoue et al., 2005) to the C terminus of mNG-iK6DHFR (mNG-iK6DHFR-Tiam1). This protein was used to activate endogenous Rac upon the addition of mDcTMP. The cell morphology was monitored by the F-actin marker Lifeact (Riedl et al., 2008) fused to mCherry (Lifeact-mCherry). (B) Representative time-lapse confocal fluorescence images of a HeLa cell coexpressing mNG-iK6DHFR-Tiam1 and Lifeact-mCherry. Images were taken before (left) and after the addition of mDcTMP (5 mM) (right). For the time-lapse movie, see Video S7. (C) PM recruitment of mNG-iK6DHFR lacking the Tiam1 domain (control experiment for B). Scale bars, 20 mm. (D) Time course of SLIPT and cell area change. The normalized fluorescence intensities of mNG-iK6DHFR-Tiam1 (or mNG-iK6DHFR) in the cytoplasm were plotted as a function of time to evaluate SLIPT (bottom). Cell areas were calculated from the Lifeact-mCherry images, and cell area changes were plotted as a function of time. Data are presented as the mean ± SD (n = 40 cells for mNG-iK6DHFR-Tiam1 and 24 cells for mNG-iK6DHFR).
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Murine: embryonic fibroblast cell line NIH3T3 Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University TKG 0299 Monkey: kidney fibroblast-like cell line Cos-7 Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University TKG 0514 Human: embryonic kidney cell line Lenti-X 293T Clontech Cat# 632180, RRID:CVCL_4401 Recombinant DNA pCMV-eDHFR-EGFP Ishida et al., 2013 N/A pPBpuro-K6-eDHFR-EGFP Nakamura et al., 2020a N/A pCMV-EGFP-K6-eDHFR This study N/A pCMV-eDHFR(69K6)-EGFP This study N/A pCMV-EGFP-eDHFR(69K6) This study N/A pCMV-eDHFR(145K6)-EGFP This study N/A pCMV-EGFP-eDHFR(145K6) This study N/A pCMV-mCherry-Giantin Suzuki et al., 2020 N/A pCMV-EGFP-eDHFR(69K6)-NES This study N/A pCMV-miRFP680-eDHFR(69K6) This study N/A pCMV-mCherry-FKBP-INPP5E This study N/A Plasmid: Lyn11-targeted FRB Inoue et al., 2005 Addgene plasmid #20147 Plasmid: GFP-C1-PLCd-PH Stauffer et al., 1998 Addgene plasmid #21179 Plasmid: Lact-C2-GFP Yeung et al., 2008 Addgene plasmid #22852 pCMV-mTagBFP2-PHOSBP This study; modified fluorescent protein from piRFP713-PH-OSBP1 (provided by Dr. Fubito Nakatsu, Niigata University).
Techniques: Control, Activation Assay, Construct, Marker
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