![Figure 2. Design, characterization, and validation of the GEF-H1 FLARE biosensor. (A) Schematic of the biosensor design, using homology with the Vav1 structure (PDBID: 3ky9), and showing the FRET pair inserted in the hinge region between the AID and DH domains. Domains colored as in Fig. 1 A. (B) Emission spectra of unmodified and hyperactivated (Δ161) GEF-H1 FLARE212 expressed in suspended HEK293T cells (excitation at 405 nm). (C) Live MDA-MB-231 cells coexpressing the GEF-H1 FLARE212 and an MT marker (EMTB). GEF-H1 FLARE212 (middle) is localized primarily on MTs (left). The activity map (right) shows that inactive GEF-H1 is on MTs, while active GEF-H1 is at the cell edge. (D) Time-lapse microscopy of COS-7 cells expressing GEF-H1 FLARE212 shows that inactive GEF-H1 localized on MT and active GEF-H1 localized in protrusions (new protrusions occurring between t = 0 and t = 18 are indicated by white arrows). (E) Nocodazole treatment of MEFs expressing GEF-H1 FLARE212 led to a global increase of GEF-H1 activity. (F) Quantification of GEF-H1 FLARE212 activity from cells treated as in E and monitored for 14 and 26 min after treatment (left panel, n = 9 and n = 5, respectively). Statistical analysis of biosensor activity upon nocodazole treatment compared with vehicle control (DMSO, right panel; paired t test, P < 0.0001 [****] for before vs. after 14-min treatment, P < 0.01 [**] for before vs. after 26-min treatment and after 14-min vs. after 26-min treatment; P < 0.0001 [****] for vehicle vs. nocodazole at 14 and 26 min after treatment; right panel). Intensity values were normalized to the activity before treatment. (G) Truncation of the first 161 residues of GEF-H1 FLARE212 led to increased overall activity in MDA-MB-231 cells. (H) Statistical analysis of cells expressing the full-length (n = 6) or truncated GEF-H1 FLARE212 (n = 8; t test, P < 0.01 [**]). Intensity values were normalized to the activity of GEF-H1 FLARE212. Error bars indicate SD. (I) Coexpression of GEF-H1 FLARE and constitutively active Gα13 (Q226L) in suspended HEK293T cells led to higher activity. Intensity values were normalized to the activity of GEF-H1 FLARE212. Error bars indicate SD of three independent replicates. (J) RhoA exchange activity of GEF-H1 biosensor constructs as assayed by NMR. Activity of WT GEF-H1 and GFP shown for reference. Error bars indicate SD of three independent replicates. Scale bar = 10 µm.](https://cdn.rupress.org/rup/content_public/journal/jcb/218/9/10.1083_jcb.201812073/8/jcb_201812073_fig2.jpeg?Expires=1770733659&Signature=SX~fIPh3IAPs-NlnfLQ8pPVB0BKZYGgMfLm0VDtxKmdlZt9oieuPQzhlVqMbstjRb1XpAwtv5aEWH8mm27vIK9rAQpcwhN1aLnLTnf-erb~FDDY2r4AR78HKnpKEKgZjC1YGb~kI1NsbFvvTDfVVjlFLh2OvV~37rtCULeSjeF1IyxbRYl8CBZI9KInYLqiZKHl6WfoT7V1cHmHqic9DuGPXEr9egFRYclpaaEp5uJDK74IVPLlrC3jPbjWF6YDOqk7BCXjnDppcqmHCE8ztxSMK9Sw4es9jYyw0e6~aucABtlztdE9Us7wGBBxbU7fAv2Q0Tplzngclff00CJ3Xzg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Design, characterization, and validation of the GEF-H1 FLARE biosensor. (A) Schematic of the biosensor design, using homology with the Vav1 structure (PDBID: 3ky9), and showing the FRET pair inserted in the hinge region between the AID and DH domains. Domains colored as in Fig. 1 A. (B) Emission spectra of unmodified and hyperactivated (Δ161) GEF-H1 FLARE212 expressed in suspended HEK293T cells (excitation at 405 nm). (C) Live MDA-MB-231 cells coexpressing the GEF-H1 FLARE212 and an MT marker (EMTB). GEF-H1 FLARE212 (middle) is localized primarily on MTs (left). The activity map (right) shows that inactive GEF-H1 is on MTs, while active GEF-H1 is at the cell edge. (D) Time-lapse microscopy of COS-7 cells expressing GEF-H1 FLARE212 shows that inactive GEF-H1 localized on MT and active GEF-H1 localized in protrusions (new protrusions occurring between t = 0 and t = 18 are indicated by white arrows). (E) Nocodazole treatment of MEFs expressing GEF-H1 FLARE212 led to a global increase of GEF-H1 activity. (F) Quantification of GEF-H1 FLARE212 activity from cells treated as in E and monitored for 14 and 26 min after treatment (left panel, n = 9 and n = 5, respectively). Statistical analysis of biosensor activity upon nocodazole treatment compared with vehicle control (DMSO, right panel; paired t test, P < 0.0001 [****] for before vs. after 14-min treatment, P < 0.01 [**] for before vs. after 26-min treatment and after 14-min vs. after 26-min treatment; P < 0.0001 [****] for vehicle vs. nocodazole at 14 and 26 min after treatment; right panel). Intensity values were normalized to the activity before treatment. (G) Truncation of the first 161 residues of GEF-H1 FLARE212 led to increased overall activity in MDA-MB-231 cells. (H) Statistical analysis of cells expressing the full-length (n = 6) or truncated GEF-H1 FLARE212 (n = 8; t test, P < 0.01 [**]). Intensity values were normalized to the activity of GEF-H1 FLARE212. Error bars indicate SD. (I) Coexpression of GEF-H1 FLARE and constitutively active Gα13 (Q226L) in suspended HEK293T cells led to higher activity. Intensity values were normalized to the activity of GEF-H1 FLARE212. Error bars indicate SD of three independent replicates. (J) RhoA exchange activity of GEF-H1 biosensor constructs as assayed by NMR. Activity of WT GEF-H1 and GFP shown for reference. Error bars indicate SD of three independent replicates. Scale bar = 10 µm.
