![Figure 1. G3BP1 and IMP1 exhibit different dynamics of protein exchange between SGs. (A) Protein interaction (gray) and RNA-binding domains (black) of human G3BP1 and IMP1 according to SMART analysis for identification of signaling domains (Schultz et al., 1998). RRM, RNA recognition motif; KH, K homology domain. Right: Western analysis of cellular lysates after transfection with PAGFP-tagged G3BP1 and IMP1 (arrowhead). Lysates were analyzed using size-based capillary electrophoresis, and electropherograms are represented as pseudoblots as described in Materials and methods. Molecular mass standards are indicated. The respective endogenous proteins are indicated by an arrow. Please note that analysis by size-based capillary electrophoresis can yield protein mobilities that differ from separation by standard SDS-PAGE. (B) Colocalization of exogenously expressed mCherry-G3BP1 with the SG marker TIA-1. Bar, 10 µm. (C) Colocalization and dynamic exchange of fluorescence-tagged G3BP1 and IMP1 in SGs of living PC12 cells. Granules were labeled with mCherry-IMP1 and PAGFP-G3BP1 was activated in one granule (dashed square). The outline of the cell and the nucleus are indicated in the red fluorescent micrographs. Fluorescence distribution was followed over time. After some seconds, photoactivated PAGFP-G3BP1 appears in SGs outside of the activated region indicating dynamic exchange of G3BP1 between SGs. Bar, 10 µm. (D) Bar plot showing the size distribution of SGs as determined from the area of mCherry-IMP1 positive granules. The box represents 50% of the population, whiskers range from 5% to 95% and crosses correspond to the minimal and maximal values (n = 34). (E) Schematic representation showing the FDAP approach to determine dynamics and binding of PAGFP-tagged G3BP1 and IMP1 in granules. Photoactivation and fluorescence recording was performed in a 3 × 5 µm region containing cytosol and granules between nucleus and plasma membrane (red box). The decay curves were fitted with model FDAP functions. (F) FDAP curves for PAGFP-IMP1, PAGFP-G3BP1, and 3×PAGFP (mean ± SEM, n = 13 [PAGFP-IMP1], n = 17 [PAGFP-G3BP1], n = 20 [3×PAGFP]) showing the different dynamics of IMP1 and G3BP1. (G) Residence time of PAGFP-tagged IMP1 and G3BP1 in granules as determined by the model FDAP function (mean ± SEM, n = 19 [PAGFP-IMP1], n = 20 [PAGFP-G3BP1], n = 20 [3×PAGFP] from two [3×PAGFP], four [PAGFP-G3BP1], and five [PAGFP-IMP1] independent experiments). Comparison between the constructs involved one-way ANOVA followed by post-hoc Tukey's test. **, P < 0.01; ***, P < 0.001 (compared with control [3×PAGFP]); +, P < 0.05. For all experiments, stress had been induced by a 20-min treatment with 0.5 mM sodium arsenite before imaging.](https://cdn.rupress.org/rup/content_public/journal/jcb/217/4/10.1083_jcb.201709007/10/jcb_201709007_fig1.jpeg?Expires=1771701958&Signature=I-wLbbN9AJSVDcFP-nnhbzQrR6puVGuzCRn6urYNQ9CDnR61p4sa6Qg-Q~lGrTM2xK8lhilsFKoixT~FkI3eK0IfjWs9kMw40Yoe2~shWqByP0kzspSnhT8qV8z9gddBplwIrCZObHxka7biKNkOoUCRHP6HvuFbEiG3EzGjk1SccXcZLhP1LXWMUh8rF6~uGezp~ipfVRgmWlOAHHQ~Wh9isB2HDcugeV3WaN7AiAT8O0CsKXaFYQdCY5K6KSfRHu4bbHzyGg1A73OLS7D9qArqEevOyyWVxxR4HPFNsAhMMn8mdHaVoyEuQv7vsKPgCBaKnoKu21qyGVdPE4r-fQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
G3BP1 and IMP1 exhibit different dynamics of protein exchange between SGs. (A) Protein interaction (gray) and RNA-binding domains (black) of human G3BP1 and IMP1 according to SMART analysis for identification of signaling domains (Schultz et al., 1998). RRM, RNA recognition motif; KH, K homology domain. Right: Western analysis of cellular lysates after transfection with PAGFP-tagged G3BP1 and IMP1 (arrowhead). Lysates were analyzed using size-based capillary electrophoresis, and electropherograms are represented as pseudoblots as described in Materials and methods. Molecular mass standards are indicated. The respective endogenous proteins are indicated by an arrow. Please note that analysis by size-based capillary electrophoresis can yield protein mobilities that differ from separation by standard SDS-PAGE. (B) Colocalization of exogenously expressed mCherry-G3BP1 with the SG marker TIA-1. Bar, 10 µm. (C) Colocalization and dynamic exchange of fluorescence-tagged G3BP1 and IMP1 in SGs of living PC12 cells. Granules were labeled with mCherry-IMP1 and PAGFP-G3BP1 was activated in one granule (dashed square). The outline of the cell and the nucleus are indicated in the red fluorescent micrographs. Fluorescence distribution was followed over time. After some seconds, photoactivated PAGFP-G3BP1 appears in SGs outside of the activated region indicating dynamic exchange of G3BP1 between SGs. Bar, 10 µm. (D) Bar plot showing the size distribution of SGs as determined from the area of mCherry-IMP1 positive granules. The box represents 50% of the population, whiskers range from 5% to 95% and crosses correspond to the minimal and maximal values (n = 34). (E) Schematic representation showing the FDAP approach to determine dynamics and binding of PAGFP-tagged G3BP1 and IMP1 in granules. Photoactivation and fluorescence recording was performed in a 3 × 5 µm region containing cytosol and granules between nucleus and plasma membrane (red box). The decay curves were fitted with model FDAP functions. (F) FDAP curves for PAGFP-IMP1, PAGFP-G3BP1, and 3×PAGFP (mean ± SEM, n = 13 [PAGFP-IMP1], n = 17 [PAGFP-G3BP1], n = 20 [3×PAGFP]) showing the different dynamics of IMP1 and G3BP1. (G) Residence time of PAGFP-tagged IMP1 and G3BP1 in granules as determined by the model FDAP function (mean ± SEM, n = 19 [PAGFP-IMP1], n = 20 [PAGFP-G3BP1], n = 20 [3×PAGFP] from two [3×PAGFP], four [PAGFP-G3BP1], and five [PAGFP-IMP1] independent experiments). Comparison between the constructs involved one-way ANOVA followed by post-hoc Tukey's test. **, P < 0.01; ***, P < 0.001 (compared with control [3×PAGFP]); +, P < 0.05. For all experiments, stress had been induced by a 20-min treatment with 0.5 mM sodium arsenite before imaging.
