. 2014 Mar 27;6(6):1153-1164.
Epub 2014 Mar 13.
The Rac-FRET Mouse Reveals Tight Spatiotemporal Control of Rac Activity in Primary Cells and Tissues
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The Rac-FRET Mouse Reveals Tight Spatiotemporal Control of Rac Activity in Primary Cells and Tissues
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The small G protein family Rac has numerous regulators that integrate extracellular signals into tight spatiotemporal maps of its activity to promote specific cell morphologies and responses. Here, we have generated a mouse strain, Rac-FRET, which ubiquitously expresses the Raichu-Rac biosensor. It enables FRET imaging and quantification of Rac activity in live tissues and primary cells without affecting cell properties and responses. We assessed Rac activity in chemotaxing Rac-FRET neutrophils and found enrichment in leading-edge protrusions and unexpected longitudinal shifts and oscillations during protruding and stalling phases of migration. We monitored Rac activity in normal or disease states of intestinal, liver, mammary, pancreatic, and skin tissue, in response to stimulation or inhibition and upon genetic manipulation of upstream regulators, revealing unexpected insights into Rac signaling during disease development. The Rac-FRET strain is a resource that promises to fundamentally advance our understanding of Rac-dependent responses in primary cells and native environments.
Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
Raichu-Rac Expression Level in Tissues and Primary Cells of the Rac-FRET Mouse Strain (A) Whole-tissue lysates were prepared from adult Rac-FRET mice and blotted with anti-GFP antibody alongside recombinant CFP standards to reveal the CFP portion of Raichu-Rac. a.u., arbitrary units; WT, wild-type. (B) Raichu-Rac expression levels in total lysates of the indicated numbers of diisopropyl fluorophosphate-treated Rac-FRET neutrophils were determined by western blotting alongside recombinant RAC1 and CFP standards using anti-RAC1 AB (green) and anti-GFP AB (red) to compare biosensor expression with endogenous RAC1. (C) Raichu-Rac expression levels in total lysates of the indicated numbers of Rac-FRET MEFs were determined by western blotting alongside recombinant RAC1 and CFP standards with antibodies as in (B).
High Rac Activity in Lamellipodial Protrusions and Membrane Ruffles of PDGF-Stimulated Primary Rac-FRET MEFs (A) Rac activity in PDGF-stimulated (50 ng/ml) Rac-FRET MEFs determined by ratiometric FRET live imaging. Frames were taken every 10 s (left: generation of ratiometric image) and analyzed at protruding (top) and nonprotruding sections (bottom) along the cell edge (magnifications of boxes in the FRET image on the left). Note that, for ratiometric FRET measurements, increase in FRET ratio equals increase in Rac activity. (B) Quantification of mean Rac activity (FRET ratio ± SEM) in 88 basal and protruding sections along the cell edge of PDGF-stimulated Rac-FRET MEFs as in (A) from 20 cells and three independent experiments.
∗∗p < 0.01 by paired Student’s t test. (C) Rac activity in insulin-stimulated (100 μg/ml) Rac-FRET MEFs determined by ratiometric FRET imaging. Consecutive frames taken every 10 s, shown from 80 s after addition of insulin (left: YFP/FRET and CFP images used to generate first ratiometric image). Arrow shows Rac activity at membrane ruffles.
High Rac Activity during Spreading and Polarization of Rac-FRET Neutrophils (A) Spatiotemporal distribution of Rac activity in Rac-FRET neutrophils during spreading and polarization on glass coverslips was determined by ratiometric FRET live imaging in the presence or absence of 75 μM NSC23766 (without preincubation), starting from the first point of contact of the cells with the coverslip. Representative FRET ratio images are shown. (B) Quantification of Rac activity (average cellular FRET ratio, normalized to control cells) in 69 control and 59 NSC23766-treated cells as in (A) from six independent experiments ± SEM p < 0.0001 by ANOVA. (C) Surface area of the cells in (B). (D) Polarization of the cells in (B) was analyzed as: time to start polarizing (i), % of fully polarized cells (as defined by their ability to locomote) (ii), and % of cells reverting to nonpolarized morphology over the 30 min of imaging (iii).
∗p < 0.05 by unpaired Student’s t test.
High Rac Activity Correlates with Protrusion Formation at the Leading Edge and Oscillates between the Front and Back of Chemotaxing Rac-FRET Neutrophils (A) Ratiometric FRET live imaging of a representative Rac-FRET neutrophil chemotaxing toward 3 μM fMLP in an Ibidi chamber; images taken at 1 s intervals over 20 s (chemoattractant source is due south). For comparison of Rac activity with cell protrusions and retractions, cell perimeters at consecutive time points T (blue) and T+1 s (red) were plotted using ImageJ plugin QuimP. (B) Enlargement of boxed sections shown in (A). The green-framed box shows a protrusion at the leading edge of the cell, the red box a retraction at the trailing edge. (C) Polar plot of Rac activity (FRET ratio) around the perimeter of the cell shown in (A) over time; perimeters depicted as perfect circles, 1 circle/1 s time frame, starting in the center (1 s) and growing eccentrically outward over time. (D) Pearson’s correlation between Rac activity (FRET ratio) at nodes around the cell perimeter and 0.4 μm from the cell edge (schematic example shown in insert) and cell edge velocity. FRET intensities were evaluated against edge speeds between T−5 s and T+5 s to test for time dependence between Rac activity and membrane protrusion/retraction speed. Red line shows average correlation from seven independent experiments with a total of 133 cells chemotaxing toward 3 μM fMLP over 100–120 s with images acquired every 1 s; gray traces show means of individual experiments. (E) Rac-FRET neutrophils chemotaxing toward 3 μM fMLP in an Ibidi chamber were live imaged at 1 s intervals, line scans performed through the central longitudinal axis, and Rac activity (FRET ratio) over time plotted as kymographs. Profiling of steep (protruding) versus flat (stalling) sections of the kymograph was achieved by averaging line scans for each segment (as detailed in Supplemental Experimental Procedures). Average speed was 15 μm/min during protruding and 4.2 μm/min during stalling phases of migration. (F) Average Rac activity (FRET ratio) in central longitudinal line scans of 25 cells from five independent experiments. Gray and black dotted lines show the distance of the peak Rac activity from the front edge during protruding and stalling phases of migration. The extent of the retrograde shift was 1.6 μm during stalling phases (mean of 25 cells; five experiments; paired t test p = 0.02). (G) Rac activity oscillates between the front and the back of chemotaxing neutrophils. Maximum Rac activity (blue) along the central longitudinal axis of a representative chemotaxing neutrophil was plotted for each 1 s time frame in order to allow an assessment of the spatial localization of the point of highest Rac activity over time, and best fit periodicity curves (purple) were applied to evaluate the oscillations. The position of the front of the cell at each time point is traced in green, that of the back in red. Data shown are from one cell representative of 19 cells analyzed.
Spatiotemporal Distribution of Rac Activity in Intestinal Tissue (A) PMA stimulation of Rac activity at the base is stronger than in distal cells of Rac-FRET duodenal crypt cultures. Representative fluorescence image of an intestinal crypt culture (left) with Raichu-Rac (blue) and corresponding FLIM-FRET fluorescence lifetime maps of Rac activity before (middle) or after (right) 200 nM PMA treatment. In the FLIM-FRET images, arrows highlight the crypt base and insets show enlarged boxed sections. Note that, for FLIM-FRET measurements, decrease in fluorescence lifetime equals increase in Rac activity. (B) Quantification of Rac activity in intestinal crypt cultures as in (A) upon stimulation with 200 nM PMA for 0–90 min. Mean fluorescence lifetime ± SEM of 21–31 cells at varying positions in the base (black) or the distal section of the crypt (gray), as indicated by the schematics, for each time point and location.
∗p < 0.05, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 by unpaired Student’s t test between indicated time and 0-time control. (C) Rac activity at the base of Rac-FRET intestinal crypts is stimulated by PMA ex vivo. Representative FLIM-FRET images before (left) and after (right) stimulation of freshly isolated intestinal crypt tissue with 200 nM PMA for 15 min. For each pair of images, the left-hand panel shows a representative fluorescence image of intestinal crypts expressing Raichu-Rac (blue), the right-hand one a corresponding fluorescence lifetime map. (D) Quantification of Rac activity (fluorescence lifetime; mean ± SEM) at the base of 20 intestinal Rac-FRET mouse intestinal crypts before and after PMA stimulation ex vivo as in (C). (E) Rac activity at the base of intestinal crypts is increased upon tissue-specific APC loss in vivo. Representative FLIM-FRET images at the base of intestinal crypts of live Vil-Cre-ER T2 Rac-FRET control mice (left) and Vil-Cre-ER T2 APC fl/fl Rac-FRET mice with intestinal tissue-specific APC deletion (right). For each pair of images, the left-hand panel shows a representative fluorescence image of intestinal crypts expressing Raichu-Rac (blue) and the second harmonic generation (SHG) signal from host ECM components (white), the right-hand one a corresponding fluorescence lifetime map. (F) Quantification of Rac activity (fluorescence lifetime; mean ± SEM) from 206 cells at the base of intestinal crypts in control mice and 197 cells in APC-deleted mice as in (E); three independent regions each. ∗∗p < 0.05 by unpaired Student’s t test.
High Rac Activity in Pancreatic Tumors (A) Rac activity is stimulated by PMA in Rac-FRET mouse pancreas ex vivo. From the left, panels show a fluorescence image of freshly isolated pancreatic tissue, an enlargement of the region analyzed, with Raichu-Rac expression in blue and SHG signal from host ECM in white, a corresponding Rac activity (fluorescence lifetime) map, and images of Rac activity before (top) and after (bottom) treatment with 200 nM PMA for 15 min. (B) Quantification of pancreatic Rac activity (fluorescence lifetime; mean ± SE) as in (A) from 30 cells in three regions before and after stimulation with 200 nM PMA for 15 min.
∗p < 0.05 by unpaired Student’s t test. ns, not significant. (C) Increased Rac activity in pancreatic tumors in vivo. Representative FLIM-FRET images comparing normal pancreas in live Rac-FRET mice and pancreatic tumors in live KPC Rac-FRET mice. For each pair of images, the left-hand panel shows a representative fluorescence image of Raichu-Rac (blue) and SHG signal from host ECM (white), the right-hand one a corresponding FLIM-FRET fluorescence lifetime map. (D) Quantification of Rac activity in Rac-FRET and KPC Rac-FRET pancreas in vivo as in (C). Mean fluorescence lifetime ± SEM of 222 Rac-FRET and 461 KPC Rac-FRET cells from three to four regions/mouse, three mice/genotype. ∗∗∗p < 0.001 by unpaired Student’s t test.
Rac Activity in Mammary Tumors Is Inhibited by NSC23766 (A) Rac activity in PyMT Rac-FRET mouse mammary tumors is inhibited by NSC23766 ex vivo. Representative FLIM-FRET images of freshly isolated mammary tumors of PyMT Rac-FRET mice without (left) or with (right) treatment with 50 μM NSC23766 for 60 min ex vivo. For each pair of images, the left-hand panel shows a representative fluorescence image of tissue expressing Raichu-Rac (blue) and SHG signal from host ECM (white), the right-hand one a corresponding Rac activity (fluorescence lifetime) map. (B) Quantification of Rac activity in PyMT mammary tumors as in (A). Mean fluorescence lifetime ± SEM of 144 control cells and 74 NSC23766-treated cells from two to three different regions/group.
∗∗∗p < 0.001 by unpaired Student’s t test. (C) Rac activity in mammary tumors is inhibited by treatment with NSC23766 in vivo. Representative FLIM-FRET images of Rac activity in mammary tumors of live PyMT Rac-FRET mice before (left) and 60 min after (right) intraperitoneal (i.p.) injection of NSC23766 (4 mg/kg). Order of images as in (A). (D) Quantification of Rac activity (fluorescence lifetime; mean ± SEM) in 210 cells within mammary tumors of PyMT Rac-FRET mice in vivo 0–90 min after i.p. injection of NSC23766. ∗∗p < 0.01 and ∗∗∗p < 0.001 by unpaired Student’s t test.
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