GTPase-activating protein DLC1 spatio-temporally regulates Rho signaling
- Institute of Cell Biology, University of Bern, Switzerland
- Graduate School for Cellular and Biomedical Sciences, University of Bern, Switzerland
Figures
Figure 1
Cytoskeletal structures in WT and DLC1 deficient cells.
(A,B) Representative immunofluorescence images of paxillin and F-actin (phalloidin) (A), and pMLC and F-actin (phalloidin) (B) immunostains are shown in inverted black and white (ibw) contrast as well as color composites (DAPI signal also included). Scale bar = 10 µm. (C) Compared to WT cells (n=243), lamella size is significantly increased in DLC1 KD cells (n=290, p-adjusted<0.001) and DLC1 KO cells (n=242, p-adjusted<0.001), while DLC1 rescue cells (n=57) show significantly lower lamella sizes than both WT (p-adjusted=0.0098) and KO cells (p-adjusted<0.001). (D) DLC1 KD cells (n=290) display a significant increase in the total area of focal adhesions per cell (p-adjusted<0.001) compared to WT cells (n=243), while DLC1 KO cells (n=242) do not show a significant difference (p-adjusted=0.09). Rescue cells show a significant decrease in the total area of focal adhesions (p-adjusted<0.001). ANOVA plus Tukey’s honestly significant difference test.
Figure 2 with 4 supplements
F-actin dynamics and cell morphodynamics in WT and DLC1 KO cells.
All time series are representative ibw contrast images of WT and DLC1 KO cells expressing Lifeact-mCherry. Dotted lines mark the area used for the kymographs shown in the right panels. (A) Cells were imaged for 10 min in 10 s intervals during spreading immediately after replating. Dotted red lines display differences in lamella size in WT and DLC1 KO cells. Time (B) One hour after reseeding, when cells are still isotropically spreading, DLC1 KO cells display increased lamellipodia size and more prominent edge protrusion retraction cycles. High-magnification insets are shown for time point 10 min and show the robust increase of contractility in DLC1 KO cells. (C) Cells were imaged for 10 hr with 15 min intervals starting from spreading. WT cells break symmetry and display episodes of polarized motility. KO cells are much less dynamic and reach a contractile phenotype much faster, without being able to polarize. (D) Box plot showing at which time point cells exhibited the elongated phenotype described in (C). DLC1 KO cells (n=42) exhibited this phenotype a mean of 90 min earlier than WT cells (n=22) p-adjusted=0.002, Student’s t-test. 17 additional WT cells failed to develop that phenotype entirely. Scale bars = 20 µm for all images.
Figure 2—figure supplement 1
Cell motility properties in WT versus DLC1 KO cells.
(A) Example fields of view of tracked WT and DLC1 KO cells. 20,000 REF fibroblasts expressing a histone H2B-miRFP marker were seeded in a 24-well plate well coated with fibronectin and stimulated with 20 ng/ml PDGF, imaged for 12 hr at 15 min interval. Tracking was performed using Stardist 37 on the H2B image. DLC1 KO cells display a large subpopulation of cells that remain extremely stationary. (B) Mean Square displacements (MSDs) for different time intervals, WT cells move more than DLC1 KO cells for all possible lag times. The plot shows directionality (exponential bit between 0.25 and 1.5 hr). For times longer than 2 hr, the curve is flat, indicating that migration has characteristics of random walk at these timescales. Thick lines: mean, thin lines: standard deviation. A dotted gray line shows delta t chosen for figure C. (C) Distribution of velocities calculated as µm/hr from Root-MSD. We again observe the bimodal distribution in the DLC1 KO cell with a big subgroup of the cells moving extremely slowly (5 µm/hr). Thick lines are mean and extrema. Two-sided t-test: (statistic = 25.1, p-value = 1.6e-131).
Figure 2—figure supplement 2
Effect of DLC1 overexpression on F-actin cytoskeleton.
Effect of DLC1 overexpression on F-actin. REF52 cells stably expressing Lifeact-mCherry were transfected with mCherry-DLC1 or mCherry plasmids, allowed to spread for 12 hr on fibronectin-coated coverslips, and imaged. Images are shown in ibw contrast. Large mCherry-DLC1 pool in the cytosol levels documents its overexpression. Note how DLC1 overexpression leads to loss of contractile F-actin structures.
Figure 2—video 1
F-actin dynamics of WT and DLC1 KO REF52 cells during late spreading (relevant to Figure 2B).
Representative ibw contrast movie of WT and DLC1 KO cells expressing Lifeact-mCherry during late spreading. Time: Min:s. Scale bar = 20 mm.
Figure 2—video 2
F-actin dynamics and edge dynamics of WT and DLC1 KO REF52 cells during acquisition of a polarized cell migration phenotype over a period of 15 hr (relevant to Figure 2C).
Representative ibw contrast movie of WT and DLC1 KO cells expressing Lifeact-mCherry for 15. Edge dynamics were color-coded with respect to time. Time: Min:s. Scale bar = 20 mm.
Figure 3 with 2 supplements
RhoA activation dynamics in WT and DLC1 KO cells.
Cells are stably expressing Lifeact-mCherry and the RhoA2G FRET sensor. RhoA localization images show RhoA2G localization that is identical to RhoA. RhoA activity images display the computed FRET ratio. Images are color-coded according to the normalized scales shown below the panels. (A) Representative images of WT and DLC1 KO cells during spreading. Kymographs for the violet lines are shown in the right panels. Note the RhoA activity band maintains constant width during spreading at the periphery in WT cells. Note increased global RhoA activity in DLC1 KO cells, with maintenance of a similar RhoA band pattern at the cell edge. (B) Box plots of FRET ratio averaged over the whole cell (right panel) or a ROI placed at the cell edge (left panel). This shows that during spreading, DLC1 KO cells (n=149) have an increased total RhoA activity compared to WT cells (n=114, p=0.005). In addition, the FRET ratio at just the cell edge is increased as well (p=0.011). (C) Representative images of WT cells and DLC1 KO cells that have transitioned in a contractile state 12 hr after plating. No difference in RhoA activity pattern can be observed between WT and DLC1 KO cells, although the latter still display slightly higher global RhoA activity levels. (D) Box plots of FRET ratio averaged over the whole cell (right panel) or a ROI placed at the cell edge (left panel). This shows that contractile DLC1 KO cells (n=103) have an increased total RhoA activity (p<0.001) and edge FRET ratio (p<0.001) compared to WT cells (n=82) compared to WT cells (n=82). Students t-test. (A,B) Scale bars = 20 µm.
Figure 3—figure supplement 1
Effect of DLC1 overexpression on RhoA activity.
Effect of DLC1 overexpression on RhoA. Cells stably transfected with the RhoA2G biosensor were transfected with mCherry-paxillin or mCherry-DLC1. mCherry signals are shown in ibw contrast. RhoA2G FRET ratio and expression levels are color-coded according to the scale. Note how low mCherry-DLC1 expression that remains associated with FA already lowers RhoA activity, while high mCherry-DLC1 expression leads to even lower RhoA activity and aberrant cell morphology.
Figure 3—video 1
F-actin and RhoA activity dynamics in WT and DLC1 KO REF52 cells during late spreading (relevant to Figure 3A).
Representative movie of WT and DLC1 KO cells expressing Lifeact-mCherry and the RhoA2G FRET biosensor during late spreading. Left panels: F-actin channel in ibw contrast. Right panels: RhoA activity emission ratio color coded according to color scale. Time: Min:s. Scale bar = 20 mm.
Figure 4
An optogenetic actuator - Rho biosensor circuit to probe Rho GTPase flux.
(A) Schematics of the optogenetic actuator - Rho biosensor to measure Rho GTPase flux. OptoLarg is based on an iLID system which does not interact with Ssb-LARG in the dark state. The iLID module is anchored to the PM by a Stargazin anchor that displays slow diffusion, allowing better focusing of optogenetic activation. Upon light exposure, ssb-LARG is locally recruited to the plasma membrane, activating Rho. Rho activity is measured by a rGBD effector binding domain. (B) Time series of REF52 cells locally stimulated with light first in a ROI at the cell top with a high stimulation frequency, and then with a ROI at the cell bottom with a low stimulation frequency. Light pulses have the same intensity. Thick and thin blue thunder symbols represent high and low optogenetic stimulation. A red dotted line is used for the kymograph shown in (C). (C) Kymographs of cells in (B). Light pulse stimulation regimes of top and bottom ROIs are shown in the upper box. Blue dotted boxes indicate the region and length of the stimulation. Note how intense optogenetic stimulation in the top ROI leads to Rho activity, assembly of contractile F-actin structures, and robust edge retraction. Upon removal of the light input, the Rho activity and F-actin resume, and edge protrusion occurs again. Lower optogenetic stimulation in the bottom ROI leads to much lower Rho activity and F-actin structures, as well as lower edge retraction. (B,C) Scale bars = 10 µm.
Figure 5 with 2 supplements
Rho GTPase activation kinetics in WT versus DLC1 KO cells.
(A) Computer vision pipeline and experiment: (1) a cell is segmented using the rGBD-dTomato channel. (2) FAs and non-FAs are detected using the paxillin channel (see Materials and methods). ROIs on FAs (blue dots, shown in the bottom left panel) and ROIs on non-FA regions in between (orange dots, shown in the upper left panel) are selected for stimulation. (3) Image of the stimulation pattern (green channel) shows that the calibrated DMD can stimulate the regions with high spatial precision (image is overexposed to show the diffraction pattern of the mirrors). (B) The DMD is used to stimulate ROIs (FAs or non-FAs) with a pulse of blue light (blue line). rGBD signal fluctuations are then measured in the ROIs. (C) Distribution of paxillin-miRFP intensities in the FA and non-FA ROIs normalized to the mean paxillin-miRFP intensity of the whole cell shows that our segmentation pipeline accurately identifies FA and non-FA ROIs. (D) Paxillin-miRFP fluorescence of stimulated ROIs, normalized to the mean paxillin intensity of the whole cell. Median and 99% CI are shown. The intensity of paxillin does not change upon stimulation, indicating that the stimulation is too weak to trigger reinforcement of focal adhesions (compare to Figure 7A–D). (E) Normalized and averaged rGBD fluorescence fluctuations upon ROI optogenetic stimulation. For each stimulated ROI, the fold change to the baseline (average activity from 0 to 150 s before optogenetic stimulation) is calculated. Median and 99% CI are shown. Regions on top of focal adhesions have a larger fold change in rGBD activity than regions between focal adhesions. DLC1 KO cells have a larger rGBD fold change in the initial time after stimulation (150–200 s), but then also fall back down to the baseline quicker. (KO FA: N=3643, KO non-FA: N=3643, WT FA: N=2144, WT non-FA: N=2,144. Mean number of regions per cell: ~16.90, Cells WT = 321, DLC1 KO = 431). The same cell can appear multiple times in the experiment, but with a relaxation time in between and new stimulation regions. (F) The different dynamics described in (E) can be robustly observed in technical replicates of the experiment. In all replicates, the rGBD recruitment is higher in FAs vs non-FAs, and we see the trend of faster accumulation and faster return to baseline of rGBD in DLC1 KO vs WT cells.
Figure 5—figure supplement 1
Quantification of optoLARG recruitment dynamics to subcellular locations.
(A) optoLARG recruitment over time shows similar dynamics to FA ROIs (N=190) and non-FA ROIs (N=184) regions (background subtracted, divided by mean optoLARG expression in the cell and normalized to baseline of first 10 frames / 100 s). Mean of all ROIs and 99% CI are shown. (B) Comparing optoLARG recruitment at peak response (t=200 s), averaging all FA/non-FA ROIs within same cell. Wilcoxon paired test shows no significant difference between the two conditions (cells N=39, p=0.419, mean FA: 1.1450±0.23, mean NON-FA: 1.1884±0.22). (C) Characterization of optoLARG recruitment dynamics. As FA and non-FA regions show similar dynamics, here we average all ROIs. To quantify the kinetics of recruitment and dissociation, we fit the mean normalized and drift-corrected time series with piecewise exponential models. The rising phase during stimulation (150–200 s) was well described by an inverse exponential function, with an amplitude of 0.166 and a half-life of 7.9 s (R²=0.991, RSS = 1.88*10⁻⁴). The decay phase (200–300 s) was well described by a single exponential decay with a half-life of 10.8 s and a slightly lower amplitude of 0.164 (R²=0.989, RSS = 3.24*10⁻⁴).
Figure 5—video 1
Characterization of the optoLARG/rGBD genetic circuit (relative to Figure 5).
REF52 cells stably expressing the optoLARG/rGBD circuit as well as Lifeact-miRFP are locally stimulated with blue light of identical intensity, first in an ROI at the cell top with a high stimulation frequency, and then with a ROI at the cell bottom with a lower stimulation frequency. The stimulation frequencies are indicated by the appearance/disappearance frequency of the ROIs. Left panel: rGBD signal, Right panel: F-actin Lifeact-miRFP. Note the more robust edge contraction, as well as increased RhoA activity in response to high versus low light frequency stimulation. Time: Min:s. Scale bar = 10 mm.
Figure 6 with 1 supplement
Mathematical Model of RhoA differential activation/deactivation dynamics in WT vs. DLC1 KO cells.
(A) Schematics of RhoA activation/deactivation system that correspond to the ODE model of RhoA dynamics. The light pulse increases GEF activation rate, which in turn increases RhoA activation. Active RhoA activates GAP, negatively auto-regulating itself. RhoA deactivation is mediated by active GAPs. A detailed description of the modeling approach is provided in the Methods section. (B) Results after fitting the ODE model to the data for both RhoA dynamics at the membrane (left panel) and at focal adhesions (right panel). WT and DLC1 KO conditions were fitted separately in each case. The fold change to the baseline of averaged and normalized rGBD intensity was taken as a datapoint for each acquisition frame. (C) Illustration of the dynamics of the other model species that are active GEF and active GAP after the light pulse trigger. The left panel shows the dynamics at the membrane, and the right panel shows the dynamics at focal adhesions.
Figure 6—video 1
DLC1 dynamics at FAs during an optoLARG-induced contractility pulse (relevant to Figure 6A–C, cell denoted by pink arrow).
REF52 KO cells rescued with mCherry-DLC1 and expressing miRFP-paxillin, and the optoLARG/rGBD circuit were imaged before, during, and after a transient optogenetic stimulation in the whole field of view shown in the movie. Time: Minutes:seconds. Scale bar = 5 mm.
Figure 7 with 2 supplements
Optogenetic control of force-dependent DLC1 interactions with Fas.
(A) Color-coded fluorescence micrographs of REF52 fibroblasts expressing miRFP-paxillin (left) and mCherry-DLC1 (right) and the optoLARG construct (not shown). The black boxes indicate the area used for close-up images in (B). The white boxes indicate the ROI for optogenetic illumination. Selected FAs denoted by the pink and green arrowheads. The black dotted lines were used for the kymograph in B. (B) Kymographs showing two selected FAs, the gray box indicates the time at which optogenetic stimulation has been applied. Optogenetic stimulation is applied on select ROIs placed over FAs with 50 ms light pulses per frame (every 15 s) for a duration of 7.5 min. (C) Close-up time series of paxillin and DLC1 signals at a single FA, denoted by the respective arrowheads. Scale bars = 10 µm. (D) Quantification of mCherry-DLC1 and miRFP-paxillin fluorescence signals during optoLARG-mediated control of FA reinforcement and relaxation. Normalized miRFP-paxillin and mCherry-DLC1 from 2 FAs shown in panels A–C. (E) Model of Rho GTPase activity modulation by DLC1 at FAs and at the plasma membrane relevant to Figure 5. Left and right panels show schematics of Rho activation dynamics in response to optoLARG optogenetic input at FAs (left panel) and plasma membrane (right panels). Top and bottom panels show schematics for Rho activation dynamics in response to optoLARG optogenetic input in control (top) and DLC1 KO (bottom) panels. (F) Model of force-dependent regulation of DLC1 at FAs relevant to this and Figure 7—figure supplement 2. Left panel, in the absence of acute mechanical input, DLC1 increases with FA assembly and decreases with FA disassembly. Central panel, upon acute local increase of mechanical stress in response to application of an optoLARG optogenetic input, DLC1 unbinds from FA in a reinforcement regime and rebinds FA in a relaxing regime when the optoLARG input is removed. Right panel, upon acute local increase of mechanical stress in response to application of an optoLARG optogenetic input, some FAs rupture after DLC1 dissociation and FA rupture.
Figure 7—figure supplement 1
DLC1 dynamics in an optoLARG stimulated FA that undergoes reinforcement followed by disassembly, as well as FA behavior in absence of optoLARG stimulus.
(A–C) document FAs that when subjected to a nearby pulse of optogenetic Rho-mediated contractility display a behavior of FA reinforcement followed by disassembly. Optogenetic stimulation is applied on select ROIs placed over FAs with 50 ms light pulses per frame (every 15 s) for a duration of 8 min. (D–F) document the dynamics of DLC1 in an ROI not stimulated with light in the same cell as shown in Figure 6A–C. (A) Color-coded fluorescence micrographs of REF52 fibroblast expressing miRFP-paxillin (top) and mCherry-DLC1 (bottom) and the optoLARG construct (not shown). The black boxes indicate the area used for close-up images in (B). The white boxes indicate the ROI for optogenetic illumination. (B) Close-ups of the ROIs (left panel) and kymographs (right panel) of selected FAs denoted by the arrowheads. The pink and green arrowheads indicate the FAs of interest in (B). The black arrowhead indicates the FA of interest in (C). The black dotted lines were used for the kymograph. In the kymograph, the gray box indicates the time at which optogenetic stimulation has been applied. (C) Close-up time series of paxillin and DLC1 signals at a single FA, denoted by the respective arrowheads. Scale bars = 10 µm. (D) Color-coded fluorescence micrographs of REF52 fibroblast expressing miRFP-paxillin (top) and mCherry-DLC1 (bottom) and the optoLARG construct (not shown). The black boxes indicate the area used for close-up images in (B,E). The white boxes indicate the ROI for optogenetic illumination. (E) Close-ups of the ROIs (left panel) and kymographs (right panel) of selected FAs denoted by the arrowheads. The pink and green arrowheads indicate the FAs of interest in (B). The black arrowhead indicates the FA of interest in (D). The black dotted lines were used for the kymograph. In the kymograph, the gray box indicates the time at which optogenetic stimulation has been applied. (F) Close-up time series of paxillin and DLC1 signals at a single FA, denoted by the respective arrowheads.
Figure 7—figure supplement 2
DLC1 dynamics at FAs in unperturbed cells.
(A–C) document FAs in an assembly state. This shows that the DLC1 signal augments concomitantly with paxillin signal during FA assembly. (D–F) document FAs in a disassembly state. This shows that the DLC1 signal decreases concomitantly with paxillin signal during FA disassembly. (A,D) Color-coded fluorescence micrographs of REF52 fibroblast expressing miRFP-paxillin (top) and mCherry-DLC1 (bottom). The white boxes indicate the area used for close-up images in (B,E). (B,E) Close-ups of the ROIs (left panel) and kymographs (right panel) of selected FAs denoted by the red arrowheads. White dotted lines were used for the kymograph. In the kymograph, the gray box indicates the time at which optogenetic stimulation has been applied. (C,F) Close-up time series of paxillin and DLC1 signals at a single FA, denoted by the respective red arrowheads. Scale bars = 10 µm.
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GTPase-activating protein DLC1 spatio-temporally regulates Rho signaling
eLife 12:RP90305.
https://doi.org/10.7554/eLife.90305.3
