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 = 10µm.

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), as well as 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.

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 minutes in 10 second 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 minutes and show the robust increase of contractility in DLC1 KO cells.

C. Cells were imaged for 10 hours with 15 minutes 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 minutes 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.

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 hours 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.

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 plasma membrane by a Stargazin anchor that displays slow diffusion, allowing better focussing 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. 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

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 Material 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 pipelines accurately identifies FA and Non-FA ROIs.

D. Normalized and averaged rGBD fluorescence fluctuations upon ROI optogenetic stimulation. For each stimulated ROIs, the fold change to the baseline (average activity from 0-150 seconds 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 seconds), but then also fall back down to the baseline quicker. (KO-FA: N=3643, KO-NON-FA: N=3643, CTRL-FA: N = 2144, CTRL-NON-FA:2144. Mean number of regions per cell: ∼16.90, Cells CTRL = 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).

E. The different dynamics described in (D) 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 CTRL cells.

Optogenetic control of force-dependent DLC1 interactions with FAs

A. Color-coded fluorescence micrographs of REF52 fibroblast 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 grey 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 seconds) for a duration of 7.5 minutes.

C. Closeups 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-paxilllin 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 panel 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 S3. Left panel, in 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.

Cell motility properties in WT versus DLC1 KO cells

A. Example fields of view of tracked WT and DLC1 KO cells. 20000 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 hours at 15 minutes 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 move more than DLC1 KO cells for all possible lag times. Plot shows directionality (exponential bit between 0.25 and 1.5 hours). For times longer than 2 hours the curve is flat indicating that migration has characteristics of random walk at these timescales. Thick lines: mean, thin lines: standard deviation. Dotted grey line shows delta t chosen for figure C.

C. Distribution of velocities calculated as µm/hour from Root-MSD. We again observe the bimodal distribution in the DLC1 KO cell with a big subgroup of the cells move extremely slowly (5 µm/hour). Thick lines are mean and extrema. Two sided t-test: (statistic=25.1, p-value=1.6e-131)

Effect of DLC1 overexpression on F-actin cytoskeleton and RhoA activity

A. Effect of DLC1 overexpression of F-actin. REF52 cells stably expressing Lifeact-mCherry were transfected with mCherry-DLC1 or mCherry plasmids, allowed to spread for 12 hours 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.

B. Effect of DLC1 overexpression of 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.

DLC1 dynamics at FAs in unperturbed cells

Panels A-C document FAs in an assembly state. This shows that the DLC1 signal augments concomitantly with paxillin signal during FA assembly. Panels 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. Closeups 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 grey box indicates the time at which optogenetic stimulation has been applied.

C,F. Closeups time series of paxillin and DLC1 signals at a single FA denoted by the respective red arrowheads.

Scale bars = 10 µm.

DLC1 dynamics in an optoLARG stimulated FA that undergoes reinforcement followed by disassembly, as well as FA behavior in absence of optoLARG stimulus

Panels 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 seconds) for a duration of 8 minutes.

Panels D-F document the dynamics of DLC1 in a ROI not stimulated with light in the same cell as in 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. Closeups 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 grey box indicates the time at which optogenetic stimulation has been applied.

C. Closeups 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. Closeups 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 grey box indicates the time at which optogenetic stimulation has been applied.

F. Closeups time series of paxillin and DLC1 signals at a single FA denoted by the respective arrowheads.