{"CAPTION FIG3-2.png": "'(3) Relative force changes (\\\\(\\\\beta\\\\) axis) for membrane tension monitored on the static tether as a function of the extending tether length (\\\\(\\\\nu\\\\) axis) upon continuous pulling. In the case of bibels or cells with heavily disassembled actin cortex (light and dark green), the tension on static tether increases as the extending tether lengthens; the however, there are no perceptible tension changes on the static tether tension from the cell body (pink, intact central) even when the other tether has extended by more than 60 \\\\(\\\\mu\\\\)m \\\\(\\\\left\\\\{ \\\\text{h} > 14,\\\\text{h} > 3\\\\right\\\\}\\\\). Graphical data represent means \\\\(\\\\approx\\\\) SDs. See also Figure S3 and Video S2.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: Actin-driven protrusions stimulate global, nearly undampened membrane tension propagation (A) A dual-tether pulling assay to simultaneously monitor membrane tension on the far end (left, trap 1 at 180\u00b0) and on the side of the cell (top, trap 2 at 90\u00b0) during light-activated protrusion. (B) Representative time traces of dual trap forces over successive cycles of light-activated protrusion show coinciding tension increases on both membrane features adjacent to (trap 2) and at the opposite cell surface from (trap 1) protrusion; light: 90 s on (shaded area), 180 s off. (C) Correlation between trap forces at the two tether positions during activation (blue) remains rocky transit from first activation cycle to the next; for comparison, minimal correlation is seen between the two tethers before osteogenic activation (gray). Dashed line: Inner progression. (D) (L-left) Time delay measured between tension rise on membrane tethers adjacent to (trap 2 at 90\u00b0, black) and opposite from trap 1 at 180\u00b0, red) cell protrusion. (Right) In most cells, the traps detect membrane tension increase on both tethers within a second or less of one another, indicating a rapid propagation of tension across the cell. (E) Averaged traces of dual trap forces before, during (light), and after activation (yreams \\\\(\\\\pm\\\\) SD; \\\\(n\\\\) > 25, N = 4). (F) Pearson correlation coefficient between dual trap forces measured at steady state, during light activation, and recovery afterward (70 s post light). Error bar: means \\\\(\\\\pm\\\\) SD; p values from Welch\u2019s unpaired Student\u2019s test in \\\\(>\\\\) 10, N \\\\(\\\\times\\\\) 4). See also Figure S2 and Video S2.\\n\\n'", "CAPTION FIG4.png": "'Figure 4: Long-range tension propagation is accompanied by directed membrane and actin flows toward the protrusion (A) Confocal images of opta-PI3K ceils expressing membrane marker (CAX-HaIaTaj): before and during light-activated protrusion. Scale bars: 5 \u03bcm. (B) Kymographs of membrane fluorescence along the normalized cell circumference (a axis) show that over time (x axis) membrane accumulates toward the protruding cell front and is depleted from the back (n > 50, N = 6; Figure S4; see STAR Methods). (C) Flow of membrane and actin during protrusion are calculated assuming optimal transport (see STAR Methods). (D) Membrane flow field referred using optimal transport from kymograph intensity changes over time: shortly after activation begins (f = 70 s, dark text traces), the magnitude of membrane flow speed increases (red dashed arrows), with positive speed for clockwise flow along the cell upper hat and negative speed for counter-clockwise flow along the bottom hat (G), all moving toward the cell protruding front (x). During recovery (f = 170 s, light green traces), the direction of membrane flow reverses (black dashed arrows). (E) Membrane flow around the cell before, during, and after (f = 30, 70, and 170 s) right-side protrusion; the flow magnitude is denoted by the arrow size (red; forward flow, blue; backward). Membrane flows toward the protrusion in the protruding phase and away from the protrusion during the recovery phase. (F) Attracting membrane diffusion assay in which we bleach the membrane marker CellMask across a wide section of the cell (sparing a small section of the membrane mask), opto-activate a portion of the cell angled 90\u00b0 from the unbleached area (or use no light as control), and monitor the diffusion pattern of the unbleached area over time. (G) (Top) Example kymograph of unbiased diffusion in a control cell (no activating light). (Bottom) Same as top but in a protruding cell, showing biased diffusion and bulk flow of the unbleached membrane signal toward the protrusion. Heatmap similar as in (B). (H) Sample fits of individual timepoints of kymograph data (points colored by respective time points) with a gaussian equation (thick curves, colored by respective time points). Shifts in the means of the gaussian fits, quantified bulk membrane flow, are shown as vertical lines (colored by respective timepoints). (I) Quantification of mean shifts fit by linear regression to assay membrane flow rate in control cells (gray, no apparent flow, u = 3.34 nm/x) and protruding cells (red, biased flow toward side of protrusion, u = 35.51 nm/x) (N = 3, n = 3). See also Figure S4 and Video S3.\\n\\n'", "CAPTION FIG3-1.png": "'Figure 3: Membrane tension does not propagate upon direct mechanical pulling on the cell membrane (A) A dual-iether assay to detect tension propagation (static tether, left) while a nearby force is exerted through the use of an optically trapped bead to pull on the membrane -2-\\\\(\\\\mu\\\\)m away (moving tether, right).\\n\\n(B) An example time trace of trap force for dual membrane tension measurements, in which one moving trap (T2, gray) dynamicaly pulls on the cell membrane by continuously pulling and extending the membrane tether, whereas the other trap controls a second static membrane tether (T1, black) to monitor nearby changes in membrane tension. The increase in the length of the actadge tether from the cell body is plotted in gray along the right y axis.\\n\\n(C) Correlation plots of normalized trap forces between the moving and static tethers. Five representative measurements from different cells are shown: dashed lines: linear regression.\\n\\n(D-F) Similar to (A\u2013C), but probing tension in bibels (membrane detached from actin cortex generated by using altrunculin B treatment to weaken the actin cortex), a high correlation is observed between static and moving tethers.\\n\\n(A\u2013B) Similar to (A\u2013C), but probing tension in cells where the actin cortex has been significantly disassembled using a combination of altrunculin B treatment and osmotic shock; a high correlation is observed between static and moving tethers even at a significant distance from one another (here, 90\\\\({}^{\\\\circ}\\\\), but in Figures S3H-S31, 180\\\\({}^{\\\\circ}\\\\)).\\n\\n(J) Pearson correlation coefficient between dual trap forces measured before perturbations (none; light gray), upon light-activated protrusions (purple; Figure 2), during cell membrane pulling (pink; A\u2013C), during membrane pulling on a bleb (light green; D\u2013F), and during cell membrane pulling in cells with nearly ds-assembled actin cortex (dark green; G\u2013I). Error bar: means +- SD; p values from Welch\u2019s unpaired Student\u2019s t test (\\\\(\\\\eta\\\\sim 15\\\\), N \\\\(>3\\\\)).\\n\\n'", "CAPTION FIG5.png": "'Figure 5.\u2014 **Optogenetically induced actomyosin contractions generate rapid long-range membrane tension propagation and membrane flows (A) Optogenetic approach for light-induced activation of isulamia-associated Rho guanine nucleotide exchange factor (LARG), resinEng in Rho GTPase activation to initiate actomyosin-driven cell contraction (see STAR Methods).**\\n\\n(B) Time-lapse confocal images of a neutrophil-like HL-60 cell expressing opto-corastruct (Opto-LARG) and membrane marker (CellMask), showing localizad membrane contraction and cell flattening upon light activation.\\n\\n(C) After light-activated contraction on one side of the cell (top), changes in membrane tension on the opposite side (bottom) are measured via a membrane tether held by an optical trap.\\n\\n(D) Aupward image trace of trap force before (steady state), during (light), and after activating cell contraction (means = SD; n > 55, N = 7).\\n\\n(E) Aupward trap force before (steady state) and during actuation. Box and whiskers: median and min to max; p values from Wilcoxon paired Student\u2019s t test.\\n\\n(F) A dual-tether pulling assay to simultaneously monitor membrane tension on the far end (left, trap 1 at 180\\\\({}^{\\\\circ}\\\\)) and on the side of the cell (top, trap 2 at 90\\\\({}^{\\\\circ}\\\\)) during light-activated contraction.\\n\\n(G) Average traces of dual trap forces before, during (left), and after activation showing coinciding tension increases on both membrane tethers adjacent to (trap 2) and at the opposite cell surface from (trap 1) contraction (means = SD; n = 25, N = 4).\\n\\n(H) Pearson correlation coefficient between dual trap forces measured at steady state and during light activation. Error bar: means = SD; p values from Welch\u2019s unpaired Student\u2019s t test (n > 20, N > 4).\\n\\n(I) Confocal images of opto-LARG cells stained with membrane marker (CellMask) before and during light-activated contraction.\\n\\n(J) Kipographs of membrane fluorescence along the normalized cell circumference (_y_ axis) show that over time (_x_ axis) membrane accumulates toward the contracting cell front and is depleted from the back (_n_ = 40, N = 3; see STAR Methods).\\n\\n(B) Membrane flow field infrared using optimal transport from lymphograph intensity changes over time: shortly after activation begins (_t_ = 120 s, basal traces), the magnitude of membrane flow speed increases (red dashed arrows), with positive speed for clockwise flow along the cell upper half and negative speed for counter-clockwise flow along the bottom half, at moving toward the site of cell contraction (_n_). During recovery (_t_ = 200 s, light green traces), the direction of membrane flow reverses (blue dashed arrows).\\n\\n(L) Membrane flow around the cell before, during, and after (_t_ = 30, 120, and 240 s) right-side contraction; the flow magnitude is denoted by the arrow size (red forward flow, blue-backward). Membrane flows toward the contraction in the contracting phase and away from the contraction during the recovery phase. Scale bars: 5 \u03bcm. See also Figure SS and Video S4.\\n\\n'", "CAPTION FIGS3.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG3.png": "'\\n\\n## References\\n\\n* [1] A. A. K. K.\\n\\n'", "CAPTION FIGS6.png": "'Figure S6. Mechanical perturbations applied on both membrane and cortex lead to rapid tension propagation across the cell, related to Figure 6\\n\\n(A) Tether pulling assay in which tethers are pulled at constant speed until they break. Maximum tether length is used as a proxy for local membrane reservoirs [16]. (B) Maximum tether length comparison of 3T3s fibroblasts versus H-6ths cells. In red are cells for which the maximum pulling length was reached on our setup, without tether breaking occurring, suggested high local membrane reservoir availability. Error bar: means +- SD; \\\\(n\\\\) > 15, N > 3. (C) Average trap force of different opto-cells (OptoT1M-based portation induction and OptoT4G-based actomyosin contractility), before and after light in the absence or presence of the Earn inhibit MSC668394 (25 mM). These data show that lowering MCA or alkyl sticky attractive membrane tension increase in protruding cells but severely impedes membrane tension increases in recontracting cells. Error bar: means +- SD; \\\\(n\\\\) values from Welch\u2019s unpaired Student\u2019s t test (n > 25, N > 30, (D) A dual-stufher pulling assay to simultaneously monitor membrane tension on the far end (bottom, trap 1 at 180-) and on the side of the cell (right, trap 2 at 30-) during micropipette aspiration (top, ~4-5 mm in tip diameter), which mechanically pulls on both the membrane and actin cortex underneath. (E) Representative time traces of dual trap forces over successive cycles of aspiration (shaded area) and relaxation; the magnitude of aspiration progressively increased in the last two cycles (+ and +; the first three cycles were also shown in Figure S6, The nearby superimposable tension rise and tail on the two membrane tethers show that membrane tension propagates rapidly across the cell upon mechanical perturbations exerted to both the cortex and membrane. Note that the profiles of tension rise upon aspiration and of tension drop during relaxation resemble those observed with light-activated actin-driven protrusions (Figure 2B). (F) Zoom-in on the first aspiration event shows that the trap force for membrane tension on the tether closer (pink) to the aspiration site started increasing slightly earlier and ended up slightly higher compared with that measured on the tether opposite from the aspiration (purple). (G) An example trace of tether tension response monitored on the opposite side of micropipette aspiration (trap 1 at 180-). Here, the recording lasted for six rounds of aspiration and relaxation. (H) Another example of dual-stufher membrane tension measurement upon micropipette aspiration; the tether in trap 2 broke (?) shortly after the aspiration stopped. (I) An example time trace of trap force for cell membrane tension exhibits robust responses over three aspiration cycles using a micropipette of slightly smaller diameter (~2 mm). (J) (Latin) Time delay measured between tension rise on membrane tethers adjacent to (trap 2 at 90-, pink) and opposite from (trap 1 at 180-, purple) cell aspiration using micropipettes. (Right) In most cells, the traps detect membrane tension increase on both tethers within a second or less of one another, indicating a rapid propagation of tension across the cell. (D) Pearson correlation coefficient between dual trap forces measured before any perturbations (steady state) and during mechanical pulling upon micropipette aspiration. Error bar: means +- SD; \\\\(n\\\\) values from Welch\u2019s unpaired Student\u2019s t test (n > 15, N > 3). (L) Correlation plots of normalized trap forces between the two tethers during micropipette aspiration. Five representative measurements from different cells are shown; dashed lines: linear regression. (M) Comparing membrane flows of light-induced protrusions at the mid and ventral plane of the cell. (N) Confocal images at a cell membrane (visualized using CAXX-HialTag) before and during protrusion at two different \\\\(z\\\\) planes (mid-section and ventral plane of the cell, Scale bar: 5 \u03bcm. (Q) Average zymograph of relative membrane fluorescence intensity along the normalized cell circumference (y axis) at the ventral and mid-plane of the cell over time (x axis) showing a decreased membrane flow at the ventral side of the cell, likely due to friction between the cell and the substrate (n > 30, N = 3). (P) Normalized membrane fluorescence intensity across the blue dotted line in (Q).\\n\\n'", "CAPTION FIGS2.png": "'Figure S2: Membrane tension propagates within seconds across the cell during actin-driven protrusion, related to Figure 2 (A) Rad and blue: averaged time traces of trap force for dual membrane tension measurements before (steady state), during (B)H, and after (recovery) activating cell protrusion. A nearly coinciding tension increase is observed between the membrane tether adjacent to (trap 2, blue) and opposite from (trap 1, red) cell protrusion. Gray: as a control, averaged trace from cells treated with actin inhibitor (10 \u03bcM latrunculin B) shows no membrane tension change upon activation (means \\\\(\\\\pm\\\\) SD; n \\\\(>\\\\) 15, 18 \u03bcM \\\\(\\\\div\\\\) 4).\\n\\n(B) Zcom-in on traces in (A): increases in membrane tension emerging on both tethers within the first 5\u201310 s of light activation.\\n\\n(C) Three example time traces of trap force for dual membrane tension measurements before, during, and after light-induced cell protrusion. At steady state, durations from the two tethers show little correlation, but they become highly correlated upon light activation (purple shaded area). During the recovery phase, we often observe a lag in time between the two tethers\u2019 tension drop, with the tether opposite from the protrusion she recovering more slowly (red).\\n\\n(D) Three example time traces of trap force for dual membrane tension measurements with cells treated with actin inhibitor (10 \u03bcM latrunculin B) before, during (purple shaded area), and after light activation of cell protrusion.\\n\\n'", "CAPTION FIGS4.png": "'Figure S4: Long-range tension propagation coincides with directed membrane flows toward the protrusion, related to Figure 4 (A and B) Apparent membrane thickness is measured based on the width of fluorescence intensity profile across the cell contour, a.g., on the side of cell protrusion (black line). At steady state (pre-activation), the cell membrane contour appears tagged (top image) and thick in width (light green curve in B). **(B)**, **(K)** due to the presence of membrane reservoirs. As the cell protrudes, the membrane intensity outside of the protruding region drops (bottom image) and becomes thinner in width (purple curve in B). **(C)** Kymograph of averaged apparent membrane thickness along the normalized cell circumference (\\\\(y\\\\) axis) over time (\\\\(x\\\\) axis): before, during, and after localized light-activated protrusion (box in white dashed line). Apart from the protruding site, apparent membrane thickness reduces on average throughout the cell, **(K)** reflecting a decrease in membrane reservoirs and a redistribution of extra membranes toward the protrusion site. **(D)** Representative confocal images of an opto-PI3K cell stained with plasma membrane (a) (CellMask) before light activation or during protrusion. **(E)** Kymograph of membrane fluorescence intensity (from cells stained with CellMask) along the normalized cell circumference (\\\\(y\\\\) axis) over time (\\\\(x\\\\) axis): before, during, and after localized light-activated protrusion (box in white dashed line; \\\\(n>25\\\\), \\\\(N=4\\\\)). **(F)** Confocal images of opto-PI3K cells expressing actin marker (actin-HaloTap: before and during light-activated protrusion. **(G)** Kymographs of actin fluorescence along the normalized cell circumference (\\\\(y\\\\) axis) show that over time (\\\\(x\\\\) axis) actin accumulates toward the protruding cell front and is depleted from the back (\\\\(n>30\\\\), \\\\(N=6\\\\); see STAR Methods). **(J)** Left, evolution of the total membrane intensity across the cell contour (means = SD; \\\\(n>30\\\\), \\\\(N=6\\\\)). Except for a small intensity decrease due to the bleaching of the fluorophore, the membrane quantity is conserved. Right, evolution of the total actin density across the cell contour (means = SD; \\\\(n>50\\\\), \\\\(N=6\\\\)). **(B)**.\\n\\n'", "CAPTION FIG6.png": "'Figure 6: Mechanical forces acting on the actin cortex drive rapid long-range membrane tension propagation in cells (A) A-3-tier composite model for membrane tension propagation in cells: membrane displacements (_k_) as a readout for tension propagation upon cortical flows (_k_), depend on the membrane elasticity (_k_) and the membrane-cortex friction \\\\(m\\\\) imposed through the adhesive linkers. (B) Model predictions of membrane tension response at moderate membrane-cortex friction (see Methods S1-f) only actin-based puffing leads to tension increase and propagation (red rectangles); external pulfing on the membrane alone is inefficient (blue circles). (C) Predicted membrane tension transmission as a function of membrane-cortex friction (_k_ axis) for different targets of force application: plasma membrane only (blue) and actin cortex only (red). (D) Membrane tension measurements during light-induced protrusions in cells with decreased MCA by using 25 uM of Ezrin inhibitor NSC66394. (E) Red: averaged time trace of trap force before steady state), during (light) and after activating cell protrusion in control cells (same data as Figure 1F). Orange: averaged trace from cells with decreased MCA by using 25 uM of Ezrin inhibitor NSC66394, showing slight defects in membrane tension propagation during light-activated protrusions (_m_ <= <= <= > = >\\n\\n'", "CAPTION FIGS3-1.png": "'Figure S3. Mechanical perturbations affecting only the plasma membrane do not result in measurable membrane tension propagation in cells but do in blebs detached from actin cortex, related to Figure 3 (A). An example time trace of trap force for dual membrane tension measurements, where one moving trap (T2, blue) mechanically perturbs on the cell membrane by continuously pulling and extending the membrane tether, and the other trap remains static (T1, red) to monitor changes and propagation in tension to a nearby membrane tether. The increase in length of the astending tether from the cell body is plotted in gray along the right y axis. \u201c\u201c annotations when the astending tether broke. Note that a sudden tension release upon breakage of the astending tether (blue, at 1~50 s) does not lead to changes in tension on the static tether (red, which is in close proximity to the astending tether (<=2 \u03bcm). This observation shows that mechanical perturbations affecting only the plasma membrane in cells are locally constrained and inadequate to generate measurable tension propagation between the two tethers. (B) Similar operations as (A) but monitoring tension propagation between two membrane tethers on cellular blebs (i.e., a vesicle-like, small section of membrane detached from actin cortex upon intravenous B treatment). The tension readouts between the extending and the static tethers on blebs appear highly correlated, (_\\n\\n'", "CAPTION FIGS5.png": "'Figure S5: Optogenetically induced actomyosin contractions generate rapid long-range membrane tension propagation and actin flows, related to Figure 5 (A and B) Representative time traces of trap force (a direct readout of cell membrane tension charge) during light-induced actomyosin contraction. Revealing robust increase in membrane tension during light-activated contractions on the opposite end of the cell; light: 90 s on (shaded area). (C) Averaged time trace of trap force before (steady state), during (light), and after activating call contraction, measured at the side (90\u00b0) of the contraction (means \u00b1 SD; n > 30, N = 7). (D) Left: Time delay measured between tension rise on membrane tethers adjacent to (trap 2 at 90\u00b0, blue) and opposite from (trap 1 at 180\u00b0, red) call contraction. (Right) In most cells, the traps detect membrane tension increase on both tethers within a second or less of one another, indicating a rapid propagation of tension across the cell. Error bar: means \u00b1 SD. (E) Confocal images of opto-LARG cells stained with actin marker (SPV650-FastAct); before and during light-activated contraction. (F) Average kymograph of relative actin fluorescence intensity along the normalized call circumference (y axis) show that over time (x axis). Actin accumulates toward the contracting call front (_n_ > 25, N = 3; see STAM Methods). (G) Actin flow field traces using optimal transport from kymograph intensity changes over time: shortly after activation begins (_t_ = 120 s, tail traces), the magnitude of membrane flow speed increases (red dashed arrows), with positive speed for clockwise flow along the cell upper half and negative speed for counter-clockwise flow along the bottom half, at moving toward the cell contracting front (_n_). During recovery (_t_ = 230 s, light yellow traces), the direction of membrane flow reverses (blue dashed arrows). (H) Actin flow around the cell before, during, and after (_t_ = 30, 80, and 230 s) right-side contraction; the flow magnitude is denoted by the arrow size (red: forward flow, blue: backward). Membrane flows toward the contraction in the contracting phase and away from the protrusion during the recovery phase. (I) Two examples of actin speckle tracking during light-induced cell contraction. Tracked actin patches are circled in red and their trajectory is represented by lines of different colors. Scale bars: 5 \u03bcm.\\n\\n'", "CAPTION TABNA.png": "'\\n\\n**Acknowledgments**'", "CAPTION FIG1.png": "'Figure 1: Local cell protrusions elicit a sharp increase in membrane tension on the opposite side of the cell within seconds (A) Optogenetic control for light-induced activation of phosphatidylinositol 3-kinase (PI3R) via localized recruitment of inter SH2 domain (ISH2), resulting in Rac GTPase activation that initiates acsin-driven cell protrusions (see STAR Methods).\\n\\n'", "CAPTION FIGS1.png": "'Figure S1: Optogenetic control of PI3K leads to local Rac activation, which triggers localized actin-driven cell protrusion and rapid membrane tension increase, related to Figure 1 (A) Membrane-anchored optogenetic control for light-induced activation of phosphoinositide 3-kinase (PI3K): upon localized 488-nm excitation, the membrane-anchored protein (LiRL-BFP-CAAX) undergoes a conformational change, which results in the binding of inter SH2 domain (SH2) to the illuminated region. ISB2 proceeds to recruit PI3K, whose lipid product (PIP3) induces the activation of Rac GTPase (Rac), Active Rac than triggers actin polymerization leading to localized membrane protrusion. By imaging the mCherry-labeled Rac biosensor (Pak-PBD-mCherry), which recognizes and binds the active GTP-bound Rac, we can monitor Rac activation during light-induced protrusions (see STAR Methods). (B) Time-lapse confocal images of HL-60 cells expressing opto-coreshrt (Opto-PI3K), membrane marker (CAAX-HaToq. imaged on top), and Rac biosensors (Pak-PBD-mCherry, imaged on bottom). Middle and right: localized recruitments of active Rac is confirmed at the site of right extinction for cell protrusion (box h). (C) Time-lapse brightfield (top) and confocal images (bottom) of an opto-PI3K cell during light activation. The specific recruitment of PI3K activator, (SH2-EGFP) to the illuminated area (box in white dashed line) is monitored upon 488-nm excitation. Within 2 s [between the first two frames], SH2 has redistributed from the cytoplasm to the plasma membrane. Scale bar: 1 \u03bcm. (D) Fluorescence intensity line scans (along the white dashed line in (C) show the enrichment of opto-construct (ISH2-EGFP) at the cell protruding site over time. (E) Kymograph of the above line scan (white dashed line in (C) shows that after SH2 is required to the membrane, the cell contour (j.e., its membrane) rapidly expands outward. (F) In red, average time trace of cells before and during light-induced protrusion. In green, apparent cell diameter (long axis) over time as proxy of cell shape change and increases in apparent surface area during protrusion. Trap force and shape change are correlated during the initial phase of the protrusion (giving phase) but when are decoupled as the cell access its membrane reservoirs limiting further increases in membrane tension (platanu phase) even as the protrusion continues to extend (means a SD; m = 15, N = 5). (G) Representative time trace of trap force measured from the tether pulling assay with a cell at steady state: membrane tension remains stable with low magnitude of stochastic fluctuations. (H) As a control, we light activate the wild-type (WT) cells, which lack opto-constructs, and use the same tether pulling assay described above to monitor membrane tension response before, during, and after 488-nm illumination (purple shaded area). Representative time trace of trap force for cell membrane tension recorded from WT cells with light activation. The activation light alone does not elicit any changes in cell morphology or membrane tension responses. (I) In another control, we light activate cells lacking the membrane anchor protein for opto-control (LiRL-BFP-CAAX) and monitor their membrane tension response upon 488-nm illumination (purple shaded area). No perceptible changes in cell morphology or membrane tension were observed. (J) Averaged time trace of trap force (red) for cell membrane tension recorded before (steady state), during (activation), and after (recovery and return to steady state) light-induced protrusion on the opposite side of the cell (see Figure 1C). Individual data traces are shown in light gray (same data as in Figure 1F, n > 60, N = 3.). (S).\\n\\n'", "CAPTION FIGS3-2.png": "'Unlike those on cell body in (A), Specifically, during the \"step-wise pulling\" to extend tether in trap 2 (blue), the static tether held in trap 1 (red) exhibits immediate stability rises in transfer, mirroring the pattern in trap 2. When a smooth increase is exerted on the extending tether by trap 2 (blue, at \\\\(\\\\sim\\\\) 13 s), the tension increase on static tether (red) accordingly becomes gradual. Furthermore, the sudden drop in tension back to initial level on the static tether (red, t \\\\(\\\\sim\\\\) 26 s)--in response to the sudden tether breakage (\\\\(\\\\overset{\\\\sim}{\\\\gamma}\\\\)) and thus tension release of the ascending tether (blue)--reflects a direct tension transmission and rapid propagation (see E) within a membrane bleb detached from the constraining actin cortex.\\n\\n(C) Average time trace of relative distance between head and cell in untreated cells and cells treated with 10 uM of the actin inhibitor lattunculin B. After tether pulling measurements, the trapping laser is turned off and the elastic recoil of the bead toward the cell is observed to confirm the absence of cytoskeleton in the tether. Similar tether recoil is observed between untreated and lntunculin-treated cells (means \\\\(\\\\pm\\\\) SD; n \\\\(>\\\\) 13, N \\\\(>\\\\) 3).\\n\\n(D) Similar to (A) but we alternate which buffer is pulling and which tether is static. Trap forces (readout of membrane tension) from static tether is concentrated to that of moving tether ?e., little to no change in tension on the static tether during pulling of the moving tether.\\n\\n(E) Similar to (C) but probing tension in blobs (membrane detached from actin cortex); here, a high correlation is observed between static and moving fathers. (F) Example zoom-in traces of dual trap forces (raw data at 78 kHz) showing the time difference between a sudden tension release upon breakage (\\\\(\\\\overset{\\\\sim}{\\\\gamma}\\\\)) of the extending tether (blue) and the subsequent reduction (\\\\(\\\\pm\\\\)) in tension on the static tether (red; traces slightly offset in \\\\(\\\\overset{\\\\sim}{\\\\gamma}\\\\) axis for illustration clarity). Typically, this time delay observed is \\\\(\\\\leq\\\\) 100 ms (measured between the inflection points, \\\\(\\\\overset{\\\\sim}{\\\\gamma}\\\\) and \\\\(\\\\overset{\\\\sim}{\\\\gamma}\\\\), on each trace), which is right around the temporal resolution of our optical trapping instrument (limited by the core frequency of a 2-\\\\(\\\\overset{\\\\sim}{\\\\gamma}\\\\)-unhead held by a trap with stiffness of \\\\(\\\\sim\\\\)0.2 pN/nm), indicating that the actual timescale of tension propagation on cellular blobs is less long to fast to be resolved in our experiments.\\n\\n(G) Representative confocal images of actin in cells using actin dye SIR-actin, comparing untreated cells as control with cells treated with either 10 uM of lntunculin B or with a combination of 10 uM of lattunculin B and in hypotonic media (+60% H2O). Scale bar: 10 uM.\\n\\n(H) Borghetti made of dual-tather pulling from opposite sides of a cell treated with a combination of 10 uM of lattunculin B and hypotonic shock.\\n\\n(H) Representative force traces of at cell treated with a combination of 10 uM of lattunculin B and a hypotonic shock showing long-range membrane tension propagation in cells with heavy depolymerated cytoskeleton.\\n\\n(J) Two example time traces of distance between head and cell in cells treated with 10 uM of the actin inhibitor lattunculin B and with an hypotonic camote shock to heavily depolymerize the actin cytoskeleton. After tether pulling measurements, the trapping laser is turned off and the elastic recoil of the bead toward the cell is observed to confirm the absence of cytoskeleton in the tether. We observe similar tether recoil as with untreated and lattunculin-treated cells.\\n\\n'"}