{"CAPTION FIG1.png": "'Figure 1: **Membrane response to stretch and osmotic changes.****(a)** Cells transfected with pETYP-memb before (top panel) and after (middle and bottom panels) applying different magnitudes of constant stretch. Yellow arrow indicates a membrane rutile flattened by stretch. (**b**) Cells transfected with DEYFP-memb before and after reducing medium osmolarity to either 50 or 0% of original medium. Cells submitted to 0% osmolarity (de-ionized water) for 3 min sometimes rounded and flattened membrane ruffles (middle panel) and sometimes underwent membrane lysis (right panel). Yellow arrows indicate membrane ruffles, which either remain or flatten after applying 50 or 0% hypo-osmotic medium, respectively. (**c**) Confocal slice showing a cell before (green) and after (red) application of medium with 50% osmolarity for 3 min. (**d**) Corresponding quantification of the increase in cell volume and required membrane area (\\\\(n=5\\\\) cells). (**e**) % of cells showing membrane tearing after 3 min of constant stretch application (\\\\(n=70\\\\) cells). (**f**) % of cells showing membrane lysis after 3 min of application of medium with different osmolarity (\\\\(n=50\\\\) cells). Scale bars, 20 \u03bcm. Error bars are mean \\\\(\\\\pm 5\\\\) s.m.\\n\\n'", "CAPTION FIG7.png": "'Figure 7: **Membrane remodelling can be described through pathways along the surface/volume phase diagram. (a-d) Different pathways tested in the phase diagram by applying stretch and hypo-osmotic shocks (red arrows, numbers refer to the corresponding image in the panel below). (e-h) Corresponding response of pEYFP-mem-transfected cells after applying stretch and somatic shocks as indicated to allow the pathways. Time flows from top to bottom. In all cases, the first application of stretch/hypo-osmotic shock (second row of cells) lasted 3 min. Subsequent steps were carried out as quickly as possible to evaluate membrane response before cells had time to actively eliminate structures. (f) In cells submitted to hypo-osmotic shock, co-localization of membrane structures formed after first restoring iso-osmotic medium (red) and then applying stretch (green). (j) Confocal vertical slices showing VLD shape before (top) and after (bottom) stretch release. Zoomed image to the right shows the superimposed shape prediction from the theoretical model in red. (k) Quantification of VLD fluorescence for cells under hypo-osmotic medium after either restoring iso-osmotic medium (blue symbols) or restoring iso-osmotic medium and then releasing stretch application (pink symbols, arrow indicates moment of stretch release). (N = 100/50 structures from 10/5 cells. (l) In cells submitted to both hypo-osmotic shock and stretch, co-localization of membrane structures formed after first releasing stretch (red) and then restring iso-osmotic medium (green). (m) Quantification of VLD diameter (_n_ = 100/50/70 structures from 10/3/3 cells, \u204e\\\\(\\\\rightarrow\\\\)P < 0.001, analysis of variance (ANOVA)) and density (_n_ = 50/30/30 regions from 8/3/3 cells, \u204e\\\\(\\\\rightarrow\\\\)P < 0.001, ANOVA/A) in cells submitted only to semantic shocks or also to de-stretch (stretch release) before or after restoring iso-osmolarity). Scale bars are 5 min in j and 20 min elsewhere. Insets show zoomed views (\\\\(10\\\\times 10\\\\,\\\\mu\\\\)m\\\\({}^{2}\\\\)) of membrane structures. Error bars are mean \u00b1 s.e.m.\\n\\n'", "CAPTION FIG3.png": "'Figure 3: **VLD formation is driven by the confinement of liquid flows at the cell-substrate interface.** Response of pEGFP-mem-transfected cells seeded on either poly-acrylamide (PA) gels or soft silicone elastomers to: (**a**) the application of 50% hypo-osmotic medium for 3 min, (**b**) the application of 6% strain for 3 min and (**c**) a fast 6% strain pulse. Insets show zoomed views (0x10 \\\\(\\\\mu\\\\)m\\\\({}^{2}\\\\)) of membrane structures. Scale bars, 2.0 \u03bcm. No significant differences were observed between any of the cases either in the diameter of reservoirs (n = 150 reservoirs from 3 cells) or in their density (\\\\(n=30\\\\) cells) regions from 3 cells. (**d**) Time sequence of VLD formation and resorption in pEGFP-mem-transfected cells exposed to dextran-labelled iso-osmotic media after 3 min incubation with 50% unlabelled hypo-osmotic media. (**e**) Zoomed insets (\\\\(20\\\\times 20\\\\) \u03bcm\\\\({}^{2}\\\\)) corresponding to red square in **d** showing the evolution of membrane and dextran fluorescence, and merged images. (**f**) Corresponding quantification of pEGFP-mem and dextran relative fluorescence levels.\\n\\n'", "CAPTION FIG2.png": "'Figure 2: **Cell membranes use different strategies to readapt to normal surface and volume.****(a)** pETFP-mem-transfected cells before, during and after constant stretch application during 3 min. **(b)** Quantification of reservoir fluorescence after stretch release (1: initial fluorescence, \\\\(\\\\overline{\\\\alpha}\\\\): background). \\\\(n=100\\\\) reservoirs from 10 cells. **(c)** Confocal vertical slice from a pEYFP-mem-transfected cell before (top) and after (bottom) application of 6% stretch for 3 min. **(d)** Staining images of cells fixed immediately after stretch release showing the membrane (pEYFP-mem transfection), pixillin and actin. **(f)** Co-localization images are shown to the right. **(e)** pEYFP-mem-transfected cells before, during and after application of 50% hypo-osmotic medium during 3 min. **(f)** Quantification of VLD fluorescence after re-application of iso-osmotic medium (\\\\(\\\\overline{\\\\alpha}\\\\): initial fluorescence, \\\\(\\\\overline{\\\\alpha}\\\\): background). \\\\(n=100\\\\) VLDs from 10 cells. **(g)** Confocal images of a pEYFP-mem-transfected cell before (top) and after (bottom) application of 50% hypo-osmotic medium for 3 min. **(h)** Staining images of cells fixed immediately after re-application of iso-osmotic medium showing the membrane (pEYFP-mem transfection), pixillin and actin. **(f)** Garaged co-localization images are shown to the right. **(f)** Quantification of mean diameter of structures formed after stretch release (reservoirs) and re-application of iso-osmotic medium (VLDs). \\\\(n=250/100\\\\) structures from 8/10 cells. **(f)** Quantification of mean density of structures formed after stretch release (reservoirs) and re-application of iso-osmotic medium (VLDs). \\\\(n=30/50\\\\) regions from 5/8 cells. **(k)** Quantification of mean height of structures formed after stretch release (reservoirs) and re-application of iso-osmotic medium (VLDs). \\\\(n=80/50\\\\) structures from 6/4 cells (**P\\\\(<\\\\)0.001, two-tailed Student\u2019s t-test). We note that reservoir heights are close to the axial resolution of our confocal microscope (0.9 \u03bcm) and thus represent upper estimates rather than accurate measurements. Scale bars are 5 \u03bcm in **c.g** and 20 \u03bcm in **d.h**. In all cases, zoomed insets (\\\\(10\\\\times 6\\\\,\\\\mu\\\\)m\\\\({}^{2}\\\\) in **c.g** and \\\\(10\\\\times 10\\\\,\\\\mu\\\\)m\\\\({}^{2}\\\\) in **d.h**) show a magnification of the area marked in the main image. Error bars are mean \\\\(\\\\pm\\\\) s.e.m.\\n\\n'", "CAPTION FIG6.png": "\"Figure 6: **I Membrane mechanical adaptation is explained by minimization of the strain and adhesion energies required to generate surface and volume containers.** (**a**) Phase diagram showing the predicted structures that require minimal energy to deform the membrane and detach it from the substrate in order to accommodate membrane surface area (upon stretch release) and liquid volume at the cell-substrate interface (upon an increase in osmolarity)[38]. Surface storage is achieved optimally with increasingly long tubules, whereas volume storage leads to the formation of spherical caps (VLDs). When both volume and surface storage are required, spherical caps 'bud' and become more invagulated. (**b**) Left: time-course sequences of cell membrane regions showing the formation of either reservoirs or increasingly long tubules after releasing different stretch magnitudes. Right: Mean reservoir/tubule length (black dots, experimental data, red line, theoretical prediction) as a function of de-stretch magnitude (for increasing stretch, \\\\(n=80/50/50\\\\) structures from 6/3/3 cells). (**c**) Left: images showing the formation of increasingly large VLDs after restoring iso-osmotic medium in cells previously exposed to different magritudes of hypo-osmotic shocks for 3 min. Right: mean VLD diameter (black dots, experimental data, red line, theoretical prediction) as a function of hypo-osmotic shock magnitude (for increasing osmotic shock, 60/100/100/50 structures from 5/5/10/3 cells). Scale bars, 5 \u03bcm. Error bars are mean \\\\(\\\\pm\\\\) s.e.m.\\n\\n\"", "CAPTION FIG4.png": "'Figure 4: **Effect of stimulus application rate on membrane structure formation.** (**a**) Cell submitted to two successive steps of 6% stretch for 3min, in which the first is released immediately and the second slowly (ISs). (**b**) Cell submitted to two successive applications of 50% hypo-osmotic media, in which iso-osmotic medium is restored first immediately and then slowly (1min). Scale bars, 20 \u03bcm. Insets show zoomed views (\\\\(10\\\\times 10\\\\,\\\\mathrm{\\\\SIUnitSymbolMicro m}^{2}\\\\)) of membrane structures.\\n\\n'", "CAPTION FIG5.png": "'Figure 5: _Membrane mechanical adaptation is a passive process followed by active recovery._ (**a**) Examples of control and ATP-depleted pYFP-mem-transfected cells before, during and after application of transfected cells before, during and after application of two 3-min pulses. (**f**) Quantification of VLD fluorescence after the first re-application of iso-osmotic medium and during application and release of the second pulse (\\\\(n=35/40\\\\) VLDs from 3/3 cells). The effect of ATP depletion was significant (P \\\\(<\\\\) 0.001, two-tailed Student\u2019s t-test). (**g**) Quantification of reservoir size in control and ATP-depleted cells (\\\\(n=150/200\\\\) reservoirs from 3/3 cells). No significant differences were observed, two-tailed Student\u2019s t-test. (**d**) Quantification of reservoir density in control and ATP-depleted cells (\\\\(n=50/50\\\\) regions from 5/5 cells). No significant differences were observed. (**e**) Examples of control and ATP-depleted pYFP-mem-transfected cells before, during and after application of 50% hypo-osmotic medium in two 3-min pulses. (**f**) Quantification of VLD fluorescence after the first re-application of iso-osmotic medium and during application and release of the second pulse (\\\\(n=35/40\\\\) VLDs from 3/3 cells). The effect of ATP depletion was significant (P \\\\(<\\\\) 0.001, two-tailed Student\u2019s t-test). (**g**) Quantification of VLD size in control and ATP-depleted cells (\\\\(n=30/35\\\\) VLDs from 3/3 cells). No significant differences were observed. (**h**) Quantification of VLD density in control and ATP-depleted cells (\\\\(n=20/25\\\\) regions from 3/3 cells). No significant differences were observed. Scale bars, \\\\(20\\\\,\\\\mathrm{\\\\SIUnitSymbolMicro m}\\\\). Insets show zoomed views (\\\\(10\\\\times 10\\\\,\\\\mathrm{\\\\SIUnitSymbolMicro m}^{2}\\\\)) of membrane structures.\\n\\n'"}