STIM-Orai1 signaling regulates fluidity of cytoplasm during membrane blebbing

Abstract

The cytoplasm in mammalian cells is considered homogeneous. In this study, we report that the cytoplasmic fluidity is regulated in the blebbing cells; the cytoplasm of rapidly expanding membrane blebs is more disordered than the cytoplasm of retracting blebs. The increase of cytoplasmic fluidity in the expanding bleb is caused by a sharp rise in the calcium concentration. The STIM-Orai1 pathway regulates this rapid and restricted increase of calcium in the expanding blebs. Conversely, activated ERM protein binds to Orai1 to inhibit the store-operated calcium entry in retracting blebs, which results in decreased in cytoplasmic calcium, rapid reassembly of the actin cortex.

Fig. 1. Highly fluid cytoplasm is formed in the expanding membrane blebs.

a, b Membrane blebbing of DLD1 cells expressing GFP-PLCδ-PH. Bin widths of the histogram (b) are 0.02 μm/s and the data presented are the means ± the standard deviation (SD) of measurements from 10 independent cells over three independent experiments. c–f QDs dynamics during bleb expansion and retraction. c Example tracks superimposed in situ (left) and centered trajectory maps showing the sum of the frame-to-frame distances over 600 msec (30 frames at 50 Hz; right). Red, yellow, and blue trajectories indicate diffusion velocities >3.3 μm/s, >1.6 μm/s and <3.3 μm/s, and <1.6 μm/s, respectively. See also Supplementary Movie 1. d Cumulative distances of QDs motion. e Mean square displacement (MSD) analysis of five representative trajectories per condition. f Diffusion coefficient (D, μm²/msec) was calculated from the slope of the fitted regression line derived by MSD analysis of (d). QDs dynamics in the cell body of vehicle- (control) or Latrunculin B (LatB)-treated cells are also shown. N = 100 particles from 10 blebs from 10 independent cells per condition. Individual data points are plotted with the means ± SD in d and f. ****P < 0.0001 (One-way ANOVA with Tukey’s post-hoc multiple comparison test). g Membrane blebbing of DLD1 cells expressing GFP-Mena. Black and white arrowheads show expanding and retracting blebs, respectively. Result shown is representative of five independent experiments. See also Supplementary Movie 2. h–k Kymograph analysis of GFP-Mena in bleb cytoplasm. h Representative kymographs of NucBlue (blue) and GFP-Mena (green) from three independent experiments. Bleb extension is shown on the vertical axis, and time is shown on the horizontal axis. i Schematic of the analysis in (j, k). Fluorescence intensities in “bleb”　(yellow) and “cell body” (white) cytoplasm were quantified and expressed as ratios. j Representative kymographs of GFP-Mena (left) and the control cytoplasm protein, RFP (right). k Fluorescence intensities of GFP-Mena and RFP were quantified as in i. Data presented are means ± SD based on the values from five independent experiments. a and g Indicated times are relative to the first image. Scale bar, 2 μm. Source data are provided as a Source Data file.

Fig. 2. The concentration of calcium ion increases in the expanding membrane blebs.

a Cytoplasmic calcium ion concentration was monitored by expressing GCaMP6s. Arrowheads show expanding bleb. Result shown is representative of five independent experiments. See also Supplementary Movie 3. b–d Kymograph analysis of GCaMP6s in bleb cytoplasm. b Representative kymographs of NucBlue (blue) and GCaMP6s (green) from three independent experiments. Bleb extension is shown on the vertical axis, and time is shown on the horizontal axis. c Schematic of the analysis in d. Fluorescence intensities in “bleb” (yellow) and “cell body” (white) cytoplasm were quantified and expressed as ratios. d Fluorescence intensities of GCaMP6s and RFP were quantified as in c. e, f Calcium ion concentration at the juxtamembrane cytoplasm was monitored with the membrane-targeted calcium probe (GCaMP6s-CAAX; e). Fluorescence intensities of GCaMP6s-CAAX were normalized to those of mCherry-PLCδ-PH (f). Data presented in d and f are means ± SD based on the values from five independent experiments. a and e Indicated times are relative to the first image. Scale bar, 2 μm. Source data are provided as a Source Data file.

Fig. 3. Artificially increasing the cytoplasmic calcium ion concentration upregulates cytoplasmic fluidity and exaggerates membrane blebbing.

a Membrane blebbing of DLD1 cells expressing GCaMP6s and Lifeact-RFP treated with the calcium ionophore 4-bromo-A23187 (10 µM). Arrowheads show blebs fusing. Result shown is representative of three independent experiments. See also Supplementary Movie 4. b–e QDs dynamics in the cell body of ionophore-treated cells. b Centered trajectory maps showing the sum of the frame-to-frame distances over 600 msec (30 frames at 50 Hz; right). Red, yellow, and blue trajectories indicate diffusion velocities >3.3 μm/s, >1.6 μm/s and <3.3 μm/s, and <1.6 μm/s, respectively. c Cumulative distances of QDs motion. d Mean square displacement (MSD) analysis of five representative trajectories per condition. e Diffusion coefficient (D, μm²/msec) was calculated from the slope of the fitted regression line derived by MSD analysis of (c). N = 110 particles from 11 blebs from 10 independent cells per condition. c, e The number (f, N = 20 cells), area (g, N = 20 blebs) and retraction velocity (h, N = 10 blebs) of membrane blebs in vehicle-treated (control) and ionophore-treated (4-bromo-A23187) DLD1 cells over 10 min from three independent experiments. i Membrane blebbing of DLD1 cells expressing GCaMP6s and Lifeact-RFP treated with the SERCA inhibitor Thapsigargin (1 µM). Arrowhead shows increased GCaMP6s signal intensity in the bleb. Result shown is representative of three independent experiments. a and i Indicated times are relative to drug treatment. Scale bar, 10 μm. c, e-h Individual data points are plotted with the means ± SD. ****P < 0.0001 (Two-sided, unpaired Student’s t test). Source data are provided as a Source Data file.

Fig. 4. The influx of calcium ions via SOCE support expansion of membrane blebs.

Membrane blebbing of DLD1 cells expressing either Sec61β-mCherry and GFP-PLCδ-PH (a, Supplementary Movie 5) or Orai1-mCherry and GFP-tagged STIM1 (b). Arrowheads show the ER-PM contact sites. Indicated times are relative to the first image. Results shown are representative of three independent experiments. Scale bar, 2 μm. c–f DLD1 cells expressing Lifeact-RFP were treated with the SOCE inhibitors AnCoA4 (50 µM) and SKF96365 (10 µM). c Representative images from three independent experiments. Indicated times are relative to drug treatment. Scale bar, 10 μm. The number (d, N = 20 cells), area (e, N = 20 blebs) and retraction velocity (f, N = 20 blebs) of membrane blebs in vehicle-treated (control) and drug-treated (4-bromo-A23187) DLD1 cells over 10 min from three independent experiments. Individual data points are plotted with the means ± SD. ****P < 0.0001 (One-way ANOVA with Tukey’s post-hoc multiple comparison test). Source data are provided as a Source Data file.

Fig. 5. The formation of STIM1-Orai1 complex promotes the expansion of blebs.

a–f Membrane blebbing in DLD1 cells expressing Lifeact-RFP with either WT or dominant negative (E106Q) Orai1. a Representative still images from five independent experiments. b Tricolor maps showing angular coordinates along the horizontal axis and time on the vertical axis. Red zones represent expansion, blue zones represent retraction, and white zones represent no movement. Results shown are representative of three independent experiments. c Fluorescence intensities of GCaMP6s in “bleb” and “cell body” cytoplasm were quantified in DLD1 cells expressing either WT or E106Q Orai1. The ratio of “bleb” to “cell body” intensities are plotted over time. Data presented are means ± SD based on the values from five independent experiments. The number (d, N = 20 cells), area (e, N = 20 blebs), and retraction velocity (f, N = 10 blebs) of membrane blebs in WT or E106Q Orai1-expressing cells. g–l Membrane blebbing in DLD1 cells expressing Lifeact-RFP with either WT or dominant active (DA, D76A) STIM1. g Representative still images from three independent experiments. Arrowhead indicate persistent ER-PM contact site. h Tricolor maps showing angular coordinates along the horizontal axis and time on the vertical axis. Red zones represent expansion, blue zones represent retraction, and white zones represent no movement. Results shown are representative of three independent experiments. i Histograms of bleb expansion and retraction velocities in WT an DA STIM1-expressing cells. Three independent measurements are plotted for each condition. The number (j, N = 20 cells), area (k, N = 20 blebs), and retraction velocity (l, N = 10 blebs) of membrane blebs in WT or DA STIM1-expressing cells. Individual data points are plotted with the means ± SD. m Schematic of PM-ER contact site formation in the early stages of bleb formation. Details of the model are described in the text. a and g Scale bar, 10 μm. d–f, j–l ****P < 0.0001 (Two-sided, unpaired Student’s t test). Source data are provided as a Source Data file.

Fig. 6. Activated Ezrin inhibits SOCE by directly binding to Orai1 and promotes retraction of membrane blebs.

a Representative still images of membrane blebbing in DLD1 cells expressing Lifeact-RFP and GFP-E-Syt1 from five independent experiments. See also Supplementary Movie 6. b, c DLD1 cells expressing Lifeact-RFP with either GFP-E-Syt1 (b) or GCaMP6s (c) were treated with the actin polymerization inhibitor Latrunculin B (LatB, 10 µM). Arrowheads show disrupted actin cortex. Results shown are representative of three independent experiments. See also Supplementary Movie 7. d–f Biochemical analyses of ezrin’s role in STIM1-Orai1 complex formation. d Orai1-HA was co-expressed with either GFP or GFP-ezrin. Inputs and HA immunoprecipitates were immunoblotted with antibodies against HA and GFP. e STIM1-FLAG and Orai1-HA were co-expressed with either GFP or GFP-ezrin. Inputs and FLAG immunoprecipitates were immunoblotted with antibodies against FLAG, HA, and GFP. f GFP expression levels (shaded in green) and co-precipitated Orai1-HA (individual measurements) were quantified and normalized to GFP-expressing control. Data presented are means ± SD based on the values from four independent experiments. g, h Proximity ligation assay (PLA) for in situ detection of Orai1-STIM1 interaction. Representative images from five independent experiments are shown in g. h Quantification of PLA signals in expanding (low Lifeact intensity) and retracting (high Lifeact intensity) blebs based on data shown in g and Supplementary Fig. 6a. STIMATE-STIM1 and Claudin-3-STIM1 are positive and negative controls, respectively. N = 25 independent blebs. Individual data points are plotted with the means ± SD. i Membrane blebbing in Ezrin KO cells expressing GFP- PLCδ-PH and Sec61β-mCherry (upper panels) and in WT DLD1 cells expressing constitutive active Rnd3 (S240A) and Sec61β-mCherry (lower panels). Arrowheads indicate persistent ER-PM contact sites. Results shown are representative of three independent experiments. j Representative images from three independent experiments of DLD1 cells expressing constitutive active Ezrin (T567E) and Sec61β-mCherry. a, g (middle and bottom panels), i, j (three right panels) Scale bar, 2 μm. b, c, g (top panels), and j (left panel) Scale bar, 10 μm. f, h ****P < 0.0001 (Two-sided, unpaired Student’s t test). Times shown are relative to drug treatment (b, c) or the first image (i). Source data are provided as a Source Data file.

Fig. 7. SOCE formation is necessary for the dissemination of amoeboid single cells from cancer spheroid in 3D environment.

a–e Role of SOCE in cell migration. a Transwell migration assays using non-transfected DLD1 cells and cells expressing either dominant negative Orai1 (E106Q) or constitutive active Ezrin (T567E). Individual data points are plotted with the means ± SD based on the values from N = 5 independent experiments. b 4T1 cell spheroids embedded in type I collagen were treated with DMSO (left panel) or DMOG (1 mM), an inhibitor of prolyl hydroxylase, to activate HIF-1, (right panel). Scale bar, 100 μm (left panel) or 200 μm (right panel). c Representative 4T1 cell spheroid embedded in type I collagen was treated with DMOG only (1 mM, left panel), or with a combination of DMOG and SOCE inhibitors AnCoA4 (50 µM, center panel) or SKF96365 (10 µM, right panel) for 72 h. Scale bar, 200 μm (left panel) or 100 μm (center and right panels). d Number of single cells migrating away from the spheroid were quantified based on the experiment shown in c. N = 10 independent spheroids per condition from three independent experiments. Individual data points are plotted with the means ± SD. e Representative amoeboid migration of 4T1 cells expressing Lifeact-GFP and stained with ER-tracker (red). after spheroid treatment with DMOG as in b from three independent experiments. Arrowheads indicate regions where the ER flows into blebs. Times shown are relative to the first image. Scale bar, 10 μm. f Schematic showing the proposed mechanism of SOCE-dependent cytoplasm solation. In the expansion phase of membrane blebbing, the influx of calcium ions via SOCE inhibits actin polymerization and promotes solation of the cytoplasm, which supports rapid bleb expansion. By contrast, during the retraction phase, activated Ezrin inhibits SOCE by directly binding to Orai1, allowing the reassembly of the actin cytoskeleton by decreasing the calcium ion concentration in the cytoplasm of the bleb. a, d ****P < 0.0001 (One-way ANOVA with Tukey’s post-hoc multiple comparison test). Source data are provided as a Source Data file.