A RhoA and Rnd3 cycle regulates actin reassembly during membrane blebbing

Abstract

The actin cytoskeleton usually lies beneath the plasma membrane. When the membrane-associated actin cytoskeleton is transiently disrupted or the intracellular pressure is increased, the plasma membrane detaches from the cortex and protrudes. Such protruded membrane regions are called blebs. However, the molecular mechanisms underlying membrane blebbing are poorly understood. This study revealed that epidermal growth factor receptor kinase substrate 8 (Eps8) and ezrin are important regulators of rapid actin reassembly for the initiation and retraction of protruded blebs. Live-cell imaging of membrane blebbing revealed that local reassembly of actin filaments occurred at Eps8- and activated ezrin-positive foci of membrane blebs. Furthermore, we found that a RhoA-ROCK-Rnd3 feedback loop determined the local reassembly sites of the actin cortex during membrane blebbing.

Keywords: Eps8; Rnd3; actin cortex; cell migration; membrane bleb.

Fig. 1.

Eps8 locally accumulates at the initial phase of membrane retraction in the membrane bleb. (A) DLD1 cells exhibit membrane blebbing when cultured in a type I collagen matrix (Right). (Scale bar, 20 μm.) (B and C) Membrane blebbing of DLD1 cells transfected with Lifeact–RFP and GFP-tagged PLCδ–PH. Timing relative to the first image is indicated in white text. Actin cortex reassembly started from multiple sites of the protruded membrane (arrowheads). (Scale bar: B, 5 μm; C, 2 μm.) (D) Localization of GFP–MRLC1 during membrane bleb expansion and retraction. MRLC1 accumulates at multiple regions of membrane blebs (arrowheads). (Scale bar, 1 μm.) (E) Localization of GFP-tagged Eps8 in membrane blebs of DLD1 cells. Eps8 accumulates in multiple foci at the protruded membrane (arrowheads). (Scale bar, 2 μm.) (F and G) Kymographs showing actin localization (red) with respect to actin cytoskeleton-related proteins (green) during bleb retraction. Bleb extension is shown on the horizontal axis, and time is shown on the vertical axis. Eps-8 (F, green) localizes to the protruded membrane before actin filaments. MRLC1 (G, green) is recruited after actin filaments. (H) Timing of arrival of Eps8 and MRLC1 relative to that of actin filaments (t = 0 s). Data are the mean ± SD.

Fig. 2.

The end-capping and actin-bundling activities of Eps8 are required for continuous membrane blebbing. (A) Expression of Eps8 is greatly reduced in Eps8-KD DLD1 cells. (B) Eps8-KD DLD1 cells are spherical and do not exhibit membrane blebbing when cultured in a type I collagen matrix. (Scale bar, 10 μm.) (C) Exogenous expression of GFP-tagged mouse Eps8 restores membrane blebbing (arrow) in Eps8-KD DLD1 cells. (Scale bar, 10 μm.) (D) Schematic drawings of mutant Eps8 constructs. The number of amino acid residues of Eps8 is shown. (E) Total cell lysates of DLD1 cells expressing each construct separated by SDS/PAGE and immunoblotted with an anti-GFP mAb. (F) The percentages of GFP-positive cells showing membrane blebbing relative to the total number of GFP-positive cells in a given field were calculated. In each experiment, the total cell number was 100 (n  =  3). Data are the mean ± SD. *P < 0.05 (Student's t test). (G) Localization of GFP-tagged mutant Eps8 in Eps8-KD DLD1 cells. Expression of the GFP-tagged Eps8 mutant lacking the proline-rich region (GFP–Eps8ΔPR) or the GFP-tagged Eps8 mutant lacking the SH3 domain (GFP–Eps8ΔSH3) restores membrane blebbing in Eps8-KD DLD1 cells (arrows). (Scale bar, 10 μm.)

Fig. S1.

Eps8 is required for continuous membrane blebbing. (A) Exogenous expression of GFP-tagged mouse Eps8 significantly restores membrane blebbing in Eps8-KD DLD1 cells. Data are mean ± SD. *P < 0.05 (Student's t test) (n = 3). (B) Parental and Eps8-KD DLD1 cells were mixed and stained for Eps8 (red) and with phalloidin (green). Eps8-KD DLD1 cells are marked with asterisks. (Scale bar, 10 μm.) (C and D) Quantification of the fluorescence intensity of actin filaments along the white line shown in B. The fluorescence intensity of phalloidin was measured at six positions per cell. The SD was calculated based on the values from four independent experiments (Student's t test, **P < 0.01). (E) The fluorescence intensity of phalloidin was quantified in Eps8-KD DLD1 cells expressing wild-type Eps8, GFP–Eps8ΔPR, GFP–Eps8ΔSH3, GFP–Eps8ΔBundle, or GFP–Eps8ΔCap. SD was calculated based on the values from four independent experiments (Student's t test, **P < 0.01, *P < 0.05). (F) Parental and Eps8-KD DLD1 cells were mixed and stained for Eps8 (red) and for phosphorylated myosin light chain (p-MLC) (green). Eps8-KD DLD1 cells are marked with asterisks. (Scale bar, 10 μm.) (G) The fluorescence intensity of the p-MLC signal was quantified in wild-type DLD1 cells and Eps8-KD DLD1 cells. (H) The fluorescence intensity of p-MLC signal was quantified in Eps8-KD DLD1 cells expressing wild-type Eps8, GFP–Eps8ΔPR, GFP–Eps8ΔSH3, GFP–Eps8ΔBundle, or GFP–Eps8ΔCap. SD was calculated based on the values from four independent experiments (Student's t test, *P < 0.05). (I) The fluorescence intensity of phalloidin was quantified in wild-type DLD1 cells and ezrin-KO DLD1 cells. (J) The fluorescence intensity of the p-MLC signal was quantified in wild-type DLD1 cells and ezrin-KO DLD1 cells.

Fig. 3.

Activation of ezrin occurs at retracting membranes and is required for the rapid retraction of membrane blebs. (A) Membrane blebbing in DLD1 cells transfected with GFP–ezrin. GFP–ezrin localizes uniformly at the protruded membrane. (Scale bar, 2 μm.) (B) DLD1 cells were fixed and stained with an anti–phospho-ERM antibody (red) and an anti-total ERM antibody (green). Nuclei were stained with DAPI (blue). The asterisk indicates a membrane bleb in which ERM proteins were not activated. (Scale bar, 2 μm.) (C) DLD1 cells were fixed and stained with an anti–phospho-ERM antibody (green) and an anti-Eps8 antibody (red). The arrowheads indicate the colocalization of Eps8 and phosphorylated ERM proteins. (Scale bar, 2 μm.) (D) DLD1 cells were fixed and stained with an anti–phospho-ERM antibody (green) and Alexa 594–phalloidin (red). The boxed area shows the membrane blebs with regrowing actin filaments. High-magnification image of the boxed area is shown in the right panels. The asterisks indicate a membrane bleb covered with actin cortex. (Scale bar, 10 μm.) (E) Total cell lysates of wild-type DLD1 cells and ezrin-KO DLD1 cells separated by SDS/PAGE and immunoblotted with an anti-total ERM antibody, an anti-ezrin antibody, an anti-Eps8 antibody, and an anti–α-tubulin antibody. (F) Membrane blebbing of wild-type DLD1 cells and ezrin-KO DLD1 cells transfected with Lifeact–RFP and GFP-tagged Eps8. (Scale bar, 10 μm.) (G) Tricolor map of membrane blebs in wild-type DLD1 cells and ezrin-KO DLD1 cells. Angular coordinates are shown on the horizontal axis, and time is shown on the vertical axis. Red zones represent expansion, blue zones represent retraction, and white zones represent no movement. (H) Histogram of bleb expansion and retraction velocities in wild-type DLD1 cells and ezrin-KO DLD1 cells. (I) The frequencies of membrane blebs in wild-type DLD1 cells and ezrin-KO DLD1 cells during 10 min were quantified. **P < 0.01 (Student's t test). (J) The sizes of membrane blebs in wild-type DLD1 cell and in ezrin-KO DLD1 cells during 10 min were quantified. **P < 0.01 (Student's t test).

Fig. S2.

Ezrin is required for the recruitment of Eps8 to protruded membrane blebs and the rapid retraction of membrane blebs. (A) Localization of GFP–Eps8 in ezrin-KO DLD1 cells transfected with wild-type ezrin, the T567A ezrin mutant, and the T567E ezrin mutant. (Scale bar, 10 μm.) (B) The percentage of cells with blebs in which GFP–Eps8 was recruited to the plasma membrane relative to the total number of GFP–Eps8–positive cells in a given field was calculated. In each experiment, the total cell number was 100 (n  =  3). Data are the mean ± SD. *P < 0.05 (Student's t test). (C) Transwell migration assays were performed using Transwell filters (8-μm pore diameter; Corning Costar) coated with 1.0 mg/dL type I collagen. Membrane filter inserts were precoated with 100 μL of 1.0 mg/dL type I collagen solution in each well before seeding cells. Then, 2 × 10⁵ cells were seeded into the upper chamber, which contained the same medium as the lower chamber. After migration for 6 h, cells on the lower side were stained with DAPI, and the number of cells per high-power field was counted. Data are the mean ± SD. *P < 0.05 (Student's t test).

Fig. 4.

The RhoA–ROCK–Rnd3 feedback loop determines the actin reassembly sites of retracting membranes. (A) GFP–ROCK-1 is recruited to the retracting protruded membrane in DLD1 cells. (Scale bar, 2 μm.) (B) GFP–ROCK is recruited to the retracting protruded membrane in ezrin-KO DLD1 cells (arrowheads). (Scale bar, 2 μm.) (C) Membrane blebbing of DLD1 cells transfected with Lifeact–RFP and GFP-tagged AHD. (Scale bar, 2 μm.) (D) Membrane blebbing of DLD1 cells transfected with Lifeact–RFP and GFP-tagged Rnd3. Rnd3 accumulation gradually disappears upon the initiation of membrane blebbing retraction (t = 25 s). (Scale bar, 2 μm.) (E) Kymographs showing the localization of actin (red) with respect to that of Rnd3 (green) during bleb retraction. Bleb extension is shown on the horizontal axis, and time is shown on the vertical axis. (F) Kymographs showing the localization of Rnd3 (red) with respect to that of Eps8 (green) during bleb retraction. Bleb extension is shown on the horizontal axis, and time is shown on the vertical axis. (G) GFP–p190B–Rho–GAP localizes only at expanding blebs that lack the actin cortex. The membrane localization of p190B Rho–GAP is gradually lost upon the initiation of actin cortex recovery. (Scale bar, 2 μm.)

Fig. S3.

Membrane localization of Rnd3 persists at the protruded plasma membranes in DLD1 cells treated with Latrunculin-B. Membrane blebbing of DLD1 cells transfected with Lifeact–RFP and GFP-tagged Rnd3. Cells were treated with 1 µM Latrunculin-B (Lat B) at t = 0. The accumulation of Rnd3 persisted at the actin cytoskeleton-free protruded membranes (arrowheads). (Scale bar, 10 μm.)

Fig. 5.

A model of Rnd3- and RhoA-mediated regulation of actin cytoskeleton during membrane-blebbing cycle. (A) Localization of Eps8 in DLD1 cells expressing GFP–wild type Rnd3 (Upper) and GFP–Rnd3 S240A mutant. (Scale bar, 10 μm.) (B) Tricolor map of membrane blebs in DLD1 cells expressing GFP–wild type Rnd3 or GFP–Rnd3 S240A mutant. Angular coordinates are shown on the horizontal axis, and time is shown on the vertical axis. Red zones represent expansion, blue zones represent retraction, and white zones represent no movement. (C) Histogram of bleb expansion and retraction velocities in DLD1 cells expressing wild-type Rnd3 or the Rnd3 S240A mutant. (D) The frequencies of membrane blebs in DLD1 cells expressing GFP–wild type Rnd3 or GFP–Rnd3 S240A mutant during 10 min were quantified. **P < 0.01 (Student's t test). (E) The sizes of membrane blebs in DLD1 cells expressing GFP–wild type Rnd3 or GFP–Rnd3 S240A mutant during 10 min were quantified. *P < 0.05 (Student's t test). (F) In the expansion phase of membrane blebbing, Rnd3 and p190B–Rho–GAP inhibit the activation of RhoA. As the protruded membrane areas become enlarged, the relative concentration of Rnd3 decreases. Sporadic activation of RhoA leads to ROCK phosphorylation of Rnd3 and removal of p190B–Rho–GAP from the membrane. Thus, RhoA activation is amplified and sustained by the positive-feedback loop. ROCK also phosphorylates ezrin and activated ezrin recruits, which leads to reassembly of the actin cortex.

Fig. S4.

Extraction of cell contour and its smoothing for spatiotemporal visualization of membrane blebs. (A) Original grayscale frame image. (B) The cell contour is extracted by Otsu’s binarization. From the binarization image, the center of gravity, $p$ (green point), and the distance between $p$ and the cell contour in the direction $\theta$, $d_{\theta ,t}$ (orange line), are determined. In the lower image, the orange line intersects with the contour three times (as indicated by orange arrows), and thus there is ambiguity to determine the distance $d_{\theta ,t}$. (C) A smoothed contour (red) is derived through an optimization algorithm based on dynamic programming. By the smoothing, any line emerged from $p$ will intersect with the contour only once, and thus the distance $d_{\theta ,t}$ is determined uniquely. (D) The process of the optimal smoothing is illustrated. The value $d_{\theta }$ is the original distance given by the binarization. The aim of the optimization is to determine $a=\widehat{d_{\theta }}$ and $b=\widehat{d_{359-\theta }}$ for $\theta =0,\dots 179$ so that $a\sim d_{\theta }$ and $b\sim d_{359-\theta }$ and $a$ and $b$ will not violate the conditions for smoothness.

Fig. S5.

Spatiotemporal visualizations of membrane blebs and their application for counting blebs. (A) An example of the distance map shows white “blobs” on it. Because a whiter part in the distance map indicates that the part is further from the center, a consecutive white region, i.e., blob, will correspond to a bleb. The horizontal width of a blob is relative to the spatial width of a bleb, and the vertical height is relative to the lifetime of a bleb. (B) A tricolor map visualizes the membrane bleb dynamics more directly. An expanding bleb is represented as a red blob, and a retracting bleb is represented as a blue blob. (C) The number of the membrane blebs emerged in a video can be measured by detecting the local peaks (green) in the distance map, because each white blob in the distance map corresponds to a bleb. (D) By using the process of the peak detection, a segmentation result of the distance map is obtained. Each segment clarifies the area of a blob and, equivalently, spatiotemporal area of a bleb. (E) The color segmentation result of D is used for showing each bleb area on the contour of a frame. A bleb is represented in the same color from the beginning of its expansion to the end of the retraction.