extracted_captions/aoki2016rhoa.json

{"CAPTION FIG1.png": "'\\nFig. 1: Epss locally accumulations at the initial phase of membrane extraction in the membrane bath. (_A_) (LDD) cells exhibit membrane blocking when cultured in a single region (_k_igth). (Scale bar, 20 \\\\(\\\\mu\\\\)m) (_B_ and C) Membrane blebbing of DLD1 cells transfected with LHeat-RFP and GFP-tagged PLCs-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 \\\\(\\\\mu\\\\)m; \\\\(C\\\\), 2 \\\\(\\\\mu\\\\)m). (_D_) Localization of GFP-MRC1 during membrane bleb expansion and retraction. MRC1 accounts/ates at multiple regions of membrane blebs (arrowheads). (Scale bar: 1 \\\\(\\\\mu\\\\)m). (_E_) Localization of GFP-tagged Epss in membrane blebs of DLD1 cells. Epss accumulates in multiple foci at the protruded membrane (arrowheads). (Scale bar: 2 \\\\(\\\\mu\\\\)m). (_F_ and _G_) Extrapogrsk showing actin localization (red) with respect to actin cytoskeleton-related proteins (green) during bleb retraction. Blob extension is shown on the horizontal axis, and time is shown on the vertical axis. Epss-B (_F_, green) localizes to the protruded membrane before actin filaments. MRC1 (_G_, green) is executed after actin filaments. (_H_) Timing of arrival of Epss and MRC1 relative to that of actin filaments (_f_ = 0 s). Data are the mean \\\\(\\\\pm\\\\) SD.\\n\\n'", "CAPTION FIG2.png": "\"\\nFig. 2: The end-tapping and actin-burdling activities of Epsl are required for continuous membrane blebbing. (A) Expression of Epsl is greatly reduced in Epsl-KD DLD1 cells. (B) Epsl-KD DLD1 cells are spherical and do not exhibit membrane blebbing when cultured in a type I collagen matrix. (Scale bar, 10 \u03bcm.) [C] Exogenous expression of GFP-tagged mouse Epsl restores membrane blebbing (arrow) in Epsl-KD DLD1 cells. (Scale bar, 10 \u03bcm.) (D) Schematic drawings of mutant Epsl constructs. The number of amino acid residues of Epsl is shown. (F) Total cell lysates of DLD1 cells expressing each construct separated by SDS9AGE 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 \\\\(\\\\pm 5\\\\). \\\\(\\\\pm P<0.05\\\\) (Student's t test). (G) Localization of GFP-tagged mutant Epsl8 in Epsl-KD DLD1 cells. Expression of the GFP-tagged Epsl mutant lacking the proline-rich region (GFP-Epsl8APR) or the GFP-tagged Epsl mutant lacking the SH3 domain (GFP-Epsl8APR34) restores membrane blebbing in Epsl8-KD DLD1 cells (arrow). (Scale bar, 10 \u03bcm.)\"", "CAPTION FIG3.png": "'Figure 3: Activation of azaft 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 \u03bcm.) (_E_) DLD1 cells were fixed and stained with an anti-phospho-ER 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 \u03bcm.) (_C_) DLD1 cells were fixed and stained with an anti-phospho-ERM antibody (green) and an anti-Epr8 antibody (red). The arrowheads indicate the colocalization of Epr8 and phosphorylated ERM proteins. (Scale bar, 2 \u03bcm.) (_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 growing 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 \u03bcm.) (_E_) Total cell lysates of wild-type DLD1 cells and azaft-KO DLD1 cells separated by SDSPAGE and immunohistolated with an anti-total ERM antibody, an anti-Epr8 antibody, and an anti-tubulin antibody. (_F_) Membrane blebbing of wild-type DLD1 cells and azaft-KO DLD1 cells transfected with Histcard-RFP and GFP-tagged Epr8. (Scale bar, 10 \u03bcm.) (_G_) Tricolor map of membrane blebs in wild-type DLD1 cells and azaft-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 azaft-KO DLD1 cells. (_I_) The frequencies of membrane blebs in wild-type DLD1 cells and azaft-KO DLD1 cells during 10 min were quantified. **_P_\\\\(<\\\\) 0.01 (Student\u2019s t test). (_J_) The sizes of membrane blebs in wild-type DLD1 cell and in azaft-KO DLD1 cells during 10 min were quantified. **_P_\\\\(<\\\\) 0.01 (Student\u2019s t test).\\n\\n'", "CAPTION FIG4.png": "'\\nFig. 4: The RhoA-ROCK-Rn3 feedback loop determines the actin reassembly sites of retracting membranes. (A) GFP-ROCK-It is recruited to the retracting protruded membrane in DLD1 cells. (Scale bar, 2 \\\\(\\\\mu\\\\)m) (B) GFP-ROCK is recruited to the retracting protruded membrane in azin-KO DLD1 cells (arrowhead). (Scale bar, 2 \\\\(\\\\mu\\\\)m). (C) Membrane blebbing of DLD1 cells transfected with Lifcat-RFP and GFP-tagged AHD. (Scale bar, 2 \\\\(\\\\mu\\\\)m) (D) Membrane blebbing of DLD1 cells transfected with Lifcat-RFP and GFP-tagged AHD. (Scale bar, 2 \\\\(\\\\mu\\\\)m). (D) Membrane blebbing of DLD1 cells transfected with Lifcat-RFP and GFP-tagged Rnds. (Rad accumulation gradually disappears upon the initiation of membrane blebbing retraction (\\\\(t=25\\\\) S. (Scale bar, 2 \\\\(\\\\mu\\\\)m).) (E) Kymographs showing the localization of actin (red) with respect to that of Rnds (green) during bleb retraction. (B) Extension is shown on the horizontal axis, and time is shown on the vertical axis. (F) Kymographs showing the localization of Rnds (red) with respect to that of Exp8 (green) during bleb retraction. (B) Extension is shown on the horizontal axis, and time is shown on the vertical axis. (G) GFP-D1908-Rho-GAP localizes only at expanding blebs that lack the actin cortex. The membrane localization of p1908 Rho-GAP is gradually lost upon the initiation of actin cortex recovery. (Scale bar, 2 \\\\(\\\\mu\\\\)m.)'", "CAPTION FIG5.png": "'\\n\\n## References\\n\\nFig. 5: A model of Rnd3- and RhoA-mediated regulation of actin cytoskeleton during membrane-bleibbing cycle. (_A_) Localization of Ep8 in DLD1 cells expressing GFP-wild type Rnd3 (Upper) and GFP-Rrd3 S240A mutant. (Scale bar, 10 \u03bcm.) (_B_) Tricolor map of membrane blobs 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 bleib expansion and retraction velocities in DLD1 cells expressing wild-type Rnd3 or the Rnd3 S240A mutant. (_D_) The frequencies of membrane bleibs in DLD1 cells expressing GFP-wild type Rnd3 or GFP-Rnd3 S240A mutant during 10 min were quantified. *_P_< 0.01 (Student\u2019s 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\u2019s test). (_P_) In the expansion phase of membrane bleibbing, Rnd3 and p1968\u2013Rho-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 p1968\u2013Rho-GAP from the membrane. Thus, RhoA activation is amplified and sustained by the positive-feedback loop. ROCK also phosphorylates earin and activated earin recruits, which leads to reassembly of the actin cortex.\\n\\n'"}