Volume 6, Issue 1, Pages 29-41 (January 2004) Polarization of Plasma Membrane Microviscosity during Endothelial Cell Migration  Amit Vasanji, Prabar K. Ghosh, Linda M. Graham, Steven J. Eppell, Paul L. Fox  Developmental Cell  Volume 6, Issue 1, Pages 29-41 (January 2004) DOI: 10.1016/S1534-5807(03)00397-6

Figure 1 Polarization of PMM in Migrating ECs (A) Confluent, quiescent ECs were subjected to wound-induced migration for 22 hr and then incubated for 15 min with 8.3 μg/ml DiI-C16. PMM was measured by FRAP at the leading (open squares), middle (filled circles), and trailing (open circles) edges of migrating ECs (mean, n = 5 cells), and normalized recovery curves are shown. Similar measurements were made in five randomly selected positions in five confluent cells behind the razor-wound (filled triangles). Inset: Calculated lateral diffusion coefficients, D (mean ± SEM, n = 5 cells). (B) Migrating ECs were labeled with DiI-C16 as in (A), and FRAP was measured at 22 membrane locations near the edge of the cell membrane spanning the length of the cell. Lateral diffusion coefficients were calculated at each bleach spot, interpolated, and indicated by a false-color; the color look-up table shows relative microviscosity. Direction of cell migration is shown by the open arrow. (C) ECs were induced to move in the presence of basic FGF (bFGF, 10 ng/ml). After labeling with DiI-C16, PMM was measured by FRAP in the leading (black bars) and trailing (gray bars) edges of ECs that had migrated into the wound area as in (A). Lateral diffusion coefficients are shown (mean ± SEM, 10 cells). (D) The effect of cell treatment with VEGF (10 ng/ml) on polarization of PMM was measured as in (C). (E) The effect of cytochalasin D (Cyt. D) on VEGF-induced polarization of PMM was measured as in (C). Migrating ECs were incubated for 20 min with Cyt. D (2 μM) just prior to FRAP determination. (F) Polarization of PMM was measured by fluorescence anisotropy. ECs were induced to move in the presence of VEGF for 22 hr, and then incubated with TMA-DPH (5 μM) for 5 min. Horizontally and vertically polarized images were acquired simultaneously and combined into anisotropy images. The open arrow indicates direction of cell movement, the dashed lines indicate forward cell extremities, and the grayscale look-up table indicates relative PMM. (G) EC were induced to move in the presence of basic FGF or VEGF for 22 hr and fluorescence anisotropy determined as in (F). The rear-to-front anisotropy ratio was determined from the mean pixel intensities in the extreme 10% of the cell surface (mean ± SEM, n = 30 cells). Developmental Cell 2004 6, 29-41DOI: (10.1016/S1534-5807(03)00397-6)

Figure 2 Role of Cholesterol in Formation of Gradient of PMM (A) Confluent aortic EC were induced to migrate by wounding and then incubated with methyl-β cyclodextrin (1%) for 30 min. After cyclodextrin removal, ECs were allowed to migrate for 22 hr. As a positive control, cells were stimulated with basic FGF (10 ng/ml). The cells were labeled with DiI-C16 and FRAP microviscosity measurements were done in the leading (black bars) and trailing (gray bars) edges as in Figure 1A and shown as calculated lateral diffusion coefficients (mean ± SEM, 10 cells). (B) Inhibition of cell migration by cyclodextrin is shown by phase contrast microscopy. (C) Lack of effect of cyclodextrin on actin filament formation was shown by staining fixed cells with Alexa 568 phalloidin (0.2 μM) and detection by fluorescence. (D) Immediately after wounding the monolayer, ECs were incubated with either NBD-cholesterol (10 μM, left panel) or with NBD-C6-HPC (10 μM, right panel) for 22 hr and imaged by fluorescence microscopy. Open arrows indicate direction of migration. Inset: NBD-cholesterol distribution in confluent ECs behind wound edge. (E) ECs were wounded and incubated with NBD-cholesterol or NBD-C6-HPC as in (B), and were simultaneously treated with basic FGF (10 ng/ml) or VEGF (10 ng/ml). Fluorescence intensity in the leading (black bars) and trailing (gray bars) regions (the outermost 10% of the cell area was measured) of migrating cells was determined densitometrically and expressed as average pixel intensity (mean ± SEM, n = 20 cells). (F) ECs were subjected to wound-induced migration for 22 hr in the absence (left panel) or presence (right panel) of basic FGF (10 ng/ml). The cells were labeled with FITC-conjugated cholera toxin (β subunit, 9 μg/ml for 20 min). Developmental Cell 2004 6, 29-41DOI: (10.1016/S1534-5807(03)00397-6)

Figure 3 Influence of PMM on Actin-Dependent Vesicle Deformation (A) LUVs encapsulating Alexa 488 actin (48 μM) were made by ether injection of a lipid solution (20 mg/ml) containing PC and phosphatidylethanolamine (4:1 molar ratio), DiI-C16 (4 μg/ml), ATP (0.2 mM), and valinomycin (0.1 mg per mg of phospholipid). Vesicles incubated in G-buffer without (left panel) or with (right panel) 100 mM KCl were imaged by confocal microscopy. (B) LUVs encapsulating unlabeled actin (48 μM) were made by ether injection of a lipid solution as in (A), but without DiI-C16, and containing cholesterol. Vesicle deformation was induced by incubation in G-buffer with 100 mM KCl, and images taken by phase contrast microscopy. (C) The aspect ratio of cholesterol-enriched vesicles in G-buffer without (gray bars) or with (black bars) 100 mM KCl was quantitated (mean ± SEM, n = 25 vesicles). (D) Actin-containing LUVs were made by ether injection as in (B) but with α-tocopherol instead of cholesterol and aspect ratios measured as in (C). F-actin in deformed liposomes was measured fluorimetrically after incubation with 0.4 μM rhodamine-phalloidin and expressed as relative intensity (gray bars). Developmental Cell 2004 6, 29-41DOI: (10.1016/S1534-5807(03)00397-6)

Figure 4 Effect of Angiogenic Growth Factors on Actin Polymerization in Lamellipodia of Migrating ECs (A) ECs transfected with pEGFP-actin (1.25 μg) were subjected to wound-induced migration for 24 hr. The cells were fixed and actin was stained with Alexa 568 phalloidin (0.2 μM). GFP-actin (left panel) and phalloidin-labeled actin (middle panel) were detected by fluorescence, and an overlay of both images is shown (right panel). Direction of EC movement is shown by open arrow. (B) EC monolayers were wounded and incubated for 24 hr with basic FGF (10 ng/ml, open squares), VEGF (10 ng/ml, filled squares), or medium alone (filled circles). Actin polymerization was determined by photobleaching GFP-actin-enriched regions in leading edge lamellipodia, and measuring fluorescence recovery at 2 s intervals. Inset: Apparent diffusion coefficients were calculated from the recovery curves and expressed as mean ± SEM for n = 10 cells. (C) ECs transfected with vector encoding GFP alone were allowed to migrate for 24 hr in the presence (black bar) or absence (gray bar) of basic FGF (10 ng/ml). Diffusion coefficients of GFP were determined by FRAP near the cell leading edge as described in (B) and expressed as mean ± SEM for n = 10 cells. (D) ECs were transfected with pEGFP-actin and induced to migrate for 24 hr. The cells were treated with jasplakinolide (1 μM, black bar) or with medium alone (gray bar) for 20 min, and actin polymerization was measured by FRAP of a region in the lamellipodia. Diffusion coefficients are expressed as mean ± SEM for n = 10 cells. (E) ECs were transfected with pEGFP-actin and induced to migrate. After 24 hr, the cells were treated with cytochalasin D (2 μM, black bar) or medium alone (gray bar) for 20 min. Actin polymerization was measured by FRAP in the lamellipodia, and diffusion coefficients expressed as mean ± SEM for n = 10 cells. Developmental Cell 2004 6, 29-41DOI: (10.1016/S1534-5807(03)00397-6)

Figure 5 Actin Nucleation in the Lamellipodium of Migrating ECs (A) ECs were transfected with pEGFP-actin and subjected to wound-induced migration for 24 hr. Cells were incubated for 60 s with rhodamine-conjugated actin (0.05 mg/ml) in G-buffer plus polymerizing buffer containing saponin (0.2 mg/ml) at 37°C, washed, and fixed. Confocal images of untreated (left) and basic FGF-treated (right) cells are shown. Open arrows indicate direction of migration. (B) The cellular regions of high nucleation activity in GFP-actin expressing, migrating cells was expressed as % of total cell area (n = 50 cells). (C) Total cellular incorporation of rhodamine-conjugated actin measured as pixel intensity per cell and expressed as mean ± SEM, n = 50 cells. Developmental Cell 2004 6, 29-41DOI: (10.1016/S1534-5807(03)00397-6)

Figure 6 Temporal Relationship between the Actin Meshwork and Lamellipodial Extensions (A) ECs were transfected with pEGFP-actin and subjected to wound-induced migration for 12 hr in the absence (top panels) or presence (bottom panels) of basic FGF (10 ng/ml). Confocal images of the cells in a temperature- and CO2-controlled chamber were taken at 3 min intervals for 8 hr using a 488 nm argon laser. The actin meshwork (open arrowheads) trailed the leading edge lamellipodium (closed arrowheads) by a variable distance. Large open arrow indicates direction of cell movement. (B) The positions (relative to the stage) of the forward-most lamellipodial extensions (solid lines) and actin meshwork (dashed lines) were determined in each frame for ECs incubated in the presence (red lines) or absence (black lines) of basic FGF. Vertical arrows indicate time of images in (A). (C) The average lifetime of the actin meshwork structure was determined for ECs treated with (black bar) or without (gray bar) basic FGF, and expressed as mean ± SEM for n = 6 cells. (D) The mean lamellipodia extension rate was determined during a 90 min interval for ECs incubated as in (C). Developmental Cell 2004 6, 29-41DOI: (10.1016/S1534-5807(03)00397-6)

Figure 7 Model for Relationship between Membrane Deformability and Actin Dynamics (A) Actin filament formation at early times after simulation of cell movement. Shown are migrating cells with leading edge plasma membranes of high (thin curved line, left panel), moderate (regular curved line, center panel), and low (thick curved line, right panel) membrane deformability. Actin monomers are shown as open circles, and crosslinking proteins (e.g., Arp2/3) are shown as gray objects. The direction of cell movement is indicated by an open arrow, and the rigid barrier property of the membrane indicated by an inverted “T.” (B) Actin filament formation at late times after simulation of cell movement as in (A). Developmental Cell 2004 6, 29-41DOI: (10.1016/S1534-5807(03)00397-6)