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First published online 12 September 2007
doi: 10.1242/jcs.005942

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Cdc42 is required for EGF-stimulated protrusion and motility in MTLn3 carcinoma cells

Mirvat El-Sibai¹, Peri Nalbant^2,*, Huan Pang¹, Rory J. Flinn¹, Corina Sarmiento³, Frank Macaluso⁴, Michael Cammer⁴, John S. Condeelis^3,4, Klaus M. Hahn² and Jonathan M. Backer^1,,*

¹ Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
² Pharmacology, University of North Carolina School of Medicine CB7365, Chapel Hill, NC 27599, USA
³ Anatomy and Structural Biology
⁴ Analytical Imaging Facility, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA

View larger version (42K): [in this window] [in a new window]   Fig. 1. Cdc42 activation at the protruding edge in response to EGF stimulation. MTLn3 cells were serum-starved for 3 hours and then stimulated with 5 nM EGF at 37°C for the indicated times. (A) GST-CRIB pull-downs of unstimulated and stimulated cells. Anti-Cdc42 western blots showing activated (GTP-Cdc42) and total Cdc42. The data are representative of three experiments. (B) GST-CRIB assay for Cdc42 activation in cells pre-treated with carrier (DMSO) or 100 nM wortmannin (Wm) for 15 minutes, then stimulated with EGF. (C) Representative micrographs of EGF-stimulated cells, fixed at various times and immunostained with anti-Cdc42 antibody. (D) Quantification of data shown in C; fluorescence intensity in the lamellipod edge (0.2-0.6 µm in from the edge of the cell) was normalized to edge intensity of non-stimulated cells. The data is the mean ±s.e.m. from three different experiments (15 cells per experiment), plotted as a function of time. (E) MTLn3 cells were starved for 3 hours and microinjected with the Cdc42 biosensor as explained in Materials and Methods. After 1 hour, cells were stimulated with 5 nM EGF for various times, or not (0 minutes), and fixed. Ratio images (I-SO:GFP) reflect the activation (GTP binding) of Cdc42 at different times after EGF stimulation. Activation was maximal at the 180 seconds, with regions of highest activity five times greater than in the unstimulated cells. (F) At each time point, the mean ratio from a region of interest (2 µm inwards from the edge to the center of the cell) is calculated as fold increase relative to unstimulated cells, and plotted as a function of time. Data are the mean ± s.e.m. from 40 different cells. Bars, 10 µm.

View larger version (65K): [in this window] [in a new window]   Fig. 2. Cdc42-siRNA-treated cells show reduced protrusion in response to EGF stimulation. (A) Representative western blot showing Cdc42 (upper panel) and Rac (lower panel) expression in cells treated with control luciferase-siRNA-treated (Luc) or Cdc42 siRNA (Cdc42 KnDn). Quantification of western blots from four different experiments (bar graph) shows an 80% decrease in the level of expression of Cdc42 in cell lysates. (B) Representative phase micrographs of MTLn3 cells transfected with control luciferase or Cdc42 siRNA (Cdc42 KnDn), starved and stimulated with EGF for 1 or 3 minutes or not (0 min). (C) Quantification of the surface area of EGF-stimulated control luciferase-siRNA-treated () and Cdc42-siRNA-treated cells () normalized to unstimulated cells. Data are the mean ± s.e.m. from at least 45 cells. Bar, 10 µm. (D) MTLn3 cells were transfected with luciferase control or Cdc42 siRNA and starved for 3 hours. Cells were then imaged using time-lapse microscopy during a 10-minute EGF stimulation with images collected every 20 seconds. Kymographs were generated by drawing a constant line in the stacked time series and the protrusion was followed over time.

View larger version (86K): [in this window] [in a new window]   Fig. 3. Cdc42-siRNA-treated cells show a loss of actin nucleation at the protruding edge in response to EGF stimulation. (A) Representative micrographs of MTLn3 cells transfected with control luciferase or Cdc42 siRNA, starved for 3 hours and stimulated with EGF for 1 or 3 minutes or not (0 min). Cells are stained with Rhodamine-phalloidin to visualize F-actin. (B) Quantification of the average F-actin fluorescence at the protruding edge (0.2-0.6 µm in from the edge of the cell) normalized to edge intensity of non-stimulated cells. Data are the mean ± s.e.m. from three different experiments (15 cells per experiment). (C) Control and Cdc42 siRNA-treated MTLn3 cells were stimulated with EGF for 1 or 3 minutes or not (0 min), permeabilized and incubated with biotin-actin, and immunostained with anti-biotin to detect the newly formed barbed ends. (D) Quantification of barbed-end formation at the protruding edge (0.2-0.6 µm in from the periphery) normalized to unstimulated cells. Data are the mean fluorescence ± s.e.m. from three different experiments (15 cells per experiment). Bars, 10 µm.

View larger version (42K): [in this window] [in a new window]   Fig. 4. Cdc42 meditates the translocation of Arp2/3 to the protruding edge in response to EGF stimulation. MTLn3 cells were transfected with control or Cdc42 siRNA, starved for 3 hours and stimulated with EGF for 1 or 3 minutes or not. The cells were then fixed and immunostained with anti-Arp3 and anti-tropomyosin antibodies. (A) Representative merged images of control luciferase-siRNA-treated (left panels) or Cdc42-siRNA-treated (right panels) cells stimulated for 3 minutes and stained for Arp3 (red) and Tropomyosin (green). Small panels show magnification of boxed areas of respective large panel. Arrows indicate the edges of Arp2/3 (red) and tropomyosin-rich (green) zones. (B) Quantification of average fluorescence pixel intensity at the edge for Arp2/3 in control luciferase-siRNA-treated (top diagram) and Cdc42-siRNA-treated (bottom diagram) cells stimulated with EGF for 1 minute ( and , respectively) or 3 minutes ( and , respectively) or left untreated ( and , respectively). Data are the mean ± s.e.m. from three different experiments (15 cells per experiment) plotted as a function of distance from the cell edge. (C) Quantification of average fluorescence pixel intensity at the edge for tropomyosin in control (top diagram) and Cdc42 siRNA-treated cells (top diagram). Cells were stimulated as in B. (D) Data from B presented as an average of the mean fluorescence in the lamellipodia (0.2-0.6 µm from the edge) normalized to unstimulated cells. Bar, 10 µm.

View larger version (148K): [in this window] [in a new window]   Fig. 5. Cdc42 is required for actin branching at the protruding edge. Rotary-shadowing transmission electron micrographs of cells treated with control luciferase (left panel) or Cdc42 (right panel) siRNA (n=10 cells). Bar, 100 nm.

View larger version (76K): [in this window] [in a new window]   Fig. 6. Cdc42 mediates the translocation of N-WASP and WAVE2 to the edge in response to EGF. Control luciferase-siRNA-transfected or Cdc42-siRNA-transfected (Cdc42 KnDn) MTLn3 cells were starved for 3 hours, and stimulated with EGF for 1 or 3 minutes, or not. (A) Representative images of cells immunostained with anti-N-WASP antibody. (B) The average N-WASP fluorescence in the lamellipodia (0.2-0.6 µm in from the edge) was measured as explained in Materials and Methods and normalized to edge fluorescence of unstimulated cells. (C) Representative images of cells immunostained with anti-WAVE2 antibody. (D) The average WAVE2 fluorescence in the lamellipodia (0.2-0.6 µm in from the edge) was measured as explained in Materials and Methods and normalized to edge fluorescence of unstimulated cells. Data in B and D are the mean ± s.e.m. from 45 cells for each staining condition. Bars, 10 µm.

View larger version (96K): [in this window] [in a new window]   Fig. 7. Cdc42 is required for activation of Rac, PI3K and IRSp53 at the protruding edge of EGF-stimulated cells. Control luciferase-siRNA-transfected or Cdc42-siRNA-transfected MTLn3 cells were starved for 3 hours, and stimulated with EGF for 1 or 3 minutes, or not. Representative images of cells immunostained against (A) Rac, (B) PtdIns(3,4,5)P3 or (C) IRSp53. Bar graphs show the average Rac, PtdIns(3,4,5)P3 or IRSp53 fluorescence in the lamellipodia (0.2-0.6 µm from the edge), normalized to edge fluorescence of unstimulated cells. Data are the mean ± s.e.m. from 45 cells. Bars, 10 µm.

View larger version (12K): [in this window] [in a new window]   Fig. 8. Model of Cdc42-mediated cytoskeletal signaling in carcinoma cells. Cdc42 and PI3K are both activated within the first minute of EGF stimulation in carcinoma cells, and might form a positive feedback loop. At later times, PI3K also receives input from Ras, which is required for protrusion. Activated Cdc42 is upstream of Rac, which regulates the formation of adhesive structures behind the protruding edge. Cdc42 regulation of WAVE2 and/or N-WASP is required for recruitment and activation of Arp2/3 and formation of branching actin structures. Cdc42 activation of N-WASP might also be involved in directional sensing (Nakahara et al., 2003; Yamaguchi et al., 2005), in tandem with PLC-mediated activation of cofilin (Mouneimne et al., 2004).

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