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First published online 2 November 2004
doi: 10.1242/jcs.01506

Journal of Cell Science 117, 5905-5912 (2004)
Published by The Company of Biologists 2004
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Migration and retraction of endothelial and epithelial cells require PHI-1, a specific protein-phosphatase-1 inhibitor protein

Nikolaos A. Tountas and David L. Brautigan*

Center for Cell Signaling, University of Virginia School of Medicine, PO Box 800577, Charlottesville, VA 22908, USA



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  		Fig. 1. Vascular smooth-muscle and endothelial localization of PHI-1 in rat brain sections. (A) {alpha}-Smooth-muscle-actin immunostaining (green). (B) PHI-1 immunostaining (red). (C) Overlay of A and B. Original magnification was 600x; bar in C, 50 µm.

 


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  		Fig. 2. Localization of endogenous PHI-1 to the trailing edge of HMVEC-L cells. (A,B) Migrating HMVEC-L cells stained for PHI-1 (green) and F-actin (red). (C) Migrating cell stained with pre-immune rabbit serum instead of anti-PHI-1 antibody, as a control. (D) Non-migrating cell stained for PHI-1 and F-actin. Magnification was 600x; bar in D, 15 µm.

 


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  		Fig. 3. PHI-1 depletion by siRNA retards HeLa cell migration. (A) Total cell lysates (20 µg per lane) from cells transfected with buffer, {alpha}4 siRNA or PHI-1 siRNA#2 resolved by SDS-PAGE and blotted for PHI-1, {alpha}4 and PP1. `C' is a band present in PHI-1 immunoblots that serves as loading control. (B) Quantitation of the distance of migration 17 hours after scraping of cells transfected with buffer, {alpha}4 siRNA or PHI-1 siRNA#2 (*P<0.05). (C) Kinetics of cell migration under PHI-1 depletion. Same as B, except that cell migration was followed for a total of 41 hours after scraping (circles represent control cells and triangles represent PHI-1-knocked-down cells).

 


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  		Fig. 4. PHI-1 depletion using siRNAs results in extremely elongated HeLa cells that fail to retract. (A) Time-lapse phase-contrast photomicroscopy of control cell exhibiting two cycles of protrusions and retractions (white arrows) in a 3-hour period. Original magnification was 160x; bar, 35 µm. (B) Time-lapse phase-contrast photomicroscopy of a cell depleted of PHI-1 extending along the direction of the double-headed arrow with no retraction events in 5-hour period. Original magnification was 100x; bar, 40 µm. See Movie 1 and 2 in supplementary material.

 


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  		Fig. 5. PHI-1 depletion by siRNAs accelerates HUVEC spreading. (A) HUVEC knocked down for PHI-1 exhibited a 13% larger surface area after 30 minutes than control cells (*P<0.05, n=30). (B) Kinetics of cell spreading for a control (squares) vs a PHI-1 knockdown HUVEC. The slopes of the lines represent the rate of cell spreading. (C) Rate of cell spreading of control vs PHI-1 knocked-down HUVECs (*P<0.05, n=3).

 


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  		Fig. 6. PHI-1 knockdown by siRNA inhibits lamellipodium retraction but not protrusion in HUVECs. (A) The surface areas of replated cells were compared by overlaying binary images of cells acquired every 30 seconds. The absolute difference between the two surfaces ({Delta}) was used to determine protrusion (P) and retraction (R) events. (B) The number of protrusion events (black bars) between control and PHI-1 knockdown HUVECs was not significantly different. However, the number of retraction events in PHI-1-knockdown cells was significantly reduced compared with control cells (29±0.3 vs 44±4.4, respectively; *P<0.05, n=3).

 


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  		Fig. 7. PHI-1 knockdown by siRNA does not affect stress fibers or the phosphorylation state of selected cytoskeleton and focal-adhesion proteins. (A) Control HeLa cells stained for actin stress fibers. (B) PHI-1-depleted HeLa cells stained for actin stress fibers. Magnifications for A and B were 600x; bar for A and B, 25 µm. (C) Immunoblots of total cell lysates (20 µg per lane) from control and PHI-1-depleted HeLa cells were resolved by SDS-PAGE and blotted for PHI-1, phosphorylated and total cofilin, phosphorylated and total ERK, phosphorylated (P) and total (T) ERM, FAK phosphorylated on Ser722, phosphorylated MLC, and paxilin. `C' is a band present in PHI-1 immunoblots that serves as loading control.

 




© The Company of Biologists Ltd 2004