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First published online September 18, 2007
doi: 10.1242/10.1242/jcs.000638

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ZBP1 enhances cell polarity and reduces chemotaxis

Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461-1975, USA

View larger version (20K): [in this window] [in a new window]   Fig. 1. ZBP1 expression causes phenotypic conversion from random walk to directed movement, increasing persistence and directionality. Movement of GFP- and GFP-ZBP1-expressing MTLn3 cells (from two independently derived stable cell lines, indicated as ZBPA and ZBPB) was examined in 5% serum. The total time interval was 30 minutes, and time between successive frames was 1 minute. (A,B) Plots of cell (A) centroid and (B) perimeter for six GFP-expressing control MTLn3 cells (CTRL, A and B top) and six GFP-ZBP1-expressing MTLn3 cells (ZBP, A and B bottom). (C,D) Differences in (C) persistence and (D) directionality were statistically significant between the populations (*P<0.005, error bars indicate ± s.e.m.). Persistence is the speed divided by the change in direction, directionality is the net path length divided by the total path length.

View larger version (14K): [in this window] [in a new window]   Fig. 2. ZBP1 expression reduces movement in the direction of a chemoattractive gradient. (A,B) Perimeter and centroid plots are from (A) GFP-expressing or (B) GFP-ZBP1-expressing MTLn3 cells when exposed to a gradient of EGF after starvation. These results are representative of cell behaviors over the time frame analyzed (1 hour at 1-minute intervals; * indicates needle position, which is the source of EGF). (C) Quantification of cell movement relative to an EGF gradient. The angle between cell movement and the direction of applied chemoattractant indicates how well a cell orients to the chemoattractant, with a smaller angle suggesting greater alignment of the directions. This is reflected in our results, which take the cosine of the angle, and for which a greater alignment would give a value closer to 1 and lower degree of alignment gives a lower value, and 0 would indicate average random motion. MTLn3 cells are efficiently stimulated to move along the gradient, whereas ZBP1-expressing cells less efficiently orient and move in response to such a gradient (*P=0.035, error bars indicate ± s.e.m.).

View larger version (28K): [in this window] [in a new window]   Fig. 3. ZBP1-expressing cells can sense and move towards an oriented gradient. ZBP1-expressing cells are capable of responding normally to a gradient of EGF, but only when the gradient is applied in the direction of their intrinsic polarity. A chemoattractant, EGF, was released from a needle placed in front of the leading edge of the cell while protrusion and retraction were measured from both the front and back of the cell. Standardized membrane protrusion is plotted versus time after the micropipette stimulation. Responses of ZBP1-expressing cells are not substantially different from controls expressing only GFP (15 cells were measured for each group, error bars indicate ± s.e.m.).

View larger version (76K): [in this window] [in a new window]   Fig. 4. ZBP1 localizes to the periphery of cells and reduces motility and orientation towards vessels in living tumors. (A) ZBP1-GFP-expressing cells imaged in vivo using multiphoton microscopy show ZBP1 (green; one cell outlined with dotted line) localized to the periphery of cells and at cell-cell junctions (arrow). Collagen is imaged by second harmonic generated polarized light in the multiphoton microscope (purple, arrowhead). (B) MTLn3 cells transfected with CFP show elongated cell morphology (arrow) and orientation towards vessels (black spaces) in a living tumor. (C) ZBP1-expressing cells show a rounded morphology (arrowhead) along vessels. ZBP1-expressing cells do not polarize in the direction of vessels. Bars, 25 µm. (D) ZBP1 cells are less motile than cells in tumors generated by MTLn3 control cells. ZBP1 cells also show decreased orientation towards vessels than cells in tumors generated by MTLn3 control cells (*P=0.0003, error bars indicate ± s.e.m.).