Potential chirality axes (A) and possible regulators of leftward polarity bias (B). (A) Schematic diagram of an unpolarized cell resting on a coverslip, showing three chirality axes. Axis 1 is the arrow drawn from the center of the nucleus through the centrosome, and axis 2 is the vertical axis, from coverslip to medium. Orthogonal to the first two axes, axis 3 is the predicted direction of polarity to be assumed by the cell after addition of uniform fMLP: the pseudopod will be on the left, and the uropod will be on the right. (B) The diagram presents postulated relations among regulatory molecules we speculate may be involved in executing the leftward polarity bias of dHL60 cells. Solid lines connecting elements in the pathway represent steps documented in regulation of polarity and centrosome orientation in astrocytes and neurons (6–10). Dotted arrows with question marks indicate speculative links between a proposed chiral template and possible target steps in the pathway.

Perturbing leftward bias. We assessed effects on leftward bias of perturbing either microtubules (A) or activities of three proteins thought to regulate polarity in other cells (B). (A) dHL-60 cells expressing GFP-N-Clip170 were subjected to no drug (Left), nocodazole (20 μM, 40 min) (Center), or 2 h after multiple washes with RPMI medium (Right), and then exposed to uniform fMLP (100 nM). Centrosome positions of the cells after polarization in response to fMLP are indicated by filled or empty blue circles, representing cells that expressed either of two centrosome markers, GFP-N-Clip170 or GFP-Arr3, respectively. For all GFP-N-Clip170-expressing cells, centrosome positions relative to the arrow were recorded at 360 s after exposure to fMLP, as described in the legend of Fig. 1D. Centrosome positions of GFP-Arr3-expressing cells (confined to the Center figure) were recorded at 180 s after exposure to fMLP, because these cells polarized more rapidly than cells expressing the other marker as described in the text. Thus the appropriate controls for the empty circles in this Center figure are the 13 GFP-Arr3-expressing cells whose positions after 180 s exposure to fMLP are depicted in Fig. 1B. Regardless of the centrosome marker used, the leftward bias of polarity was not detected after treatment with nocodazole (Center) but was restored after nocodazole was removed (Right). (Scale bar, 20 μm.) (B) dHL-60 cells were transiently cotransfected with GFP-N-Clip170 and one of following mutant constructs: PKCζ-KD (Left), p50-dynamitin (Center) GSK3β -S9A (Right), and then exposed to uniform fMLP (100 nM). Centrosome positions 360 s later were assessed as described in the legend to Fig. 1B. Whereas both PKCζ-KD and p50-dynamitin abolish the leftward bias, GSK3β -S9A reverses it. (Scale bar, 20 μm.)

Leftward bias of differentiated HL-60 (dHL-60) cell polarity. The bias of cell polarity was assessed by observing the locations of an individual cell's centrosome at two different times: before exposure to uniform fMLP by using GFP-Arr3 (A and B) or GFP-N- Clip170 (C and D) as markers and again when polarity was complete (180 or 360 s after application of fMLP in A and B or C and D, respectively). (A) dHL-60 cells were transiently transfected with GFP-Arr3 and uniformly stimulated with fMLP (100 nM) for the times indicated. GFP-Arr3 fluorescence (green) and nuclei (blue) are shown in the Upper images, and the corresponding Nomarski images are shown in the Lower images. (B) Leftward bias of cell polarity. In the first figure (far left), the cell outline from A is shown in blue (0 s) and purple (180 s after fMLP); centrosome positions are indicated by the red and blue dots (at 0 and 180 s, respectively) and are linked with a straight blue line; the red arrow is drawn through the center of the nucleus (light green), pointing to the centrosome, at 0 s. The second figure (second from the left) shows cell outlines and centrosome positions, at 0 and 180 s, rotated so that the arrow points directly upward. The third figure (second from the right) shows outlines of 13 GFP-Arr3 expressed cells at 180 s, all corrected so that their red arrows point in the same direction. The fourth figure (far right) shows the locations of centrosomes at 180 s. Only 1 of 13 cells polarized to the right side of the arrow. (C) (Upper) dHL-60 cells were transiently transfected with GFP-N-Clip170, and uniformly stimulated with fMLP (100 nM) for the times indicated. GFP-N-Clip170 fluorescence is green; nuclei are in blue. (Lower) The corresponding Nomarski images. (D) Outlines and centrosome positions of the cell in C at 0 and 360 s after fMLP addition are shown exactly as described in B. Of 11 GFP-N-Clip170-expressing cells, only 1 turned to the right of the arrow. (Scale bars, 10 μm.)