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First published online July 2, 2007
doi: 10.1242/10.1242/jcs.007732

Journal of Cell Science 120, 2318-2327 (2007)
Published by The Company of Biologists 2007
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Possible roles of the endocytic cycle in cell motility

David Traynor and Robert R. Kay*

MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK


Figure 1
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  		Fig. 1. Endocytosis of membrane and fluid. Ax2 cells, grown in axenic medium, were washed free of growth medium and used immediately (vegetative cells) or starved for approximately 5 hours with periodic cAMP pulses after the first hour (aggregation competent cells). (A) Aggregation-competent cells stained with FM1-43: after a few seconds labelling is restricted to the cell surface, but by 100 seconds, there is additional punctate labelling within the cell, presumably as a result of endocytosed membrane (confocal sections). (B) Membrane endocytosis by vegetative ({square}) and aggregation-competent ({circ}) cells determined using FM1-43, with 10 mM azide added as indicted (Figure 1, Figure 1). Results are given as the fraction of the cell surface taken up by normalizing to the first measurement after mixing dye and cells. Inset shows a continuous trace made by adding FM1-43 directly to the cuvette illustrating the nearly instantaneous increase in fluorescence. (C) Fluid-phase endocytosis by vegetative ({square}) and aggregation-competent cells ({circ}) determined using FITC dextran. Values are mean ± s.d. of three experiments.

 

Figure 2
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  		Fig. 2. Chemotaxis of temperature-sensitive NSF cells. Control Ax2 and temperature sensitive NSF cells (strain HM1067, nsfA2) were made aggregation-competent at 20°C, then shifted to the restrictive temperature of 28°C for at least 20 minutes, before being filmed moving towards a micropipette containing cAMP. The left-hand panels show stills from the films (Movies 1 and 2 in supplementary material) taken after 20-30 minutes. The insets show a typical elongated chemotaxing Ax2 cell and a rare nsfA2 cell that is chemotaxing efficiently but maintains a smooth profile and is only moderately elongated. The movement speeds of 68 (Ax2) or 80 (nsfA2) randomly chosen cells from each condition are tabulated in the right hand panels. Bar, 20 µm.

 

Figure 3
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  		Fig. 3. Temperature-sensitive NSF cells can be polarized by cyclic-AMP gradients. Each panel shows temperature sensitive NSF cells (HM1067) expressing the appropriate GFP fusion proteins to act as reporters for cell polarization, were made aggregation-competent and stimulated with cAMP from a micropipette, at either the permissive temperature of 20°C (shown only for ABD-GFP) or the restrictive temperature of 28°C. Reporters: CRAC, reports PtdIns(3,4,5)P3 in the membrane; PTEN, normally bound to the membrane but repelled from the leading edge; ABD, binds to F-actin, thus reporting sites of actin polymerization. Wild-type cells behaved in a similar way at both temperatures, and did not round up at 28°C. When the pipette was moved to a new position (white circle) the image shown is the first taken at the new location; time in seconds after introducing the pipette is shown in each image; bar, 10 µm. See Movies 4-6 in supplementary material.

 

Figure 4
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  		Fig. 4. Tracks of spots photobleached in the plasma membrane of moving cells. Spots were photobleached on the underside of cells expressing cAR1-GFP, and tracked until they faded. They were made either in the area where a pseudopod appeared (left and middle panels), or at the posterior of the cell (right panel `posterior'). The positions of the cells at various times is shown in the diagrams below. Graphs quantify displacement of the spot centre from the starting position in parallel with the leading edge (pseudopods) or the uropod (posterior) are shown at the bottom. Ax2 cells transformed with cAR1-GFP were made aggregation-competent and induced to migrate under a thin layer of agarose to constrain movement in the z-axis. Photobleaching was for 0.16, 0.79 or 1.32 seconds, giving spots of initial diameter 2.5 µm (pseudopods) or 2.0 µm (uropod); the frame rate was 1 frame per 0.8, 0.6 or 0.5 seconds; times are given in seconds; bar, 10 µm. See Movie 8 in supplementary material.

 

Figure 5
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  		Fig. 5. Track of a spot photoactivated in the plasma membrane of a moving cell. A spot was photoactivated on the underside of a cell expressing the cAMP receptor coupled to photoactivatable GFP and followed as a pseudopod extended. Ax2 cells transformed with plasmid pDT18 were made aggregation-competent and induced to migrate under a thin layer of agarose to constrain movement in the z-axis. Cell outlines were traced from the accompanying DIC image. Photoactivation was for 0.77 seconds and frames were taken every 0.59 seconds; times are given in seconds in the images; scale bar is 10 µm. To aid visualization, the contrast/brightness of these images was altered in ImageJ by conversion to 8-bit then changing the min/max pixels from 0-255 to 11-137. See Movie 9 in supplementary material.

 

Figure 6
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  		Fig. 6. Surface reconstruction of a cell moving under agarose. Ax2 cells expressing cAR1-GFP were induced to chemotax under 0.6% agarose containing cAMP. Stacks of confocal sections were taken at the indicated times and reconstructed using the Volocity package. (A) A single confocal section through a cell at different times; (B) a maximum density projection of the z-section stack; (C) the 3D reconstruction viewed from 30° to the substratum; (D) the 3D reconstruction viewed from -30° to the substratum. The relative surface area and volume (A/V) of the cell are given in C. See Movie 11 in supplementary material.

 

Figure 7
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  		Fig. 7. Surface area and volume of a cell moving under agarose. Ax2 cells expressing cAR1-GFP were induced to chemotax under 0.6% agarose towards cAMP. Bursts of three sets of confocal stacks (12 sections taking three seconds) were taken at approximately 25 seconds intervals and the areas and volumes obtained from each burst treated as repeat measurements at the same time point, thus allowing statistical analysis (1-way ANOVA, with Tukey's test). (A) Relative surface area and volume of the cell body (without filopodia); (B) number and total length of filopodia, measured manually; (C) relative surface area of cell body and filopodia (assumed to be 0.1 µm in diameter); plotted to the same scale, but note the different origins. The increase in surface area at 0-69 seconds and the subsequent decline at 69-174 seconds are both significant at P<0.001, whereas none of the volume changes are (P>0.05). The cell is the same one as shown in Fig. 6 with an initial surface area of the body of 698 µm2 and of the filopodia of 3.5 µm2 (0.5 % of the total surface area); and a volume of 586 µm3.

 

Figure 8
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  		Fig. 8. Surface area and volume of cells moving under buffer. Ax2 cells expressing soluble GFP were brought to aggregation-competence and filmed by taking confocal stacks every 9-13 seconds; the reconstructed cells are viewed at a 30° angle to the substratum. The surface area and volume of the cell are given above each image (A, V, respectively), normalized to the values at the start of the sequence.(A) A randomly moving cell (at 0 seconds the surface area=567 µm2; volume=547 µm3); (B) chemotaxing cell moving to the right towards a micropipette containing cAMP (506 µm2; 562 µm3); (C) a quiescent cell, stimulated with a micropipette containing cAMP (to the right) at the start of the sequence (397 µm2; 361 µm3); (D) a quiescent cell, not stimulated (211 µm2; 191 µm3). Bars, 15 µm. Selected time points are shown. See Movies 12-14 in supplementary material.

 

Figure 9
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  		Fig. 9. Quantification of cell surface area changes during movement under buffer. Aggregation-competent cells, expressing soluble GFP, were filmed by taking confocal stacks at 15-30 second intervals. Changes in relative surface area of representative cells (A) moving randomly; (B) chemotaxing steadily towards a micropipette containing 1-2 µM cAMP; (C) quiescent, but stimulated to move at the start of the sequence with a micropipette containing cAMP; (D) quiescent, but unstimulated.

 

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© The Company of Biologists Ltd 2007