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Propagating chemoattractant waves coordinate periodic cell movement in Dictyostelium slugs

School of Life Sciences, Division of Cell and Developmental Biology, Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, UK

View larger version (66K): [in a new window]   Fig. 1. Wave propagation in a slug of Dictyostelium discoideum. The slug shown is from a mutant AX2 strain that has been transformed with different variants of GFP expressed under the control of prestalk-specific promotors to enable cell movement analysis (see movie on CD ‘GFP in Motion’ Trends Cell Biol.). (A) Movement of cells inside the slug. GFP-labelled cells were tracked in both the prestalk and in the prespore region over 20 images (400 seconds). The tracks are superimposed on the brightfield image of the slug, the direction of cell movement is indicated by black arrows. The slug is migrating towards the left. (B) Image subtraction of the corresponding brightfield images taken at 100 second intervals reveals the wave front as dark band in the middle of the slug indicated by a white arrow. (C) The same slug 200 seconds later. The wave (white arrow) has moved backwards. Scale bar: 100 µm.

View larger version (69K): [in a new window]   Fig. 2. Correlation of wave propagation and cell movement in streamer NP377 slugs. (A) Brightfield image of a slug shown from below. To detect the optical density waves the average brightness was measured in small squares at the position of the cells. Four squares are shown (1-4); the cells are visible in the corresponding fluorescence image (B). The long rectangle over the midline of the whole slug indicates the position of the measuring window for the time-space plot (C). (B) Fluorescence image of the slug showing the GFP-labelled cells. The positions of the four cells at different time points of the sequence are indicated by white dots. (C) The time-space plot reveals the presence of several waves propagated from the tip to the back of the slug over a period of 46 minutes. The waves are visible as dark sloping bands; the white arrows point at three waves just before they seem to disappear in the rear part of the slug. To enhance the visibility of the waves, this time-space plot was created by using a sequence of subtracted images. (D) Graph showing the changes of velocity and average brightness of cell 1. The velocity is plotted in black, the intensity in grey. Owing to a drift in the brightfield signal, the intensity data were corrected by subtracting the values of a calculated linear regression line from the original data. The curves were smoothed by a running average over three data points. Broken lines mark the intensity peaks. (E) Periodic movement of cells 1, 5 and 6 from (B). (F) Cross-correlation analysis of the cell velocities. Scale bars: 100 µm.

View larger version (105K): [in a new window]   Fig. 3. Wave propagation in isolated slug tips. (A) Brightfield image of an isolated tip from a slug of strain NP377. The tip was separated from the prespore region with a fine syringe needle. (B) Subtracted image showing the same tip 16 minutes later. The black arrow points at the dark appearing wave front. (C) Time-space plot over the whole sequence of subtracted images. The white arrows mark five propagated waves. Scale bars: 100 µm.

View larger version (64K): [in a new window]   Fig. 4. Effect of tip removal on cell movement in slugs. (A) The tracks of three cells are indicated by black dots and superimposed on the brightfield image of the first time point. The arrow indicates the direction of cell movement. The pattern of dots in each track shows that cells go through phases of fast and slow movement. (B) Velocity plot of four cells, including the three cells from A. The periodic movement changes are obvious. (C) Tracks of four cells from the same slug after the tip was removed. The broken line marks the position where the tip was cut off. (D) Velocity profiles of the four cells shown in C. The coordinated behaviour of the cells is lost. Scale bar: 100 µm.

View larger version (99K): [in a new window]   Fig. 5. Microinjection in slugs. (A) Subtracted image of a slug showing two wave fronts (arrows), the position of the microelectrode containing 10-2 M cAMP is indicated. (B) The waves have disappeared after the cAMP pulse, the microelectrode had been removed after the injection. (C) Time-space plot showing the disappearance of the waves after cAMP injection. The arrow indicates the position of micro-pipette and the time of the injection. (D) Brightfield image of a slug with an inserted micro-electrode (outline) containing phosphodiesterase (5 mg/ml). (E) Subtracted image of the same slug. The arrows point at two wave fronts coming from the slug tip. (F) Time-space plot. The waves stop around the injection site. The arrow indicates where and when the micro-pipette was inserted. Scale bars: 100 µm.

View larger version (92K): [in a new window]   Fig. 6. Optical density waves in strain NP377 during different stages of development.

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