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Inducible nuclear translocation of a STAT protein in Dictyostelium prespore cells: implications for morphogenesis and cell-type regulation

Dirk Dormann, Tomoaki Abe, Cornelis J. Weijer and Jeffrey Williams

School of Life Sciences, University of Dundee, Wellcome Trust Biocentre, Dow Street, Dundee DD1 5EH, UK

Fig. 2A

Induction of Dd-STATa nuclear translocation in the slug rear by injection of cAMP

The A15-GFP:STATa transformed slug was viewed from below through the agar. The micro-pipette containing 10^-2M cAMP was inserted from a dorsal position into the prespore region of the slug. The bright-field insert shows the position of the pipette. cAMP was injected at the indicated time point ('pulse !'), leading to rapid nuclear translocation within minutes.

Fig. 3

Induction of Dd-STATa nuclear translocation by injection of cAMP in slugs expressing GFP:STATa under the control of the pspA promoter

The slug is moving from left to right, the prestalk/prespore boundary is visible at the right-hand side of the fluorescence image since only prespore cells express the GFP fusion protein. The insert shows the corresponding bright-field images and the position of the micro-pipette. At the indicated time point a single cAMP pulse was applied. After 1 minute the first signs of nuclear localization are visible, the brightness of the nuclei increases further and peaks after about 6 minutes. The micro-pipette was removed 4.5 minutes after the pulse.

Fig. 4

Quantitative analysis of Dd-STATa nuclear translocation in prespore cells

A slug with cells expressing pspA-GFP:STATa was transferred to a water agar plate containing 5mM caffeine. Recording started 7 minutes after the transfer. cAMP (10^-2M cAMP) was injected once as indicated. Following cAMP stimulation GFP:STATa translocates rapidly to the nuclei of the two cells shown, depleting the cytoplasm of the fusion protein. The graph shows the change of brightness of the nuclei over time.

Fig. 5

Effect of the cAR1 inhibitor IPA on the cAMP induced accumulation of GFP:STATa

GFP:STATa was expressed under the prespore specific pspA promotor. The slugs were pulsed with the different compounds at the beginning of the sequence. Injection of 5x10^-3M cAMP leads to a dramatic increase of nuclear localization in large parts of the prespore region (upper sequence). Co-injection of the same amount of cAMP with a fivefold excess (over cAMP) of the cAR1 inhibitor IPA reduces the number of cells showing a positive response and nuclear localization remains restricted to the cells directly surrounding the injection site (middle sequence). Injection of the inhibitor IPA (25mM ) alone shows a very localized nuclear translocation similar to the effects induced by mechanical disturbance (lower sequence).

Fig. 6A

Accumulation of nuclear GFP:STATa protein in response to mechanical stimulation

Accumulation of GFP:STATa in nuclei of cells surrounding an empty micro-pipette. The microelectrode was inserted into the slug as shown in the bright-field sequence.

Fig. 6C

Accumulation of nuclear GFP:STATa protein in response to mechanical stimulation

Accumulation of nuclear A15-GFP:STATa protein in slugs injected with a bubble of nitrogen. N₂ gas was directly injected into the slug as indicated in the bright-field sequence. Within minutes the nuclear translocation of GFP:STATa is clearly visible in cells surrounding the nitrogen bubble.

Fig. 7

Accumulation of nuclear GFP:STATa protein in bissected slugs

A cut was made through the prespore region of the slug with the tip of an empty glass pipette using a micromanipulator. After 2 minutes the first nuclei become discernible around the cutting site, translocation increases further and peaks at about 5 minutes. The nuclear translocation is localised to the cells surrounding the cutting site.

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