Figure 3.
Figure 3. Two functions of sGC: as a protein and as an enzyme producing cGMP. The strains used were gc null cells, gc null cells expressing full-length sGC, gc null cells expressing sGCΔN with a deletion of the N-terminal 877 amino acids, and gc null cells expressing sGCΔCat with a point mutation rendering the protein catalytic inactive. (A) Localization of sCGΔCat-GFP and sGCΔN-GFP in a cAMP gradient in the absence or presence of LY + BPB. (B) Colocalization of sCGΔCat-GFP and LimEΔcoil-RFP in a cAMP gradient in the presence of LY + BPB (same cell as in A). Bar, 10 μm. (C) cGMP response. (left) The kinetics of the response of wild-type cells at 5 (circle) and 7 h (triangle) of development. (right) The cGMP response at 12 s after stimulation of the indicated strains with 1 μM cAMP. The data shown are the means and SD of three determinations. (D) Formation of pseudopodia in cAMP gradients. Cells were placed in a gradient delivered by a modified Zigmond chamber with 1 μM cAMP at the source. Videos were recorded at a rate of one frame per second and analyzed for where and how pseudopodia are extended and retracted. We recorded the formation of pseudopodia in the front (f) or back (b) half of the cell, their formation by splitting of an existing pseudopod (s) or de novo from an area devoid of pseudopod activity (n), and whether pseudopodia are retracted (r) or maintained (m). From the eight categories possible, we never observed splitting in the back of the cell, leaving the six categories as presented. The rate of pseudopod formation per min is 2.04 ± 0.31 for sGC, 2.13 ± 0.23 for sGCΔN, 2.46 ± 0.38 for sGCΔCat, and 2.45 ± 0.30 for gc null cells. The data shown are the means and SE for 36 pseudopodia from three independent experiments. (E) Chemotaxis to 1 μM cAMP was measured with the small population assay. The difference between gc null cells and gc null cells expressing mutant sGC is significant at P < 0.001 (***), at P < 0.01 (**), or is not significant at P > 0.5 (ns). The data presented are the means and SEM of at least three independent measurements.

Two functions of sGC: as a protein and as an enzyme producing cGMP. The strains used were gc null cells, gc null cells expressing full-length sGC, gc null cells expressing sGCΔN with a deletion of the N-terminal 877 amino acids, and gc null cells expressing sGCΔCat with a point mutation rendering the protein catalytic inactive. (A) Localization of sCGΔCat-GFP and sGCΔN-GFP in a cAMP gradient in the absence or presence of LY + BPB. (B) Colocalization of sCGΔCat-GFP and LimEΔcoil-RFP in a cAMP gradient in the presence of LY + BPB (same cell as in A). Bar, 10 μm. (C) cGMP response. (left) The kinetics of the response of wild-type cells at 5 (circle) and 7 h (triangle) of development. (right) The cGMP response at 12 s after stimulation of the indicated strains with 1 μM cAMP. The data shown are the means and SD of three determinations. (D) Formation of pseudopodia in cAMP gradients. Cells were placed in a gradient delivered by a modified Zigmond chamber with 1 μM cAMP at the source. Videos were recorded at a rate of one frame per second and analyzed for where and how pseudopodia are extended and retracted. We recorded the formation of pseudopodia in the front (f) or back (b) half of the cell, their formation by splitting of an existing pseudopod (s) or de novo from an area devoid of pseudopod activity (n), and whether pseudopodia are retracted (r) or maintained (m). From the eight categories possible, we never observed splitting in the back of the cell, leaving the six categories as presented. The rate of pseudopod formation per min is 2.04 ± 0.31 for sGC, 2.13 ± 0.23 for sGCΔN, 2.46 ± 0.38 for sGCΔCat, and 2.45 ± 0.30 for gc null cells. The data shown are the means and SE for 36 pseudopodia from three independent experiments. (E) Chemotaxis to 1 μM cAMP was measured with the small population assay. The difference between gc null cells and gc null cells expressing mutant sGC is significant at P < 0.001 (***), at P < 0.01 (**), or is not significant at P > 0.5 (ns). The data presented are the means and SEM of at least three independent measurements.

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