Figure 2—figure supplement 2

Phosphoinositide 3-kinase (PI3K) activity in myocardial cells.

(A–B) Three-dimensional confocal reconstructions of the myocardium at 20s in DMSO- (A) and LY- (B) treated Tg(myl7:lck-egfp) embryos in which a myl7:PH-mkate2 plasmid was injected to mosaically visualize PI3K activity. PH-mkate2 translocates to the membrane when PI3K activity produces PIP3. In DMSO-treated embryos PH-mkate2 was often found at the membrane enriched asymmetrically (A'–A''') indicating PI3K activity in the myocardium, while in LY-treated embryos PH-mkate2 was defuse in the cytoplasm of myocardial cells (B''' arrowhead) or enriched in subcellular organelles (B''' arrow). A'–A''', B'–B''' are magnifications of A, B, respectively, showing both lck-emgfp and PH-mkate2 (A', B'), PH-mkate2 only (A'', B''), or PH-mkate2 intensities as a heat-map (A''', B'''). (C–E) Graphs depict the average fluorescent intensity for lck-emGFP (green) and PH-mkate2 (red) at different points across the length of a labeled cell, starting with the side of highest mkate2 fluorescence, in DMSO- (C) and LY- (D) treated embryos. The fluorescent intensity of mkate2 normalized to emGFP fluorescence at each point along the line (E) reveals an asymmetrical enrichment of PH-mkate2 at the membrane (0–20%) in DMSO-treated embryos, compared to LY-treated embryos in which PH-mkate2 is enrich in the cytoplasm, the middle of the cell (20–70%). 17 and 13 cells from four DMSO- and four LY-treated embryos were analyzed. Scale bars, 30 μm. Raw data and full p-values included in the source file.