Fig 4. Amino acid residue changes affecting the maintenance of Step at furrow membranes.

(A) Time points from FLIP analyses of GFP-Step[WT] and GFP-Step[I319E] at early cellularization. 0s shows the embryo before the first photobleaching. In the second column, the white bar shows where the embryo was photobleached, as well as cell compartments in Row 1 and Row 2 away from the cell row being bleached. The same positions were bleached at 60s intervals (arrows), and the embryos are shown 45s after each bleaching. The red asterisks indicate example Row 2 furrows that maintain their signal for GFP-Step[WT] or become depleted of signal for GFP-Step[I319E]. The images are also shown with an inverted Fire look up table (Image J) to show that a gradient of both cytosolic and membrane signal depletion arises with the repeated GFP-Step[I319E] photobleaching, but not for GFP-Step[WT]. (B) Quantification of the responses at Row 2 furrows as averages of three sites per embryo normalized to the signals at 0s (each line is data from one embryo). (C) Quantification of the same responses at Row 2 furrows as in B, but as averages of each single site over time points 225-405s normalized to the signals at 0s (each circle is data from one furrow). For B and C, note that Step[I319E] displayed the greatest increase in mobility versus Step[WT].