Fig. 5

Spatiotemporal Control of Pard3 Activity

(A) Single z slice through an EVL cell of an 18-somite embryo, labeled with Pard3-EGFP-PIF6 and PHYB-MCherry-CAAX. The EGFP signal is shown alone and is pseudo-colored with the Fire look-up table. (i) In the unbound state under 740 nm light, Pard3-EGFP-PIF6 is distributed around the whole cell membrane. (ii) Position of ROI (white circle) before 633 nm illumination. (iii) 633 nm light was applied specifically within the ROI with a background uniform 740 nm light for 23 min. Pard3-EGFP-PIF6 specifically accumulated to the ROI (star) and was depleted from the surrounding cell membrane (e.g., arrow). (iv) Uniform 740 nm light for 9 min. Highest levels of Pard3-EGFP-PIF6 were redistributed along the cell membrane. Some intracellular membranes are also labeled with EGFP in these images.

(B) (i) Single z slice through the EVL of a 15-somite embryo labeled with Pard3-EGFP-PIF6, PHYB-CAAX, and Pard6-MCherry. The position of the ROI (white rectangle) before 633 nm illumination is shown. (ii) Grayscale images of EGFP and MCh signals showing recruitment of Pard3-EGFP-PIF6 and its partner Pard6-MCh to the ROI (white rectangle) following 633 nm illumination. Areas outside the ROI receive 750 nm light.

(C) Quantification of fluorescent intensity differences illustrated in (B) for Pard3-EGFP-PIF6 and Pard6-MCh between membranes (data shown in purple) inside and outside the 633 nm ROI, and between cytoplasm (data shown in blue) inside and outside the ROI. Following 633 nm light illumination, intensity sample areas were placed at the membrane and in the cytoplasm from regions both inside and outside the 633 nm ROI. Fluorescence intensity was normalized to mean levels outside the 633 nm ROI. One-way ANOVAs with Tukey’s multiple comparison tests were carried out (***p < 0.001, **p < 0.01, *p < 0.05). Error bars denote SEM.

(D) Time lapse of a neural progenitor cell undergoing division within a 13-somite zebrafish embryo neural keel. The cell is expressing Pard3-EGFP-PIF6 and PHYB-MCherry-CAAX, but only the Pard3-EGFP-PIF6 signal is shown and pseudo-colored with the Fire look-up table. (i) Under 740 nm illumination, Pard3-EGFP-PIF6 was distributed around the cell membrane and also expressed in the cytoplasm. (ii) Pard3-EGFP-PIF6 is recruited to the right-hand side of the cell membrane using an ROI (white rectangle) illuminated with 633 nm light for 6 min. (iii) The majority of Pard3-EGFP-PIF6 is inherited into the right-hand daughter following division. Right-hand daughter is within 633 nm ROI (white rectangle), left-hand daughter is outlined by dashed white line.

See also related Figure S3.