Adapting the Phytochrome System for Use in a Vertebrate Embryo

(A) 50-µm z projections through the hindbrains of 14-somite zebrafish embryos labeled with PHYB-MCherry-CAAX fusion proteins with different PHYB truncations. Dotted lines denote basal edges. N-PAS2-GAF-PHY-PAS contains a PAS domain at the C terminus and was not successfully expressed in six of six embryos. N-PAS2-GAF-PHY does not contain a PAS domain at the C terminus and was robustly expressed in cell membranes in nine of nine embryos.

(B) (i) Normal PHYB covalently binds PCB chromophore. Energy from far-red (FR) light causes photoisomerization of PCB and the allosteric transition of PHYB from its inactive (Pr) to its active (Pfr) state. This is reversible by infrared (IR) light exposure (not shown). The Pfr state can bind PIF. (ii) Conjugation of PCB with Y276H mutant PHYB creates an activated holoprotein that can directly bind PIF, without the need for far-red light illumination (Su and Lagarias, 2007). Energy from far-red illumination causes infrared fluorescence.

(C) An oblique confocal slice through the overlying EVL and the underlying neuroepithelium of a 14 hpf embryo, labeled with Y276H PHYB and H2A-GFP and bathed in PCB. Only the EVL fluoresces under far-red light (magenta), demonstrating binding of PCB to Y276H PHYB.

(D) A horizontal confocal slice through the neuroepithelium of an 18 hpf embryo, labeled with Y276H PHYB-CAAX and PIF6-EGFP and injected with PCB. Anterior is up. The membranes of all labeled cells fluoresced under far-red light, demonstrating binding of PCB to Y276H PHYB-CAAX. PIF6-EGFP was also recruited to the membrane in these cells. PIF6-EGFP recruitment to the membrane was not reversed after 5 min exposure to 750 nm light.

See also related Figure S1

Light-Controlled Shuttling of Protein between Cytoplasm and Membrane In Vivo

(A) Sequential images of a horizontal confocal slice through the developing neuroepithelium of a 15-somite embryo labeled with PHYB-MCherry-CAAX and PIF6-EGFP. The embryo was illuminated with alternating 5-min exposures to 650- and 750 nm light. PIF6-EGFP was recruited to the membrane after 650 nm illumination and released from the membrane into the cytoplasm after 750 nm illumination.

(B) (i) Relative PIF6-EGFP intensity after alternating 650- and 750 nm illumination, normalized to mean 750 nm levels. Error bars denote SEM. (ii) Illustration of PIF6-EGFP intensity sampling areas in membrane and cytoplasm. (iii) Raw image of (ii).

See also related Figure S2.

Rapid Kinetics of PHYB-PIF6 Binding and Unbinding

(A) Time sequence of a sagittal confocal slice through NE cells of a 15-somite embryo labeled with PHYB-MCherry-CAAX and PIF6-EGFP. Only the PIF6-EGFP signal is shown. At 0 s, 740 nm illumination was replaced with BF illumination and PIF6-EGFP was rapidly recruited to the membrane as illustrated by 8- and 36-s time points.

(B and C) Quantification of PIF6-EGFP intensity over time following BF (B) and 740 nm (C) illumination. EGFP intensity was normalized to mean levels at t = 0 s. One-phase exponentials (shown in blue) were fitted to EGFP levels, generating time constants (τ) for binding (B) and unbinding (C) of PIF6 to PHYB. τ = time taken for EGFP levels to either decrease by a factor of 1/e (approximately 36.8% of the original amount) or to increase by a factor of 1  1/e (approximately 63.2% of the asymptotic value). Error bars denote SEM. Black lines denote 95% confidence bands. (B) (i) Quantification was from the cells shown in (A). (ii) Illustration of PIF6-EGFP sampling areas. (C) Quantification of unbinding from NE cells in a 22-somite embryo.

Subcellular Control of Protein Localization

(A–J) Sequences of single confocal slices through two EVL cells of a 14-somite embryo (A–E) and a collection of NE cells (F–J). Cells were labeled with PIF6-EGFP and PHYB-MCherry-CAAX. Dotted line denotes developing midline of the neural rod. Only the PIF6-EGFP signal is shown and is pseudo-colored with the Fire look-up table. (A and F) PIF6-EGFP distribution in uniform BF light. (B and G) PIF6-EGFP distribution in uniform 740 nm light for 2 min (EVL) or 4 min (NE). (C and H) Position of ROIs (white rectangle and circles) before 633 nm illumination. (D and I) PIF6-EGFP distribution after 633 nm light was specifically delivered within the ROIs for 30 s (EVL) and 15 min (NE). A uniform background illumination of 740 nm light was also present. PIF6-EGFP was specifically recruited to regions of 633 nm light in three of three EVL cells and eight of eight NE cells. (E and J) PIF6-EGFP distribution following uniform 633 nm light for 1 min (EVL) or BF light for a few seconds (NE).

(K) (i) Quantification of PIF6-EGFP intensity inside (IN) and outside (OUT) the 633 nm ROI from the image depicted in (D). EGFP intensity was normalized to mean levels outside the 633 nm ROI. A one-way ANOVA with Tukey’s multiple comparison test was carried out (***p < 0.001, **p < 0.01). Error bars denote SEM. EGFP intensity at the membrane (memb) inside the 633 nm ROI was significantly higher than all other regions of the cell. EGFP intensities in other regions of the cell were not significantly different to one another. (ii) Illustration of the sample areas of membrane (shown in purple) and cytoplasm (shown in blue) assessed for EGFP intensity inside and outside the 633 nm ROI (white rectangle). (iii) Raw image of (ii).

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.

Related to Figure 5: Pard3 localisation and BFP-ERK2 negative control.

A. Cartoon illustrating the localisation of Pard3 (green) during the maturation of neuroepithelial cells within the developing neural tube. Red dotted box shows the region imaged in figure 5D. At keel stages Pard3 broadly localises to the tissue centre before division. Midline crossing divisions (C-divisions) occur at the centre of the tissue and localise Pard3 more specifically to their cleavage planes, helping to produce a precise localisation of Pard3 at the tissue midline at rod stages. The neural tube lumen then cavitates from this midline, resulting in the opening of the neural tube. Later, NE cells undergo further rounds of division at the Pard3-labelled apical surface, resulting in the production of both NE cells and neurons.

B. En face images of the neuroepithelial apical surface labelled with anti-Pard3 antibody or Pard3-EGFP-PIF6.

C. En face images of EVL cells labelled with anti-Pard3 antibody or Pard3-EGFP-PIF6. Pard3 is present at membranes around the perimeter of cells. A small amount of fusion protein is also present in intracellular membrane inclusions.

D. Single confocal slice through the EVL of an embryo labelled with Pard3- EGFP-PIF6, PHYB-CAAX and BFP-ERK2. Signal for Pard3-EGFP-PIF6, PHYB-CAAX and BFP-ERK2 are shown in (i) and (ii) respectively. Pard3-EGFP-PIF6 is robustly recruited to the 633nm ROI, but there is no concomitant recruitment of BFP-ERK2 to this region.

E. Normalised fluorescence intensities for Pard3-EGFP-PIF6 and BFP-ERK2 from experiment shown in (D). Oneway ANOVAs with Tukey’s multiple comparison tests were carried out. Error bars denote standard error of the mean.The experiment was repeated 5 times and a significant increase in membrane intensity within the 633nm ROI when compared with membrane regions in the same cells outside the ROI was seen in 5/5 cases for EGFP, but 0/5 cases for BFP. In 2/5 cases there was also a significant increase in EGFP intensity within the cytoplasm in the 633nm ROI, but no concomitant increase in cytoplasmic BFP-ERK2.