RICS analysis reveals a dorsal-ventral asymmetry in the mobility of Dl.

(A) Representative image of the Dl gradient in an nc 14 embryo. Scale bar = 25 μm. (B) Representative image of Dl-GFP used for RICS analysis. Scale bar = 1 μm. (C,D) Plots of the autocorrelation function (ACF) of the image in A. (C) 3D plot of the ACF. Blue and red open circles represent the slice of the 3D surface for the fast and slow directions, respectively. (D) Plot of the fast and slow slices of the 3D ACF. Blue and red open circles (experimental data) correspond to those found in (C). The experimental data (open circles) are fit using a diffusion model (solid curves). The data and fit are separated into two components, the “fast” (ξ, blue) and “slow” (η, orange) directions. (E-G) Plots of the diffusivity of Dl-GFP vs NCR, measured using RICS on the entire imaging frame (nuclear plus cytoplasmic) (E), nuclear regions only (F), or cytoplasmic regions only (G). (H) Boxplot of the diffusivity of Dl-GFP in mutant embryos measured using RICS on the entire imaging frame (nuclear plus cytoplasmic) (“tot”; left side), nuclear regions only (“nuc”; center, gray), or cytoplasmic regions only (“cyt”; right side). In (E-H), blue represents ventral/“ventral-like”, orange represents lateral/“lateral-like”, and yellow represents dorsal/“dorsal-like” measurements. Solid dots represent individual measurements. Black lines represent a linear regression fitted to the data. p-values for the slope of the trendline being zero are indicated on the graph. Sample sizes indicated on graph.

One- and two-component diffusion models quantify the relationship between DNA binding and mobility of Dl.

(A) Illustration of the two-component model. Dl can diffuse and reversibly bind DNA. Dl bound to DNA is immobile. In contrast to the one-component diffusion model, in which Dl is freely diffusible and does not bind to DNA. (B) Comparison of one-component (dashed gray curve) and two-component (solid orange curve) diffusion models fit to the slow direction of the experimental RICS data (orange open circles). (C,D) Plots of the fraction of immobilized Dl (φ) vs. NCR measured using RICS on nuclear regions only (C) or cytoplasmic regions only (D). (E) Boxplot of φ in mutant embryos measured using RICS on nuclear regions only (“nuc”; left side, gray) or cytoplasmic regions only (“cyt”; right side). In (C-E), blue represents ventral/“ventral-like”, orange represents lateral/“lateral-like”, and yellow represents dorsal/“dorsal-like” measurements. Solid dots represent individual measurements. Black lines represent a linear regression fitted to the data. p-values for the slope of the trendline being zero are indicated on the graph. Sample sizes indicated on graph.

Cross-correlation RICS analysis of DNA-bound Dl.

(A,B) Cross-correlation between Dl-GFP and H2Av-RFP, with nuclear mask, on the ventral side (A) or the dorsal side (B). (C) Example of autocorrelation function of H2Av-RFP, with nuclear mask. (D) Plot of ψ, the fraction of Dl bound to DNA, in wildtype embryos vs. NCR. (E) Boxplot of ψ in Toll signaling mutant embryos. In (D,E), blue represents ventral/“ventral-like”, orange represents lateral/“lateral-like”, and yellow represents dorsal/“dorsal-like” measurements. Solid dots represent individual measurements. Black lines represent a linear regression fitted to the data. p-values for the slope of the trendline being zero are indicated on the graph. (F) Boxplot of ψ, the fraction of Dl bound to DNA, in either wildtype (wt) embryos or embryos overexpressing a mutated form of Dl with reduced DNA binding (R063C). Left side of plot (gray): ventral side; right side of plot (white): dorsal side. p-values for differences in means of the distributions are indicated on the graph. In (D-F): Sample sizes indicated on graph.

pCF analysis reveals a dorsal-to-ventral asymmetry in nuclear export of Dl.

(A) Representative images of Dl-GFP on the ventral (top left image) and dorsal (top right image) sides of the embryo with their respective pCF carpets (bottom left and bottom right). Scale bar = 2um. White arrow represent the direction of the line scan. Magenta circles in dorsal side image outline the two nuclei crossed by the line scan. White dashed lines represent areas where Dl-GFP movement is measured out of the nucleus. Orange, dashed lines in the carpet label arches which indicate delayed Dl-GFP movement, while green, solid lines demarcate black regions of the carpet which indicate no Dl-GFP movement. (B) Percent of embryos that have high (Movement Index (MI) > 0.5, orange) and low (MI ≤ 0.5, blue) Dl-GFP movement either out of (out) or into (in) the nucleus. * denotes p<0.07, ** denotes p<0.05, Chi-squared test with likelihood ratio.

Variation of Dl mobility along the DV axis is due to presence of Cact in the nuclei.

(A) (top left) Depiction of the different embryo domains, ventral (blue region), lateral (orange region), and dorsal (yellow region). (bottom left) Schematic of ventral side of embryo. High levels of activated Toll (magenta receptors) result in the destruction of Cact and phosphorylation of Dl. Free Dl enters the nucleus, so GFP fluorescence is mostly nuclear. Free Dl in the nucleus binds to DNA, which lowers the overall mobility of Dl species, and exits the nucleus at a slow rate. (bottom right) Schematic of lateral domain of embryo. Lower levels of Toll activation lead to a mixture of free and Cact-bound Dl. Nuclear and cytoplasmic levels of GFP fluorescence are roughly equal. Dl has a higher mobility due to lower fraction of Dl bound to the DNA. Dl exits the nucleus faster than on the ventral side. (top right) Schematic of the dorsal domain of the embryo. Little-to-no free Dl is present, so Dl-GFP fluorescence is mostly cytoplasmic. High mobilities stem from no DNA binding. (B,C) Boxplots of ψ, the fraction of Dl bound to DNA for the UAS-dlwt-gfp (control), UAS-dlS234P-gfp (reduced Cact binding), and UAS-dlS317A-gfp (reduced Toll phosphorylation) alleles on the ventral (B) or dorsal (C) sides. Solid dots represent individual measurements. p-values for pairwise comparisons to the control are indicated on the graph. Sample sizes indicated on graph.