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. 2010 May 11;20(9):811-7.
doi: 10.1016/j.cub.2010.04.016. Epub 2010 Apr 29.

Modulation of fat:dachsous binding by the cadherin domain kinase four-jointed

Michael A Simon  1 , Aiguo Xu, Hiroyuki O Ishikawa, Kenneth D Irvine
Affiliations

Modulation of fat:dachsous binding by the cadherin domain kinase four-jointed

Michael A Simon et al. Curr Biol. .

Abstract

In addition to quantitative differences in morphogen signaling specifying cell fates, the vector and slope of morphogen gradients influence planar cell polarity (PCP) and growth. The cadherin Fat plays a central role in this process. Fat regulates PCP and growth through distinct downstream pathways, each involving the establishment of molecular polarity within cells. Fat is regulated by the cadherin Dachsous (Ds) and the protein kinase Four-jointed (Fj), which are expressed in gradients in many tissues. Previous studies have implied that Fat is regulated by the vector and slope of these expression gradients. Here, we characterize how cells interpret the Fj gradient. We demonstrate that Fj both promotes the ability of Fat to bind to its ligand Ds and inhibits the ability of Ds to bind Fat. Consequently, the juxtaposition of cells with differing Fj expression results in asymmetric Fat:Ds binding. We also show that the influence of Fj on Fat is a direct consequence of Fat phosphorylation and identify a phosphorylation site important for the stimulation of Fat:Ds binding by Fj. Our results define a molecular mechanism by which a morphogen gradient can drive the polarization of Fat activity to influence PCP and growth.

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Figures

Figure 1
Figure 1. Fj has a dual effect of Fat-Ds binding
A-D) Histograms showing the results of binding of AP-tagged Fat or Ds proteins to cells expressing Fat or Ds. The histograms show the average of two replicate binding assays with bound AP activity expressed as milli-OD/min. Bars indicate the deviation between the replicates. To correct for endogenous cellular phosphatase activity, binding activity was reduced by the activity seen using control condition S2 media. The proteins expressed by the binding cells and the cells used to produce the conditioned media are indicated. A) Binding assays using Ds:AP conditioned media and cells. B) Binding assays using Fat:AP conditioned media and cells. A) The effects of Fj mutant on Ds binding to Fat. D) The effects of Fj mutant on Ft binding to Ds. E) Concentration dependence of Ds:AP binding to cells expressing Fat or Fat and Fj:V5. The concentration of Ds:AP is indicated below. Bound AP activity is given in mill-OD/min.
Figure 2
Figure 2. Effect of Fj on Fat-Ds localization
Confocal images of pupal wing discs stained with either anti-Fat or anti-Ds as indicated. In each panel, the approximate center of the mutant cells at the edge of the clone are indicated by the white circles. The images are oriented with distal towards the right. A) Two clones of fat cells over-expressing Fj. Fat in the wild-type cells immediately adjacent to the Fj over-expressing cells fails to accumulate along the border with the Fj over-expressing cells. B) A clone of fat cells that do not overexpress Fj. In the absence of Fj over-expression, Fat is still present at the clone border. C) A clone of ds cells over-expressing Fj. Note that Ds in the adjacent wild-type cells is preferentially drawn to the border of the Fj over-expressing cells. This can be seen by the reduced Ds staining at the cell-cell boundaries between the wild-type cells that border the Fj over-expressing cells. D) A clone of ds cells that do not over-express Fj. No effect on Ds localization is seen in the adjacent wild-type cells. Similar effects on Fat and Ds localization can be observed on all sides of Fj over-expressing clones. The genotypes are A) hsFLP/+; TubGal80, FRT40A/ftG-rv, FRT40A; Tub-Gal4/UAS-Fj, B) hsFLP/+; P[Ubi-GFP]2L, FRT40A/ftG-rv, FRT40A, C) hsFLP/+; TubGal80, FRT40A/ds38k, FRT40A; Tub-Gal4/UAS-Fj and D) hsFLP/+; P[Ubi-GFP]2L, FRT40A/ds38k, FRT40A.
Figure 3
Figure 3. Molecular characterization of the influence of Fj on Fat-Ds binding
Histograms show the results (averages of from two to four replicate experiments) of binding of AP-tagged Ds proteins, with bound AP activity expressed as milli-OD/min. Error bars indicate standard deviation. A) In vitro Fat-Ds binding assays, using conditioned medium from cells expressing Ds1-10:AP (2400 mOD/min) and beads loaded with Fat1-10:FLAG from S2 cells, Fat1-10:FLAG from S2 cells co-transfected to express Fj, or conditioned medium from S2 cells transfected with empty vector, as indicated. Where indicated, beads were phosphorylated in vitro with Fj or treated with phosphatase. For simplicity of display, the “−” samples here display averages from the mock treated (no enzyme) kinase and phosphatase experiments. Inset shows the results of Western blotting (anti-FLAG) on Fat1-10:FLAG from S2 cells without or with (+Fj) Fj co-expression to show that the amounts of Fat1-10 are similar. B) In vitro Fat-Ds binding assays, using conditioned medium from cells expressing Ds1-10:AP (2400 mOD/min) and beads loaded with Fat1-10:FLAG from S2 cells with or without Fj co-transfection, or conditioned medium from S2 cells, as indicated. Where indicated by superscripts, Fat1-10:FLAG with point mutations in the Fj site in Cadherin domain 3 were used. C) Cell based binding assays, using conditioned medium from cells expressing full length DS:AP or Ds1-10:AP (1200 mOD/min), with or without Fj co-expression, as indicated. Cells were transfected to express full length Fat, Fat and Fj together, Fat and kinase-dead Fj (FjGGG), or empty vector (S2). D) In vitro Fat-Ds binding assays, using conditioned medium from cells expressing Ds1-10:AP (2400 mOD/min) with or without Fj co-expression, or as a control Fc:AP, as indicated, and beads loaded with Fat1-10:FLAG from S2 cells co-transfected to express Fj.
Figure 4
Figure 4. Schematic model for polarization of Fat activity in response to a Fj gradient
Rounded rectangles represent cells at different points in an Fj expression gradient, illustrated by different levels of shading. A) Fj acts cell autonomously to decrease the Fat-binding activity of Ds (block arrows) and increase the Ds-binding activity of Fat (pointed arrows). B) As a consequence of these different binding activities, strong signaling (large red arrows) occurs from cells with lower Fj to cells with higher Fj, and weak signaling (small red arrows) occurs from cells with higher Fj to cells with lower Fj C) The action of this mechanism at each cell boundary results in polarized Fat activity (asterisks) within each cell. D) If Fj were only to act on Fat, Fat activity would vary across the tissue, but would not be polarized within indivdual cells. E) If Fj were only to act on Ds, cells would have to be able to discriminate both between relatively high levels of Fat (right) and between relatively low levels of Fat (left) in order to polarize. See text for details.
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References

    1. Rogulja D, Irvine KD. Regulation of cell proliferation by a morphogen gradient. Cell. 2005;123:449–461. - PubMed
    1. Simon MA. Planar cell polarity in the Drosophila eye is directed by graded Four-jointed and Dachsous expression. Development. 2004;131:6175–6184. - PubMed
    1. Matakatsu H, Blair SS. Interactions between Fat and Dachsous and the regulation of planar cell polarity in the Drosophila wing. Development. 2004;131:3785–3794. - PubMed
    1. Ma D, Yang CH, McNeill H, Simon MA, Axelrod JD. Fidelity in planar cell polarity signalling. Nature. 2003;421:543–547. - PubMed
    1. Yang C, Axelrod JD, Simon MA. Regulation of Frizzled by Fat-like Cadherins during Planar Polarity Signaling in the Drosophila Compound Eye. Cell. 2002;108:675–688. - PubMed

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