Letter | Published:

Endocytosis-mediated downregulation of LIN-12/Notch upon Ras activation in Caenorhabditis elegans

Abstract

The coordination of signals from different pathways is important for cell fate specification during animal development. Here, we define a novel mode of crosstalk between the epidermal growth factor receptor/Ras/mitogen-activated protein kinase cascade and the LIN-12/Notch pathway during Caenorhabditis elegans vulval development. Six vulval precursor cells (VPCs) are initially equivalent but adopt different fates as a result of an inductive signal mediated by the Ras pathway and a lateral signal mediated by the LIN-12/Notch pathway1. One consequence of activating Ras is a reduction of LIN-12 protein in P6.p (ref. 2), the VPC believed to be the source of the lateral signal. Here we identify a ‘downregulation targeting signal’ (DTS) in the LIN-12 intracellular domain, which encompasses a di-leucine-containing endocytic sorting motif. The DTS seems to be required for internalization of LIN-12, and on Ras activation it might mediate altered endocytic routing of LIN-12, leading to downregulation. We also show that if LIN-12 is stabilized in P6.p, lateral signalling is compromised, indicating that LIN-12 downregulation is important in the appropriate specification of cell fates in vivo.

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Acknowledgements

We thank M. Stern for the egl-17p::LacZ plasmid and helpful advice on making the modified egl-17 promoter; R. Waterston for anti-MH27 antibody; M. Han for the sur-2(ku9) strain; H. Fares for reagents and much helpful advice; K. Simpson for help with backcrossing integrated arrays; B. Grant for anti-sera against endocytic markers; D. Levitan for advice on immunostaining; R. Ruiz for technical assistance; and B. Grant, O. Hobert, S. Jarriault, X. Karp, G. Struhl and A. Tomlinson for discussion and critical reading of the manuscript. D.S. was supported by a National Science Foundation predoctoral fellowship and an NIH National Institute on Aging training grant, coordinated by R. Liem. I.G. is an Investigator of the Howard Hughes Medical Institute.

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Correspondence to Iva Greenwald.

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The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Information 1: The downregulation targeting signal (DTS) is conserved in other nematode LIN-12/Notch proteins (DOC 77 kb)

Supplementary Information 2: LIN-12(γDTS)::GFP behaves like LIN-12(+)::GFP in cell fate decisions other than VPC specification. (DOC 55 kb)

Supplementary Information 3: Some LIN-12(+)::GFP containing vesicles are early endosomes (DOC 388 kb)

Supplementary Information 4: VPC fate specification is altered in lin-12g::LIN-12(γDTS)::GFP hermaphrodites (DOC 61 kb)

Supplementary Information 5: Genotypes of transgenic animals in figure 2: (DOC 48 kb)

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Further reading

Figure 1: VPC fate specification and cell fate markers.
Figure 2: Strategy for defining the LIN-12 downregulation targeting signal (DTS).
Figure 3: Identification of the LIN-12 downregulation targeting signal (DTS).
Figure 4: Failure to downregulate LIN-12, either by DTS deletion or mutations in sur-2, inhibits lateral signalling.
Figure 5: Model for the mechanism and role of endocytosis-mediated downregulation of LIN-12 in response to Ras activation in P6.p.

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