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First published online 19 October 2005
doi: 10.1242/dev.02076

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LIN-12/Notch trafficking and regulation of DSL ligand activity during vulval induction in Caenorhabditis elegans

Daniel D. Shaye^1,2,* and Iva Greenwald^1,3,

¹ Howard Hughes Medical Institute, Columbia University, New York, NY 10032, USA
² Department of Genetics and Development, Columbia University, New York, NY 10032, USA
³ Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA

View larger version (49K): [in a new window]   Fig. 1. Relevant background information. (A) In the third larval stage, an inductive signal (black arrow) from the gonadal anchor cell (AC) and a lateral signal (gray arrows) from P6.p impose a 3°-3°-2°-1°-2°-3° pattern of cell fates on six equivalent VPCs, P3.p-P8.p. Cell fates can be distinguished by appropriate markers (see text and legends of Figs 5, 6). (B) Full-length LIN-12, with hallmark regions of EGF-like (EGF), LIN-12/Notch repeat (LNR), cdc10/ankyrin (ANK) repeat and PEST sequence (P). The position of the green fluorescent protein (GFP) tag is also shown. `Region 1' (r1) was shown to be sufficient to promote downregulation of membrane tethered GFP in P6.p (Shaye and Greenwald, 2002). (C) Canonical endocytic downregulation. Sub-apical adherens junctions (AJ), marked by the protein AJM-1 (Koppen et al., 2001), separate the apical surface from the basolateral surface. A receptor (white bar: extracellular domain; hatched bar: intracellular domain) marked for downregulation is internalized and trafficked to early endosomes (EE). From EEs, receptors that are tagged for downregulation are trafficked to late endosomes (LE)/multi-vesicular endosomes (MVE). An MVE-sorting step, by which the receptor is sorted into invaginating lumenal vesicles, removes the intracellular domain from the cytosol. MVE lumenal vesicles are delivered to the lysosome. (D) The first 45 amino acids of region 1 from LIN-12 and GLP-1 in C. elegans (Ce), C. briggsae (Cb) and C. remanei (Cr) (Rudel and Kimble, 2001; Rudel and Kimble, 2002) have regions of conservation. The DTS is boxed, and conserved residues analyzed in this study are marked by asterisks. The conserved CSL-binding region (Kovall and Hendrickson, 2004) is also indicated. Alignments were obtained with the ClustalW feature of MacVector (Accelrys).

View larger version (69K): [in a new window]   Fig. 2. Cis-determinants required for LIN-12 downregulation. GFP (green), and AJM-1 (red) were visualized by immunofluorescence (see Materials and methods). Left panels show lateral views, with the apical surface toward the bottom. Right panels show ventral views, with the apical surface surrounded by AJM-1 staining. Throughout this study, the effect on downregulation was assessed in the daughters of the VPCs – the Pn.px stage – to ensure that P6.p has been induced and that there has been sufficient time for downregulation. (A,B) Schematic representation of VPCs at the Pn.px stage; green indicates LIN-12 accumulation in the apical domain, dashed lines the basolateral domain. Note that LIN-12 remains apical wherever it is detected in VPCs and their descendants. (C,D) LIN-12(+)::GFP is downregulated in the 1° lineage (P6.px), and can be seen at the apical membrane and in endocytic puncta (arrowheads) in the 2° lineage (P5.px and P7.px). (E,F) The DTS di-leucine is required for internalization and degradation. (G,H) The DTS serine/threonine residues are required for internalization and degradation. We note that these residues may regulate other aspects of LIN-12 trafficking or localization, as we sometimes see some basolateral accumulation of LIN-12(S/TtoA)::GFP (arrowheads in G). (I,J) LIN-12(KtoA)::GFP is internalized but not degraded in the 1° lineage; it accumulates in large and pleiomorphic endocytic puncta (arrowheads), suggestive of late endosomes (see also Fig. 4C). (K) Quantification of downregulation of different LIN-12 proteins. Scale bar: 10 µm.

View larger version (63K): [in a new window]   Fig. 3. The phosphomimicking mutant LIN-12(S/TtoD)::GFP. (A,B) LIN-12(S/TtoD)::GFP is downregulated normally in the 1° lineage. Enhanced internalization and increased accumulation in endocytic puncta (white arrowheads) and the basolateral plasma membrane (yellow arrowheads) is seen in the 2° lineage. (C,D) Loss of sur-2 activity prevents LIN-12(S/TtoD)::GFP downregulation in the 1° lineage and causes accumulation in the basolateral plasma membrane (yellow arrowheads). (E) Quantification of LIN-12(S/TtoD)::GFP downregulation. Scale bars: 10 µm.

View larger version (23K): [in a new window]   Fig. 4. Trans-acting factors required for LIN-12 downregulation. (A) ß-Galactosidase filter lift assay showing that ALX-1 interacts with LIN-12(region 1) in the yeast two-hybrid system. Four independent transformants carrying the activation domain (AD) and DNA-binding domain (DBD) fusions indicated are shown. (B) Domain structure of Ce ALX-1, Saccharomyces cerevisiae (Sc) Bro1p and H. sapiens (Hs) Alix, showing the N-terminal BRO1 domain, which mediates localization to late endosomes (Kim et al., 2005), a highly conserved Src kinase phosphorylation consensus site, one or two coiled-coils and a C-terminal proline-rich domain (PRD). (C) A GFP::ALX-1 translational reporter is highly and widely expressed (data not shown), and is seen in all the VPCs. This is a lateral view, with AJM-1 (red) detected by immunofluorescence, while native fluorescence of fixed GFP::ALX-1 is visualized without the need for anti-GFP staining. Note that GFP::ALX-1 appears to accumulate in vesicular structures (arrowheads) that are probably LE/MVEs. (D) Domain structure of Ce WWP-1, D. melanogaster (Dm) Su(dx) and Mus musculus (Mm) Itch, showing the C2 phospholipid-binding motif, WW motifs, and the ubiquitin-conjugating HECT domain (reviewed by Ingham et al., 2004). (E) Phylogenetic relationship among Nedd4-family members from C. elegans, Drosophila and mouse. Neighbor-joining analysis of the full-length proteins was done with MacVector (Accelrys), using an uncorrected `p' setting for distance calculation and 1000 bootstrap repetitions to calculate the percent branch support, shown above each branch. CeD2085.4 (HECT domain only) and CeF45H7.6 (WW repeats and HECT domain) were used to anchor the tree. Scale bar: 10 µm.

View larger version (79K): [in a new window]   Fig. 5. wwp-1 and alx-1 are required for LIN-12 degradation. All pictures are ventral views. In panels A-F, experiments were carried out with hermaphrodites that express LIN-12(+)::GFP. (A) The negative control lacZ(RNAi) does not affect LIN-12 downregulation in the 1° lineage. (B) lacZ(RNAi) does not prevent lateral signaling, as evidenced by the presence of AJM-1-positive granddaughters of P5.p and P7.p. (C) alx-1(RNAi) inhibits degradation, but not internalization (arrowheads). (D) alx-1(RNAi) does not prevent lateral signaling. (E) wwp-1(RNAi) inhibits degradation but not internalization, as LIN-12(+)::GFP accumulates in endocytic puncta (arrowheads). (F) wwp-1(RNAi) does not prevent lateral signaling. (G,H) For comparison, LIN-12(DTS)::GFP is not downregulated, and inhibits lateral signaling. (I) Quantification of downregulation and lateral signaling in RNAi experiments. Each set was performed in parallel three different times. Each time, 30 hermaphrodites at the Pn.px and Pn.pxx stages were scored for presence of GFP (gray bars) and 2° fate defects (black bars). Scale bars: 10 µm.

View larger version (88K): [in a new window]   Fig. 6. Mechanism of lateral signaling inhibition by persistent LIN-12 in the 1° lineage. All pictures are ventral views. In left panels, GFP (green), AJM-1(blue) and nuclear ß-galactosidase expressed from egl-17p::lacZ (red) are visualized in Pn.px stage hermaphrodites. In right panels, AJM-1 (teal) and egl-17p::LacZ (red) are visualized in Pn.pxx stage hermaphrodites. (A) LIN-12(EDTS)::GFP is an activated form of LIN-12 that is not downregulated. Most of the GFP is nuclear localized. A' shows magnification of GFP staining, and A" shows overlapping nuclear ß-galactosidase and GFP. (B) LIN-12(EDTS)::GFP does not affect VPC fates. The 1° fate is assessed by the presence of P6.pxx cells marked with egl-17p::LacZ and AJM-1. The 2° fate is scored as described in Fig. 5B. (C) LIN-12(extra)::TM::GFP is not downregulated, and accumulates at the apical plasma membrane. C' shows magnification of GFP staining, while C" shows that most of the GFP is enclosed inside the AJM-1 boundary (pseudocolored red). (D) LIN-12(extra)::TM::GFP does not affect the 1° fate, but inhibits lateral signaling as evidenced by loss of AJM-1-marked granddaughters of P5.p/P7.p. (E,F) Adding region 1 promotes downregulation of LIN-12(extra)::TM::GFP and allows for normal lateral signaling. (G) Adding the DTS to LIN-12(extra)::TM::GFP does not promote downregulation. G' shows magnification of GFP staining, while G" shows that most of the GFP is outside the AJM-1 boundary (pseudocolored red), consistent with basolateral accumulation. (H) LIN-12(extra)::TM::DTS::GFP does not inhibit lateral signaling. (I-L) Structure of GFP-tagged proteins expressed above, and quantification of downregulation (gray bars) and 2° fate defects (black bars).

View larger version (41K): [in a new window]   Fig. 7. Conservation of cis-acting determinants of LIN-12 trafficking in vertebrate Notch proteins. The relevant portion of LIN-12 region 1 was aligned with the equivalent region from Dm Notch, Danio rerio (Dr) Notch1-3, Mm Notch 1-3, Rattus norvegicus (Rn) Notch1-3 and Hs Notch 1-3. The requirement for the leucines and upstream serine/threonines in internalization has been verified experimentally for the LIN-12 DTS. We have marked the conserved di-leucine and serine residues in red, with shading. The conserved di-leucine in Dr Notch2 and Dr Notch3 and the di-leucine-like `LM' in other Notch3 proteins are shaded, but not marked in red to emphasize the lack of upstream negative or phospho-accepting amino acids that may be important for function as an internalization signal. Most vertebrate Notch proteins also have a canonical (D/E)XXXLL consensus motif (Bonifacino and Traub, 2003); the key residues are marked in red without shading. Conserved lysines that have been verified experimentally as being involved in degradation of LIN-12 are marked in blue.

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