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First published online 10 January 2007
doi: 10.1242/dev.02767

Development 134, 691-702 (2007)
Published by The Company of Biologists 2007
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SEL-2, the C. elegans neurobeachin/LRBA homolog, is a negative regulator of lin-12/Notch activity and affects endosomal traffic in polarized epithelial cells

Natalie de Souza1, Laura G. Vallier1,2, Hanna Fares1,3 and Iva Greenwald1,*

1 Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, 701 W. 168th Street, Hammer Health Sciences, New York, NY 10032, USA.
2 Department of Biology, Hofstra University, Gittleson Hall Room 103, Hempstead, NY 11549, USA.
3 Department of Molecular and Cellular Biology, University of Arizona, Life Sciences South, Room 531, 1007 E. Lowell Street, Tucson, AZ 85721, USA.


Figure 1
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  		Fig. 1. sel-2 loss of function enhances lin-12(d) activity in VPCs. (A) Late L4 stage N2 (left) and sel-2(n655) lin-12(n302) (right) hermaphrodites. Top panels show Nomarski photomicrographs, with the arrow(s) indicating a normal vulva (N2) or pseudovulval invaginations; bottom panels show clusters of cells expressing egl-17::cfp-lacZ (arIs92). (B) Schematic view of the predicted sel-2 operon, indicating the regions used for rescue and RNAi experiments. (C) The sel-2 genomic region and a sel-2 translational reporter `rescue' the enhancement of lin-12(n302) by sel-2(n655). Arrays are described in Materials and methods. Multivulva (Muv) is defined as ≥3 pseudovulvae. The number of worms scored is indicated above each bar. (D) sel-2(RNAi) enhances lin-12(d). pFV denotes the empty RNAi feeding vector. Except for mab-21(RNAi), each bar represents the mean of at least two independent experiments; error bars are the s.e.m. Muv is defined as ≥2 pseudovulvae; a minimum of 100 animals was scored for each treatment.

 

Figure 2
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  		Fig. 2. sel-2 sequence analysis. (A) Comparison of SEL-2 with mammalian neurobeachin and LRBA. The % amino acid identity is indicated for the BEACH and WD40 domains, and for other regions of high homology designated `A', `B' and `C' (aa 164-1036, 1059-1306, and 1407-1828 in SEL-2, respectively). Region C includes the predicted PH domain of neurobeachin and LRBA (see text). We note that neurobeachin and DAKAP550 have been shown to bind to the regulatory domain (RII) of PKA with high affinity in vitro (Han et al., 1997Go; Wang et al., 2000bGo), whereas LRBA does not (Wang et al., 2000bGo). The RII-binding region of neurobeachin lies in the unconserved interval between homology regions A and B. (B) Phylogenetic bootstrap tree constructed from an alignment of the C-terminal regions (see Materials and methods) of C. elegans (C.e.) SEL-2, VTB23.5 and T01H10.8 and their homologs. Numbers at the nodes are bootstrap values, indicating the frequency of occurrence of a given partition in the 100 replicate trees. Boxed areas represent subfamilies of BEACH domain-containing proteins. Species are designated as follows: D.m., Drosophila melanogaster; H.s., Homo sapiens; M.m., Mus musculus; D.d., Dictyostelium discoideum.

 

Figure 3
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  		Fig. 3. sel- 2 expression. The 5' flanking region of sel-2 drives expression of NLS-YFP in several cell types, including VPCs in an L2 or L3 larva (A,B) and intestinal epithelial cells in an L4 larva (C,D). SEL-2::GFP expression is shown in the rectal epithelia in a live L2 or L3 larva (E,F).

 

Figure 4
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  		Fig. 4. LIN-12::GFP and LET-23 localization. (A,B) Schematic depiction of the localization of an apical or a basolateral protein in the VPCs relative to the adherens junction (AJ) in a lateral (A) or a ventral view (B). (C) Lateral and (D) ventral views of the distribution of LIN-12::GFP expressed from arIs41 (green) relative to the AJ (red). (E) Lateral and (F) ventral views of LIN-12::GFP (green) and LET-23 (red). (G) Lateral view of LIN-12::GFP distribution (green) in rme-6(b1018) relative to the AJ (red). (H) Quantification of LIN-12::GFP fluorescence intensity in sel-2(+) and sel-2(-) hermaphrodites (see Materials and methods). In photomicrographs, `gon' marks LIN-12::GFP staining in the gonad, which is close to the basolateral surface of the VPCs, and arrows indicate basolateral LIN-12::GFP. P5.px, P6.px and P7.px are indicated.

 

Figure 5
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  		Fig. 5. LIN-12::GFP targeted to the basolateral surface of the VPCs. (A) Lateral views of Pn.px hermaphrodites showing LIN-12::GFP-BL (upper panel) and LIN-12(n302)::GFP-BL (lower panel) in green, and the adherens junctions (AJM-1) in red. `gon' marks LIN-12::GFP staining in the gonad. The basolateral surface of some cells is indicated with arrows. P5.px, P6.px and P7.px are indicated. (B) Each bar represents an independent transgenic line expressing the indicated protein. (C) The percentage of Muv animals in three independent transgenic lines carrying LIN-12(n302)::GFP-BL in sel-2(+) and sel-2(n655) backgrounds is plotted. The number above each bar is the number of animals scored. Muv is defined as ≥3 pseudovulvae.

 

Figure 6
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  		Fig. 6. Regulated endocytosis in sel-2(ar219). (A) The fraction of animals with LIN-12::GFP or LIN-12::GFP-BL downregulated in P6.px is plotted. (B) The fraction of animals with LET-23 downregulated in P5.px and P7.px is plotted. For both graphs, numbers above the bars are the number of animals scored. In (C), endogenous LET-23 is shown in green and the adherens junction (AJM-1) is in red. Arrows indicate persistent LET-23 expression. P5.p, P6.p and P7.p descendants are indicated.

 

Figure 7
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  		Fig. 7. Accumulation of FM4-64 in the intestine after injection into the pseudocoelom (basolateral delivery). (A) FM4-64 fluorescence in sel-2(+) and sel-2(ar219). (B) FM4-64 fluorescence (middle panel, red) is shown relative to autofluorescent granules (left panel, green); co-localized signals are shown in yellow in the merge (right panel). Arrows indicate irregularly shaped, FM4-64 labelled compartments that are not coincident with autofluorescence in sel-2(ar219). (C) Quantification of the `sel-2' mutant phenotype (see text). Rescue of the phenotype with an extrachromosomal array carrying the sel-2 genomic region and phenocopy with sel-2(RNAi) is also shown. All scoring was done blind to genotype. Each bar is the mean of at least two independent experiments, and error bars are the s.e.m. The total number of worms scored is indicated on each bar.

 

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© The Company of Biologists Ltd 2007