|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 10 January 2007
doi: 10.1242/dev.02767
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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.
* Author for correspondence (e-mail: dev.gree{at}cancercenter.columbia.edu)
Accepted 30 November 2006
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: LIN-12, Notch, BEACH, Endocytosis, Caenorhabditis elegans
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Miaczynska et al., 2004;
Le Roy and Wrana, 2005).
Endocytic traffic of non-activated receptors plays a part in controlling their
steady-state level and can thereby affect the sensitivity of cells to ligands.
Mutations that alter this process can have profound phenotypic effects. For
example, defects in multivesicular endosome sorting can cause inappropriate
activation of LIN-12/Notch, leading to cell fate change
(Shaye and Greenwald, 2005) or
neoplastic transformation and ectopic mitogenic signal production
(Moberg et al., 2005;
Thompson et al., 2005;
Vaccari and Bilder, 2005;
Herz et al., 2006) in vivo. In
the case of the epidermal growth factor receptor (EGFR), endocytic traffic is
required both for attenuation of receptor activity
(Sorkin and Waters, 1993) as
well as for full activation (Burke et al.,
2001; Vieira et al.,
1996).
LIN-12/Notch and EGFR (LET-23) regulate cell fate specification in the
developing Caenorhabditis elegans vulva
(Greenwald, 2005;
Sundaram, 2006). Initially,
six vulval precursor cells (VPCs), P3.p-P8.p, each have the potential to adopt
fates called `1°', `2°' and `3°'. The 1° and 2° fates are
vulval, meaning that they divide to produce cells that form the mature vulva,
whereas the 3° fate is non-vulval. An EGF-like inductive signal from the
anchor cell in the overlying gonad is followed by a LIN-12/Notch-mediated
lateral signal between P6.p and its neighbors, P5.p and P7.p. As a result of
inductive and lateral signaling, P3.p-P8.p take on an invariant pattern of
3°-3°-2°-1°-2°-3° fates. Mutations that affect
LIN-12/Notch or LET-23/EGFR activity alter this cell fate pattern, resulting
in profound phenotypic consequences that have enabled the genetic
identification of core components and modulators of the signal transduction
pathways.
The VPCs are polarized epithelial cells, with distinct apical and
basolateral domains delineated by circumferential adherens junctions
(Kim, 1997). LIN-12/Notch and
LET-23/EGFR are type I transmembrane proteins that are initially present on
the surface of all six VPCs; LIN-12 is present on the apical surface
(Levitan and Greenwald, 1998;
Shaye and Greenwald, 2002),
whereas LET-23 is present basolaterally
(Kaech et al., 1998;
Whitfield et al., 1999).
During the patterning of the VPCs, there appears to be reciprocal modulation
of endocytosis and trafficking of both LET-23 and LIN-12. LET-23 is activated
maximally in P6.p, leading to LIN-12 internalization via endocytosis and
degradation via multivesicular endosomes
(Shaye and Greenwald, 2002;
Shaye and Greenwald, 2005), as
well as to the production of ligands that activate LIN-12 in the neighboring
cells, P5.p and P7.p (Chen and Greenwald,
2004). When LIN-12 is activated in P5.p and P7.p, LET-23 is
downregulated (Whitfield et al.,
1999; Stetak et al.,
2006). LIN-12 target genes include factors that may negatively
regulate LET-23/EGFR activity by promoting its endocytosis and degradation
(Yoo et al., 2004;
Yoo and Greenwald, 2005).
In this study we report the characterization of sel-2, identified
as a negative regulator of lin-12 activity in the VPCs. We show that
SEL-2 is the single C. elegans homolog of two closely related human
proteins, neurobeachin (also referred to as BCL8B) and LRBA (also referred to
as BGL or CDC4L) (Wang et al.,
2000b; Wang et al.,
2001; Dyomin et al.,
2002). Neurobeachin is expressed most strongly in the nervous
system in mouse and human, whereas LRBA is broadly expressed
(Wang et al., 2000b;
Dyomin et al., 2002). In
humans, the neurobeachin locus has been linked to an idiopathic case of
non-familial autism (Castermans et al.,
2003) and spans a common fragile site on chromosome 13
(Savelyeva et al., 2006); mice
lacking neurobeachin die at birth because of a lack of evoked neuromuscular
transmission and a consequent inability to breathe
(Su et al., 2004). LRBA is
upregulated in a number of human tumor cell lines; it has been shown to lower
the sensitivity of cells to apoptosis and to positively regulate the activity
of the EGF receptor in tissue culture
(Wang et al., 2004). In
Drosophila, mutations in the single neurobeachin/LRBA homolog
rugose (also referred to as DAKAP550) cause defects in eye
development consistent with abnormalities in Notch and EGFR signaling
(Schreiber et al., 2002;
Shamloula et al., 2002;
Wech and Nagel, 2005), but the
basis for these defects is unclear.
Here, we have investigated the function of SEL-2/neurobeachin/LRBA on LET-23 and LIN-12 accumulation and subcellular localization, and other aspects of endocytic trafficking in polarized epithelial cells. Our observations suggest that SEL-2/neurobeachin/LRBA is involved in endosomal traffic and perhaps in efficient delivery of cell surface proteins to the lysosome in polarized cells, and may thereby potentiate lin-12 activity in the VPCs.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Alleles: LG III-daf-2(e1370), dpy-17(e164), sel-2(ar219, n655), lin-12(n302, n676, n379), mab-21(bx53), emb-5(hc61), unc-79(e1068), pha-1(e2123); LG V-sel-10(ar41), him-5(e1490); LG X-rme-6(b1018). Information about these alleles may be obtained via Wormbase at http://www.wormbase.org.
Transgenes: arIs41[lin-12::gfp] expresses LIN-12::GFP under the
control of lin-12 regulatory sequences
(Levitan and Greenwald, 1998);
LIN-12::GFP is visualized by antibody staining. zcIs4[hsp-4p::gfp] is
a marker for ER stress (Urano et al.,
2002). arIs92[egl-17p::cfp-lacZ] may be used as a marker
for the VPC 2° fate by assessing descendants at the L4 stage
(Burdine et al., 1998;
Inoue et al., 2002).
arIs12[lin-12(intra)] expresses the intracellular domain of LIN-12
under the control of lin-12 regulatory sequences
(Struhl et al., 1993).
arEx551 and arEx562 are extrachromosomal arrays carrying a
PCR fragment spanning the sel-2(+) genomic region. arEx545
and arEx546 are extrachromosomal arrays carrying PCR fragments
encoding SEL-2::GFP (see below). arEx553 and arEx554 are
extrachromosomal arrays carrying LIN-12::GFP with a basolateral targeting
sequence at the carboxy terminus [LIN-12::GFP-BL].
Mutagenesis, mapping and molecular identification
sel-2(n655) was identified after EMS mutagenesis of the
weak hypermorph lin-12(n302) and initially mapped to the
mab-21-emb-5 interval (data not shown). Then,
sel-2(n655) lin-12(n302);
him-5(e1490) males were crossed to hermaphrodites of a hybrid
strain carrying sequences from Hawaiian CB4856 between daf-2 and
dpy-17, and Daf non-Dpy animals were picked in the F2 generation. Daf
non-Dpy worms that retained sel-2(+) were examined for the
presence of SNP F10f2. Fourteen out of 16 such recombinants kept SNP F10f2 but
two out of 16 lost it, thus placing sel-2 to the left of SNP F10f2
and narrowing the genomic interval containing sel-2 to a region with
four predicted genes. Sequence analysis of sel-2(n655)
identified a nonsense mutation (W1012stop) in exon 12 of the gene F10F2.1.
sel-2(ar219) was identified in a non-complementation screen: EMS-mutagenized dpy-17(e164) hermaphrodites were crossed with sel-2(n655) lin-12(n302); him-5(e1490) males, and an F1 Muv hermaphrodite segregated sel-2(ar219), associated with a nonsense mutation (Q330stop) in exon 8 of F10F2.1.
Sequence analysis and phylogenetic trees
The carboxy terminal region (extending from the BEACH domain to the end of
the protein) of three C. elegans BEACH-WD40 proteins (SEL-2, VT23B5.2
and T01H10.8) and their homologs in human, mouse, Drosophila, and
Dictyostelium were aligned using ClustalW
(Chenna et al., 2003) and
T-Coffee (Notredame et al.,
2000), and maximum parsimony phylogenetic trees were found via
heuristic search with PAUP version 4.0 beta 10
(Swofford, 2003). Trees were
visualized with TreeView version 1.6.6
(Page, 1996). Robustness of
the partitions was assessed by constructing a bootstrap consensus tree with
100 replicates.
Reporters and extrachromosomal arrays
Unless otherwise stated, all injection mixes included the
pha-1(+) gene (50 ng/µl) and were injected into
pha-1(e2123) hermaphrodites. Injected animals generated F1
progeny at 15°C for 4-5 days, and the plates were then shifted to the
restrictive temperature of 25°C to select for F2 animals carrying
transgenes. When unsequenced PCR products were used to generate transgenes, a
mixture of at least six independent reactions was used.
The sel-2 genomic region (extending until the next gene on either side) was PCR amplified and injected (10 ng/µl) into sel-2(n655) hermaphrodites together with myo-3p::gfp (20 ng/µl) or ttx-3p::gfp (60 ng/µl) as the only co-injection markers. Rescue of the sel-2(-) lin-12(n302) Muv phenotype was assessed by crossing each array into the double mutant and scoring the number of pseudovulvae in animals with or without the array.
The transcriptional reporter for sel-2 was generated by overlapping PCR: the 5' flanking region extending until the next gene was fused to the coding sequence of YFP containing two nuclear localization sequences (2XNLSYFP) and injected at 50 ng/µl.
We generated the large (22 kb) translational reporter for sel-2
using two PCR fragments covering the rescuing sel-2 genomic region,
such that they overlapped by 
1 kb. The upstream fragment included the
5' flanking region of sel-2 followed by approximately half the
sel-2 gene, and the downstream fragment included the other half of
the sel-2 gene with the coding sequence for GFP inserted just before
the stop codon, followed by the sel-2 3' flanking region.
Either one or both fragments were injected at 5 ng/µl together with
ttx-3::gfp at 60 ng/µl. Transgenic arrays generated by
co-injecting both fragments rescued the Muv phenotype of
sel-2(n655) lin-12(n302) (3/7 lines) and
expressed GFP, presumably because of recombination in the overlapping regions
of the co-injected fragments, whereas arrays including only one of the two
fragments did not (0/7 lines for the N-terminal fragment, 0/6 lines for the
C-terminal fragment).
lin-12(n302)::gfp was prepared by subcloning the
relevant PCR-amplified region from lin-12(n302)
hermaphrodites into the lin-12::gfp construct
(Levitan and Greenwald, 1998),
and was injected at 5 ng/µl. Basolaterally targeted lin-12::gfp
and lin-12(n302)::gfp were generated by fusing the
coding sequence for the carboxy-terminus of the LET-23 receptor - upstream
primer: 5'-CCCCAACTTTTCCTGGAAAATTC, downstream primer:
5'-AAGACAAGTTTCCTTTTGTGATAC - to the 3' end of
lin-12::gfp (Levitan and
Greenwald, 1998) or lin-12(n302)::gfp
(this study) and injected at 5 ng/µl.
RNAi
For sel-2(RNAi), the genomic region covering exons 8-21
was amplified and cloned into the double T7 promoter plasmid pPD129.36
(Timmons et al., 2001). For
asb-1(RNAi) and apc-11(RNAi), the entire
genomic region of each gene was amplified and cloned into pPD129.36. As a
negative control, the coding sequence of GFP was used. These constructs were
transformed into HT115 bacteria and used for RNAi by feeding as described
(Timmons et al., 2001) except
that overnight cultures were used directly without IPTG induction in liquid
culture and NGM agar plates contained fresh IPTG at 5 mmol/l. L4 larvae were
placed onto lawns of the relevant bacteria and their progeny were scored.
Immunofluorescence staining and microscopy
The procedure of Bettinger et al.
(Bettinger et al., 1996) was
used, except that 1% formaldehyde and 0.1% Triton was used for staining of
LET-23. Eggs were collected by bleaching and grown at 20°C for 41 hours
before fixation of larvae. Anti-GFP (Molecular Probes) was used at 1:300; MH27
(Developmental Studies Hybridoma Bank, University of Iowa) was used at 1:1000
for the purified antibody and at 1:10 for unpurified supernatant; anti-LET 23
(Whitfield et al., 1999),
kindly provided by S. Kim, was used at 1:2000. All fluorescently labeled
secondary antibodies (Jackson Immunochemicals) were used at 1:300 dilution.
Image acquisition was on a Zeiss Axioplan 2 microscope with a Hamamatsu
ORCA100 camera, a Zeiss Z1 microscope with an ApoTome attachment and a
Hamamatsu ORCA-ER, or a Biorad MRC 1024 ES confocal set-up attached to a Nikon
Eclipse E800 microscope. All images subject to comparison were identically
acquired and processed.
|
As there is some variability between worms in the size and shape of the Pn.px cells, the number of Z-slices varied slightly from animal to animal (13-20 slices). In all three genotypes, the cells quantified were P4.px, P5.px, P7.px or P8.px, either in a ventral or lateral view. There is no significant difference in fluorescence intensity between secondary and tertiary cells within a particular genotype. To avoid bias, images of the first five Pn.px stage worms on each slide were acquired for quantification, and the acquisition was carried out blind to genotype.
Endocytosis assays in the intestine
To assess endocytosis from the apical surface of the intestine, young adult
hermaphrodites were incubated in a drop of TMRE-dextran (0.1 mg/ml) or FM4-64
(0.4 mmol/l) in buffer (118 mmol/l NaCl, 48 mmol/l KCl, 2 mmol/l
MgCl2, 2 mmol/l CaCl2, 10 mmol/l HEPES, pH 7.4) for 4-5
hours, allowed to recover on NGM plates seeded with OP50 bacteria for 120
minutes in the dark, then mounted in M9 supplemented with 10 mmol/l levamisole
and imaged. To assess endocytosis from the basolateral surface of the
intestine, the dyes were prepared as above, injected into the pseudocoelom of
10 L4 hermaphrodites at a time and the animals were mounted and imaged
10 minutes after injection (Fares and
Grant, 2002; Hermann et al.,
2005).
|
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Greenwald
et al., 1983). The phenotype caused by these mutations in C.
elegans hermaphrodites largely reflects the degree of constitutive
activity of the receptor. When LIN-12 constitutive activity is low, the
gonadal anchor cell is not specified and all of the VPCs take on the
non-vulval 3° fate, resulting in a `Vulvaless' (Vul) phenotype. However,
when LIN-12 constitutive activity is high, all VPCs become 2°, even though
there is no anchor cell. The descendants of each 2° VPC undergo
morphogenesis to form a pseudovulva, resulting in a `Multivulva' (Muv)
phenotype. sel-2(n655) was isolated after EMS mutagenesis of lin-12(n302), an allele that normally causes a Vul phenotype. Although in the somatic gonad there may be slight suppression (<1%) of the lack of an AC caused by lin-12(n302) (data not shown), the striking feature of the sel-2(n655) lin-12(n302) double mutant is that it displayed a completely penetrant Muv phenotype (Table 1; Fig. 1A). This result suggests that sel-2(n655) enhances lin-12(n302) activity in the VPCs.
|
Genetic evidence suggests that sel-2 is not involved in the same
processes that are affected by the other negative regulators of
lin-12 studied so far: proteasomal degradation (sel-10),
endoplasmic reticulum (ER) associated degradation (sel-1) and quality
control at the ER (sel-9). sel-2(-), unlike
sel-10(-) (Hubbard et
al., 1997), did not enhance the activity of
lin-12(intra), a constitutively active cytosolic form of
LIN-12 (Table 1) (N.d.S., H.F.
and I.G., unpublished). sel-2(-), unlike
sel-1(-) (Urano et al.,
2002), did not cause upregulation of ER chaperones at the
transcriptional level (data not shown). Finally, sel-2(-),
unlike sel-9(-) (Wen and
Greenwald, 1999), did not suppress the phenotype of
glp-1(q415), in which the LIN-12 paralog GLP-1 accumulates
intracellularly in the gonad at the restrictive temperature (data not shown).
Instead, as described herein, sel-2 may function as a negative
regulator of lin-12 activity through an effect on endocytic
traffic.
Molecular identification of sel-2
sel-2 was mapped to a region encompassing four predicted ORFs (see
Materials and methods). Sequencing of these genes in
sel-2(n655) identified a nonsense mutation (W1012stop) in
exon 12 of F10F2.1, and sel-2(ar219) has a nonsense
mutation (Q330stop) in exon 8 (Fig.
2A). F10F2.1 is the first gene in a predicted operon, which
includes asb-1, a homolog of ATP synthase B, and apc-11, a
subunit of the anaphase promoting complex
(Fig. 1B). mab-21
(Chow et al., 1995) is a small
gene located in the opposite orientation in the 22nd intron of F10F2.1. Three
lines of evidence indicate that F10F2.1 is sel-2. First, both
sel-2 alleles contain stop codons in this predicted gene (see also
below). Second, extrachromosomal arrays carrying the genomic region of F10F2.1
rescued the Muv phenotype of sel-2(-)
lin-12(n302) (Fig.
1C). Third, reduction of sel-2 activity, but not of
asb-1, apc-11 or mab-21 activity, by RNAi resulted in a Muv
phenotype in lin-12(n302) or lin-12(n379)
animals (Fig. 1D).
|
; Gebauer et al.,
2004). A WD40 motif folds into a `ß-propeller blade', and
tandem copies form a multibladed propeller structure that is thought to
mediate protein-protein interactions
(Sondek et al., 1996;
Wall et al., 1995). The
BEACH-WD40 region is positioned at the carboxy terminus of the protein. Both
sel-2 mutants are predicted to encode truncated proteins that would
lack the BEACH-WD40 region (Fig.
2A). Reciprocal BLAST searches and sequence alignments using either the entire protein or only the carboxy terminal BEACH-WD40 domain indicated that SEL-2 has homologs in several eukaryotes, including Drosophila DAKAP550 and mammalian neurobeachin and LRBA. There are several regions of high conservation between SEL-2 and its mammalian homologs throughout the length of the protein (Fig. 2A), with 64-65% amino acid identity in the BEACH domain, as well as 48% identity in `homology region C', which includes the PH domain that was structurally defined for neurobeachin and LRBA. We could not conclude whether SEL-2 is more `neurobeachin-like' or `LRBAlike' based on sequence analysis (Fig. 2A,B).
SEL-2/neurobeachin/LRBA appears to form a subfamily within a larger family
of BEACH-WD40 domain-containing proteins. There are three other C.
elegans BEACH-containing proteins
(Fig. 2B and data not shown).
The most closely related to SEL-2 is VT23B5.2, the ortholog of human ALFY
(WDFY3 - Human Gene Nomenclature Committee)
(Simonsen et al., 2004) and
Drosophila Blue Cheese (Finley et
al., 2003), consisting of little else but the BEACH domain and
five WD40 motifs, plus a C-terminal FYVE domain. The
VT23B5.2(ok912) null allele (kindly provided by the Knockout
Consortium) did not have a readily discernable visible phenotype on its own or
in a double mutant with sel-2(-), and did not cause a Muv
phenotype in combination with lin-12(n379), suggesting that
SEL-2 and VT23B5.2 are not functionally redundant (data not shown). The two
other BEACH-containing proteins in C. elegans are T01H10.8, the
apparent ortholog of mammalian LYST (Nagle
et al., 1996), although it (unlike LYST) does not have predicted
WD40 motifs, and F52C9.1, which has only two WD40 motifs.
SEL-2 expression and subcellular distribution
A transcriptional reporter for sel-2 in which the 5'
upstream region drives nuclearly localized YFP is expressed in the VPCs and
their descendants (Fig. 3A,B),
as well as in many other cell types, including the epithelial cells of the
intestine (Fig. 3C,D) and the
rectum, the seam cells, many cells in the head and the tail, and the cells of
the ventral nerve cord (not shown). In live worms, expression of a rescuing
SEL-2::GFP translational reporter (Fig.
1C; also see Materials and methods) was strongest in the rectal
epithelial cells (Fig. 3E,F)
and in the seam cells (not shown), where it appeared to be distributed
throughout the cytoplasm, but particularly concentrated close to the nucleus.
This distribution is consistent with partial membrane association of
SEL-2::GFP, as has previously been proposed for its mammalian homologs
neurobeachin and LRBA (Wang et al.,
2001; Wang et al.,
2000b).
sel-2 affects the subcellular localization of LIN-12::GFP in VPCs
We examined the subcellular localization of LIN-12::GFP in the VPCs and
their descendants in sel-2(+) and sel-2(-)
hermaphrodites (Fig. 4), using
the adherens junction protein AJM-1 and LET-23/EGFR for comparison. The
adherens junctions are circumferential, close to the apical surface of the
VPCs. LET-23 is localized basolaterally at the Pn.px stage, except in P6.px,
where it is also present at the apical surface
(Whitfield et al., 1999). It
is expressed strongly in P6.px and weakly in the other VPC descendants at this
stage.
In sel-2(+), LIN-12::GFP was mostly at the apical surface of the VPCs or in puncta close to the apical surface (Fig. 4C,D, top panel; 36% of animals had visible non-apical LIN-12::GFP, n=292), and did not co-localize appreciably with the basolateral LET-23/EGFR (Fig. 4E,F, top panel). By contrast, in sel-2(-), LIN-12::GFP was not restricted to the apical surface but was also present basolaterally (Fig. 4C,D, bottom panel, see arrows; 89% of animals had visible non-apical LIN-12::GFP; n=154) and indeed appeared to co-localize with LET-23 at the basolateral membrane (Fig. 4E,F, bottom panel, see arrows). The VPCs seemed to be polarized normally in sel-2(-), as LET-23/EGFR displayed its typical distribution (Fig. 4E,F; red channel), and the adherens junctions appeared normal (Fig. 4C,D; red channel).
In addition to being mislocalized to the basolateral surface, the overall level of LIN-12::GFP was elevated in sel-2(-) in fixed and stained (Fig. 4) or live, unstained animals (not shown). Indeed, LIN-12::GFP was often undetectable in the VPCs of unstained sel-2(+) animals in VPCs and their descendants but could be detected at both apical and basolateral surfaces of the VPCs in live sel-2(-) animals. Quantification of LIN-12::GFP fluorescence intensity showed significantly higher levels in VPC descendants of sel-2(-) versus sel-2(+) animals at the Pn.px stage (Fig. 4H; see Materials and methods).
|
) to LIN-12::GFP. LIN-12::GFP-BL accumulated
basolaterally (Fig. 5A) and was
able to rescue lin-12(0) defects (not shown), indicating
that the added amino acids do not eliminate lin-12 activity.
Hermaphrodites expressing LIN-12::GFP-BL were not Muv
(Fig. 5B), suggesting that
basolateral localization is not sufficient to activate the receptor. We also tested whether basolateral targeting of a constitutively active form of LIN-12 makes it refractory to loss of sel-2 activity, as might be the case if sel-2(-) enhances lin-12(n302) activity simply by misrouting it to the basolateral domain. Hermaphrodites expressing LIN-12(n302)::GFP or LIN-12(n302)::GFP-BL were Muv to the same extent, consistent with the results described above suggesting that basolateral targeting per se does not activate LIN-12 (Fig. 5B). Moreover, the Muv phenotype of LIN-12(n302)::GFP-BL animals was further enhanced in a sel-2(n655) background (Fig. 5C). As LIN-12(n302)::GFP-BL was significantly basolateral in all VPCs in the majority of sel-2(+) animals examined, the results suggest that the effect of sel-2 on lin-12 activity is not solely due to the basolateral accumulation of LIN-12 in sel-2(-), subject to the caveat that an effect on untagged LIN-12(+) protein may be contributing to this enhancement.
|
).
LIN-12::GFP was downregulated to the same extent in sel-2(+)
and sel-2(-) hermaphrodites
(Fig. 6A), suggesting that
sel-2 is not required for LET-23-dependent internalization or
downregulation of LIN-12 from the apical surface. However, sel-2
activity appears to be required for efficient downregulation of basolateral
LIN-12::GFP-BL, as a greater proportion of
sel-2(-)hermaphrodites retain substantial fluorescence in
P6.px (Fig. 6A).
We also examined downregulation of endogenous LET-23 in the VPC
descendants. When LIN-12 is activated in P5.p and P7.p by the lateral signal,
LET-23/EGFR is downregulated (Stetak et
al., 2006; Whitfield et al.,
1999). By contrast, in a sel-2(-) mutant,
LET-23/EGFR appears to accumulate aberrantly in P5.p and P7.p
(Fig. 6C). At both the Pn.px
and the Pn.pxx stage, a smaller fraction of sel-2(ar219)
worms (44%, n=52 at Pn,px) as compared to wild type (74%,
n=79 at Pn.px) had no detectable LET-23 in the descendants of P5.p
and/or P7.p (Fig. 6B). A
similar effect was seen in sel-2(n655) (data not shown).
However, we did not detect any evidence of ectopic LET-23/EGFR activity in
P5.p or P7.p, as assayed by ectopic expression of a LET-23 target gene (data
not shown).
In addition to these regulated events that occur in response to
intercellular signals, LIN-12 appears to be constitutively internalized, and
recycled or degraded at a basal level in all VPCs
(Shaye and Greenwald, 2005).
To examine the effect of reducing constitutive endocytosis, we removed the
activity of rme-6, a regulator of Rab5 found in coated pits
(Sato et al., 2005). We found
that lin-12 activity was enhanced by loss of rme-6: 21% of
lin-12(n302); rme-6(b1018) were Muv
(n=106), compared with 2% of lin-12(n302) animals
(n=104). However, LIN-12::GFP did not accumulate at the basolateral
surface of the VPCs (Fig. 4G),
suggesting that rme-6, which functions in traffic from the plasma
membrane to early endosomes, behaves differently from sel-2.
sel-2 and endocytic traffic in the intestine
sel-2 is expressed in cells of the intestine, which like the VPCs,
are polarized epithelial cells. Endocytic traffic from either the apical or
the basolateral surfaces of the intestinal epithelium may be observed after
the delivery of marker dyes by feeding (apical) or injection into the
pseudocoelom (basolateral) (Fares and
Grant, 2002). A prominent feature of intestinal cells is the
presence of autofluorescent `gut granules', which have been shown to be
lysosomes (Clokey and Jacobson,
1986; Grant et al.,
2001). The membrane-intercalating dye FM4-64 accumulates in these
structures when delivered apically or basolaterally
(Grant et al., 2001;
Fares and Grant, 2002;
Hermann et al., 2005).
Fluid-phase markers accumulate in these structures when delivered apically,
but not basolaterally due to rapid recycling to the pseudocoelom
(Fares and Grant, 2002).
FM4-64 delivered to the pseudocoelom concentrated in a different pattern in the intestine of sel-2(n655) and sel-2(ar219) compared with wild type (Fig. 7), suggesting an alteration in endocytic traffic from the basolateral surface. In wild-type animals imaged 10-30 minutes after injection, FM4-64 concentrated in defined punctate structures, which partially overlap with autofluorescent gut granules. By contrast, FM4-64 compartments in sel-2(-) were irregularly shaped, largely not overlapping with autofluorescent granules, and predominantly clustered at the apical surface of the intestinal epithelium (Fig. 7A,B). These experiments were performed on mixed populations of sel-2(+) and sel-2(-) hermaphrodites, blind to genotype. sel-2(+) worms were identified after scoring by the presence of ttx-3::GFP expressed in a single neuron in the head of the worm: 85% of sel-2(ar219)(n=33) and 89% of sel-2(n655) (n=9) had the `sel-2 phenotype' compared with 0% of sel-2(+) (n=42) (Fig. 7C). Furthermore, the phenotype was partially rescued by an extrachromosomal array expressing the sel-2 genomic region (1 out of 2 lines) and also partially reproduced in wild-type animals by sel-2(RNAi) (43%, n=23) but not by control GFP(RNAi) (0%, n=25) conducted in parallel (Fig. 7C). These observations suggest that there is a perturbation in endosomal traffic or in endosomal membrane organization in the intestinal epithelium of sel-2 (-) animals.
|
;
Fares and Grant, 2002). There was no obvious difference in the distribution of FM4-64 when delivered to the apical surface of the intestinal epithelium of sel-2(-) hermaphrodites by feeding (not shown). However, it is possible that the timescale of these experiments (feeding of dye for 4 hours, followed by a `chase' of 2 hours to clear excess dye from the gut lumen) makes it difficult to see a small difference in rates of traffic between wild-type and sel-2 animals. Feeding the fluid-phase marker also did not reveal any difference between wild-type and sel-2(-) hermaphrodites, with the same potential caveat about timescale.
The intestines of sel-2(-) worms appeared visually
normal, and the distribution of markers for the early (RAB-5)
(Zerial and McBride, 2001),
late (RAB-7) (Chen et al.,
2006) and recycling (RAB11, RME-1)
(Casanova et al., 1999;
Grant et al., 2001;
Chen et al., 2006) endosomal
compartments appeared qualitatively normal (not shown). Further, there is no
significant perturbation in acidic organelles, as assessed by Lysotracker
staining, in sel-2(-) (data not shown). These observations
at steady-state are consistent with the loss of sel-2, resulting in a
modest effect on traffic rates.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
Many of these defects appear to point to a role for SEL-2 in endocytosis from the basolateral surface of polarized epithelial cells. At this time, we have no evidence that loss of sel-2 activity also compromises endocytic traffic from the apical surface of polarized epithelial cells. As mentioned above, endocytic downregulation of LIN-12::GFP from the apical surface of P6.p occurred normally in sel-2(-) mutants. Furthermore, apically delivered fluorescent lipids appeared to be distributed normally, although the long incubation time required for this assay may obscure a small effect on rates.
One model that can account for many of our observations is that in
sel-2(-) mutants there is a primary defect in the rate of
internalization from the basolateral surface of polarized epithelial cells. As
LIN-12 is not generally seen in the basolateral domain in wild-type
hermaphrodites but is seen in the basolateral domain in sel-2
mutants, this model requires the assumption that at least some LIN-12 normally
transits through the basolateral domain, and therefore can be seen in
sel-2 mutants because internalization is slowed. However, removing
the activity of rme-6, a regulator of RAB-5 that mediates traffic
from the plasma membrane to early endosomes, did not result in a similar
accumulation of LIN-12 at the basolateral surface of the VPCs. As
rme-6 is associated with clathrin-coated vesicles
(Sato et al., 2005) and is
likely to be involved in both apical and basolateral endocytosis, this result
suggests that a simple reduction in internalization does not lead to
basolateral accumulation of LIN-12.
An alternative model that may better account for all of our observations is
that aberrant basolateral protein accumulation in sel-2(-)
results from problems in endosomal sorting. Surface proteins from the apical
and basolateral domains of polarized mammalian cells are initially taken up
into separate early endosomes but enter a common endosome before recycling
back to their respective plasma membrane domains
(Brown et al., 2000;
Wang et al., 2000a).
Basolateral proteins are transported directly from the common endosome back to
the basolateral membrane, while apical proteins first enter the apical
recycling endosome and are then delivered to the apical plasma membrane
(Mostov et al., 2003;
Hoekstra et al., 2004).
Moreover, some fraction of surface proteins traffic to late endosomes and
lysosomes for degradation, at steady state
(Stahl and Barbieri, 2002).
Increased traffic between the common endosome and the basolateral surface in
sel-2 mutants could result in the net accumulation of LIN-12 and
LET-23 there. However, as we have also observed a reduced rate of lipid
delivery to lysosomes in the intestinal epithelium of sel-2 mutants,
we favor the view that in the absence of sel-2 activity there is a
defect in traffic to late endosomes and lysosomes, rather than in recycling
per se. For example, SEL-2 may function in sorting endosomes for the
specification of membrane domains that will mature into late endosomes. A
defect in this process would result in the slowed delivery of endocytosed
membrane lipid to late endosomes and lysosomes. Furthermore, in the absence of
SEL-2, transmembrane proteins such as LIN-12 and LET-23 may then segregate
into tubular domains of sorting endosomes
(Bonifacino and Rojas, 2006)
that are destined for recycling back to the plasma membrane. Additional
support for this view comes from the observation that certain mutant forms of
LIN-12 that can be internalized from the apical surface but not degraded also
accumulate at the basolateral surface of the VPCs
(Shaye and Greenwald,
2005).
We note that acidic compartments in the intestine of
sel-2(-) are qualitatively indistinguishable from those in
wild type, consistent with a defect in traffic in the endosomal system rather
than defective lysosomes themselves. Further, neither inhibition of sorting
into internal vesicles of multivesicular (i.e. late) endosomes with
alx-1(RNAi), nor mutation of LIN-12 lysine residues that are
putative ubiquitination sites, cause basolateral localization of LIN-12::GFP
(Shaye and Greenwald, 2005),
suggesting that sel-2 acts at a different step in this process. Taken
together, our results suggest defective or slowed traffic in the endosomal
system in sel-2(-). The resultant increased accumulation of
LIN-12 could account for the enhancement of LIN-12 signaling revealed by the
genetic interactions between sel-2(-) and lin-12
mutations.
SEL-2 and regulation of lin-12 activity
The genetic interactions between sel-2 and lin-12 suggest
that sel-2 behaves as a negative regulator of lin-12
activity. In particular, loss of sel-2 activity dramatically enhances
the effect of certain lin-12 alleles that cause constitutive
activity. Loss of sel-2 only enhances constitutive activity resulting
from point mutations in the extracellular domain, i.e. forms of LIN-12 that
still need to undergo proteolytic cleavage events to signal, and not the
constitutive activity resulting from a truncation that mimics the end product
of the cleavage events. These genetic results are consistent with a role for
sel-2 in LIN-12 trafficking either before ligand activation or in the
cleavage events that occur upon ligand binding.
The observation that sel-2 mutants are phenotypically wild type
raises the question of the normal contribution of sel-2 in regulating
lin-12 activity in VPCs. We did not see any evidence that VT23B5.2,
the closest BEACH-WD40 paralog encoded in the C. elegans genome, is
functionally redundant with SEL-2 (N. de S. and I.G., unpublished). However,
it is possible that another protein is functionally redundant, or that there
is an independent but redundant regulatory mechanism. Alternatively,
sel-2 may be involved in finetuning the level of lin-12
activity by contributing to proper LIN-12 trafficking. In both well-studied
paradigms of LIN-12 signaling in C. elegans, the AC/VU decision in
the developing gonad or VPC fate specification in the developing vulva, the
control of relative levels of the receptor in different cells is a key
component of fate specification. In the AC/VU decision, regulation of LIN-12
levels occurs principally by modulating lin-12 transcription
(Wilkinson et al., 1994;
Greenwald, 2005). In the VPCs,
LIN-12 traffic and stability, rather than lin-12 transcription, is
modulated. It may be that in the VPCs, post-transcriptional mechanisms are
better able to coordinate LIN-12 activity in lateral signaling with
LET-23/EGFR activity in inductive signaling. Perhaps sel-2 activity
contributes to the delicate balance and mutual feedback that operates during
fate specification in these cells.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bettinger, J. C., Lee, K. and Rougvie, A. E. (1996). Stage-specific accumulation of the terminal differentiation factor LIN-29 during Caenorhabditis elegans development. Development 122,2517 -2527.[Abstract]
Bonifacino, J. S. and Rojas, R. (2006). Retrograde transport from endosomes to the trans-Golgi network. Nat. Rev. Mol. Cell Biol. 7, 568-579.[CrossRef][Medline]
Brown, P. S., Wang, E., Aroeti, B., Chapin, S. J., Mostov, K. E. and Dunn, K. W. (2000). Definition of distinct compartments in polarized Madin-Darby canine kidney (MDCK) cells for membrane-volume sorting, polarized sorting and apical recycling. Traffic 1,124 -140.[CrossRef][Medline]
Burdine, R. D., Branda, C. S. and Stern, M. J. (1998). EGL-17(FGF) expression coordinates the attraction of the migrating sex myoblasts with vulval induction in C. elegans. Development 125,1083 -1093.[Abstract]
Burke, P., Schooler, K. and Wiley, H. S.
(2001). Regulation of epidermal growth factor receptor signaling
by endocytosis and intracellular trafficking. Mol. Biol.
Cell 12,1897
-1910.
Casanova, J. E., Wang, X., Kumar, R., Bhartur, S. G., Navarre,
J., Woodrum, J. E., Altschuler, Y., Ray, G. S. and Goldenring, J. R.
(1999). Association of Rab25 and Rab11a with the apical recycling
system of polarized Madin-Darby canine kidney cells. Mol. Biol.
Cell 10,47
-61.
Castermans, D., Wilquet, V., Parthoens, E., Huysmans, C.,
Steyaert, J., Swinnen, L., Fryns, J. P., Van de Ven, W. and Devriendt, K.
(2003). The neurobeachin gene is disrupted by a translocation in
a patient with idiopathic autism. J. Med. Genet.
40,352
-356.
Chen, C. C., Schweinsberg, P. J., Vashist, S., Mareiniss, D. P.,
Lambie, E. J. and Grant, B. D. (2006). RAB-10 is required for
endocytic recycling in the Caenorhabditis elegans intestine. Mol.
Biol. Cell 17,1286
-1297.
Chen, N. and Greenwald, I. (2004). The lateral signal for LIN-12/Notch in C. elegans vulval development comprises redundant secreted and transmembrane DSL proteins. Dev. Cell 6, 183-192.[CrossRef][Medline]
Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T. J.,
Higgins, D. G. and Thompson, J. D. (2003). Multiple sequence
alignment with the Clustal series of programs. Nucleic Acids
Res. 31,3497
-3500.
Chow, K. L., Hall, D. H. and Emmons, S. W. (1995). The mab-21 gene of Caenorhabditis elegans encodes a novel protein required for choice of alternate cell fates. Development 121,3615 -3626.[Abstract]
Clokey, G. V. and Jacobson, L. A. (1986). The autofluorescent "lipofuscin granules" in the intestinal cells of Caenorhabditis elegans are secondary lysosomes. Mech. Ageing Dev. 35,79 -94.[CrossRef][Medline]
Dyomin, V. G., Chaganti, S. R., Dyomina, K., Palanisamy, N., Murty, V. V., Dalla-Favera, R. and Chaganti, R. S. (2002). BCL8 is a novel, evolutionarily conserved human gene family encoding proteins with presumptive protein kinase A anchoring function. Genomics 80,158 -165.[CrossRef][Medline]
Fares, H. and Grant, B. (2002). Deciphering endocytosis in Caenorhabditis elegans. Traffic 3, 11-19.[CrossRef][Medline]
Finley, K. D., Edeen, P. T., Cumming, R. C., Mardahl-Dumesnil,
M. D., Taylor, B. J., Rodriguez, M. H., Hwang, C. E., Benedetti, M. and
McKeown, M. (2003). blue cheese mutations define a novel,
conserved gene involved in progressive neural degeneration. J.
Neurosci. 23,1254
-1264.
Gebauer, D., Li, J., Jogl, G., Shen, Y., Myszka, D. G. and Tong, L. (2004). Crystal structure of the PH-BEACH domains of human LRBA/BGL. Biochemistry 43,14873 -14880.[CrossRef][Medline]
Gonzalez-Gaitan, M. (2003). Signal dispersal and transduction through the endocytic pathway. Nat. Rev. Mol. Cell Biol. 4,213 -224.[CrossRef][Medline]
Grant, B., Zhang, Y., Paupard, M. C., Lin, S. X., Hall, D. H. and Hirsh, D. (2001). Evidence that RME-1, a conserved C. elegans EH-domain protein, functions in endocytic recycling. Nat. Cell Biol. 3,573 -579.[CrossRef][Medline]
Greenwald, I. (2005). LIN-12/Notch signaling in C. elegans. In WormBook (ed. The C. elegans Research Community), doi/10.1895/wormbook.1.10.1, http://www.wormbook.org.
Greenwald, I. and Seydoux, G. (1990). Analysis of gain-of-function mutations of the lin-12 gene of Caenorhabditis elegans. Nature 346,197 -199.[CrossRef][Medline]
Greenwald, I. S., Sternberg, P. W. and Horvitz, H. R. (1983). The lin-12 locus specifies cell fates in Caenorhabditis elegans. Cell 34,435 -444.[CrossRef][Medline]
Han, J. D., Baker, N. E. and Rubin, C. S.
(1997). Molecular characterization of a novel A kinase anchor
protein from Drosophila melanogaster. J. Biol. Chem.
272,26611
-26619.
Hermann, G. J., Schroeder, L. K., Hieb, C. A., Kershner, A. M.,
Rabbitts, B. M., Fonarev, P., Grant, B. D. and Priess, J. R.
(2005). Genetic analysis of lysosomal trafficking in
Caenorhabditis elegans. Mol. Biol. Cell
16,3273
-3288.
Herz, H. M., Chen, Z., Scherr, H., Lackey, M., Bolduc, C. and
Bergmann, A. (2006). vps25 mosaics display non-autonomous
cell survival and overgrowth, and autonomous apoptosis.
Development 133,1871
-1880.
Hoekstra, D., Tyteca, D. and van IJzendoorn, S. C.
(2004). The subapical compartment: a traffic center in membrane
polarity development. J. Cell Sci.
117,2183
-2192.
Hubbard, E. J., Wu, G., Kitajewski, J. and Greenwald, I.
(1997). sel-10, a negative regulator of lin-12 activity in
Caenorhabditis elegans, encodes a member of the CDC4 family of proteins.
Genes Dev. 11,3182
-3193.
Inoue, T., Sherwood, D. R., Aspock, G., Butler, J. A., Gupta, B. P., Kirouac, M., Wang, M., Lee, P. Y., Kramer, J. M., Hope, I. et al. (2002). Gene expression markers for Caenorhabditis elegans vulval cells. Gene Expr. Patterns 2, 235-241.[CrossRef][Medline]
Jogl, G., Shen, Y., Gebauer, D., Li, J., Wiegmann, K., Kashkar, H., Kronke, M. and Tong, L. (2002). Crystal structure of the BEACH domain reveals an unusual fold and extensive association with a novel PH domain. EMBO J. 21,4785 -4795.[CrossRef][Medline]
Kaech, S. M., Whitfield, C. W. and Kim, S. K. (1998). The LIN-2/LIN-7/LIN-10 complex mediates basolateral membrane localization of the C. elegans EGF receptor LET-23 in vulval epithelial cells. Cell 94,761 -771.[CrossRef][Medline]
Kim, S. K. (1997). Polarized signaling: basolateral receptor localization in epithelial cells by PDZ-containing proteins. Curr. Opin. Cell Biol. 9, 853-859.[CrossRef][Medline]
Le Roy, C. and Wrana, J. L. (2005). Clathrin- and non-clathrin-mediated endocytic regulation of cell signalling. Nat. Rev. Mol. Cell Biol. 6, 112-126.[CrossRef][Medline]
Levitan, D. and Greenwald, I. (1998). LIN-12 protein expression and localization during vulval development in C. elegans. Development 125,3101 -3109.[Abstract]
Miaczynska, M., Pelkmans, L. and Zerial, M. (2004). Not just a sink: endosomes in control of signal transduction. Curr. Opin. Cell Biol. 16,400 -406.[CrossRef][Medline]
Moberg, K. H., Schelble, S., Burdick, S. K. and Hariharan, I. K. (2005). Mutations in erupted, the Drosophila ortholog of mammalian tumor susceptibility gene 101, elicit non-cell-autonomous overgrowth. Dev. Cell 9,699 -710.[CrossRef][Medline]
Mostov, K., Su, T. and ter Beest, M. (2003). Polarized epithelial membrane traffic: conservation and plasticity. Nat. Cell Biol. 5,287 -293.[CrossRef][Medline]
Nagle, D. L., Karim, M. A., Woolf, E. A., Holmgren, L., Bork, P., Misumi, D. J., McGrail, S. H., Dussault, B. J., Jr, Perou, C. M., Boissy, R. E. et al. (1996). Identification and mutation analysis of the complete gene for Chediak-Higashi syndrome. Nat. Genet. 14,307 -311.[CrossRef][Medline]
Notredame, C., Higgins, D. G. and Heringa, J. (2000). T-Coffee: a novel method for fast and accurate multiple sequence alignment. J. Mol. Biol. 302,205 -217.[CrossRef][Medline]
Page, R. D. (1996). TreeView: an application to display phylogenetic trees on personal computers. Comp. Appl. Biosci. 12,357 -358.[Medline]
Sato, M., Sato, K., Fonarev, P., Huang, C. J., Liou, W. and Grant, B. D. (2005). Caenorhabditis elegans RME-6 is a novel regulator of RAB-5 at the clathrincoated pit. Nat. Cell Biol. 7,559 -569.[CrossRef][Medline]
Savelyeva, L., Sagulenko, E., Schmitt, J. G. and Schwab, M. (2006). The neurobeachin gene spans the common fragile site FRA13A. Hum. Genet. 118,551 -558.[CrossRef][Medline]
Schreiber, S. L., Preiss, A., Nagel, A. C., Wech, I. and Maier, D. (2002). Genetic screen for modifiers of the rough eye phenotype resulting from overexpression of the Notch antagonist hairless in Drosophila. Genesis 33,141 -152.[CrossRef][Medline]
Shamloula, H. K., Mbogho, M. P., Pimentel, A. C.,
Chrzanowska-Lightowlers, Z. M., Hyatt, V., Okano, H. and Venkatesh, T. R.
(2002). rugose (rg), a Drosophila A kinase anchor protein, is
required for retinal pattern formation and interacts genetically with multiple
signaling pathways. Genetics
161,693
-710.
Shaye, D. D. and Greenwald, I. (2002). Endocytosis-mediated downregulation of LIN-12/Notch upon Ras activation in Caenorhabditis elegans. Nature 420,686 -690.[CrossRef][Medline]
Shaye, D. D. and Greenwald, I. (2005).
LIN-12/Notch trafficking and regulation of DSL ligand activity during vulval
induction in Caenorhabditis elegans. Development
132,5081
-5092.
Simonsen, A., Birkeland, H. C., Gillooly, D. J., Mizushima, N.,
Kuma, A., Yoshimori, T., Slagsvold, T., Brech, A. and Stenmark, H.
(2004). Alfy, a novel FYVE-domain-containing protein associated
with protein granules and autophagic membranes. J. Cell
Sci. 117,4239
-4251.
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 
