1 Dipartimento di Biochimica e Biotecnologie Mediche, Università degli
Studi di Napoli Federico II, Via S. Pansini, 5, I-80131, Napoli, Italy
2 International Institute of Genetics and Biophysics, Consiglio Nazionale delle
Ricerche, Via G. Marconi, 10, I-80125, Napoli, Italy
Author for correspondence: (e-mail: zambrano{at}unina.it )
Accepted 3 January 2002
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Summary |
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We generated and isolated a deletion allele of feh-1, and the corresponding homozygous mutants arrest as late embryos or as L1 larvae, demonstrating for the first time an essential role for a Fe65-like gene in vivo. The pharynx of homozygous larvae does not contract and the worms cannot feed. Analysis of pharyngeal pumping in heterozygous worms and in feh-1 RNA-interfered worms indicates that dosage of feh-1 function affects the rate of pharyngeal contraction in C. elegans. Interference with apl-1 double-stranded RNA showed a similar effect on pharyngeal pumping, suggesting that FEH-1 and APL-1 are involved in the same pathway. The non-redundant system of the nematode will prove useful for studying the basic biology of the Fe65-APP interaction and the molecular events regulated by this evolutionarily conserved system of interacting proteins.
Key words: Protein-protein interaction, Alzheimer's disease, RNAi
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Introduction |
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Binding of several proteins to the cytosolic domain of APP influences APP
proteolytic processing (Sabo et al.,
1999; Borg et al.,
1998
; Sastre et al.,
1998
; Guènette et al.,
1999
; Ando et al.,
2001
). Some of these APP-interacting proteins belong to the family
of the Fe65s, which includes Fe65, Fe65-L1 and Fe65-L2
(Fiore et al., 1995
;
Guènette et al., 1996
;
Duilio et al., 1998
;
Russo et al., 1998
). The
common modular structure of the Fe65s, composed of a WW domain and two
independent phosphotyrosine-binding domains (PTB), PTB1 and PTB2, suggests the
function of molecular adaptors for these proteins. The PTB2 domain of the
three proteins interacts with the cytosolic region of APP and of related
proteins APLP1 and APLP2 at the level of a YENPTY sequence, which is common to
all these APPs (Guènette et al.,
1996
; Duilio et al.,
1998
; Russo et al.,
1998
, Zambrano et al.,
1997
). Other than the Fe65s, additional PTB-domain-containing
adaptors, the X11 family members and m-Dab1, bind to the APP cytodomain
(Borg et al., 1996
;
Howell et al., 1999
;
Homayouni et al., 1999
). In
turn, these adaptors interact with other proteins; in the case of Fe65, the WW
domain binds to several proteins, including Mena
(Ermekova et al., 1997
) and
the non-receptor tyrosine kinase Abl
(Zambrano et al., 2001
). In
the latter case, Fe65 recruits active Abl close to APP, which, upon
phosphorylation of its tyrosine 682, binds the Abl SH2 domain
(Zambrano et al., 2001
). The
PTB1 domain may interact at the membrane level, with the low-density
lipoprotein receptor-related protein LRP
(Trommsdorff et al., 1998
),
and in the nucleus, with the transcription factor CP2/LSF/LBP1
(Zambrano et al., 1998
) and
the histone acetyltransferase Tip60 (Cao
and Sudhof, 2001
).
Most of the genes encoding the proteins taking part in this complex
molecular machinery have been isolated and, in some instances, characterised
in C. elegans. In fact, apl-1 is the nematode orthologue of
the APP gene family; the structural properties of the encoded protein suggest
that APL-1 adopts the same topology as APP, and the similarity of the
C-terminal cytosolic domain of APL-1 with that of APP is strikingly high
(Daigle and Li, 1993),
suggesting an evolutionary conservation of the functions of this domain. Two
different presenilin genes, sel-12 and hop-1, are present in
C. elegans, where they control Notch signalling
(Levitan and Greenwald, 1995
;
Li and Greenwald, 1997
).
Nicastrin, and its nematode orthologue aph-2, have been proposed as
molecular links between presenilin and APP machineries
(Yu et al., 2000
).
lin-10 is the C. elegans orthologue of X11, and it is needed
for proper localisation of the let-23 gene product during vulval
development (Rongo et al.,
1998
). m-Dab and Mena orthologues have been first described in
Drosophila, where they genetically interact with the tyrosine kinase
abl gene (Gertler et al.,
1993
; Gertler et al.,
1995
), and their orthologues are also present in the C.
elegans genome.
The existence of three Fe65 proteins in mammals, all interacting with APP, renders the use of mouse genetic manipulation to study the functional role of Fe65s and of their complexes with APPs difficult. On the contrary, the lower complexity of the nematode genome enables the use of reverse genetic approaches in C. elegans to study the functions of the orthologues of mammalian gene families in a simpler, and in many cases, non-redundant genetic system. On the basis of this evidence, we attempted to identify, in the C. elegans genome, putative Fe65-like genes. In silico screening allowed us to find only one gene, which we call feh-1 (Fe65 homolog-1), encoding a protein with high degree of similarity to mammalian Fe65s. In fact, FEH-1 protein possesses the same modular organisation of the Fe65s, and its PTB2 domain interacts with the cytosolic domain of APL-1. Reverse genetics analyses demonstrated that the FEH-1 protein provides essential functions in C. elegans. Analysis of heterozygous worms, and of the phenotypes induced by RNA interference targeting either feh-1 or apl-1 transcripts, suggests that both genes are involved in a common molecular mechanism, whose alteration affects the rate of pharyngeal contractions.
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Materials and Methods |
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Bioinformatic tools, molecular methods and transgenic lines
Homology alignments were performed with the ClustalW protocol, available
from EBI (Thompson et al.,
1994). Blast searches (Altschul
et al., 1990
) were performed through the Sanger Centre Blast
server. Analysis of the genomic sequences was performed with Gene Finder,
which is available through the Baylor College of Medicine
(Favello et al., 1995
). In
silico isolation of the feh-1 gene was accomplished as described: the
HVFRCEAPAKNIATSLHEICSKIMSERR sequence was used to test C. elegans
databases with the TBLASTN protocol, and the expressed sequence tag, yk23f9.5
was identified. Its sequence allowed the isolation of the C. elegans
genomic sequence Y54F10. Analysis of this sequence with GeneFinder allowed the
identification of the coding regions of a putative feh-1 gene. The
yk423e6 clone was obtained by Y. Kohara and fully sequenced by the Sequenase
kit (Amersham Pharmacia Biotech). The resulting sequence was submitted to EMBL
database (Accession #AJ345015).
Standard methods were used for generation and propagation of the
recombinant constructs (Sambrook et al.,
1989). Restriction and modifying enzymes were from Roche;
oligonucleotides were from CEINGE. Polymerase chain reactions (PCR) for
cloning purposes were performed with Pfu DNA polymerase (Promega) or Gold Taq
(Perkin Elmer) for mutant screening.
The feh-1 cDNA regions encoding the WW and the PTB2 domains were amplified by PCR, using the yk423e6 clone as a template; the APL-1 cytodomain was obtained by amplification of Bristol N2 genomic DNA. The primers were:
WW-F: 5'-AGTGGATCCCCGAAAGATTTACCACCAGG-3'; WW-R: 5'-AGTGAATTCGGTCTCACGGTTCACTGGT-3'; PTB2-F: 5'-ACGGGATCCGCTATCGAATCAGGAGAAAAGA-3'; PTB2-R: 5'-ACGGAATTCGGCGTCGAGCACTTTTTGGT-3'; APL-1F 5'-ATCGGATCCACCAACGCTCGTCGTCGC-3'; APL-1R 5'-ACT-GAATTCGGCCTTCGAGTCGAAGAATG-3'.
The purified and digested products were cloned into the pGEX2TK vector (Amersham Pharmacia Biotech) for expression in Escherichia coli.
The 5' region of feh-1 (positions -2352 to +1311, +1 is at the A residue of the first methionine codon) was obtained by PCR amplification of Bristol N2 genomic DNA with the following primers: EXP-F1X: 5'-AAAATCTAGAGTATGTGTACGAGATTATCGCCT-3'; EXP-R1B: 5'-AATAAGGATCCCGATGTTCGGTCATAATTGT-TGTATC-3'.
The product was digested with XbaI and BamHI enzymes and
cloned into the pPD21.28 and pPD95.75 vectors, kindly provided by A. Fire
(Fire et al., 1990), to
generate, respectively, the 5'-feh-1::lac z, or the
5'-feh-1::GFP constructs. For the FEH-1 expression construct,
which was used to rescue the mutant phenotype (fl-feh-1::HA), the
complete gene, starting from the 5' end used in the
5'-feh-1::GFP constructs, to codon 693, was cloned in pPD95.75.
The primers were: EXPR-F1-P:
5'-AAAACTGCAGGTATGTGTACGAGATTATCGC-CT-3'; EXPR-R2-B:
5'-ACGGGATCCCGTTGTGCCGACCTAG-AAGGTACC-3'.
Green fluorescent protein (GFP) expression from the resulting construct was abolished by cloning, upstream of GFP, a 550 bp amplification product containing the hemagglutinin epitope (HA), a stop codon and the 3' end of the gene. The primers used were: F-HA-10.8: 5'-ACAGGATCCCTACCCATATGATGTTCCAGATTACGC-TGCACAACGACTTTGAATTCTTCAT-3'; 11250R-B: 5'-ACAGG-ATCCCATACCAGCCTGGTTACCTACC-3'.
Recombinant constructs were co-injected into the ovary of young adult
hermaphrodites to establish transformed lines; the transformation markers were
the wild-type lin-15 for injections into temperature-sensitive
lin-15(n765), multivulva (Muv) worms
(Clark et al., 1994). The
expression of wild-type lin-15 allowed reversion of Muv phenotype at
20°C and the identification of recombinant lines bearing the arrayed
constructs. Injections leading to phenotypic rescue were performed with the
elt-2::GFP vector (Fukushige et
al., 1999
) into feh-1 (+/gb561) worms.
Histochemical detection of ß-galactosidase
(Edgar, 1995) was performed on
acetone-fixed worms in a buffer containing 0.2 M sodium phosphate buffer, pH
7.5, 1 mM magnesium chloride, 0.004% (w/v) sodium dodecyl sulfate, 10 mM each
of potassium ferricyanide and potassium ferrocyanide, 0.4% X-gal.
Antibodies, pull-down assays and immune detections
The recombinant proteins used were obtained by isopropyl-thio galactoside
(IPTG) induction of bacterial cultures harbouring the corresponding constructs
in the pGEX2TK vector. Affinity purification of the glutathione S-transferase
(GST) fusion proteins was performed on glutathione-sepharose resin (Amersham
Pharmacia Biotech). FEH-1 and APL-1 antisera were obtained by immunisation of
rabbits with the GST-FEH-1-WW domain protein or with a peptide-ovalbumin
conjugate of the juxtamembrane sequence of APL-1 (PRIMM). To obtain FEH-1
purified antibodies, immune sera were deprived of GST antibodies on a GST
column, then purified on an antigen column. For APL-1 antibody purification, a
specific peptide column was set. The GST and GST-FEH-1-WW columns, as well as
the APL-1 peptide resin, were obtained by crosslinking the purified antigens
to CNBr-activated sepharose (Amersham Pharmacia Biotech), following the
instructions of the manufacturer. Fe65 antibodies have been described
(Zambrano et al., 1997). For
preadsorption control, the FEH-1 antibody was challenged with 10 µg of
purified antigen, then used in a western blot. Phosphatase treatment was
performed on FEH-1 immunoprecipitates from 500 µg of C. elegans
proteins as described (Zambrano et al.,
1998
). For the pulldown experiments, equal amounts of fusion
proteins (10 µg) were bound to glutathione-sepharose beads (10 µl) and
challenged with nematode or mouse brain extracts obtained by lysis in a buffer
containing 50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol,
50 mM NaF, 1mM Na vanadate, with a protease inhibitor cocktail (Complete,
EDTA-free, Roche). The extracts were clarified at 16,000 g at
4°C, and the protein concentration determined by the Bio-Rad protein assay
according to manufacturer's instructions. Unbound proteins were removed by
washing the beads three times with lysis buffer, whereas retained proteins
were resolved by SDS-PAGE, electroblotted to polyvinylidene fluoride
Immobilon-P membrane (Millipore) and analysed by western blot with FEH-1 and
APL-1 antibodies. Signals were detected with the ECL system (Amersham
Pharmacia Biotech). Loading of the GST proteins was checked by staining the
filters with Ponceau S (Sigma).
Whole-mount immunohistochemical detection
(Miller and Shakes, 1995) was
performed in a buffer containing 1% (w/v) bovine serum albumin, 0.1% NP-40
(v/v), 1 mM EDTA, in phosphate buffered saline (PBS) and appropriate dilutions
of the FEH-1 and MH27 antibodies. The latter was kindly provided by M. C.
Hresko and R. H. Waterston (Francis and
Waterston, 1991
). Fluorescent secondary antibodies (Jackson
Immunoresearch Lab) were diluted 1:200. Texas-red conjugated phalloidin
(Molecular probes), 1:20, was incubated for 15 minutes with worms previously
stained with FEH-1 antibodies to detect pharyngeal muscles.
Mutant generation and RNA-mediated interference
Young adult worms were mutated using standard protocols
(Yandell et al., 1994).
Briefly, Bristol N2 worms fed to DH5
E. coli cells were treated
with 4, 5', 8-trimethylpsoralen, 30 µg/ml, for 15 minutes, then
irradiated for 1 minute with 366 nm UV source. 24 hours later, F1 eggs were
allowed to hatch, then split at 1250 individuals per plate onto 96 NGM/agarose
plates. Identification of mutant addresses was performed by PCR on DNA
prepared from aliquots of nematode pools. Nested PCR was performed with two
pairs of oligonucleotides. The outer set was: F454:
5'-CTCCAGATCATTCCCGTAGAGGA-3'; R4734:
5'-TGAAGCTTTCCGATGAGGTTTGC-3'. The inner set was: F756:
5'-ACGACTCTCGTGGTTACTCTTCG-3'; R4232:
5'-CAGCAGCTCTACATCATCCCTAC-3'. Optimal reaction conditions tested
to detect preferentially the deleted allele were: 94°C, 30 seconds;
56°C, 30 seconds; 72°C, 90 seconds for 30 cycles. Nested PCR was
performed on 2 µl of a 1:100 dilution of the first amplification following
the same scheme. The wild-type allele was detected as a 3.5 kb band. One
population, giving rise to a shorter, 2-kilobase product, was subjected to
four cycles of sibling selection for isolation of the mutated clone. The
isolated heterozygous line was balanced by crossing with dpy-1(s2170)
males. This allele allows us to distinguish among +/+ and +/gb561
worms; +/+ worms have a dumpy phenotype, whereas +/gb561 appear
normal. 250 +/+ and 250 +/gb561 individuals, as well as 500
growth-arrested L1s, were individually collected from the offspring of
heterozygous worms and lysed in SDS buffer (100 mM Tris/HCl, pH 6.8, 4% SDS,
100 mM DTT) for detection of FEH-1 protein by western blot.
For double-stranded RNA-mediated interference (RNAi), feh-1 or apl-1 transcripts obtained by T7 RNA polymerase transcription of PCR-amplified products (RiboMax system, Promega) were injected into the ovary of young adult hermaphrodite worms. The primer pairs used for feh-1 were: T7-F1: 5'-taatacgactcactataggAAGCCGTGACGGAGCCATCTCT-3'; T7-R1: 5'-taatacgactcactataggCACCTTCATGTTTCTCCCATCC-3', which amplify the third exon of the gene; T7-F2:5'-taatacgactcactataggTCGCCTACGTGTCCCGTGATCG-3'; T7-R2:5'-taatacgactcactataggGATATCTAACTCTGCACTCGGC-3', which amplifies the seventh exon of feh-1. Lower case letters indicate the T7 promoter sequence. As a control for RNAi efficacy, the progeny of feh-1 dsRNA-injected worms, as well as a comparable number of mixed-stage individuals were lysed in SDS buffer for FEH-1 immunoblot.
For RNAi by feeding, the feh-1 cDNA from the yk423e6 construct was excised and cloned into pPD129.36 vector. The apl-1 cDNA region spanning positions 961-2045 was generated by RT-PCR on mixed-stage RNA samples with Superscript reverse transcriptase (Invitrogen) and Pfu polymerase (Promega). The reverse primer used for cDNA synthesis and PCR amplification was: apl-1R-X: 5'-ATATTCTAGACCTTCGAGTCGAAGAATGAGTACGT-3'; the forward primer was: apl-1F-H: 5'-ATCAAGCTTGAGATCGAGGCGGTTCATGAGGAG-3'.
The PCR product was digested with HindIII and XbaI
enzymes and cloned into pPD129.36. This vector contains a polylinker flanked
by two T7 RNA polymerase promoters. The corresponding apl-1 construct
was also used to amplify, with a T7 promoter primer, the template for
synthesis of the dsRNA used for apl-1 RNAi by injection. E.
coli cultures harbouring the generated feh-1 and apl-1
constructs in the HT115(DE3) strain were used as source for RNAi by feeding
(Timmons and Fire, 1998).
Bristol N2 embryos or L1 larvae were deposited onto NGM plates containing 10
µg/ml tetracycline, 100 µg/ml ampicillin, 1 mM IPTG, and IPTG-induced
bacteria and allowed to lay eggs. F1 individuals were assayed for phenotype.
Control worms were grown onto pPD129.36-transformed bacteria, without insert,
producing vector-encoded dsRNA.
Pharngeal pumping was determined on 30 cloned individuals from each line or RNAi population per experiment, blindly. Such populations were scored four times during 48 hours of observation. Each experiment was repeated three times. Standard deviation was calculated for each group. For statistical comparisons, Student's t test was used.
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Results |
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A rabbit antibody, which was raised against the recombinant WW domain of
FEH-1, fused to GST, was affinity purified and used in a western blot to
detect FEH-1 in worm extracts. Its specificity was tested by probing C.
elegans protein lysate with pre-immune serum and with antibody
pre-adsorbed on the antigen (Fig.
2A, lanes 1-2). Two groups of heterogeneously migrating bands were
detected with the FEH-1 antibody, a fast one ranging from 60 to 70 kDa, and a
slower one from 80 to 90 kDa (Fig.
2A, lane 3). The presence of two discrete groups of polypeptides
could be explained as deriving from translation starting from the two
methionines, 1 and 142 (Fig.
1A). Accordingly, expression of the cDNA contained in clone
yk423e6, which is devoid of methionine 1, in COS7 cells generated a protein
with a migration corresponding to the fastest group of bands (data not shown).
The complex heterogeneity of migration found in both groups of bands may be
interpreted as owing to post-translation modifications. Also, the migration of
mammalian Fe65, shown in lane 4, produces a complex pattern on SDS-PAGE, which
has previously been shown to be due, at least in part, to phosphorylation
(Zambrano et al., 1998). In
order to demonstrate that, similarly to Fe65, FEH-1 is phosphorylated, we
immunoprecipitated the protein from worm lysates and treated them with
alkaline phosphatase. As shown in lane 6 of
Fig. 2A, the mobility of FEH-1
is increased upon phosphatase treatment, compared with the untreated sample
(lane 5), suggesting that FEH-1 is indeed phosphorylated in nematode
cells.
|
The similarity of FEH-1 to mammalian Fe65s suggests that it may interact
with APL-1, the C. elegans homolog of mammalian APP
(Daigle and Li, 1993). To
address this point, we performed a pull-down assay by using the cytosolic
domain of APL-1 fused to GST. The coprecipitation assay clearly shows that
FEH-1 in worm lysates specifically interacts with the APL-1 cytodomain
(Fig. 2B). Conversely, the PTB2
domain of FEH-1, fused to GST, is able to coprecipitate with native APL-1
(Fig. 2C).
Null feh-1 mutants are homozygous lethal
The feh-1 gene structure derived from our analysis differs
slightly from the predicted coding sequence Y54F10AM.2, which is available
through WormBase (genetic map position: III:-14.02). In fact, both by sequence
analysis of yk423e6 cDNA clone and by RT-PCR analysis (data not shown), we
have not been able to detect in the feh-1 transcript any sequence
corresponding to that of the exon V, predicted in the Y54F10AM.2 sequence. In
addition, the small predicted exon X is contained in a larger exon (IX in the
actual gene), which is the last exon of the gene, as it contains the
translation stop codon (Fig.
3A).
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In order to analyse the function of feh-1 in the nematode, we generated and characterized a deletion mutant of the gene, gb561. A genomic fragment containing this deleted allele, obtained by PCR amplification from the mutagenised line and fully sequenced, allowed us to characterise the deletion. The latter consisted of a double deletion in feh-1, removing regions from intron II to the 5' end of intron IV, and from 3' end of intron IV to most of intron V (Fig. 3A, bottom). The resulting rearrangement of feh-1 will remove exons III to V, completely eliminating the WW domain, and the N-terminal region of PTB1 domain. The encoded, truncated protein will terminate with five extra amino acids after residue 147, as the fusion between exons II and VI will generate an out of frame transcript. Therefore, the gb561 allele is a null mutant as its locus can only encode a truncated FEH-1 devoid of the WW, PTB1 and PTB2 domains. This is confirmed by the western blot of Fig. 3B, in which we assayed protein lysates from wild-type, +/gb561 and gb561/561 worms for the levels of FEH-1. Reduced levels of protein are present in heterozygous worms, whereas no signal is detected in lysates from homozygous mutant worms.
The mutant worms present a recessive embryonic/larval lethal phenotype (Table 1). About 75% of the offspring of heterozygous hermaphrodites (+/gb561) reaches the adult stage, and these worms always belong to the +/+ and +/gb561 genotypes, as assessed by single worm PCR analysis, indicating a lethal effect of the homozygous gb561/gb561 phenotype. These last worms (22.9%), confirmed to be gb561/gb561 by PCR, either do not hatch (n=25) or arrest as L1s (n=31) (Table 1). Nomarski observation of the development of eggs laid by heterozygous hermaphrodites shows embryos that do not progress normally through development, and arrest during morphogenesis (Fig. 4A).
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Hatched homozygous mutant larvae arrest as L1. They have apparently normal morphology and movement and can remain alive on the plate for several days (up to 5-10 days). No pharyngeal pumping can be detected in these worms, suggesting that their growth and development may be blocked, at least in part, because they are not feeding efficiently.
Two approaches prove that the phenotypic defects of feh-1 gb561/gb561 embryos and larvae are indeed due to the disruption of the feh-1 gene and not to some other mutation present in the gb561 strain. First, we rescued the phenotype of the mutation with the fl-feh-1::HA construct (see experimental procedures) in which expression of the full-length FEH-1 protein is driven by 2.35 kb of sequences upstream of feh-1. The construct was injected in heterozygous feh-1 (+/gb561) worms together with a transformation marker, the elt-2::GFP construct, which drives a strong expression of GFP in gut cells beginning early in embryogenesis. We isolated three independent viable lines of feh-1 (gb561/gb561) worms carrying the transgenic array. The viability of worms from these lines was strictly dependent on the extra-chromosomal array, as all the adults express the GFP marker and the loss of GFP expression was always associated with embryonic lethality or larval arrest and with lack of detectable staining with an anti-FEH-1 antibody (data not shown). The second approach used to prove the phenotypes observed in gb561/gb561 worms are due to feh-1 ablation was based on the injection of feh-1 double-stranded RNA into wild-type young adult hermaphrodites, which resulted in F1 individuals presenting the same defects observed in feh-1 (gb561/gb561) mutants. 25 worms arrested as embryos, whereas 33 were arrested and pumping-impaired L1s (Table 1). Also in this case, the presence of the phenotypes correlates well with a decreased amount of FEH-1 in the offspring of feh-1 dsRNA-injected worms (Fig. 3C). The arrested embryos seen in the offspring of injected worms show a phenotype similar to that observed for the gb561/gb561, non viable embryos (Fig. 4A,B).
feh-1 is expressed in the neuromuscular structures of the
pharynx and in the nervous system
To study the expression of feh-1 in C. elegans and to try
to correlate it with the phenotype observed in the mutant and in
RNA-interfered worms, we used immune-detection of the protein and a reporter
expression approach. FEH-1 antibodies in whole-mount immunofluorescence
analysis showed a largely prominent localisation of the protein in the pharynx
at all stages of development, from three-fold stage embryos to adults. Barely
detectable staining could be observed also in some neurons in the nerve ring
and in the nerve chord and in the tail (not shown). In the pharynx the
fluorescent signals start from the most terminal end of the organ and extend
posteriorly to the second pharyngeal bulb and the attachment to the intestine
(Fig. 5A). Part of the protein
appears to uniformly fill the cytoplasm of the expressing cells but an
important fraction appears to associate with some sub-cellular
compartment/structure in the shape of filaments that form, at the level of the
second bulb, a characteristic basket-like structure. Confocal microscopy
analysis of worms double stained with anti-FEH-1 and anti-MH27 antibodies,
which recognizes JAM-1, a component of adherens junctions of epithelia, tends
to exclude FEH-1 staining from pertaining to the processes of epithelial
cells, as the two signals never overlap and appear topologically complementary
(Fig. 5B). Confocal microscopy
of worms stained with FEH-1 antibody and Texas-Red-conjugated phalloidin,
which decorates actin-rich muscle cells, indicates, instead, that FEH-1, at
least in part, colocalizes with actin in muscle cells in some areas of the two
pharyngeal bulbs and in some areas just anterior to the second bulb
(Fig. 5C). However, costaining
with Texas-Red phalloidin also shows association of FEH-1 with structures not
stained by phalloidin. The majority of these structures have a filamentous
shape and may resemble neuronal processes. The staining associated with the
anterior end of the basket-like structure of the second bulb appears to be
clearly different from that in the actin-containing muscle cells
(Fig. 5C).
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The C. elegans pharynx is a complex organ in which muscle,
neurons, epithelia and glands are tightly packed. In several cases a single
cell type takes up functions and morphological features usually segregated to
different ones, as in the case of the mioepithelial cells that serve a
contractile role for pumping but also secrete the cuticle that lines the lumen
of the pharinx. The shape of these cells is, as a consequence, often
unconventional and it is difficult to identify, in whole mounts, the cells in
which a protein is expressed. We thus turned to the study of worms that were
transgenic for constructs in which expression of the reporters
ß-galactosidase or GFP were driven by the sequences at the 5' end
of the feh-1 gene, which allowed the rescue of the gb561
mutant phenotype (see above). Furthermore, in the
ß-galactosidase-expressing construct, a nuclear localisation signal
allowed us to identify the nuclei of the cells expressing the transgene. The
genomic fragment used for all of our expression constructs comprises 2352
nucleotides upstream of the starting ATG codon and extends 1311 nucleotides
into the third exon (see Materials and Methods). The resulting constructs,
5'-feh-1::GFP and 5'-feh-1::NLS::lacZ,
were injected along with a selectable marker in the gonad of young adult
hermaphrodites and lines of transformed worms were established. Expression of
reporters confirmed that the main site of synthesis is the pharynx, but it is
also expressed in some neurons in the nerve ring, in the ventral chord and in
the tail (Fig. 6). During
embryogenesis, the expression of the constructs is detectable unambiguously
only after proliferation has ceased and the embryo has elongated to the two-
to three-fold stage. As already indicated by immunodetection, expression of
the reporters does not change substantially from embryogenesis and hatching to
larval stages and adults. Expression is strong in several neurons and in
pharyngeal cells. The GFP clearly outlines the shape of the expressing neurons
with their processes (e.g. in the ventral nerve chord,
Fig. 6A-C). On the contrary,
the very strong GFP signal detected in the pharynx did not allow the
identification of feh-1-expressing cells among those forming this
organ. The analysis of nuclear-targeted ß-galactosidase, expressed under
the control of the feh-1 gene promoter, allowed us to clearly
identify, in agreement with GFP reporter activity, the nuclei of some
extrapharyngeal neurons (Fig.
6D), as well as neurons of the ventral nerve chord (F) and of the
tail (G). Furthermore, in the procorpus and in the first pharyngeal bulb, the
nuclei of m3 and m4 muscle cells (Albertson
and Thomson, 1976) are also stained
(Fig. 6E). The staining of the
nuclei in the second bulb, where the density is higher, does not allow us to
unambiguously assign the feh-1-expressing cells; as shown in
Fig. 6E, two main spots of
ß-galactosidase activity can be seen, which may correspond to the m5
nuclei.
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The effect of FEH-1 dosage on pharyngeal pumping rate and the
interaction with apl-1
In arrested feh-1 (gb561/gb561) L1s, which completely lack FEH-1,
no visible contraction of the pharynx can be observed even with
high-resolution optical microscopy. To elucidate the role of FEH-1 in pumping,
we analysed pharyngeal contractions in heterozygous feh-1(+/gb561)
worms. As expected, since gb561 is recessive, contractions can be
observed; however, somewhat surprisingly, the rate of pharyngeal pumping of
these heterozygous worms is always and significantly higher than that of
wild-type N2 individuals (Fig.
7A). We also analysed feh-1(gb561/gb561) worms in which
the larval arrest phenotype of the mutation has been rescued by the
fl-feh-1::HA construct (see above). In these worms the rate of
pharyngeal contractions is also completely rescued to wild-type levels by the
transgene (Fig. 7A), indicating
that the effect on pumping depends on feh-1 dosage. We next asked if
we could reproduce the dosage effect on pumping rate observed in worms
heterozygous for the feh-1-null allele gb561 by reducing,
instead of completely abolishing, the expression of feh-1 and thus
generating worms hypomorph for feh-1. For this, we used RNA
interference through feeding. With this approach, it is possible to obtain a
decrease in the levels of the corresponding mRNA and, in turn, of the cognate
protein (Timmons and Fire,
1998). In fact, by feeding adult N2 hermaphrodites with bacteria
producing feh-1-specific dsRNA, we obtained RNA-interfered adult F1
individuals presenting an enhanced pumping rate
(Fig. 7B), similar to that of
heterozygous worms. It is interesting to note that, with RNA interference
obtained by feeding, we have not been able to produce the embryonic or larval
arrest phenotypes. The latter was instead obtained by microinjection of
synthetic feh-1 dsRNA, which reproduces the phenotype of the null
mutants (see above; Table 1).
These results suggest that reduced levels of FEH-1, although compatible with
feeding and larval development, have a clear effect on the rate of pharyngeal
contractions, whereas complete loss of this function, in feh-1
(gb561/gb561) homozygous larvae, results in the absence of pharyngeal
contraction and larval development arrest, probably because feeding is
impaired.
|
A crucial point that should be addressed is whether APL-1, which we have shown in this paper to physically interact with FEH-1, is involved in the same molecular pathways as FEH-1. To analyze this point, we injected into the worms a dsRNA of apl-1. This experiment resulted in a larval developmental arrest, similar to that observed in the case of feh-1 RNAi by injection (data not shown). Then, we generated a construct to perform RNA interference by feeding with bacteria producing apl-1-specific dsRNA. Worms grown on these bacteria show a normal development, with no obvious major abnormalities. However, their rate of pharyngeal pumping is significantly increased to the same extent observed in feh-1(+/gb561) heterozygous worms and in the feh-1 dsRNA-interfered worms, whereas the pumping rate of worms fed to control bacteria was not distinguishable from that of wild-type worms (Fig. 7B).
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Discussion |
---|
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---|
As discussed by Daigle and Li (Daigle
and Li, 1993), APP, APP-like proteins and APL-1 share high
sequence similarity at the level of three different protein regions: two of
them are located in the extracytosolic domain and contain the Cys-rich
regions, whereas the third region of high similarity resides in the short,
cytosolic domain. We have calculated, in the last 32 amino acids of the APL-1
cytodomain, corresponding to the APP region necessary for the interaction with
Fe65s and other PTB-containing adaptors, that 60% of the residues are
identical to human APP; this percentage gets to 75% if we consider the
conserved substitutions occurring in this region between human and nematode
proteins. This elevated degree of similarity suggests that the functional
conservation of the APL-1 cytodomain in the nematode system, and the evidences
provided in this study, confirms, both at the biochemical and at the
functional level, this possibility.
A 2.35 kb region present in the 5' region of feh-1 is able
to direct a strong expression of the gene in various neurons throughout the
body axis and in the pharynx. In the latter case, feh-1 expression is
present both in muscular and nerve cells. In mammals, Fe65 and
related genes show distinct expression patterns; in fact, Fe65 is
highly expressed in the central and peripheral nervous systems
(Duilio et al., 1991;
Simeone et al., 1994
), whereas
Fe65-L1 (Guènette et al.,
1996
) and Fe65-L2
(Duilio et al., 1998
) show a
broader expression in several organs. Fe65-L1 transcripts are
particularly abundant in skeletal and cardiac muscles, as well as in the brain
and kidney, whereas the Fe65-L2 gene is highly expressed in the brain
and testis. In the nematode, the expression of feh-1 in the nervous
system and in the pharynx, which is a neuromuscular organ, is in agreement
with the complex expression of the three genes found in mammals, in which,
however, the nervous system is a preferential site of expression.
On the basis of the functional redundancy of Fe65 gene products in mammals, we undertook reverse genetics approaches to reveal the functional roles of feh-1 in the nematode. We have shown, by analysis of both feh-1-null and RNA-interfered worms, that the absence or the marked reduction of FEH-1 is not compatible with proper development of C. elegans. Embryonic arrest phenotype results in severe developmental defects, leading to disorganised embryos, from which cells either detach or degenerate. One possible explanation for this evidence may be interpreted as owing to cell adhesion defects, although the complexity of this phenotype does not allow us to precisely interpret its molecular basis. The larval arrest phenotype has been characterised: the arrested worms were impaired in larval development because of the absence, or the severe reduction, of pharyngeal activity. The increased pharyngeal activity shown by the worms that were heterozygous for the deleted allele, or worms with milder reduction of feh-1 transcripts, obtained through RNAi by feeding, strongly associate feh-1 function with the control of the rate of pharyngeal contractions. Contraction of the pharynx results from the coordinated activity of its muscle cells, and the rate of this activity (pumping rate) is set by a cellular system functioning as pace-maker. An explanation of why reduction of FEH-1 function increases the rate of pumping, whereas complete loss results in no pumping, must await the elucidation of the precise role of FEH-1 in pumping. At present, a simple hypothesis to explain this apparent paradox is that FEH-1 is involved in negatively modulating the rate of pumping. In the complete absence of FEH-1, the rate would be set so high that, because of the intrinsic properties of its muscle cells, coordinated contraction of the pharynx is not possible anymore and therefore results in no visible or functional pumping.
The results obtained by RNA interference by feeding with either feh-1 or apl-1 dsRNAs combined with the documentation of the physical interaction between FEH-1 and APL-1, strongly suggest that the products of these genes are involved in a common molecular pathway, which controls pharyngeal pumping in C. elegans.
Many ion channels regulating the electrical events necessary for the
activity of this neuromuscular organ have been identified
(Fleischhauer et al., 2000;
Davis et al., 1999
;
Dent et al., 1997
;
Maryon et al., 1998
). In
addition, products of genes that regulate vesicular trafficking at the level
of the neuromuscular junctions have also been shown to be important for
pharyngeal function (Nonet et al.,
1998
). The mechanisms through which FEH-1 and APL-1 influence the
contraction rate of the pharynx remain to be understood. One potential
mechanism could act by regulating the proper localisation of ion channels at
the neuromuscular junction, but other possibilities can be hypothesised, such
as a possible role of the FEH-1/APL-1 complex in determining the correct
events of vesicle recycling at the pre-synaptic compartment. Interestingly, a
phenotype similar to that obtained with feh-1 mutants has been shown
in worms bearing a gain-of-function mutation in exp-2, a gene coding
for a Kv-type ion channel. In this case, heterozygous worms presented an
elevated rhythm of pharyngeal contractions and hyperactive head movements,
whereas worms homozygous for the gain-of-function allele died as L1 larvae
because of feeding abnormalities (Davis et
al., 1999
). Recent results suggest two different and apparently
unrelated roles for the FE65-APP complex. One concerns the possible
involvement of mammalian Fe65 and APP in the regulation of gene expression. In
fact, we have previously demonstrated that Fe65 is efficiently translocated
into the nucleus and that APP functions as a cytosolic anchor for Fe65
(Minopoli et al., 2001
). These
observations suggested a role for the APP processing machines in regulating
the nuclear translocation of Fe65; it could affect gene transcription by
interacting with transcriptional factors, such as CP2/LSF/LBP1
(Zambrano et al., 1998
) or
with histone acetyl transferase Tip60 (Cao
and Sudhof, 2001
). It is not excluded that a similar scenario may
be also acting in C. elegans, as presenilin orthologues, which might
be responsible for proteolytic processing of APL-1, are present in the
nematode, where they control a well-defined pathway centred on the maturation
of Notch membrane proteins, whose cytosolic domain is translocated to the
nucleus and regulates gene transcription
(Kimble and Simpson, 1997
;
De Strooper et al., 1999
;
Struhl and Greenwald, 1999
).
The availability of mutant worms null for feh-1 will represent an
ideal tool to identify the array of genes regulated by FEH-1. On the other
hand, it has been recently discovered that the Fe65-APP complex is involved in
the regulation of cell movement (Sabo et
al., 2001
), and unpublished observations have been mentioned,
concerning a possible role of APP and Fe65 in neuronal growth cone. This
observation deserves further experiments to analyse the possible role of FEH-1
and APL-1 in the regulation of neural cell movements, which can be affected in
mutant worms.
![]() |
Acknowledgments |
---|
This work was supported by grants from MURST-PRIN 2000, to N.Z., the V Framework program (Contract QLK6-1999-02238), the CNR-Italy `Programma Biotecnologie MURST L95/95' and `Progetto strategico: Basi biologiche delle malattie degenerative del sistema nervoso centrale' and Biogem, to T.R., the Associazione Italiana per la Ricerca sul Cancro, to P.B. and the Programma MURST, Cluster 02, to I.I.G.B.
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Albertson, D. G. and Thomson, J. N. (1976). The pharynx of Caenorhabditis elegans. Phil. Trans. R. Soc. Lond. B Biol. Sci. 275,299 -325.[Medline]
Altschul, S. F., Gish, W., Webb Miller, W., Myers, E. W. and Lipman, D. J. (1990). Basic local alignment search tool. J. Mol. Biol. 215,403 -410.[Medline]
Ando, K., Iijima, K., Elliot, J. I., Kirino, Y. and Suzuki,
T. (2001). Phosphorylation-dependent regulation of the
interaction of amyloid precursor protein with Fe65 affects the production of
beta-amyloid. J. Biol. Chem.
276,40353
-40361.
Borg, J. P., Ooi, J., Levy, E. and Margolis, B. (1996). The phosphotyrosine interaction domains of X11 and FE65 bind to distinct sites on the YENPTY motif of amyloid precursor protein. Mol. Cell. Biol. 16,6229 -6241.[Abstract]
Borg, J. P., Yang, Y. N., De Taddéo-Borg, M., Margolis,
B. and Turner, R. S. (1998). The X11alpha protein slows
cellular amyloid precursor protein processing and reduces Abeta40 and Abeta42
secretion. J. Biol. Chem.
273,14761
-14766.
Cao, X. and Sudhof, T. C. (2001). A
transcriptively active complex of APP with Fe65 and histone acetyltransferase
Tip60. Science 293,115
-120.
Clark, S. G., Lu, X. and Horvitz, H. R. (1994). The Caenorhabditis elegans locus in lin-5, a negative regulator of a tyrosine kinase signaling pathway, encodes two different proteins. Genetics 1370,987 -997.
Cuff, J. A. and Barton, G. J. (1999). Evaluation and improvement of multiple sequence methods for protein secondary structure prediction. Proteins 34,508 -519.[Medline]
Daigle, I, and Li, C. (1993). apl-1, a Caenorhabditis elegans gene encoding a protein related to the human beta-amyloid protein Proc. Natl. Acad. Sci. USA 90,12045 -12049.[Abstract]
Davis, M. W., Fleischhauer, R., Dent, J. A., Joho, R. H. and
Avery, L. (1999). A mutation in the C. elegans EXP-2
potassium channel that alters feeding behavior.
Science 286,2501
-2504.
Dent, J. A., Davis, M. W. and Avery, L. (1997).
avr-15 encodes a chloride channel subunit that mediates inhibitory
glutamatergic neurotransmission and ivermectin sensitivity in
Caenorhabditis elegans. EMBO J.
16,5867
-5879.
De Strooper, B., Annaert, W., Cupers, P., Saftig, P., Craessaerts, K., Mumm, J. S., Schroeter, E. H., Schrijvers, V., Wolfe, M. S., Ray, W. J., Goate, A. and Kopan, R. (1999). A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain. Nature 398,518 -522.[Medline]
De Strooper, B. and Annaert, W. (2000).
Proteolytic processing and cell biological functions of the amyloid precursor
protein. J. Cell Sci.
113,1857
-1870.
Duilio, A., Zambrano, N., Mogavero, A. R., Ammendola, R., Cimino, F. and Russo, T. (1991). A rat brain mRNA encoding a transcriptional activator homologous to the DNA binding domain of retroviral integrases. Nucleic Acids Res. 19,5269 -5274.[Abstract]
Duilio, A., Faraonio, R., Minopoli, G., Zambrano, N. and Russo, T. (1998). Fe65L2: a new member of the Fe65 protein family interacting with the intracellular domain of the Alzheimer's beta-amyloid precursor protein. Biochem. J. 330,513 -519.[Medline]
Edgar L. G. (1995). Blastomere culture and analysis. In Methods in Cell Biology, vol.48 (ed. H. E. Epstein and D. C. Shakes), pp.303 -321. San Diego: Academic Press.[Medline]
Ermekova, K. S., Zambrano, N., Linn, H., Minopoli, G., Gertler, F., Russo, T. and Sudol, M. (1997). The WW domain of neural protein FE65 interacts with proline-rich motifs in Mena, the mammalian homolog of Drosophila enabled. J. Biol. Chem. 272,32769 -32778.
Favello, A., Hillier, L. and Wilson, R. K. (1995). Genomic DNA sequencing methods. Methods Cell Biol. 48,551 -569.[Medline]
Fiore, F., Zambrano, N., Minopoli, G., Donini, V., Duilio, A.
and Russo, T. (1995). The regions of the Fe65 protein
homologous to the phosphotyrosine interaction/phosphotyrosine binding domain
of Shc bind the intracellular domain of the Alzheimer's amyloid precursor
protein. J. Biol. Chem.
270,30853
-30856.
Fire, A., Harrison, S. and Dixon, D. (1990). A modular set of lacZ fusion vectors for studying gene expression in Caenorhabditis elegans. Gene 93,189 -198.[Medline]
Fleischhauer, R., Davis, M. W., Dzhura, I., Neely, A., Avery, L. and Joho, R. H. (2000). Ultrafast inactivation causes inward rectification in a voltage-gated K(+) channel from Caenorhabditis elegans.J. Neurosci. 15,511 -520.[Abstract]
Francis, R. and Waterston, R. H. (1991). Muscle cell attachment in Caenorhabditis elegans. J. Cell Biol. 114,465 -479.[Abstract]
Fukushige, T., Hendzel, M. J., Bazett-Jones, D. P. and McGhee,
J. D. (1999). Direct visualization of the elt-2
gut-specific GATA factor binding to a target promoter inside the living
Caenorhabditis elegans embryo. Proc. Natl. Acad. Sci.
USA 96,11883
-11888.
Gertler, F. B., Hill, K. K., Clarck, M. J. and Hoffmann, F. M. (1993). Dosage-sensitive modifiers of Drosophila abl tyrosine kinase function: prospero, a regulator of axonal outgrowth, and disabled, a novel tyrosine kinase substrate. Genes Dev. 7,441 -453.[Abstract]
Gertler, F. B., Comer, A. R., Juang, J. L., Ahern, S. M., Clark, M. J., Liebl, E. C. and Hoffmann, F. M. (1995). enabled, a dosage-sensitive suppressor of mutations in the Drosophila Abl tyrosine kinase, encodes an Abl substrate with SH3 domain-binding properties. Genes Dev. 9,521 -533.[Abstract]
Goddard, J. M., Weiland, J. J. and Capecchi, M. R. (1986). Isolation and characterization of Caenorhabditis elegans DNA sequences homologous to the v-abl oncogene. Proc. Natl. Acad. Sci. USA 83,2172 -2176.[Abstract]
Guènette, S. Y., Chen, J., Jondro, P. D. and Tanzi, R.
E. (1996). Association of a novel human FE65-like protein
with the cytoplasmic domain of the beta-amyloid precursor protein.
Proc. Natl. Acad. Sci. USA
93,10832
-10837.
Guènette, S. Y., Chen, J., Ferland, A., Haass, C., Capell, A. and Tanzi, R. E. (1999). hFE65L influences amyloid precursor protein maturation and secretion. J. Neurochem. 73,985 -993.[Medline]
Heber, S., Herms, J., Gajic, V., Hainfellner, J., Aguzzi, A.,
Rulicke, T., Kretzschmar, H., von Koch, C., Sisodia, S., Tremml, P., Lipp, H.
P., Wolfer, D. P. and Muller, U. (2000). Mice with combined
gene knock-outs reveal essential and partially redundant functions of amyloid
precursor protein family members. J. Neurosci.
20,7951
-7963.
Homayouni, R., Rice, D. S., Sheldon, M. and Curran, T.
(1999). Disabled 1 binds to the cytoplasmic domain of amyloid
precursor-like protein 1. J. Neurosci.
19,7507
-7515.
Howell, B. W., Lanier, L. M., Frank, R., Gertler, F. B. and
Cooper, J. A. (1999). The disabled 1 phosphotyrosine-binding
domain binds to the internalization signals of transmembrane glycoproteins and
to phospholipids. Mol. Cell. Biol.
19,5179
-5188.
Kimble, J. and Simpson, P. (1997). The LIN-12/Notch signaling pathway and its regulation. Annu. Rev. Cell Dev. Biol. 13,333 -361.[Medline]
Levitan, D. and Greenwald, I. (1995). Facilitation of lin-12-mediated signalling by sel-12, a Caenorhabditis elegans S182 Alzheimer's disease gene. Nature 377,351 -354.[Medline]
Li, X. and Greenwald, I. (1997). HOP-1, a
Caenorhabditis elegans presenilin, appears to be functionally
redundant with SEL-12 presenilin and to facilitate LIN-12 and GLP-1 signaling.
Proc. Natl. Acad. Sci. USA
94,12204
-12209.
Maryon, E. B., Saari, B. and Anderson, P.
(1998). Muscle-specific functions of ryanodine receptor channels
in Caenorhabditis elegans. J. Cell Sci.
111,2885
-2895.
Miller, D. M. and Shakes, D. C. (1995). Immunofluorescence microscopy. In Methods in Cell Biology, vol. 48 (ed. H. E. Epstein and D. C. Shakes), pp. 365-394. San Diego: Academic Press.[Medline]
Minopoli, G., de Candia, P., Bonetti, A., Faraonio, R.,
Zambrano, N. and Russo, T. (2001). The beta-amyloid precursor
protein functions as a cytosolic anchoring site that prevents Fe65 nuclear
translocation. J. Biol. Chem.
276,6545
-6550.
Nonet, M. L., Saifee, O., Zhao, H., Rand, J. B. and Wei, L.
(1998). Synaptic transmission deficits in Caenorhabditis
elegans synaptobrevin mutants. J. Neurosci.
18, 70-80.
Rongo, C., Whitfield, C. W., Rodal, A., Kim, S. K. and Kaplan, J. M. (1998). LIN-10 is a shared component of the polarized protein localization pathways in neurons and epithelia. Cell 94,751 -759.[Medline]
Russo, T., Faraonio, R., Minopoli, G., De Candia, P., De Renzis, S. and Zambrano, N. (1998). Fe65 and the protein network centered around the cytosolic domain of the Alzheimer's beta-amyloid precursor protein. FEBS Lett. 434,1 -7.[Medline]
Sabo, S. L., Lanier, L. M., Ikin, A. F., Khorkova, O.,
Sahasrabudhe, S., Greengard, P. and Buxbaum, J. D. (1999).
Regulation of beta-amyloid secretion by FE65, an amyloid protein
precursor-binding protein. J. Biol. Chem.
274,7952
-7957.
Sabo, S. L., Ikin, A. F., Buxbaum, J. D. and Greengard, P.
(2001). The Alzheimer amyloid precursor protein (APP) and FE65,
an APP-binding protein, regulate cell movement. J. Cell
Biol. 153,1403
-1414.
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor University Press.
Sastre, M., Turner, R. S. and Levy, E. (1998).
X11 interaction with beta-amyloid precursor protein modulates its cellular
stabilization and reduces amyloid beta-protein secretion. J. Biol.
Chem. 273,22351
-22357.
Simeone, A., Duilio, A., Fiore, F., Acampora, D., De Felice, C., Faraonio, R., Paolocci, F., Cimino, F. and Russo, T. (1994). Expression of the neuron-specific FE65 gene marks the development of embryo ganglionic derivatives. Dev. Neurosci. 16, 53-60.[Medline]
Struhl, G. and Greenwald, I. (1999). Presenilin is required for activity and nuclear access of Notch in Drosophila.Nature 398,522 -525.[Medline]
Sulston, J. E. and Hodjkin, J. G. (1988). Methods. In The nematode Caenorhabditis elegans (ed. W. B. Wood), pp. 587-606. Cold Spring Harbor, NY: Cold Spring Harbor University Press.
Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl. Acids Res. 22,4673 -4680.[Abstract]
Timmons, L. and Fire, A. (1998). Specific interference by ingested dsRNA. Nature 395, 854.[Medline]
Trommsdorff, M., Borg, J. P., Margolis, B. and Herz, J.
(1998). Interaction of cytosolic adaptor proteins with neuronal
apolipoprotein E receptors and the amyloid precursor protein. J.
Biol. Chem. 273,33556
-33560.
von Koch, C. S., Zheng, H., Chen, H., Trumbauer, M., Thinakaran, G., van der Ploeg, L. H., Price, D. L. and Sisodia, S. S. (1997). Generation of APLP2 KO mice and early postnatal lethality in APLP2/APP double KO mice. Neurobiol. Aging 18,661 -669.[Medline]
Wolfe, M. S. and Haass, C. (2001). The Role of
presenilins in gammasecretase activity. J. Biol. Chem.
276,5413
-5416.
Yandell, M. D., Edgar, L. G. and Wood, W. B. (1994). Trimethylpsoralen induces small deletion mutations in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 91,1381 -1385.[Abstract]
Yochem, J. and Greenwald, I. (1993). A gene for a low density lipoprotein receptor-related protein in the nematode Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 90,4572 -4576.[Abstract]
Yu, G., Nishimura, M., Arawaka, S., Levitan, D., Zhang, L., Tandon, A., Song, Y. Q., Rogaeva, E., Chen, F., Kawarai, T. et al. (2000). Nicastrin modulates presenilin-mediated notch/glp-1 signal transduction and betaAPP processing. Nature 407,48 -54.[Medline]
Zambrano, N., Buxbaum, J. D., Minopoli, G., Fiore, F., De
Candia, P., De Renzis, S., Faraonio, R., Sabo, S., Cheetham, J., Sudol, M. and
Russo, T. (1997). Interaction of the phosphotyrosine
interaction/phosphotyrosine binding-related domains of Fe65 with wild-type and
mutant Alzheimer's beta-amyloid precursor proteins. J. Biol.
Chem. 272,6399
-6405.
Zambrano, N., Minopoli, G., de Candia, P. and Russo, T.
(1998). The Fe65 adaptor protein interacts through its PID1
domain with the transcription factor CP2/LSF/LBP1. J. Biol.
Chem. 273,20128
-20133.
Zambrano, N., Bruni, P., Minopoli, G., Mosca, R., Molino, D.,
Russo, C., Schettini, G., Sudol, M. and Russo, T. (2001). The
beta-amyloid precursor protein APP is tyrosine-phosphorylated in cells
expressing a constitutively active form of the Ab1 protoncogene. J.
Biol. Chem. 276,19787
-19792.
Zheng, H., Jiang, M. H., Trumbauer, M. E., Sirinathsinghji, D. J. S., Hopkins, R., Smith, D. W., Heavens, R. P., Dawson, G. R., Boyce, S., Conner, M. W. et al. (1995). beta-Amyloid precursor protein-deficient mice show reactive gliosis and decreased locomotor activity. Cell 81,525 -531.[Medline]