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First published online 3 August 2006
doi: 10.1242/dev.02503
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1 Department of Molecular Pharmacology and Neurobiology, Yokohama City
University Graduate School of Medicine, 3-9 Fukura, Kanazawa-ku, Yokohama,
236-0004, Japan.
2 CREST, JST, Kawaguchi Center Building, 4-1-8, Honcho, Kawaguchi-shi, Saitama
332-0012, Japan.
* Author for correspondence (e-mail: kenogura{at}med.yokohama-cu.ac.jp)
Accepted 20 June 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Caenorhabditis elegans, Axon guidance, Autophagy, Netrin, Kinase
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Yu and Bargmann,
2001; Dickson,
2002). Many of the molecules responsible for axon pathfinding
during development have been identified - however, little is known about how
these molecules are regulated to generate appropriate connections.
The axon guidance molecule Netrin was originally identified in
Caenorhabditis elegans as UNC-6
(Ishii et al., 1992). Some
axons are attracted to Netrin, but others are repelled by it
(Hedgecock et al., 1990;
McIntire et al., 1992;
Serafini et al., 1994;
Colamarino and Tessier-Lavigne,
1995). In C. elegans, Netrin/UNC-6 is secreted from
ventral cells (Wadsworth et al.,
1996), and it is thought to form a ventral-to-dorsal concentration
gradient (Wadsworth, 2002).
Two C. elegans Netrin receptors, UNC-5 and UNC-40 (mammalian DCC
homolog), are known (Chan et al.,
1996; Leung-Hagesteijn et al.,
1992). Both belong to the immunoglobulin superfamily, have a
single transmembrane domain and are expressed in neurons that respond to
Netrin (Chan et al., 1996;
Su et al., 2000). Both UNC-5
and UNC-40 are required for dorsally extending axons to be repulsed by ventral
Netrin (Hedgecock et al.,
1990; McIntire et al.,
1992). However, the ventrally extending axons are attracted to
ventral Netrin, and require only UNC-40 for this response. In mammals, Netrin
binds the Ig domain of UNC-5, and the fibronectin type III domain of
UNC-40/DCC (Kruger et al.,
2004).
In the unc-51 and unc-14 C. elegans mutants, there are
many neurons with guidance defects
(Hedgecock et al., 1985;
McIntire et al., 1992). In
addition, abnormal membrane structures (e.g. abnormally large varicosities or
cisternae-like structures) have been observed in their axons. UNC-51 is a
serine/threonine kinase homologous to yeast Atg1, which is required for
autophagy, a form of catabolic vesicle trafficking
(Ogura et al., 1994;
Matsuura et al., 1997;
Straub et al., 1997). UNC-14
is a novel protein that contains a RUN domain
(Ogura et al., 1997). While
the function of the RUN domain in UNC-14 is not known, RUN domains are
predicted to play important roles in the Rap and Rab family GTPase signaling
pathways that affect vesicle trafficking
(Callebaut et al., 2001).
UNC-51 and UNC-14 are expressed in many neurons, and UNC-51 directly binds
UNC-14; however, their molecular functions on axon guidance are largely
unknown.
Here, we report that UNC-51 and UNC-14 regulate the subcellular localization of the Netrin receptor UNC-5. We propose that UNC-5 uses a unique localization mechanism, which probably regulates its function.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The mutants used in this paper are shown below: LG I: unc-14(e57), unc-73(e936), unc-40(n324), unc-11(e47), hT2[qIs48] and unc-101(m1);
LG II: unc-104(e1265), rrf-3(pk1426) and juIs76(unc-25p::gfp);
LG III: hT2[qIs48];
LG IV: unc-5(e53), unc-44(e362), unc-33(mn407), juIs1(unc-25p::snb-1::gfp) and nuIs24(glr-1::gfp);
LG V: unc-51(e369), unc-51(ks38::Tc1) and unc-51(ks49);
LG X: lin-15(n765ts), unc-6(ju152), unc-6(ev400), kyIs156(str-1p::odr-10::gfp), nuIs9(unc-5::gfp) and oxIs12(unc-47p::gfp).
Molecular analysis
The unc-25 promoter region
(Jin et al., 1999) was
amplified by PCR from wild-type genomic DNA using KOD-Plus (TOYOBO). The
unc-25 promoter region was cloned into the GFP expression vector
pPD95.77. The resultant plasmid p77-u25KP was used to express several GFP
fusion proteins in DD/VD neurons. We also used monomeric red fluorescent
protein (mRFP) for double labeling
(Campbell et al., 2002). To
construct mRFP fusion proteins, the GFP-coding region of pPD95.77 was swapped
with the mRFP ORF. The unc-25 promoter region was cloned into the
resulting plasmid. This plasmid (pu25P-mR1) was used to express several mRFP
fusion proteins. Full-length ORFs were amplified by PCR from EST clones or
RT-PCR products. The EST clones used for the analysis were yk668c8
(unc-51), yk459e7 (unc-14), yk678c6 (unc-115),
yk484d2 (unc-40), yk344c5 (mig-2), yk643f1
(ced-10), yk622a3 (rab-5), yk648d7 (rab-7), yk664d6
(rab-11) and yk281f2 (lgg-1). The EST clones were kindly
provided by Y. Kohara.
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). MT8189
[lin15(n765ts) X] was used as the transformation strain, and pJM23
was used as the transformation marker. For the expression analysis, each
sample of 10
50 ng/µl DNA and pJM23 was injected into the adult gonad
(final concentration of DNA=100 ng/µl).
RNA-mediated interference
EST clones yk668c8 (unc-51/ATG1), yk656b6
(bec-1=ATG6), yk281g8 (M7.5=ATG7), yk281f2
(lgg-1=ATG8) and yk515b7 (F41E6.13=ATG18) were used as
templates for double-stranded RNA synthesis. Although RNAi is not generally
effective in C. elegans neural cells, rrf-3 mutants are
hypersensitive to RNAi (Sijen et al.,
2001), and RNAi can be used in these mutants very effectively to
silence at least axon guidance genes (e.g. unc-51, unc-14, unc-33,
max-1 and unc-73, and data not shown). We used
rrf-3(pk1426) mutants for our all RNAi analysis.
For the injection method, using the PCR-amplified insert, both RNA strands
were simultaneously synthesized with T3 and T7 RNA polymerase (Promega), and
the RNA mixture was heat-denatured and annealed to form double-stranded RNA.
Microinjection of the RNA was performed as described
(Mello et al., 1991;
Fire et al., 1998) at a
concentration of 34 mg/ml in TE. For the feeding method
(Kamath et al., 2000),
PCR-amplified inserts were cloned into pPD129.36 at the NotI site.
HT115(DE3) was used for the host strain.
Visualization of DD/VD axons
DD/VD neurons were visualized by using oxIs12
(=unc-47p::gfp)
(McIntire et al., 1997) or
juIs76 (=unc-25p::gfp)
(Huang et al., 2002). To
analyze the axon guidance defects in mutants, we counted the number of DD/VD
axons that could reach the dorsal nerve cord in L4 larvae or young adults. At
least 20 worms were examined and averaged for each strain.
Imaging analysis
An LSM510 confocal microscope (Zeiss) was used to collect the images.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), and
their cell bodies are located ventrally
(White et al., 1986). During
axon growth, they first extend their axons anteriorly, but, after some
extension, their axons branch into a short anterior branch and a dorsally
extending branch. When the dorsally extending branch reaches the dorsal nerve
cord, they branch again, with a short branch extending anteriorly and a longer
one extending posteriorly. Many genes required for DD/VD axon guidance have
been identified (McIntire et al.,
1992; Forrester and Garriga,
1997; Huang et al.,
2002; Wu et al.,
2002; Lundquist et al.,
2001).
We speculated that UNC-51 and UNC-14 might regulate the localization of
molecules required for DD/VD axon guidance, probably by vesicle trafficking,
for the following reasons: (1) mutants of unc-51 and unc-14
have defects in DD/VD axon guidance
(McIntire et al., 1992)
(Fig. 1C,D); (2) Atg1, a yeast
homolog of UNC-51, is required for autophagy
(Matsuura et al., 1997;
Straub et al., 1997); (3)
UNC-14 has a RUN domain, which is predicted to play important roles in the Rap
and Rab family GTPase signaling pathways for vesicle trafficking
(Callebaut et al., 2001); (4)
unc-51 and unc-14 are expressed in DD/VD neurons
(Ogura et al., 1997).
We first investigated the localization of the Netrin receptor UNC-5 in
DD/VD neurons, because their mutant phenotypes are similar to those in
unc-51 and unc-14
(McIntire et al., 1992)
(Fig. 1F), and UNC-5 is
expressed in the DD/VD neurons (Su et al.,
2000). In wild-type animals, functional UNC-5::GFP, which can
rescue the guidance defects of unc-5, is found associated with small
vesicles that are uniformly distributed in axons and cell bodies
(Killeen et al., 2002)
(Fig. 2A). In unc-51,
UNC-5::GFP accumulated in the neuronal cell bodies
(Fig. 2B,
Fig. 3B), but small amounts of
UNC-5::GFP were found in the axons (Fig.
2B). We examined UNC-5::GFP in unc-14 mutants, and
observed the similar abnormal accumulation in neuronal cell bodies
(Fig. 2D,
Fig. 3C). The accumulation of
UNC-5::GFP was never observed in wild type but commonly observed in the
unc-51 and unc-14 mutants.
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In the unc-51 and unc-14 mutants, cell-body accumulation UNC-5::GFP was observed in many neurons that normally express UNC-5 throughout development and adulthood (data not shown). Interestingly, the distribution of UNC-5::GFP seemed normal in non-neural distal tip cells (DTCs) (Fig. 4A-C) and excretory cells (Fig. 4D-F) in these mutants, suggesting that UNC-51 and UNC-14 regulate the subcellular localization of UNC-5 only in neurons.
UNC-51 and UNC-14 do not generally regulate the localization of proteins in neurons
We next examined the localization of another Netrin receptor, UNC-40, in
unc-51 and unc-14 mutants. UNC-40 is also required for the
axon guidance of the DD/VD neurons (Fig.
4G). In wild-type animals, functional UNC-40::GFP is localized to
the cell surface of DD/VD neurons (Chan et
al., 1996) (Fig.
4H); this was a different pattern to that of UNC-5::GFP (small
vesicles, Fig. 2A). The
UNC-40::GFP localization in unc-51 and unc-14 mutants
appeared to be normal (Fig.
4I,J).
There are many genes required for DD/VD axon guidance. To analyze whether
or not UNC-51 regulates the localization of proteins in DD/VD neurons
generally, we examined the localization of UNC-33 (CRMP)
(Li et al., 1992), UNC-76
(FEZ) (Bloom and Horvitz,
1997), UNC-115 (abLIM)
(Lundquist et al., 1998),
UNC-73 (Trio) (Steven et al.,
1998), CED-10 (Rac) (Reddien
and Horvitz, 2000) and MIG-2 (Rac)
(Zipkin et al., 1997), which
are required for DD/VD axon guidance. We also examined the localization of
VAB-8 (Wolf et al., 1998),
which is required for posterior axon outgrowth. We found their localization
were normal in the unc-51 and unc-14 mutants
(Table 1).
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) in the
AWB neurons, and GLR-1 (AMPA-class glutamate receptor)
(Maricq et al., 1995;
Hart et al., 1995) in several
interneurons of the head (Table
1). Their localizations were also normal in the unc-51
and unc-14 mutants. We also examined the localization of RAB-11
(recycling endosome marker) (Grant and
Hirsh, 1999) and LGG-1 (autophagosome marker)
(Melendez et al., 2003), in
DD/VD neurons. Their localization was not affected in the unc-51 and
unc-14 mutants. As abnormally large synapses are observed in
unc-51 and unc-14 mutants
(McIntire et al., 1992;
Crump et al., 2001), we
examined the SNB-1::GFP localization in these mutants. We found abnormal
accumulation of SNB-1::GFP in the DD/VD cell bodies of unc-51 and
unc-14 mutants (Table
1). These results suggest that UNC-51 and UNC-14 regulate the localization of UNC-5 and SNB-1, at least in regard to its accumulation in the cell body, but do not appear to regulate the localization of proteins generally in neurons.
Localization of UNC-5::GFP is normal in other guidance mutants, autophagy-related mutants, kinesin mutants and clathrin adaptor mutants
To distinguish the UNC-5 accumulation in unc-51 and
unc-14 mutants from secondary defects caused by the abnormal
path-finding of the DD/VD neurons, we examined the localization of UNC-5::GFP
in other mutants with guidance defects in the DD/VD neurons, including
unc-73, unc-44, unc-33, unc-40, unc-6 and max-1
(Table 2) and found that the
UNC-5::GFP localization was normal. These results suggest that the
accumulation of UNC-5::GFP in neuronal cell bodies was not a secondary effect
of improper axon guidance.
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).
Interestingly, UNC-5::GFP localization was normal in these RNAi worms
(Table 2). In addition, we did
not find any obvious co-localization of UNC-5::mRFP and the autophagosome
marker GFP::LGG-1 (data not shown). We concluded that conventional autophagy
was not involved in the accumulation of UNC-5.
In yeast, Atg1 is required for both cytoplasm-to-vacuole targeting (Cvt)
and autophagy. The Atg1 kinase activity is required only for Cvt but not for
autophagy (Abeliovich et al.,
2003). As unc-51(e369) has a mutation at the C-terminal
region (probable regulatory region), the kinase domain is intact. One
possibility is that, in unc-51(e369) mutants, the kinase activity of
UNC-51 may be unregulated by the mutation at the probable regulatory region,
resulting in abnormal Cvt. If this possibility is correct,
unc-51(RNAi) should show different phenotypes to those of
unc-51(e369). In addition, RNAi to unc-51(e369) mutants
should rescue the phenotypes. We found that unc-51(RNAi) showed an
unc-51-like phenotype with respect to UNC-5 vesicles (data not
shown). We also found that unc-51(RNAi) in the unc-51(e369)
mutant made the guidance defects more severe: the number of axons reaching the
nerve cord in unc-51(369); control (RNAi) hermaphrodites was
5.9, whereas in unc-51(e369); unc-51(RNAi) hermaphrodites it
was 3.8 (P<0.01, Bonferroni correction). These analyses indicate
that the phenotypes observed in unc-51(e369) do not result from
unregulated UNC-51 (abnormal Cvt).
We next examined the localization of UNC-5::GFP in mutants of motor
proteins or vesicle components, including kinesin mutants (unc-104
and unc-116) (Otsuka et al.,
1991; Patel et al.,
1993) and clathrin adapter mutants (unc-101 and
unc-11) (Lee et al.,
1994; Nonet et al.,
1999). UNC-5::GFP localization was also normal in these mutants
(Table 2), suggesting that
these proteins are not required for its localization.
Genetic interaction among unc-5, unc-6, unc-51 and unc-14
To elucidate the mechanism of accumulation of UNC-5 in the DD/VD neural
cell bodies in unc-51 and unc-14 mutants, we looked for
genetic interactions among unc-5, unc-6, unc-51 and unc-14
that would affect DD/VD axon guidance. First, we examined genetic interactions
between unc-5 and unc-51, and between unc-5 and unc-14.
unc-5(e53) is a null allele in which 100% of the axons are prevented from
reaching the dorsal nerve cord (Fig.
1F, Fig. 5A). As
e53 is a recessive mutant, e53/+ heterozygotes are identical
to wild-type animals (Fig. 5A,
e53/+). Using the unc-5(e53) heterozygotes, we found that
the low dose of unc-5 strongly enhanced the defects of both
unc-51 and unc-14 (Fig.
5A). These results indicate that unc-5 interacts
genetically with unc-51 and unc-14 and are consistent with
our hypothesis that UNC-51 and UNC-14 regulate the localization of UNC-5.
Overexpression of unc-5 in unc-51 or unc-14 mutants
had no effect on the guidance defects (data not shown).
We next examined the effect of genetic interactions between Netrin/unc-6 and unc-51, and between Netrin/unc-6 and unc-14 on DD/VD axon guidance. unc-6(ev400) is a null allele in which 100% of the axons are prevented from reaching the dorsal nerve cord (Fig. 1E; Fig. 5B). unc-6(ju152) is a very weak allele, and only 15.0% of the axons were blocked (Fig. 5B, wild type: 5.6%). We found that unc-6(ju152) strongly enhanced the defects of both of unc-51 and unc-14 (Fig. 5B), suggesting that unc-6 interacts genetically with unc-51 and unc-14.
We examined the effect of genetic interactions between another Netrin receptor/unc-40 and unc-51 on DD/VD axon guidance. unc-40(n324) is a null allele in which 80% of the axons are prevented from reaching the dorsal nerve cord (Fig. 4G, Fig. 5C). As n324 is a recessive mutant, n324/+ heterozygotes are identical to wild-type animals (Fig. 5C, n324/+). Using the unc-40(n324) heterozygotes, we found that the low dose of unc-40 did not enhance the defects of unc-51(ks38::Tc1) (Fig. 5C). These results are consistent with our results that UNC-51 did not regulate the localization of UNC-40.
We finally examined the effect of genetic interactions between unc-51 and unc-14 on DD/VD axon guidance. unc-51(e369) is a severe allele and unc-14(e57) is a null allele. We found that the phenotypes of the double mutant unc-51(e369); unc-14(e57) were identical to those of unc-51(e369) (Fig. 5A), indicating that unc-51 and unc-14 function in the same genetic pathway. This result is consistent with our previous work showing that UNC-51 directly binds UNC-14. UNC-51 and UNC-14 probably cooperate to regulate the localization of UNC-5 in DD/VD neurons.
The combination of the transmembrane and cytoplasmic domains are necessary and sufficient for the regulation by UNC-51
We thought that the association of UNC-5 with small vesicles might be
related to its abnormal cell-body accumulation in unc-51 and
unc-14 mutants, given that the cell-surface localization of UNC-40
was normal in these mutants. We tried identifying the localization signal that
targeted UNC-5 to small vesicles.
First, we found that neither the extracellular nor cytoplasmic domain was essential for the localization to small vesicles, as deletion of these domains did not affect it (Fig. 6B,J,C,L). In order to avoid the effect of endogenous UNC-5 in our experiments, we also examined the deleted UNC-5 localization in unc-5(e53) null mutants. The results were identical to those in a wild-type background (data not shown), suggesting that these deleted UNC-5 proteins were capable of localizing at small vesicles by themselves. We next examined the importance of the transmembrane domain of UNC-5, and found that it was essential for the localization, as the deletion of the transmembrane domain completely abolished the localization of UNC-5 to small vesicles (Fig. 6D,N,E,O). We examined the localization of the transmembrane domain alone, and found that it did not localize to small vesicles at all (Fig. 6F,P). These results suggest that the targeting of UNC-5 to small vesicles is regulated redundantly by the extracellular and cytoplasmic domains.
As UNC-5 does not have any obvious N-terminal hydrophobic signal sequence for insertion into the membrane, the lack of the signal sequence could result in its unique localization to small vesicles. We tried adding the signal sequence from UNC-40 to UNC-5 in the hope that the resulting protein would convert its localization to the cell surface. However, this change did not affect the localization at all (Fig. 6G,Q), suggesting that the localization control of UNC-5 that locates it to small vesicles is stronger than the targeting of the signal sequence to the cell-surface membrane.
We next examined the localization of these modified UNC-5 proteins in unc-51(e369) mutants. We found that although both the UNC-5 protein with the deleted cytoplasmic domain and the UNC-5 protein with the deleted the extracellular domain localized at small vesicles in wild type (Fig. 6J,L), only the UNC-5 protein with the deleted the extracellular domain accumulated at the cell bodies in the unc-51(e369) mutants (Fig. 6K). These results suggested that the combination of the transmembrane and cytoplasmic domains were necessary and sufficient for the regulation by UNC-51.
In the course of the experiments, we found that the cytoplasmic domain of UNC-5 strongly localized at nuclei of the DD/VD neurons (Fig. 6N). UNC-5 might work at nuclei for the regulation of transcription of the genes required for axon guidance.
UNC-5 and UNC-51 or UNC-5 and UNC-14 are partly co-localized in DD/VD neurons
We next examined the co-localization of UNC-5, UNC-51 and UNC-14 in DD/VD
neurons. UNC-51::GFP and UNC-14::GFP often appeared as puncta in the axons or
cell bodies (Fig. 7A,D). We
found that UNC-51::GFP and UNC-5:mRFP or UNC-14::GFP and UNC-5::mRFP were
partly co-localized in DD/VD neurons (Fig.
7C,F, arrowheads).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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UNC-51 and UNC-14 regulate the proper localization of UNC-5 in neurons by an unknown mechanism
We have shown that, in unc-51 and unc-14 mutants, the
Netrin receptor UNC-5 accumulated in the cell bodies of DD/VD neurons.
Cell-body accumulations of UNC-5::GFP were also observed in many neurons that
express UNC-5 at all stages of development and adulthood (data not shown).
This UNC-5::GFP accumulation did not result as a secondary effect of the
misrouted axons, as other guidance mutants (unc-73, unc-44, unc-33,
etc.) have normal localization of UNC-5::GFP in DD/VD neurons. A simple
explanation is that UNC-51 and UNC-14 regulate the subcellular localization of
UNC-5. We have also shown that the localization of other GFP fusion proteins
(UNC-76, UNC-44, UNC-33, etc.) was normal in unc-51 and
unc-14 mutants, except for SNB-1, suggesting that UNC-51 and UNC-14
regulate the localization of UNC-5 and SNB-1.
In unc-51 and unc-14 mutants, we did not find an abnormal accumulation of UNC-5::GFP in non-neural cells (DTCs or excretory cells), suggesting that UNC-51 and UNC-14 regulate the localization of UNC-5 only in neurons. These results are consistent with the fact that guidance defects are not observed in non-neuronal cells (DTCs or excretory cells) in unc-51 or unc-14 mutants.
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UNC-51 and UNC-14 cooperate to regulate the localization of UNC-5
We have shown that unc-5 and unc-6 interacted genetically
with unc-51 and unc-14 to affect DD/VD axon guidance, as
low-dose unc-5 or a very weak allele of unc-6 strongly
enhanced the defects of unc-51 and unc-14. In addition,
UNC-5, UNC-51 and UNC-14 partly co-localized in the DD/VD neurons. These are
consistent with our hypothesis that UNC-51 and UNC-14 regulate the subcellular
localization of UNC-5. From these findings, we concluded that the
unc-51 and unc-14 defects in the dorsal axon guidance of the
DD/VD neurons is explained at least in part by the abnormal localization of
UNC-5. That is, the abnormal localization of UNC-5 in the DD/VD neurons in
unc-51 and unc-14 mutants probably leads to a paucity of
UNC-5 at the cell membrane of growth cones, so that they respond weakly if at
all to Netrin/UNC-6, resulting in abnormal axon guidance.
We have also shown that the phenotypes of unc-51(e369); unc-14(e57) double mutants were identical to those of unc-51(e369), indicating that unc-51 and unc-14 function in the same genetic pathway. This is consistent with our previous data that UNC-51 can bind UNC-14 directly. We think that UNC-51 and UNC-14 cooperate to regulate the localization of UNC-5.
The transmembrane and cytoplasmic regions are important for regulation by UNC-51
We have shown that none of the major protein domains of UNC-5, the
extracellular, transmembrane or cytoplasmic domains, was sufficient to target
UNC-5 to small vesicles. The combination of the extracellular domain and the
transmembrane domain, or of the transmembrane domain and the cytoplasmic
domain, was sufficient for this targeting. From these results, we conclude
that the localization of UNC-5 to small vesicles is probably regulated
redundantly by the extracellular and cytoplasmic domains.
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Possible function of UNC-51 and UNC-14 in UNC-5 localization
Our working model of the localization of the Netrin receptor UNC-5 is shown
in Fig. 8A. In our model, UNC-5
is associated with a small vesicle that is transported to the axon by an
unknown mechanism. UNC-51 and UNC-14 cooperate to regulate the formation,
selection or transport of the vesicle.
For the reasons given below, we think that UNC-51 and UNC-14 may cooperate
with unknown motor proteins to regulate the transport of UNC-5. (1) Synaptic
vesicle components are abnormally localized in the kinesin mutants
unc-104 and unc-116 (Byrd
et al., 2001; Sakamoto et al.,
2005). UNC-14 cooperates with kinesin UNC-116 for synaptic vesicle
transport. (2) UNC-51 can bind VAB-8, a kinesin-like protein, and
phosphorylate VAB-8 in vitro (Wolf et al.,
1998; Lai and Garriga,
2004). Overexpression of vab-8 partially suppresses the
guidance defects of unc-51 mutants seen in the posterior outgrowth of
CAN. Although VAB-8 probably cooperates with UNC-51 for the posterior
outgrowth of CAN, we think that VAB-8 is unlikely to regulate UNC-5
localization, because the dorsal axon guidance that requires UNC-5 is normal
in vab-8 mutants (Wightmann et al., 1996). There are 21 kinesins, six
dyneins and 17 myosins in the C. elegans genome
(The C. elegans Sequencing
Consortium, 1998). An unknown motor protein may function with
UNC-51 and UNC-14 to regulate UNC-5 localization.
In mammals, UNC51.1, a mouse homolog of UNC-51, binds SynGAP and Syntenin,
and their complex regulates Rab5-mediated endocytosis in the formation of
parallel fibers in cerebellar granule neurons
(Tomoda et al., 1999;
Tomoda et al., 2004). By
analogy, UNC-51 and UNC-14 may regulate Rab5-mediated endocytosis to affect
the regulation of UNC-5 localization. Unfortunately, we could not test this
possibility, as rab-5(RNAi) causes embryonic death. Conversely, again
by analogy, an attractive hypothesis is that UNC51.1 regulates the
localization of the mammalian homolog of UNC-5 in neurons.
In C. elegans, autophagy is essential for dauer development and
life-span extension (Melendez et al.,
2003). Autophagy-related genes (unc-51/ATG1,
bec-1/ATG6, M7.5/ATG7, lgg-1/ATG8 and F41E6.13/ATG18)
are required for this process. In our RNAi analysis using rrf-3
mutants hypersensitive to RNAi, except for unc-51/ATG1,
these autophagy-related genes were not required for DD/VD axon guidance or the
localization of UNC-5. We think that conventional autophagy is not related to
the localization of UNC-5, and that UNC-51/Atg1 has another unknown function
in its localization.
UNC-51 and UNC-14 may be required for the maturation of UNC-5. Mutating the
extracellular domain of GLP-1 (a Notch-like receptor) results in the
accumulation of GLP-1 in the cell body
(Wen and Greenwald, 1999).
This accumulation is suppressed by a mutant of sel-9, whose product,
SEL-9, is thought to function in the quality control of the endoplasmic
reticulum-Golgi transport of GLP-1. In the unc-51 and unc-14
mutants, UNC-5 could accumulate in neural cell bodies because of a transport
failure owing to quality control.
UNC-5 may use a unique transport mechanism, silencing, for its surface localization at the growth cone
In Drosophila melanogaster, the surface expression of Robo, a
receptor for the repulsive guidance molecule Slit, is regulated by Comm
(Georgiou and Tear, 2002;
Keleman et al., 2002). If Comm
and Robo are present in the same neuron, Comm sorts Robo into vesicles bound
for late endosomes and lysosomes (Keleman
et al., 2005). If Robo is present without Comm, Robo is packaged
into vesicles delivered to the growth cone.
UNC-5 may also use a unique transport mechanism for its surface
localization at the growth cone. In C. elegans, DD/VD neurons have
`C'-shaped axons, of which the first anterior branch does not respond to
Netrin/UNC-6 (Fig. 1B,
Fig. 8B). This `silencing' of
UNC-5 function is probably not owing to the transcriptional regulation of
unc-5, given that when unc-5 expression was under control of
the unc-25 promoter, which drives expression from the birth of the
DD/VD neurons into adulthood (Jin et al.,
1999), the `C'-shaped axonal morphology was not affected (data not
shown). Silencing of UNC-5 function has already been reported for the
migration of hermaphrodite DTCs (distal tip cells)
(Su et al., 2000); however,
the molecular mechanism of this silencing is unknown.
We think that, throughout the extension of the first axonal branch, UNC-5
may be packaged in an UNC-51- and UNC-14-mediated manner into small vesicles,
thus silencing the UNC-5 (Fig.
7B). Some unknown `UNC-5 surfacing signal' may localize UNC-5 to
the cell membrane at the right times and places for the growth cone to respond
to Netrin/UNC-6 with dorsal extension. The regulation of UNC5H1 (UNC-5
homolog) surface expression has been reported for mammalian cells
(Williams et al., 2003).
Here we reported that the localization of the Netrin receptor UNC-5 was cooperatively regulated by UNC-51 and UNC-14. However, the molecular mechanisms underlying the localization of UNC-5 and the transport mechanism used are still unclear. The isolation and characterization of new mutants that have defects in UNC-5 localization or suppress the unc-51 and unc-14 phenotypes, will help us understand the precise mechanism of UNC-5 regulation. Analysis of the effect of UNC51.1 (the mammalian homolog of UNC-51) on the localization of the homolog of UNC-5 will reveal its evolutionarily conserved roles.
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ACKNOWLEDGMENTS |
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