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First published online 4 April 2007
doi: 10.1242/dev.02842
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1 Department of Cell and Developmental Biology, School of Dentistry, Seoul
National University, Seoul 110-740, Republic of Korea.
2 School of Biological Sciences, College of Natural Sciences, Seoul National
University, Seoul 151-742, Republic of Korea.
3 Department of Cell and Developmental Biology, Vanderbilt University Medical
Center, Nashville, TN 37232-2175, USA.
4 Department of Physiology, School of Dentistry, Seoul National University,
Seoul 110-740, Republic of Korea.
* Author for correspondence (e-mail: seunglee{at}snu.ac.kr)
Accepted 22 February 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: kra, shot, Translation, Axon guidance, CNS, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Dickson, 2002). As growth
cones encounter these cues, they adapt their responses. When reading a
chemical gradient, growth cones continuously reset their threshold sensitivity
to the cue so that higher concentrations elicit cytoskeletal responses
required for motility and guidance (Song
and Poo, 2001). Locally presented cues may also qualitatively
change growth cone responsiveness, enabling them to proceed to the next
cellular target in the pathway (Song and
Poo, 2001).
Growth cone adaptation may require new local protein synthesis
(Dickson, 2002). Axonal or
dendritic transport may not be fast enough to supply proteins to growth cones
at a distance from the cell body. Axotomy experiments with protein synthesis
inhibitors indicate that new protein synthesis in axons or their growth cones
is required for turning in vitro in response to extracellular gradients of
guidance cues (Campbell and Holt,
2001; Ming et al.,
2002). Local protein synthesis is also required for growth cones
to change their responsiveness to specific local cues. In the spinal cord of
vertebrates, translation of ephA2 receptor mRNA is locally regulated,
occurring in commissural growth cones only after they cross the midline
(Brittis et al., 2002).
Translational regulation in axons and their growth cones may involve
rapamycin- or MAP kinase (MAPK)-sensitive pathways
(Campbell and Holt, 2001;
Campbell and Holt, 2003;
Ming et al., 2002), and also
cytoplasmic polyadenylation element (CPE)-dependent mechanisms
(Brittis et al., 2002).
However, the mechanism connecting receptor signaling to translation is largely
unknown.
In the Drosophila embryo, the midline repellent Slit prevents
longitudinally directed CNS axons from crossing the midline
(Kidd et al., 1999) and
directs them into particular longitudinal pathways through its Robo, Robo2 and
Robo3 receptors on the CNS growth cones
(Kidd et al., 1999;
Simpson et al., 2000). As we
show here, the spectraplakin Short stop (Shot; also known as Kakapo) is also
required for midline axon repulsion. Mutations in shot lead to
ectopic midline crossing of Fas II-positive axons and dominantly enhance the
slit or robo heterozygous loss-of-function phenotypes,
suggesting that shot may function in the same guidance process as
slit and robo.
Shot mediates direct interactions between F-actin and microtubules (MTs)
required for initial sensory axon extension and motor axon extension to target
muscles (Lee and Kolodziej,
2002b). We provide evidence that the role of Shot in midline
guidance involves translational regulation. Shot physically interacts with
Krasavietz (Kra; also known as Exba), an evolutionarily conserved putative
translation factor, and this interaction is required for Shot activity to
support midline axon repulsion. Kra contains a C-terminal W2 domain found in
the translation initiation factors eIF5, eIF2B
, DAP-5 and eIF4G. The W2
domains in these translation initiation factors have been shown to mediate
protein-protein interactions among translation factors that are required for
preinitiation complex assembly (Preiss and
Hentze, 2003). As suggested by the presence of W2 domain, Kra
binds to eIF2ß and 40S ribosomal subunits. We also show that Kra inhibits
translation in vitro. Finally, we show that in kra or
eIF2ß mutant embryos some Robo-expressing axons lose the ability
to respond to the midline repellent Slit. Taken together, our data suggest
that Shot functions as a platform for translational control of midline axon
guidance. Through this proposed Shot-Kra-eIF2ß circuit, the translation
of mRNAs encoding proteins essential for axon guidance at the midline may be
closely tied to the pace of cytoskeletal assembly.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), a derivative of the pUAST vector
(Brand and Perrimon, 1993), to
give UAS-HA-kraWT. The wild-type
kra cDNA was manipulated using the QuikChange Multi kit (Stratagene)
to introduce the 12A and 7A mutations (Fig.
1B). The insert of
UAS-HA-kraWT was replaced with
kra12A and kra7A cDNAs to give
UAS-HA-kra12A and
UAS-HA-kra7A, respectively. To overexpress
eIF2ß in the fly embryo, the entire coding sequence was tagged
with green fluorescent protein (GFP) and introduced into pUASTNEXT.
Genetics
Two P-element insertions in the kra locus, l(3)j9b6
(Bloomington Stock Center, Bloomington, IN) and EP(3)0428 (Szeged
Stock Center, Hungary), were obtained and mobilized by standard methods to
generate kra1 and kra2, respectively.
For the kra rescue experiments, transgenic flies expressing
UAS-HA-kraWT, UAS-HA-kra7A
and UAS-HA-kra12A cDNAs were obtained as
previously described (Robertson et al.,
1988). These transgenes were expressed in the
kra1/kra2 mutant embryos under the control of
da-GAL4 (Wodarz et al.,
1995), C155-GAL4 (Lin
and Goodman, 1994), repo-GAL4
(Sepp et al., 2001) or
slit-GAL4 (Scholz et al.,
1997). shot rescue experiments were performed as
previously described (Lee and Kolodziej,
2002b). slit2 and robo2
were obtained from the Bloomington Drosophila Stock Center.
Cell culture and double-stranded RNA interference
S2 cells were grown at 29°C in Schneider's medium (Invitrogen)
supplemented with 10% heat-inactivated (30 minutes, 55°C) fetal calf
serum. S2 cells were transfected using Cellfectin (Invitrogen) according to
the manufacturer's instructions.
Double-stranded RNA interference (dsRNAi) was performed in six-well tissue
culture plates containing 2x106 S2 cells for 3 days as
previously described (Clemens et al.,
2000). Briefly, DNA templates containing T7 promoter sequences at
their 5' and 3' ends were amplified by PCR and transcribed with
the Megascript T7 transcription kit (Ambion) to generate dsRNAs. Primers
contained T7 promoter sequences upstream of the following: kra sense
primer, 5'-TTGGTCCACCATCATGTCATT-3', kra antisense
primer, 5'-ACTGAAGCCATTCGACAAAC-3'; gfp sense primer,
5'-ACGTAAACGGCCACAAGTTC-3', gfp antisense primer,
5'-GTCCTCCTTGAAGTCGATGC-3'. For the assay, dsRNAs were used at a
final concentration of 37 nM.
Antibodies and immunohistochemistry
In order to raise antibodies against the full-length Kra and eIF2ß
proteins, GST-Kra and MBP-eIF2ß were expressed in Escherichia
coli BL21 (Stratagene) and purified with glutathione-Sepharose 4B
(Amersham Pharmacia) and amylose resin (NEB), respectively. GST-Kra was
digested with PreScission protease (Amersham Pharmacia). Protein samples were
subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the bands
representing 50 kDa Kra and 82 kDa MBP-eIF2ß were excised for the
immunization of guinea pigs and rats, respectively.
Monoclonal antibodies (mAbs) against Fasciclin II (1D4), Repo (8D12),
Wrapper (10D3), Robo (13C9) and Slit (C555.6D) were purchased from the
Developmental Studies Hybridoma Bank (DSHB). Additional antibodies used in
this study were rabbit anti-eIF4E
(Nakamura et al., 2004), 3F10
rat monoclonal anti-HA (Roche), rabbit anti-ß-galactosidase (Cappel),
rabbit anti-GFP (Abcam), rabbit anti-horseradish peroxidase (HRP) (MP
Biochemicals) and goat anti-L28 (Santa Cruz).
Whole-mount staining of embryos was performed as previously described
(Lee et al., 2000;
Spencer et al., 1998).
Binding experiments
For GST pull-down assays, GST fusion proteins of Kra and eIF5 were produced
in E. coli and purified using glutathione-Sepharose 4B (Amersham
Pharmacia). eIF2ß and the C-terminal regions of the Shot long isoforms
were synthesized using an in vitro transcription/translation kit (Promega) in
the presence of [35S]methionine. The binding of GST-Kra to the
C-terminal regions of Shot was performed in 20 mM Tris-HCl (pH 7.5), 1 mM
CaCl2, 1% NP-40, 150 mM NaCl, 5 mM DTT and 10% glycerol, or in 20
mM Tris-HCl (pH 7.5), 1 mM EGTA, 0.1% NP-40, 150 mM NaCl, 5 mM DTT and 10%
glycerol. The additional binding experiment was performed in a binding buffer
containing 20 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM EDTA and 0.5% NP-40.
For immunoprecipitation, fly embryos or S2 cells coexpressing HA-Kra with GFP-eIF2ß, Shot L(A)-GFP or C-Shot L-GFP were homogenized in immunoprecipitation buffer [50 mM Tris-HCl (pH 8.0), 1% NP-40, 150 mM NaCl, 2 mM Na3VO4, 10 mM NaF, 10% glycerol, protease inhibitors], and then centrifuged at 12,000 g for 25 minutes at 4°C. Supernatants were precleared by incubation with protein G-Sepharose (Pierce) for 1 hour at 4°C. The samples were incubated with anti-GFP or anti-HA for 4 hours at 4°C and then incubated with protein G-Sepharose for 2 hours at 4°C. Beads were washed three times with immunoprecipitation buffer and boiled in SDS sample buffer. The eluates were subjected to western blotting.
In vitro translation assays
For in vitro translation, rabbit reticulocyte lysate (Promega) was first
preincubated with 2.4 µM bovine serum albumin (BSA), GST, GST-Kra, Kra or
Kra-7A. Each translation reaction contained 50% rabbit reticulocyte lysate,
1xTNT reaction buffer (Promega), 40 µM AA mixture without methionine
(Promega), 10 µCi [35S]methionine (Amersham Pharmacia), 20 U of
RNase inhibitor (Promega) and 70 nM luciferase mRNA. After incubating
the reaction for 90 minutes at 30°C, 20% of each reaction was removed and
analyzed by SDS-PAGE and autoradiography to monitor the translation
output.
Sucrose gradient analysis
S2 cell extracts were prepared in lysis buffer [50 mM Tris-HCl (pH 7.5),
250 mM NaCl, 50 mM MgCl2, 100 µg/ml heparin, 1 mM DTT and 0.5
mg/ml cycloheximide]. Cell lysates were layered on 7-47% (w/v) linear sucrose
gradients in the lysis buffer, and centrifuged at 270,000 g
for 3 hours at 4°C in a Beckman SW41 rotor. Fractions were collected from
the top of the gradient and analyzed for absorbance at 260 nm to locate the
ribosomal subunits (40S, 60S and 80S) and polysomes. Portions of fractions
were further analyzed by SDS-PAGE and western blotting.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
These isoforms contain both an N-terminal F-actin binding domain (ABD) and a
C-terminal domain (C-Shot L) with two putative Ca2+-binding
(EF-hand) and microtubule-binding (GAS2) motifs
(Fig. 1A), all of which are
essential for axon extension (Lee and
Kolodziej, 2002b). To investigate the function of the C-terminal
motifs, we performed a yeast two-hybrid screen with a Drosophila
embryonic cDNA library using C-Shot L (AAF24343, residues 4688 to 5201) as a
bait. Two of the interacting clones encoded the C-terminal domain (residues
315 to 422) of a novel protein, Krasavietz (Kra), which has been annotated in
FlyBase as CG2922. The predicted Kra protein is 51-52% identical to its
Xenopus (AAH41729), zebrafish (AAH58875), mouse (AAH05466) and human
(hKra/BZAP45; BAA02795) homologs (Fig.
1B and data not shown). However, no Kra homologs have been
identified in yeast or Caenorhabditis elegans.
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EF-GAS2, which removes both the EF-hand and the GAS2 motifs
from C-Shot L, did not bind to GST-Kra
(Fig. 1D). The importance of
the EF-hand and GAS2 motifs for Shot-Kra interaction was further evaluated in
S2 cells. We found that binding of Shot L(A)-GFP to HA-Kra was reduced to 5%
by deletion of the EF-hand motifs and to 35% by deletion of the GAS2 motif
(see Fig. S1 in the supplementary material). These observations indicate that
the EF-hand motifs are the major Kra-binding sites, and that the GAS2 motif is
also required for strong interactions. Interestingly, Shot L(A)-EF-GFP
bound endogenous 
-tubulin at comparable levels to Shot L(A)-GFP (see
Fig. S1 in the supplementary material), suggesting that Kra binding through
the EF-hand motifs does not impair the ability of Shot to bind microtubules
through the GAS2 motif.
The two-hybrid screen result indicates that the C-terminal domain of Kra
contains the Shot binding site. A database search revealed that the region
belongs to the W2 domain family found in translation initiation factors.
Multiple alanine substitutions (12A and 7A in the AA-boxes 1 and 2,
respectively) in the W2 domain of eIF5 disrupt its interactions with
eIF2ß and eIF3-NIP1 (Asano et al.,
1999). To pinpoint the Shot binding site with regard to possible
translation initiation factor binding sites, we made similar alanine
substitutions in Kra (Fig. 1B).
The Kra-12A mutant, as well as the wild-type Kra, efficiently bound C-Shot L
in S2 cells, whereas the binding of C-Shot L with the Kra-7A mutant was
minimal, appearing only at background levels
(Fig. 1E). These data suggest
that the evolutionarily conserved residues in the AA-box 2 motif of Kra are
essential for Shot-Kra interaction.
Kra protein strongly accumulates in the embryonic CNS
We developed an antibody against full-length Kra, which detects a single
50-kDa band on western blots containing extracts of S2 cells
(Fig. 2A). Levels of the
recognized protein were significantly reduced by kra dsRNAi, but not
by gfp dsRNAi, suggesting the specificity of our anti-Kra antibody
(Fig. 2A). Immunostaining using
anti-Kra revealed that Kra was highly expressed in early stage embryos (e.g.
stage 3; Fig. 2B) because of a
significant maternal contribution. It was detected in the CNS and epidermis by
stage 11, and in the gut by stage 13 (Fig.
2C). High expression in the CNS was maintained until the end of
embryogenesis (Fig. 2D).
We inspected the embryonic CNS by double labeling with anti-Kra and
anti-Elav, a neuronal marker (Lin and
Goodman, 1994), and found that Kra is expressed in most or all
post-mitotic neurons (Fig.
2F-H). Kra localized primarily to the cytoplasm of those cells,
but was not clearly detectable in CNS axons
(Fig. 2H). Kra was also
expressed in many CNS glial cells (Fig.
2I-K), as assessed by double labeling with anti-Kra and anti-Repo,
a glial marker that visualizes CNS glial cells, except for the midline glia
(Halter et al., 1995). To
assess the expression of Kra in midline glial cells, we used a midline glial
marker anti-Wrapper (Noordermeer et al.,
1998). However, because of ubiquitous expression of Kra around the
midline, we were not able to determine its presence in these cells. We next
wished to examine the localization of Kra at single axon resolution. We
therefore expressed UAS-HA-KraWT in a small
subset of embryonic CNS neurons with the aid of a neuronal driver
apterous (ap)-GAL4
(O'Keefe et al., 1998). HA-Kra
was strongly detected in the cell body and the axon of the Ap neurons
(Fig. 2L), suggesting that Kra
can be localized into axons.
|
). To determine its role in neuronal development, we generated
molecularly defined kra-null mutants. We first verified that the
transposons of l(3)j9B6 and EP(3)0428 were inserted at -1636
and -1228, respectively, of the kra open reading frame (ORF), which
is common to its multiple transcript variants
(Fig. 3A). We then mobilized
these P-elements, recovered 200 independent excision lines from each parental
allele, and determined the breakpoints of 30 lethal alleles by PCR and DNA
sequencing. In particular, we found that kra1, derived
from l(3)j9B6, deletes 2068 bp (-1729 to +338), whereas
kra2, derived from EP(3)0428, deletes 1404 bp
(-1242 to +161). Both deletions remove most of the 5'-UTR and the
translation start site, and extend into the ORF
(Fig. 3A). Homozygotes and
transheterozygotes for these deletion alleles died at the pupal stage. The
anti-Kra antibody cleanly detected the Kra protein in wild-type, but not in
kra1/kra2 mutant larvae or pupae
(Fig. 3B and data not shown),
suggesting that both kra1 and kra2 are
null alleles for kra. The kra gene is located 463 bp upstream of the putative gene CG1427, whose function is unknown. Reverse transcription (RT)-PCR analysis showed that levels of CG1427 mRNA were normal in kra1/kra2 third instar larvae (Fig. 3C), suggesting that kra1 and kra2 mutations do not affect the expression of CG1427.
kra is required in neurons for CNS axon pathfinding
We next investigated whether axon extension and guidance are defective in
kra-null mutant embryos. No defects in sensory and motor extension
were observed in kra1/kra2 embryos stained with
mAb 22C10 or 1D4 (data not shown). We then examined axon phenotypes in the
CNS. In wild-type embryos at early stage 13, mAb 1D4 labeled the pCC axon that
pioneers the ipsilateral pCC pathway without crossing the midline
(Fig. 4A). In
kra1/kra2 embryos, the same axon aberrantly
often crossed the midline (Fig.
4B). This early-stage axon pathway defect indicates that
kra is required for accurate growth cone migration. This phenotype
becomes more obvious in later stage embryos. In wild-type embryos at stage 16,
mAb 1D4 labeled three longitudinal axon pathways on each side of the midline;
axons in these pathways did not cross the midline
(Fig. 4D). In
kra1/kra2 embryos, axons from the innermost
(pCC) pathway ectopically crossed the midline in 18% of CNS segments
(Fig. 4E,L). Kra is
ubiquitously expressed in glial cells and neurons of the embryonic CNS. To
determine where Kra functions in midline axon guidance, we examined CNS axon
development in kra1/kra2 embryos that express
UAS-HA-kraWT under a neuron-specific driver
C155-GAL4. In these embryos, only 6% of CNS segments
exhibited the midline crossing defect (Fig.
4F,L), suggesting that Kra functions, at least in part, in neurons
to enable growth cones to avoid the midline. By contrast, glial-specific
expression of UAS-HA-kraWT using
slit-GAL4 or repo-GAL4 did not improve the kra
midline phenotype (19% with slit-GAL4, n=464; 18% with Repo-GAL4,
n=512).
kra-null mutant embryos contain substantial amounts of maternally
contributed Kra protein. To generate embryos that lack maternally and
zygotically contributed Kra, we used the FRT/ovoD1 method
(Chou et al., 1993) to generate
females containing homozygous kra1 germline clones. These
females did not lay eggs, and dissected germlines were blocked in oogenesis
(data not shown). Kra therefore appears to be required during oogenesis,
precluding isolation of embryos lacking maternally and zygotically contributed
Kra.
kra and shot together control midline axon repulsion
In shot3 mutant embryos, motor axons extend outward
from the CNS, choosing the right pathways but then stalling short of their
muscle targets (Lee et al.,
2000). Sensory axons also extend appropriately during early parts
of their trajectory but fail to advance
(Lee et al., 2000). CNS axon
phenotypes for shot have not been previously described. In the
present study, we found that Fas II-positive CNS axons ectopically cross the
midline in 
16% of segments in shot3 embryos
(Fig. 4C,G), suggesting the
role of Shot in midline axon guidance. Neuronal expression of Shot L(A)-GFP
rescued the midline crossing phenotype of shot3 embryos
(Fig. 4L). We have shown
previously that the F-actin-microtubule crosslinking activity of Shot is
essential for axon extension (Lee and
Kolodziej, 2002b), and therefore we investigated whether this
would still be the case for midline axon guidance. The microtubule-binding
domain mutant Shot L(A)-GAS2-GFP rescued the CNS phenotype of
shot3 embryos (Fig.
4H,L), although the same transgene did not rescue their axon
extension phenotypes as was previously described
(Lee and Kolodziej, 2002b). By
contrast, the F-actin-binding mutant Shot L(C)-GFP
(Lee and Kolodziej, 2002b) was
unable to rescue the loss of shot function in the embryonic CNS
(Fig. 4I,L). These results
suggest that midline axon repulsion requires the F-actin-binding activity of
Shot, but neither the microtubule binding nor the F-actin-microtubule
crosslinking activity. Shot L(A)-EF-GFP showed only partial activity to
improve the midline crossing defect in shot3 mutant
embryos (Fig. 4L), suggesting
that Kra binding through the EF-hand motifs is also important for the role of
Shot in midline axon repulsion.
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kra and shot interact with the midline repellent pathway
The phenotype observed in kra and shot mutants is
qualitatively identical to that seen in robo mutants
(Kidd et al., 1998),
suggesting a link between Kra/Shot and the Slit/Robo repellent pathway. To
test this hypothesis, we examined transheterozygous interactions of
kra and shot with robo. In a control experiment,
neither kra2/+ nor shot3/+ embryos
showed significant axonal defects at the midline
(Fig. 5A,B). In embryos
heterozygous for robo2, we rarely observed ectopic midline
crossing of Fas II-positive axons (Fig.
5C). However, the phenotype was dramatically enhanced in
transheterozygous robo2/+; kra2/+ or
shot3,robo2/+ embryos (>17-fold;
Fig. 5D,E,G). The phenotype was
further increased in embryos transheterozygous for shot, kra and
robo (Fig. 5F,G).
Similar genetic interactions were also observed between kra, shot and
slit (see Fig. S2 in the supplementary material). Thus, kra
and shot genetically interact with robo and slit,
suggesting that all these genes may function in the same guidance process.
The ability of Robo-positive axons to respond to Slit is impaired in kra mutant embryos
To further define the role of Kra in midline axon repulsion, we performed
detailed phenotypic analyses of kra mutant embryos. In the embryonic
CNS, glial cells provide neurons with cues and substrata for growth cone
migrations (Chotard and Salecker,
2004). Therefore, the axon phenotype observed in kra
could, in principle, be because of defects in CNS glia. This possibility led
us to examine kra mutant embryos for glial defects. For this purpose,
we also examined the axonal trajectories of the Ap-expressing neurons by
introducing an apC-tau-lacZ transgene
(Lundgren et al., 1995) into
the wild-type and kra mutant backgrounds. Because Ap axons express
Robo (Rajagopalan et al.,
2000b), they provide an excellent opportunity to investigate the
midline behaviors of Robo-positive axons at single axon resolution. In stage
16 wild-type embryos, the Ap axons remained ipsilateral without crossing the
midline (see Fig. S3A,B in the supplementary material)
(Lundgren et al., 1995).
However, in stage 16 kra1/kra2 embryos, the Ap
axons ectopically crossed the midline in 13% of segments despite the
apparently normal arrangement of glial cells as defined by anti-Repo and
anti-Wrapper (see Fig. S3C,D in the supplementary material). Thus, the ectopic
midline crossing of CNS axons in kra mutant embryos cannot be
ascribed to gross defects in the CNS glia.
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). In the
same embryos, the Ap axons remained ipsilateral within the Robo-positive
longitudinal tracts (Fig.
6B,C,H,I). However, in stage 16
kra1/kra2 embryos, the Ap axons ectopically
crossed the midline despite normal Slit expression along the midline
(Fig. 6D-F). Surprisingly, Robo
protein was aberrantly detected in some commissural axon tracts
(Fig. 6J). The commissural
tracts expressing Robo frequently contained Ap axons that were ectopically
crossing the midline (Fig.
6K,L), suggesting that these axons contribute to aberrant Robo
expression. By contrast, Robo expression in the longitudinal axon tracts of
kra mutant embryos was approximately the same as that of wild-type
embryos (Fig. 6G,J). Taken
together, these data support an essential role of Kra in maintaining the
sensitivity of Robo-expressing growth cones to the midline repellent Slit.
However, we cannot exclude the possibility that Robo misexpression by axons
normally crossing the midline also contributes to aberrant Robo expression in
kra mutant embryos.
|
). To
determine whether Kra also associates with 40S ribosomal subunits,
Drosophila S2 cells were treated with cycloheximide to arrest
translation, and their extracts were fractionated on sucrose gradients. Kra
cosedimented with the mRNA cap-binding protein eIF4E, but not with the
ribosomal protein L28, suggesting that it is associated with 40S, but not 60S,
subunits (Fig. 7A).
The W2 domains of eIF5 and eIF2B bind directly to the initiation
factor eIF2ß (Asano et al.,
1999), the ß-subunit of eIF2 whose GTPase activity is
essential for 40S and 60S subunits joining into 80S complexes. eIF5 is a
GTPase activating protein (GAP), and eIF2B is a GDP exchange factor
(GEF) for eIF2. We therefore asked whether Kra also binds to eIF2ß
through its W2 domain. We found that GST-Kra binds to eIF2ß in vitro
(Fig. 7B). We observed this
interaction in the fly embryo as well (Fig.
7C). Both 12A and 7A mutations significantly impaired Kra binding
to eIF2ß (Fig. 7C),
suggesting that the binding of Kra to eIF2ß requires the residues in its
W2 domain previously identified as necessary for eIF5 binding to eIF2ß
(Asano et al., 1999). Because
the same W2 domain also binds to Shot, we tested whether the formation of a
multiprotein complex containing Shot, Kra and eIF2ß is possible. In S2
cells, C-Shot L-GFP coimmunoprecipitated with endogenous Kra and eIF2ß
(Fig. 7D). As C-Shot L does not
directly bind to eIF2ß (data not shown), this result suggests that Kra
can simultaneously bind to Shot and eIF2ß in vivo.
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for eIF2ß. We therefore tested whether Kra inhibits
translation in vitro. Translation of luciferase mRNA in reticulocyte
lysate was specifically inhibited by Kra, but not by BSA and GST control
proteins (Fig. 7E).
Importantly, we found that these proteins do not affect the stability of
luciferase mRNA (Fig.
7E). Translation inhibition by Kra was dose-dependent at
concentrations ranging from 240 nM to 2.4 µM
(Fig. 7E). Interestingly, the
7A mutation significantly impairs Kra activity to inhibit translation
(Fig. 7E), suggesting that
protein-protein interactions through the AA-box 2 of Kra may be essential for
its activity to inhibit translation. The extreme insolubility of Kra-12A in
E. coli or insect cells prevented us from including it in this
assay.
Protein-protein interactions through the W2 domain of Kra are essential for midline axon repulsion
Our biochemical data suggested that the AA-boxes in the W2 domain of Kra
are crucial for the formation of complexes containing Shot and eIF2ß. To
directly test the importance of these protein-protein interaction domains in
axon guidance, we tested whether mutations in the AA-boxes would impair the
ability of Kra to rescue kra mutant phenotypes in the embryonic CNS.
Neuronal expression of Kra12A or Kra-7A failed to rescue the midline guidance
defect in kra1/kra2 embryos
(Fig. 8). The expression levels
and solubility of Kra-12A and Kra-7A were comparable to those observed for the
wild-type Kra (data not shown). Thus, the ability of Kra to form protein
complexes with Shot and eIF2ß is essential for its function in midline
axon repulsion.
To confirm the implication of the Kra binding partner eIF2ß in midline axon repulsion, we looked at CNS axons in eIF2ß mutant embryos. We observed ectopic midline crossing by the Ap axons and axons from the pCC ipsilateral pathway (see Fig. S4 in the supplementary material), which is reminiscent of the kra loss-of-function phenotypes. This observation further supports a role for eIF2ß in Kra-mediated midline axon repulsion and provides in vivo evidence for the requirement of protein translation in axon guidance.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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for the common
binding partner eIF2ß, thus inhibiting the assembly of functional
preinitiation complexes. A similar mode of translation inhibition has been
proposed for DAP-5/p97, which may compete with its homolog eIF4G for eIF3 and
eIF4A, thus reducing both cap-dependent and -independent translation
(Imataka et al., 1997;
Yamanaka et al., 1997).
However, the step in translation initiation that is regulated by Kra remains
to be addressed experimentally. Kra-mediated translational repression appears to be an important mechanism underlying midline axon guidance. In the kra mutant embryos, Fas II-positive CNS axons that normally remain ipsilateral cross the midline ectopically. This phenotype is observed with the pCC axons from early stages (stages 12 and 13) of axogenesis when they pioneer one of the Fas II pathways. The introduction of multiple alanine substitutions (12A and 7A) into Kra significantly reduces its ability to bind eIF2ß and abolishes its activity to rescue the kra mutant phenotype, suggesting that the function of Kra in axon guidance depends on its interaction with eIF2ß. Consistent with this conclusion, mutations in the eIF2ß gene also lead to the ectopic midline crossing of Fas II-positive axons.
Shot couples cytoskeleton reorganization to translational control during neuronal morphogenesis
There is a growing body of evidence that F-actin and microtubules are
coordinately assembled to each other during axon extension and guidance
(Kalil and Dent, 2005;
Schaefer et al., 2002;
Zhou et al., 2002).
Interactions of filopodial actin bundles and microtubules are key features of
filopodial maturation into an axon (Sabry
et al., 1991) and of growth cone turning
(Zhou et al., 2002). Shot, a
conserved molecule that scaffolds F-actin, microtubules and the microtubule
plus end-binding protein EB1 (Lee and
Kolodziej, 2002b; Subramanian
et al., 2003), is a strong candidate to bring microtubule plus
ends into contact with F-actin bundles. Indeed, Shot is required for the
extension of sensory and motor axons (Lee
and Kolodziej, 2002b), and a mammalian homolog of Shot, ACF7, is
required for microtubules to track along F-actin cables towards the leading
edge of spreading endodermal cells (Kodama
et al., 2003). Thus, previous studies have suggested that
Shot/ACF7 coordinately organizes F-actin and microtubules to support the
motility of neuronal growth cones and nonneuronal cells.
Our findings suggest that Shot also functions together with the translation inhibitor Kra to control midline axon repulsion. Shot physically associates with Kra in vivo. The shot loss-of-function phenotype at the CNS midline is reminiscent of the kra loss-of-function phenotype. The major Kra-binding domain in Shot is required for its role in midline axon repulsion. Moreover, shot and kra genetically interact in a dosage-sensitive manner for the midline phenotype. Our data also support the idea that cytoskeletal assembly and translational regulation can occur in a coordinated way. We found that midline axon repulsion requires both the activity of Kra to recruit eIF2ß and the activity of Shot to bind to F-actin. Thus, it is likely that local levels of eIF2ß available for protein synthesis can be spatially regulated with regard to actin cytoskeleton remodeling during axon guidance.
Shot/Kra mode of action in midline axon repulsion
In Drosophila, Slit is the key ligand driving midline axon
repulsion (Kidd et al., 1999),
and therefore midline crossing of CNS growth cones is primarily controlled by
the Robo receptor of Slit (Rajagopalan et
al., 2000a). How then do neurons regulate levels of Robo on the
surface of their axons and growth cones? The transmembrane protein
Commissureless (Comm) has been shown to dynamically regulate Robo expression
(Keleman et al., 2002;
Myat et al., 2002). Comm
functions as an intracellular sorting receptor to target newly made Robo for
lysosomal degradation, thereby blocking its transport to the growth cone that
is crossing the midline (Keleman et al.,
2002; Keleman et al.,
2005).
Translational regulation has also been shown to alter the responsiveness of
growth cones to the midline repellents
(Dickson, 2002). In vitro
studies of cultured embryonic retinal ganglion cells (RGCs) provided an
insight into how translation is regulated in axons and growth cones in
response to midline guidance cues. Treatment of these neurons with netrin-1
leads to the rapid activation of signaling pathways that phosphorylate the
translation initiation factor eIF4E and its binding protein eIF4E-BP1 and thus
induces axonal protein synthesis (Campbell
and Holt, 2001; Campbell and
Holt, 2003). Our data presented here indicate that the role of Kra
in midline axon repulsion depends on its ability to recruit the translation
initiation factor eIF2ß to Shot. Thus, protein complexes containing Shot,
Kra and eIF2ß may function as additional targets for signaling systems
that critically control axon guidance at the CNS midline. Regulation of
Shot-Kra-eIF2ß complexes may occur in neuronal cell bodies, where Kra is
concentrated, or in axons and growth cones, which may require local protein
synthesis to meet developmental requirements. In the latter case, as Kra is
not detectable in the CNS axons of the Drosophila embryo, even a low
amount of Kra may be sufficient for guiding axons.
Intriguingly, Robo was aberrantly detected on commissural axons in
kra1/kra2 mutant embryos. Given the increased
frequency of ectopic crossovers in these embryos, as well as the documented
role of Robo in preventing axons from crossing the midline
(Kidd et al., 1998), this
finding is somewhat paradoxical. One possibility is that Kra, induced by
interaction with Shot and eIF2ß, could repress the synthesis of as yet
unidentified proteins that transduce or modulate Robo signaling. In
shot or kra mutant embryos, perhaps this translational
regulatory circuit is not activated, and thus Robo-expressing growth cones
abnormally cross the midline because of a decrease in the strength of Robo
signaling output. Alternatively, Kra may function to finely tune the
expression levels of their multiple targets that mediate attractive or
repulsive responses. In this scenario, impairment of the Shot-Kra-eIF2ß
circuit could disturb the precise balance between repulsion and attraction
signaling at the midline, thereby decreasing the overall sensitivity of
Robo-expressing growth cones to Slit. Therefore, efforts to reveal the direct
targets of Kra-mediated repression in the future may provide better insights
into the immediate mechanisms by which translational regulation plays an
essential role for midline axon repulsion.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/9/1767/DC1
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ACKNOWLEDGMENTS |
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