Download Organization and translation of mRNA in sympathetic axons

Research Article
4467
Organization and translation of mRNA in sympathetic
axons
Sun-Kyung Lee and Peter J. Hollenbeck*
Department of Biological Sciences, Purdue University, West Lafayette, IN 47906, USA
*Author for correspondence (e-mail: [email protected])
Accepted 7 July 2003
Journal of Cell Science 116, 4467-4478 © 2003 The Company of Biologists Ltd
doi:10.1242/jcs.00745
Summary
Many axons carry out the synthesis of macromolecules
independent of their cell bodies but the nature,
organization and magnitude of axonal protein synthesis
remain unclear. We have examined these features in axons
of chick sympathetic neurons in cell culture. In situ
hybridization showed that poly(A) mRNA is abundant and
non-uniformly distributed in nearly all axons. The specific
transcripts for β-actin and actin-depolymerizing factor
(ADF) were also present and non-uniformly distributed
in axons, with an approximately hundredfold higher
concentration in growth cones, branch points and axonal
varicosities than in the axon shaft. Immunoprecipitation
using specific antibodies indicates that β-actin, ADF and
neurofilament protein (NF) are translated in axons
independently of cell bodies. Quantification of the
distribution of β-actin and ADF mRNAs showed that their
ability to enter the axon was likely to be a property of the
neuron as a whole rather than of individual axons. To
Introduction
The long-term survival of most axons relies upon an
uninterrupted connection to the neuronal cell body (Cajal,
1991), which provides a continuous supply of proteins and
organelles generated there (Grafstein and Forman, 1980; Vallee
and Bloom, 1991). However, axons do carry out autonomous
synthesis of many macromolecules, notably lipids (Vance et
al., 1991; De Chaves et al., 1995) and proteins (Koenig and
Giuditta, 1999; Alvarez et al., 2000). Thus, it seems likely that,
when physiological changes occur in the axon, often at a great
distance from the cell body, the neuron responds not only by
altering synthesis in and transport from the cell body
(Moskowitz et al., 1993; Moskowitz and Oblinger, 1995;
Gillen et al., 1997) but also by regulating synthesis nearby,
within the axon itself (Tobias and Koenig, 1975; Eugenin and
Alvarez, 1995). However, the relative contributions of axonal
and somatic translation and their relative responses to events
in the distal axon are almost entirely unknown.
Vertebrate axons contain mRNA (Koenig and Giuditta,
1999) along with organized ribosomes (Koenig and Martin,
1996; Koenig et al., 2000), and both the delivery of mRNA to
the axon and the maintenance of its distribution there are
microtubule dependent (Bassell et al., 1994b; Olink-Coux and
Hollenbeck, 1996). Transport of mRNA into the axon must be
selective, because axons do not contain the full neuronal set of
compare the distribution of axonally translated protein
to that of mRNA, we performed 35S metabolic labeling
with axons separated from their cell bodies. Axonally
synthesized proteins were distributed throughout the axons
and their synthesis was inhibited by cycloheximide but
not by chloramphenicol. Proteins translated mainly or
exclusively in axons or cell bodies were both detected by
metabolic labeling. Axons separated from their cell bodies
synthesized up to 5% as much protein in a 3-hour period
as did intact neurons. Because axons in our culture
conditions contain ~50% of the non-nuclear volume of the
neurons, we estimate that axoplasm of sympathetic neurons
has a protein synthetic capacity per unit volume equal to
10% that of cell body cytoplasm.
Key words: Axon, mRNA, Protein synthesis, β-Actin, Actindepolymerizing factor, Neurofilament, In situ hybridization
transcripts (Olink-Coux and Hollenbeck, 1996). In particular,
transcripts for major cytoskeletal proteins have been detected
in spinal nerve roots of the rat (Koenig, 1991) and in axons of
rat brain neurons (Bassell et al., 1998; Litman et al., 1993),
goldfish retinal ganglion cells (Koenig, 1989), goldfish
Mauthner cells (Weiner et al., 1996) and chick sympathetic
neurons (Olink-Coux and Hollenbeck, 1996). In addition, the
recruitment of β-actin mRNA into the axon is regulated by cell
signaling events (Bassell et al., 1998; Zhang et al., 1999) and
might require the same RNA localization sequence that
mediates its asymmetric distribution in polarized fibroblasts
(Zhang et al., 2001).
Axonal translation of numerous polypeptides has been
demonstrated by metabolic labeling in vertebrate axons
(Koenig and Adams, 1982; Koenig, 1989; Koenig, 1991) and
the translation of two major cytoskeletal proteins (actin and βtubulin) has been demonstrated specifically in axons of rat
sympathetic neurons (Eng et al., 1999). However, is axonal
protein synthesis essential for any axonal function? There are
conflicting reports for axonal growth: inhibition of axonal
translation has been reported to inhibit axonal regeneration in
rodent nerve (Remgård et al., 1992; Edbladh et al., 1994; Gaete
et al., 1998) but to have no effect on the growth rate of cultured
rat sympathetic neurons (Eng et al., 1999). However, recent
reports show that the response to guidance cues of growth
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Journal of Cell Science 116 (21)
cones of Xenopus retinal ganglion cell neurons depends
completely on the synthesis and degradation of proteins in the
distal axon, on a time scale of minutes (Campbell and Holt,
2001; Ming et al., 2002), and synapse formation in Aplysia
neurons also requires axonal protein synthesis (Schacher and
Wu, 2002).
To determine exactly how neurons regulate the supply
of new macromolecules, it is essential to understand the
organization and magnitude of the axon’s own synthetic
capacity. Which mRNAs are exported to the axon, where do
they reside and where is axonally synthesized protein
deployed? What is the capacity of axonal translation, what
proportion of total neuronal translation synthesis can be carried
out there? We have tried to resolve these questions by
examining, in a cultured vertebrate neuron that produces bona
fide axons, the presence, distribution and translation of total
mRNA and of two individual transcripts: β-actin and actindepolymerizing factor (ADF) in particular. We find that mRNA
is delivered very efficiently to axons, that total mRNA and
specific species have a similar, non-uniform distribution, and
that axonally translated protein is distributed throughout the
axon. We immunoprecipitate newly synthesized β-actin, ADF
and neurofilament protein from axons free of cell bodies, and
show that a significant proportion of the neuron’s protein
synthesis can occur in axons.
Materials and Methods
Materials
All reagents and supplements were obtained from Sigma (St Louis,
MO) unless otherwise specified. All culture media were obtained from
Fisher Scientific (Pittsburgh, PA) unless otherwise specified.
Cell culture
Chick sympathetic neurons were cultured as described previously
(Hollenbeck, 1993a; Olink-Coux and Hollenbeck, 1996). Sympathetic
chain ganglia were isolated from 9- to 12-day-old chick embryos and
dissociated. For in situ hybridization, 18 mm round No. 1 coverslips
were coated with 1% poly-L-lysine (Mr 70,000-120,000) for 12-18
hours, followed by 0.1 µg ml–1 laminin for 1-18 hours. Coverslips
were placed in 12-well plates and dissociated neurons were grown on
them in C medium [Leibowitz L-15 medium supplemented with 10%
fetal bovine serum (FBS; Gibco BRL, Rockville, MD), 100 U ml–1
penicillin and 100 µg ml–1 streptomycin (Gibco BRL, Rockville,
MD), 0.6 mM glucose, 2 mM L-glutamine, 50 ng ml–1 2.5-S mouse
nerve growth factor (Alomone Labs, Jerusalem, Israel) and 0.5%
methylcellulose (Dow Chemical Company, Midland, MI)] at 37°C. To
grow axonal halos for metabolic labeling experiments, 60 mm tissue
culture dishes were coated with 1% poly-L-lysine in 0.1 M boric acid
buffer (pH 8.4) overnight at 4°C, then approximately 20 to 25 intact
sympathetic ganglia were plated onto each dish and grown in C
medium in the presence of 0.6 µm Ara-C (cytosine 1-Darabinofuranoside) for 4-5 days at 37°C to yield axons 4-5 mm in
length. For direct autoradiography of 35S metabolically labeled axonal
halos, ganglia were cultured on 22 mm square coverslips coated with
1% poly-L-lysine.
Probe preparation
Oligonucleotide probes
Synthetic oligo(dT) 32-mer and oligo(dA) 32-mer were 3′ end-labeled
with digoxigenin-11-dUTP using terminal transferase according
to the manufacturer’s protocol (Roche Molecular Biochemicals,
Indianapolis, IN). Probes were purified through Quick Spin columns
(Roche Molecular Biochemicals, Indianapolis, IN). Labeling
efficiency was confirmed by dot blot analysis using digoxigeninlabeled DNA standards, anti-digoxigenin alkaline-phosphatase
conjugate and CSPD, a chemiluminescent substrate for alkaline
phosphatase (Roche Molecular Biochemicals, Indianapolis, IN).
RNA probes to detect β-actin and ADF transcripts
The 1690 bp chick β-actin coding sequence plus its 3′ untranslated
region (UTR) (nucleotides 36-1726, GenBank L08165) was amplified
by PCR from a chick brain cDNA library and subcloned into pGEMT easy (Promega, Madison, WI) at the SacI and BamHI sites. The 537
bp chick β-actin 3′ UTR (nucleotides 1189-1726, GenBank L08165)
was amplified by PCR using a 5′ primer with SalI site and a 3′ primer
with a BamHI site, and subcloned into pGEM-T easy. The 723 bp
fragment containing the whole coding region and a part of the 3′ UTR
of chick ADF (provided by J. R. Bamburg, nucleotides 88-811,
GenBank J02912) was subcloned into pGEM-3Z at the BamHI and
EcoRI sites. Each construct was linearized with an appropriate
restriction enzyme to make a template for in vitro transcription.
Linearized constructs with 3′ overhang were filled by treatment with
Klenow fragment to avoid generating undesirable turn-around
transcripts (Rong et al., 1998). Antisense and sense RNA probes were
synthesized and labeled by incorporation of digoxigenin-UTP using
a DIG RNA labeling kit (SP6/T7) (Roche Molecular Biochemicals,
Indianapolis, IN). Labeled RNA probes were purified and labeling
efficiency was confirmed as described above. Denaturing agarose gel
electrophoresis was used to determine whether probes had the correct
size.
In situ hybridization
Neurons were fixed with 4% paraformaldehyde in PBS containing 4%
sucrose for 30 minutes, washed with PBS three times and then
permeabilized in 0.3% Triton X-100 in PBS for 5 minutes at room
temperature. Poly(A) mRNAs were detected using a modification of
the in situ procedure of Olink-Coux and Hollenbeck (Olink-Coux and
Hollenbeck, 1996). Fixed, permeabilized cells were hybridized with
10 ng oligonucleotide probe per coverslip, dissolved in hybridization
buffer [2× SSC: 0.2% bovine serum albumin (BSA, molecular biology
grade, Roche Molecular Biochemicals, Indianapolis, IN), 10 mM
vanadyl adenosine complex, 5 mM EDTA, 10% dextran sulfate, pH
7.0, 15% formamide, 20 µg Escherichia coli tRNA (Roche Molecular
Biochemicals, Indianapolis, IN)] in a final volume of 30 µl, overnight
at 37°C. Coverslips were incubated in malate buffer (100 mM sodium
malate, pH 7.0, 150 mM NaCl) for 5 minutes at room temperature
and then in 1% blocking solution in malate buffer (Roche Molecular
Biochemicals, Indianapolis, IN). After washing, bound probe was
detected with the 2-hydroxy-3-naphtoic acid-2′-phenylanilide
phosphate (HNPP) fluorescent detection system (Roche Molecular
Biochemicals, Indianapolis, IN), according to the manufacturer’s
protocol. Alkaline-phosphatase-conjugated anti-digoxigenin antibody
was added and the enzymatic reaction was performed by adding
HNPP and Fast Red Texas Red. Precipitated fluorescent HNP-Fast
Red Texas Red signal was visualized with a Nikon microscope using
Texas-Red filter set and a 60× objective and photographed with Kodak
TMAX 400 film.
To detect β-actin or ADF transcripts specifically, fixed
permeabilized cells were hybridized with 10 ng antisense probe per
coverslip in hybridization buffer containing 20 µg E. coli tRNA, 50%
formamide, 2× SSC, 0.2% BSA, 5 mM EDTA, 0.1% Tween 20 and
0.1 mg ml–1 heparin sodium salt. Hybridization was performed a
chamber humidified with 50% formamide/2× SSC at 65°C for 5
hours. Coverslips were washed once in 50% formamide/2× SSC for
20 minutes at room temperature and then twice in 2× SSC for 10
minutes at room temperature. Hybridized probes were detected by an
Protein synthesis in axons
indirect tyramide signal amplification system (NEN, Boston, MA)
according to the manufacturer’s protocol. Horseradish-peroxidaseconjugated
anti-digoxigenin
antibody
(Roche
Molecular
Biochemicals, Indianapolis, IN) was added and then first round
enzymatic amplification was performed by adding tyramide-biotin.
After addition of horseradish-peroxidase-conjugated streptavidin, a
second round of enzymatic amplification was performed by adding
tyramide-Cy3. Fluorescent Cy3 signals were observed through a Cy3
filter set and photographed with Kodak Technical Pan film or
collected as digital images using a Hamamatsu cooled CCD camera
(C4742-95) and Metamorph imaging software (Universal Imaging,
West Chester, PA). Kodak TMAX 400 or Kodak Technical Pan
negatives were scanned using a Sprint Scan 35 (Polaroid, Cambridge,
MA) and images were captured with Adobe Photoshop graphic
software (Adobe Systems, San Jose, CA). The contrast and
brightness of all fluorescent images in each figure were adjusted
identically to avoid distortion of relative signal intensities.
Several negative control experiments were carried out to confirm
the specificity of in situ hybridization. First, to test for nonspecific
binding of RNA probes, sense RNA probes were hybridized in parallel
to antisense probes. Second, antisense probes of the neomycin
resistance gene (expressed only in prokaryotes) were hybridized.
Third, fixed cultures were treated with an RNase mixture (0.2 mg ml–1
RNase A, 0.2 mg ml–1 RNase T1, 100 U ml–1 RNase T2) or RNase
One (Promega, Madison, WI) for 30 minutes to 2 hours at 37°C prior
to hybridization of antisense probes. Fourth, labeled antisense probes
that gave positive signals were subjected to competition with
unlabeled antisense RNA probes in the same hybridization solution.
Statistical analysis
Neurons having at least one axon longer than 100 µm were scored in
randomly selected fields from at least two different experiments using
60× objective lens and epifluorescent optics. Specific fluorescent
puncta were counted in four different regions of each axon: branch
points, varicosities, growth cones and the rest of the axonal shaft. The
proportion of the puncta found in each subcellular region was
calculated. In order to find the number of puncta per unit area or unit
volume of each region, actual numbers of puncta at each subcellular
region were divided by the average area or volume of each subcellular
region calculated from digital images using Metamorph imaging
software (Universal Imaging, West Chester, PA). A two-tailed
Student’s t test was performed to compare the axonal shaft with other
subcellular regions and an ANOVA was performed to compare all
subcellular regions with each other. For neurons with more than one
axon, the number of axons having signal puncta was counted in each
cell and a ψ2 goodness-of-fit test was performed to see whether signal
puncta were distributed randomly among axons.
Metabolic labeling of ganglia and axonal halos
After explanted ganglia had elaborated extensive radial halos of
axons, each cell body mass was removed by microsurgery essentially
as previously described (Koenig and Adams, 1982; Olink-Coux and
Hollenbeck, 1996). Ganglia were washed three times with
methionine/cysteine-free DMEM supplemented with 10% dialysed
FBS, 100 U ml–1 penicillin, 100 µg ml–1 streptomycin, 0.6 mM
glucose, 2 mM L-glutamine and 50 ng ml–1 2.5-S mouse nerve growth
factor. The cell body mass was then excised at a distance of at least
two ganglion diameters, sacrificing some axonal mass but ensuring
that no cell bodies were included in the axonal halo. After the surgery,
axonal halos were incubated in methionine/cysteine-free DMEM
containing 0.5-3.33 mCi ml–1 EasyTag EXPRE35S35S protein
labeling mix (NEN, Boston, MA) for 2-5 hours and then washed with
Hank’s balanced salt solution buffer. Axonal halos were detached
from dish by gentle pipetting, harvested into microtubes and
dissociated in hot SDS sample buffer. Whole ganglia were also labeled
4469
and collected in parallel to axonal halos. As controls, metabolic
labeling was also performed in the presence of 1 mM chloramphenicol
or 1 mM cycloheximide.
Immunoprecipitation
Radioactively labeled ganglia or axonal halos were collected in lysis
buffer (Roche Molecular Biochemicals, Indianapolis, IN) containing
50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P40, 0.5% sodium
deoxycholate, and passed ten times through a 25-gauge needle. To
immunoprecipitate labeled β-actin, AC-15 antibody (Sigma, St Louis,
MO), which is specific for β-actin, was incubated with labeled cell
extract. In case of ADF immunoprecipitation, rabbit antiserum against
chick ADF (provided by J. Bamburg) was used. For neurofilament
protein, the lysis buffer was supplemented with 0.1% SDS, 100 µg
ml–1 RNase A and 30 U ml–1 DNase, and the cell extract was heated
for 10 minutes before the addition of monoclonal antibody NR-4
(Sigma), which binds all three neurofilament proteins (NF-L, NF-M
and NF-H). The antigen-antibody complex was precipitated using
protein-A or protein-G coupled to agarose or Sepharose (Roche
Molecular Biochemicals, Indianapolis, IN; Pierce, Rockford, IL) and
analysed by SDS-PAGE. Dried gels were exposed to Kodak Biomax
MR film.
Fluorography and autoradiography
Polyacrylamide gels containing radioactive proteins were fixed in
30% acetic acid, 10% methanol for 1 hour and completely dried in a
vacuum gel dryer. Fluorography was performed using Kodak Biomax
MR film and an intensifying screen at –80°C. For direct
autoradiography of axonal halos, fixed axonal halos were dehydrated
by successive incubation in 50%, 75%, 90%, 95% and 100% ethanol.
Mounted coverslips were dipped in 42°C Kodak NTB2 emulsion
solution and air dried for 3 hours under a safe light. Emulsion solution
coated coverslips were stored in a light-tight box with desiccant for 2
weeks at 4°C and then developed with Kodak D-19. Coverslips were
observed under 10× objective using dark field optics and
photographed with Kodak TMAX 100 film.
Results
Distribution of total poly(A) mRNAs in sympathetic
neurons
We have previously demonstrated the presence of poly(A)
mRNA in axons of sympathetic neurons using fluorescence in
situ hybridization (Olink-Coux and Hollenbeck, 1996). We first
re-examined these cells using a detection method that is
significantly more sensitive and has a higher signal-to-noise ratio
(Takizawa et al., 1997) (see Materials and Methods) to allow us
to determine the distribution of total mRNA. The poly(A)
mRNA signal was bright in all cell bodies and also in most
axons, where its pattern could be clearly seen to be punctate (Fig.
1A,B). The mRNA was distributed along the length of the axons
in an irregular pattern and, although it tended to be denser in the
more proximal regions of the axon, it was also found in the most
distal regions, including the growth cones. The specificity of the
antisense (oligo dT) probe for mRNA was shown by its
elimination by RNase treatment and its absence when a sense
probe was used (Fig. 1C-F). In addition, signal was still detected
in cell bodies and proximal axons when RNase T2, which
preferentially cleaves at the 3′ end of A residues (Egami and
Nakamura, 1969), was omitted from RNase mix (not shown).
This indicates that the fluorescent signal resulted from specific
binding of antisense oligo (dT) probes to poly(A) tracts.
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Journal of Cell Science 116 (21)
β-Actin and ADF mRNAs are present in
axons of sympathetic neurons
A range of transcripts have been identified
in the axoplasm of several kinds of neurons
(reviewed by Van Minnen, 1994; Mohr,
1999; Mohr and Richter, 2000; Alvarez
et al., 2000) but, to understand their
physiological role in the axon, it is essential
to determine the presence, abundance and
distribution of specific transcript species.
Because the structure that is most remote
from the cell body in a growing axon is the
dynamic, actin-rich growth cone, actin and
its accessory proteins are good candidates
for local axonal translation during outgrowth
(Bamburg and Bray, 1987; Bassell et al.,
1998). We have previously detected β-actin
mRNA in axons of chick sympathetic
neurons indirectly using RT-PCR (OlinkCoux and Hollenbeck, 1996), and Bassell et
al. (Bassell et al., 1998) have detected its
presence directly in axons of rat cortical
neurons. Here, we set out to examine
directly the axonal localization and
subcellular distribution of mRNAs for βactin and ADF, an actin binding protein that
is essential for normal actin organization and
is involved in growth cone function
(Bamburg, 1999; Meberg and Bamburg,
2000).
Using antisense probes and two-stage
enzymatic amplification, we performed in
Fig. 1. In situ hybridization shows the punctate distribution of poly(A) mRNAs
situ hybridization against β-actin and ADF
throughout chick sympathetic neurons. Fixed neurons were hybridized with an oligo d(T)
mRNAs. Two RNA probes were used to
probe and fluorescent signals were developed using alkaline-phosphatase enzymatic
detect β-actin transcripts: a 537 bp RNA
amplification. Phase-contrast images (A,C,E) and fluorescent images (B,D,F) are shown
probe complementary to the β-actin 3′ UTR
for each field. Bright, punctate signals were detected along the length of the axons, along
with bright staining of the cell bodies (B), which are also phase-dense because of the
and a 1690 bp RNA probe complementary
accumulation of HNP/Fast-Red-Texas-Red fluorescent material. Neurons treated with
to the entire β-actin sequence. The former
RNase prior to hybridization (D) or hybridized with sense oligo d(A) probes (F) show no
was used because its sequence is unique and
signal in the axons and only trace levels of background staining in cell bodies. Scale bar,
thus lacks similarity to that of other actin
20 µm.
isoforms; the latter, because its greater
length provides increased signal. The
points, varicosities and growth cones, but also along the length
hybridization conditions were more stringent than those used
of the axon shaft (Fig. 2B,D). As with oligo dT probes, the
for the oligo d(T) or oligo d(A) probes in order to denature the
signal was strongest in the 100 µm or so of axon nearest the
long RNA probes completely and thereby maximize the
cell body but was found along the length of longer axons. The
specificity of binding. Both the 1690 bp and the 537 bp probes
overall pattern of staining was indistinguishable between the
successfully detected β-actin transcripts in axons of chick
1690 bp and 537 bp probe, implying that the signals resulted
sympathetic neurons (Fig. 2A-D). Cell bodies were very
from hybridization to the same pool of mRNA (see also Table
brightly stained and discrete, punctate signals were detected in
2). The higher signal intensity provided by the 1690 bp was
axons. These appeared most commonly at axonal branch
Table 1. Percentages of mRNA puncta found in four different subcellular regions
Hybridization
probe (size)
β-actin (1690 bp)
β-actin 3′UTR (537 bp)
ADF (723 bp)
Number of cells
having signals out
of total 100 cells
Branch points
Growth cones
Varicosities
Axonal shaft
Total
81
73
70
478 (47%)
331 (35%)
161 (45%)
78 (8%)
71 (8%)
55 (16%)
290 (29%)
327 (35%)
86 (24%)
165 (16%)
203 (22%)
54 (15%)
1011 (100%)
932 (100%)
356 (100%)
Numbers of puncta were counted in each subcellular region of 100 randomly selected cells from each hybridization experiment and summed.
ADF, actin depolymerizing factor.
Protein synthesis in axons
4471
probably due to the increased signal
provided by the longer probe, rather than
cross-hybridization with γ-actin transcripts,
which have been reported to be absent from
the axon (Bassell et al., 1998).
The ADF antisense RNA probe of 723 bp
was complementary to 500 bp of coding
sequence and 223 bp of 3′ UTR. This region
was selected because of its unique
sequence. As for β-actin, ADF probes
revealed a discrete, punctate mRNA
distribution in axons, with signal detected
at branch points, varicosities, growth cones
and along the axonal shaft (Fig. 2E,F).
However, the number and intensity of the
signal puncta were less than those seen with
β-actin probes (Table 1). Staining of the cell
body was less intense than β-actin staining
as well, probably because of the lower level
of expression of ADF in chick sympathetic
neurons. The specificity of our singletranscript detection was confirmed by
RNase treatment (Fig. 2G,H) and sense
probe controls (Fig. 2I,J), as well as by a
complete lack of signal when an antisense
RNA probe for the bacterial neo gene was
used, or excess amount of unlabeled
antisense RNA probe was added to compete
out labeled antisense probe (not shown).
Subcellular distribution of β-actin and
ADF mRNAs
β-Actin and ADF mRNAs were detected in
all of the cell bodies and most, but not all,
of the axons in sympathetic cultures (7081%; Table 1). Because recent studies have
indicated that the presence of β-actin
mRNA in axons of cultured neurons is
not constitutive but is regulated by
neurotrophin signaling (Zhang et al., 1999),
we examined the distribution of mRNA in
sympathetic neurons containing more than
one axon. In cells selected at random, each
axon was scored for whether or not it
contained β-actin or ADF mRNA (Table 2).
If the presence or absence of mRNA in an
axon is a property of the neuron as a whole
then there should be a bias towards neurons
having either all or none of their axons
containing mRNA. However, if the entry of
mRNA into each axon is a property of the
axon alone then the pattern of presence or
absence of mRNA in axons of multipleaxon neurons should follow a binomial
distribution. We found that the data were
biased significantly towards neurons having
all or none of their axons containing β-actin
and ADF mRNAs (Table 2).
The distribution of total mRNA and
recent results on the arrangement of
Fig. 2. β-Actin and ADF mRNAs are detected in axons by in situ hybridization with
species-specific probes. Fixed neurons were hybridized with probes for the whole βactin sequence (A,B), the β-actin 3′ UTR (C,D) and the ADF partial sequence (E,F).
Bright, punctate signals were detected in branch points, varicosities and growth cones,
and also along the axonal shaft (Tables 1, 2). Neither cells treated with RNase prior to
antisense probe hybridization (G,H) nor cells hybridized with sense probe (I,J) showed
any signal. The controls shown were carried out with the full length β-actin probe;
separate control experiments for the β-actin 3′ UTR and ADF probes gave identical
results. Scale bar, 20 µm.
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Journal of Cell Science 116 (21)
Table 2. mRNA is not randomly distributed among axons of multi-axon neurons
Number of cells
Axons containing
mRNA signal
β-actin (P=0.57)
β-actin 3′UTR (P=0.53)
ADF (P=0.34)
O
E
O
E
O
E
2-axon neurons
0
1
2
Total
7*
10
18*
35
6.6
17.2
11.2
35
10*
9
9*
28
6.2
14.0
7.8
28
12
29*
11*
52
22.7
23.3
6.0
52
3-axon neurons
0
1
2
3
Total
6*
6
14*
5
31
2.5
9.9
12.9
5.6
31
9*
4
17*
9*
39
4.1
13.7
15.4
5.8
39
6
14*
5
0
25
7.2
11.1
5.7
1.0
25
Neurons having 2 and 3 axons were selected, and the number of axons containing detectable mRNA counted in each category. The probability of an axon being
positive for mRNA signal (P) was calculated from the whole population of axons for each hybridization probe. Then, the fraction of the cells expected (E) to have
each possible distribution of positive and negative axons was calculated assuming that each axon of a given cell has the same independent probability of being
positive, and a chi-square goodness-of-fit test was performed to see if the distribution of mRNA (O, observed) was random. In all cases, P<0.05, and the
distribution is skewed toward neurons with all or none of their axons containing mRNA (asterisks represent that the observed value is higher than expected), thus
suggesting that the entry of mRNA into an axon is a property of the entire neuron and not the individual axon. A chi-square goodness-of-fit test was also
performed between β-actin and ADF, or between β-actin 3′UTR and ADF, using observed values to see whether they have similar distribution of puncta. In both
cases, P<0.05; however, when compared between β-actin and β-actin 3′UTR, P>0.2. Thus, the spatial distribution of ADF mRNA seems to be slightly different
from that of β-actin.
ribosomes in the axon (Koenig and Martin, 1996; Koenig et al.,
2000) both suggested that the distribution of individual mRNA
species in the axon would be non-uniform. We examined our
data to test the hypothesis that mRNA for β-actin and ADF are
not only distributed irregularly but are also concentrated
specifically in actin-rich regions of the axon. The signal was
clearly concentrated in two such regions, axonal branch points
and growth cones, as well as in varicosities (Fig. 2). We
quantified this distribution in 100 randomly selected neurons for
each hybridization protocol and found that 78-85% of the βactin and ADF mRNA was concentrated in these three regions,
with the remainder distributed along the entire axon shaft (Table
1). To quantify the β-actin and ADF mRNA concentrations in
these different regions of the axon, we measured the projected
areas occupied by each region in sympathetic cultures and
calculated the concentrations of mRNA signal per µm2 of
projected area. The concentrations in branch points, growth
cones and varicosities were all significantly higher than that in
the axon shaft (Table 3). In addition, when the relative thickness
of the growth cone was factored in to calculate the relative
concentrations of mRNA signal per µm3, the branch points,
growth cones and varicosities all had β-actin and ADF mRNA
concentrations approximately 100 times higher than those of the
axon shaft as a whole (Table 3).
the cell body mass (see Materials and Methods). When labeled
cultures were subjected to emulsion autoradiography, dense
silver grains were detected throughout not only the intact
ganglia (Fig. 3E) but also the axonal halos that had been
exposed to [35S]-methionine/cysteine in the absence of cell
bodies (Fig. 3F). There was no obvious nonuniform
distribution of axonally synthesized proteins along the length
of axons in the halos. Autonomous axonal protein synthesis
was abolished when axonal halos were treated with the
eukaryotic translation inhibitor cycloheximide, but remained at
control levels when they were treated with the prokaryotic
and mitochondrial translation inhibitor chloramphenicol (Fig.
3G,H). Thus, the axonal protein synthesis results from
cytoplasmic, not mitochondrial, translation.
Profile of newly synthesized proteins in axon was
complex and different from that of cell body
To compare mRNA translation in the axons versus cell bodies
we subjected the explant cultures, either with or without removal
of their cell body mass, to [35S]-methionine/cysteine metabolic
Table 3. Number of puncta per µm2 in subcellular regions
Probe
Metabolic labeling with [35S]-methionine/cysteine and
protein synthesis in axonal halos
When whole sympathetic ganglia are cultured for 4-5 days,
they extend radial halos of axons that extend 4-5 mm (Fig. 3).
By maintaining the ganglia on a poly-L-lysine-treated
substratum and exposing them intermittently to the DNA
synthesis inhibitor Ara-C, the presence of non-neuronal cells
in the halos can be suppressed (Fig. 3A-D). In order to
determine whether sympathetic axons are capable of
translating their mRNA independently of the cell body, we
performed [35S]-methionine/cysteine metabolic labeling on
ganglion explants with and without prior surgical removal of
β-actin (1690 bp)
β-actin 3′UTR (537 bp)
ADF (723 bp)
Branch
points
Varicosities
Growth cones
Axonal
shaft
2.55
2.26
2.28
2.30
2.26
1.62
1.30 (5.20)
1.32 (5.28)
0.96 (3.84)
0.04**
0.03**
0.02**
Axonal mRNA is highly concentrated in branch points, varicosities and
growth cones. The number of puncta per µm2 of projected area was calculated
for four subcellular regions: The average projected area of each subcellular
region was measured from 20 randomly selected cells, and then the number of
puncta was divided by the average area to give the values for mRNA density
in each region. To obtain relative values for puncta per unit volume, the
values for the growth cone were multiplied by 4 to reflect the thickness of the
growth cone relative to the axon shaft. The resulting values are shown in
parentheses in the growth cone column. ** PⰆ0.001, Student’s t-test
comparison to other subcellular regions. PⰆ0.001, F-test of analysis of
variance (ANOVA).
Protein synthesis in axons
labeling for 2-3 hours and then harvested the explants. When
these were analysed for their content of newly synthesized
proteins by SDS-PAGE and autoradiography, dozens of
polypeptides were apparent in both preparations (Fig. 4A, lanes
1,2). By this measure, new protein synthesis in both whole
ganglia and axonal halos was eliminated by cycloheximide
treatment (Fig. 4A, lanes 3,4) during [35S]-methionine/cysteine
exposure but not by chloramphenicol treatment (Fig. 4A, lanes
5,6). Thus, many individual mRNA species must be both present
and actively translated in these axons. However, the proteins
synthesized in the two preparations were not identical:
autoradiograms showed that synthesis of several proteins was
confined to or highly enriched in the cell bodies; there were also
several proteins whose synthesis was nearly or entirely confined
to the axonal compartment (Fig. 4B). This indicates that certain
transcripts are selectively and almost entirely transported into the
axon, that they are selectively translated there or both. The
identities of these proteins are not known at present but they
include two prominent polypeptides with estimated sizes of
24 kDa and 62 kDa. As described below, the stoichiometry
of synthesis of the neurofilament proteins also differed
substantially between cell bodies and axons (Fig. 5).
What proportion of the sympathetic neurons’ total capacity
for protein synthesis resides in the axons of these explant
cultures? In several experiments, we determined the number of
[35S]-methionine/cysteine-labeled axonal halos necessary to
equal the amount of labeled protein in a single [35S]methionine/cysteine-labeled whole ganglion (Fig. 4, lanes 1,2).
This number ranged between 20 and 68, meaning that axonal
protein synthesis in these explants accounted for 1.5-5.0% of
total neuronal protein synthesis. This estimate is conservative
for the same reason that it is variable: in removing the cell body
masses from the ganglion explants, we worked under a
microscope and were careful to remove all of the cell bodies,
even at a sacrifice of a variable portion of the axonal halo. The
radius of the axonal halo averaged 5 mm and we removed on
average the most proximal 0.5 mm of the halo in the course of
excising the cell body mass. Thus, 1.5-5.0% places a lower
limit on the contribution of axonal protein synthesis to these
neurons. We calculate the specific translation activity per unit
volume of axonal cytoplasm as follows: in our cultures,
micrographs show that the average length of our axonal halos
after removal of the cell body mass was 4.5 mm, the average
diameter of the cell body is 12 µm and the average nuclear
diameter is 8 µm; electron micrographs show that the average
axon diameter is 0.4 µm (Hollenbeck, 1993b). Therefore, the
non-nuclear volume of cytoplasm in the cell body is 637 µm3,
or 53% of total cytoplasmic volume in the neuron, whereas that
of the axon is 565 µm3, or 47% of the total volume. Thus, in
these sympathetic cultures, the cytoplasmic volume of the axon
is approximately equal to that of the cell body. Because these
axons contain 1.5-5.0% of the total protein synthesis in
approximately 50% of the non-nuclear volume of the cell, the
amount of protein synthesis per unit volume in the axoplasm
is 3-10% that of the cell body cytoplasm.
β-Actin and ADF are synthesized in the axons
independently of cell bodies
Although there is significant amount of olfactory marker
protein mRNA in axons of rat olfactory neurons, there is no
4473
Fig. 3. Axons of ganglion explants can synthesize protein
autonomously. Proliferation of non-neuronal cells was suppressed by
culture conditions (A-D). When chick sympathetic ganglia were
grown for 4-5 days on a poly-L-lysine substratum with intermittent
Ara-C treatment, they produced a large radial halo of axons (A) that
was virtually devoid of cell bodies or non-neuronal cells, as revealed
by DAPI staining (B). The dotted line in (B) indicates the
approximate position where the cell body mass would be removed
for metabolic labeling experiments. When grown on a laminin
substratum and in the absence of Ara-C treatment, axonal halos
contained abundant non-neuronal cells, revealed by their nuclei
(C,D). Normal ganglia grown under the conditions shown in (A,B)
were incubated with [35S]-methionine/cysteine, subjected to
emulsion autoradiography and viewed by dark-field microscopy.
They revealed a bright signal in the cell body mass and a fainter one
in the axonal halo (E). Axonal halos with the cell body mass
meticulously removed prior to metabolic labeling also showed newly
synthesized protein throughout the axons (F). This signal was
eliminated by cycloheximide treatment (G) but was undiminished
when chloramphenicol treatment accompanied metabolic labeling
(H). Conditions of radiolabeling, autoradiography and photography
were uniform, except that the photographic exposure of (E) was 25%
that of F-H owing to the intensity of signal from the cell body mass.
The dark area in the center of the cell body mass in E is an artefact of
high radioactive signal damaging the emulsion. Scale bars, 200 µm
(in D, for A-D; in H, for E-H).
evidence that they are actually translated to produce protein
(Wensley et al., 1995). In order to see whether β-actin and
ADF transcripts detected in axons are actively translated to
4474
Journal of Cell Science 116 (21)
Fig. 4. Cycloheximide-sensitive axonal
translation produces a complex
population of proteins. (A) SDS-PAGE
and autoradiography of newly
synthesized proteins in the axonal halo
vs entire ganglion. Lanes 1,3,5 contain
protein from 17 axonal halos; lanes
2,4,6 contain protein from 0.25 whole
ganglia. Protein synthesis in whole
ganglia and in axon halos (lanes 1,2)
was eliminated by treatment with
cycloheximide (lanes 3,4) but not with
chloramphenicol (lanes 5,6). (B) The
protein composition of axonal halos is
different from that of entire ganglia
(lanes1,2, enlarged area at right). The
arrow at left indicates a protein whose
synthesis is highly enriched in the axon
and the arrows at right indicate two
proteins whose synthesis is highly enriched in the cell bodies. (C) Close examination using 4-15% gradient SDS-PAGE and autoradiography
identifies the molecular weight of prominent proteins enriched in the axon (arrows at left) or cell bodies (arrowheads at right). 40 axonal halos
(lane 1) and 1 ganglion (lane 2) were loaded.
produce their corresponding proteins, immunoprecipitation
was performed using 35S-labeled axoplasmic extract. After
surgery to remove their cell body masses, axonal halos were
incubated in [35S]-methionine/cysteine-containing medium for
Fig. 5. β-Actin, ADF and neurofilament protein are synthesized in
axons separated from their cell bodies. Metabolically labeled whole
ganglia (lanes 1,3,5) or axonal halos without cell bodies (lanes 2,4,6)
were subjected to immunoprecipitation using the anti-β-actin
antibody AC-15 (lanes 1,2), antiserum against chick ADF (lanes 3,4)
or the neurofilament antibody NR-4 (lanes 5,6). Newly synthesized
43 kDa β-actin and 18.5 kDa ADF were precipitated not only from
ganglia but also from axonal halos. Some actin was precipitated with
ADF from the ganglia extract (denoted by filled circle). Newly
synthesized neurofilament subunits NF-L, NF-M and NF-H were
precipitated from whole ganglia (indicated by arrows to the right of
lane 6) but axons synthesized mainly the NF-L subunit during the
metabolic labeling period (lanes 5,6). For lanes 1 and 3, cell lysates
from five ganglia were used after 5 hours incubation with [35S]methionine/cysteine, and the exposure time of the autoradiogram was
1 hour for β-actin and 3 hours for ADF. For lanes 2 and 4,
approximately 40-50 axonal halos were used and the exposure time
was 6 hours for β-actin and 7 days for ADF. For lanes 5 and 6,
lysates from four ganglia and 75 halos, respectively, were used and
the exposure times were 6 hours and 7 days, respectively. In case of
lane 6, metabolic labeling period was extended up to 10 hours.
5 hours, then lysed and subjected to immunoprecipitation and
autoradiography to detect specific newly synthesized axonal
proteins. We were able to detect newly synthesized 43 kDa βactin in both ganglia (Fig. 5, lane 1) and axons (Fig. 5, lane 2)
using AC-15 antibody, which is specific for β-actin (North et
al., 1994). This result contrasts with previous studies that
reported no β-actin synthesis in rat sympathetic axons (Eng et
al., 1999). An unknown protein with a molecular weight of 55
kDa precipitated with β-actin. Rabbit antiserum against chick
ADF also successfully precipitated labeled 18.5 kDa ADF
from both ganglia (Fig. 5, lane 3) and axons (Fig. 5, lane 4),
along with a minor band of 43 kDa, presumably actin (Fig. 5,
round dot).
To demonstrate the axonal translation of an exclusively
neuronal protein, we also immunoprecipitated neurofilament
protein from metabolically labeled ganglia and axonal halos.
The monoclonal antibody NR-4 binds to all three
neurofilament subunit proteins: NF-L (apparent molecular
weight 66 kDa); NF-M (95-100 kDa); NF-H (110-115 kDa)
(Lee and Cleveland, 1996). This was also confirmed by
immunoprecipitation experiments with cold chick brain extract
(data not shown). All three subunit proteins were synthesized
in whole ganglia, with NF-M being present in highest
stoichiometry and NF-L in the lowest, representing a very
small proportion of the neurofilament protein synthesized in
the metabolic labeling period (Fig. 5, lane 5). However, in
axons, the major neurofilament protein synthesized during the
labeling period was the NF-L, with NF-M and NF-H virtually
undetectable (Fig. 5, lane 6). This result not only confirms that
synthesis in axonal halos is not due to non-neuronal cells but
also reinforces the finding (Fig. 4) that the pattern of synthesis
in the axons differs from that in cell bodies.
Discussion
Even the prodigious translation capacity of the neuronal cell
body and the large volume of anterograde axonal transport of
protein (Grafstein and Forman, 1980; Vallee and Bloom, 1991)
cannot entirely support the needs of the axon. Important events
Protein synthesis in axons
in the distal axon require local protein synthesis that is locally
regulated (Campbell and Holt, 2001; Ming et al., 2002;
Schacher and Wu, 2002). In this respect, the axon might have
more than previously imagined in common with dendrites,
where local protein synthesis plays a pivotal role in synaptic
plasticity (Steward, 1997; Schuman, 1999; Martin et al., 2000).
But how is mRNA organized in the axon to support local
synthesis?
We have previously demonstrated that the axons of
sympathetic neurons contain poly(A) mRNA and that its
presence there requires intact microtubules (Olink-Coux and
Hollenbeck, 1996). Here, we have shown that neurons
transport mRNA into their axons very efficiently, despite their
extreme asymmetry and the absolute distances involved. We
detected both poly(A) mRNA and individual transcripts in 70100% of axons, often at a distance of hundreds or thousands
of micrometers from the cell body (Figs 1,2; Olink-Coux and
Hollenbeck, 1996). Thus, neurons display a more spatially
polarized distribution of mRNA than is seen in cell types of
more modest dimensions, such as migrating fibroblasts, in
which β-actin mRNA is distributed preferentially towards the
leading edge of the lamellipodia in 30-60% of the cells
(Sundell and Singer, 1990; Kislauskis et al., 1994). Neurons
probably achieve this by using microtubule-based axonal
transport of mRNA over long distances (Olink-Coux and
Hollenbeck, 1996), as they do for the transport of organelles
(Lane and Allan, 1998; Goldstein and Yang, 2000). This is
similar to the mechanism of myelin basic protein mRNA
transport in oligodendrocytes (Carson et al., 1997) and several
maternal mRNAs in Drosophila early embryo (Yisraeli et al.,
1989; Theurkauf and Hazelrigg, 1998; Lall et al., 1999;
Brendza et al., 2000; Wilkie and Davis, 2001), but contrasts
with the proposed F-actin-based mechanism of β-actin mRNA
transport in fibroblasts (Sundell and Singer, 1991; Taneja et al.,
1992; Bassell et al., 1994a) and the brown alga fucus (Bouget
et al., 1996), and with Ash1 mRNA transport in yeast
(Takizawa et al., 1997; Takizawa and Vale, 2000).
Which of the thousands of transcripts produced in neurons
are present in axons? Judging from the SDS-PAGE analysis of
metabolically labeled neurons, many of them: axons showed
dozens of newly synthesized axonal polypeptides. Indeed,
relatively few major polypeptides were synthesized mainly or
exclusively in the cell body (Fig. 4). More surprising is that a
similar small number were limited to the axon (Fig. 4). Other
studies have detected many axonally synthesized polypeptides
in rat motor (Eng et al., 1999) and sensory (Koenig, 1991)
neurons, although they reported no significant difference
between the compositions of newly synthesized proteins in the
axons and cell bodies. However, Zheng et al. reported several
proteins whose synthesis is enriched in either axons or cell
bodies (Zheng et al., 2001) with molecular weights of 40-65
kDa, as seen in this study (Fig. 4), despite the different species
used. Our immunoprecipitation results with newly synthesized
neurofilament protein (Fig. 5) show that, even when the same
group of proteins is translated in both axons and cell bodies,
the pattern of synthesis may differ substantially. However,
despite the number and diversity of axonal translation products,
there are many proteins that are unlikely to be synthesized
there, including those involved in nuclear structure, in
chromatin formation and in endoplasmic-reticulum-dependent
translation and protein folding.
4475
In the case of cytoskeletal proteins, we have previously
shown by reverse-transcription PCR that mRNA encoding one
major protein (β-actin) was present in axons, whereas that
encoding α-tubulin was not (Olink-Coux and Hollenbeck,
1996). Here, we have shown directly that mRNAs for β-actin
and ADF are present and widespread in most axons, and that
they, along with the NF-L transcript, are actively translated to
produce their respective proteins. Because β-actin (Herman,
1993) is abundant in neuronal growth cones (Bassell et al.,
1998), as it is at the leading edge of other moving cells (Hoock
et al., 1991; Bassell et al., 1998), the presence of its mRNA
and its active translation there probably reflect a requirement
for rapid regulation of β-actin translation where its
concentration and dynamics are greatest (Lawrence and Singer,
1986; Kislauskis et al., 1997). The presence and translation of
both β-actin and ADF mRNA in the axon of sympathetic
neurons supports this idea, because ADF, like β-actin, is
concentrated in the growth cone (Bamburg and Bray, 1987),
where it is believed to modulate the behavior of the actin
cytoskeleton (Bamburg, 1999; Meberg and Bamburg, 2000).
However, are mRNAs limited to the growth cone region of
the distal axon, or to the regions where the proteins that they
encode are most concentrated? Our data show that the densities
of both β-actin and ADF transcripts are about two orders of
magnitude higher in the growth cone region than they are in
the axon as a whole. However, they are also highly
concentrated in axonal branch points and small varicosities,
sometimes at a considerable distance from the growth cone
(Fig. 2, Table 3). This mRNA could have been in transit toward
the distal axon at the time of fixation. Axonal mRNA transport
is microtubule dependent, whereas these regions are actin rich
and microtubule poor. Thus, mRNAs could dwell longer there,
as do many organelles undergoing axonal transport. It is also
possible that mRNAs are specifically docked in actin-rich
regions, as has been proposed in fibroblasts (Bassell et al.,
1994b), Xenopus oocytes (Yisraeli et al., 1989) and cortical
zone of goldfish Mauthner cells (Muslimov et al., 2002),
although axonal mRNA distribution has been shown to be
largely dependent on microtubules in the chick sympathetic
neurons (Olink-Coux and Hollenbeck, 1996).
Our data make it unlikely that the distribution of mRNA
indicates the sites within the axon where protein synthesis
occurs. The total mRNA, β-actin mRNA and ADF mRNA that
we detected are highly concentrated in a few regions (Table 3),
whereas the axonally synthesized protein is uniformly
dispersed along the axon (Fig. 3). However, mRNA exists in
neurons in several forms and the distribution of actively
translated mRNA could be different from the distribution of
total mRNA found here. Morphological studies have shown
that complexes of ribosomes reside in the cortical region of
vertebrate axons and perhaps contain translatable mRNA
(Koenig et al., 2000). In addition, mRNA granules detected by
microscopy in rat cortical neurons (Knowles et al., 1996) and
mouse oligodendrocytes (Barbarese et al., 1995) contain both
mRNA and components of the translation machinery such as
ribosomes and elongation factor. mRNA-containing structures
such as these could be either translationally active or quiescent;
some evidence suggests that they might be competent for
translation upon an appropriate physiological cue (Krichevsky
and Kosik, 2001; Zhang et al., 2001). Finally, evidence from
live neurons indicates that mRNA and other translation
4476
Journal of Cell Science 116 (21)
components can undergo rapid transport in a particulate form
(Knowles et al., 1996). The mRNA that we have detected in
axons could be in one or more of these forms.
Neurons with several axons were more likely than expected
by chance assortment to have β-actin or ADF transcripts in all
or none of their axons. This suggests that the transport of
individual transcripts into the axon is a property of the entire
neuron rather than an autonomous feature of each axon.
Furthermore, it suggests that the proposed mechanism for
recruiting β-actin transcripts to the axon, neurotrophin
signaling (Zhang et al., 1999; Zhang et al., 2001), is likely to
act at the level of the cell body rather than on individual axons.
This would be consistent with evidence that some intracellular
signaling events stimulated by neurotrophin treatment of
sympathetic axons are mediated through changes occurring in
the cell body (MacInnis and Campenot, 2002).
Whatever the nature of the signal that stimulates mRNA
transport into the axon, what qualifies a particular transcript to
respond? Cis-acting elements in mRNAs play a key role in
their transport and localization in a range of oocytes, embryos
and somatic cell types (Palacios and Johnston, 2001; Jansen,
2001). In particular, β-actin localization in leading edge of
fibroblast requires specific sequences in the 3′ UTR, the
‘zipcode’ (Kislauskis et al., 1994), and these sequences might
be responsible for the transport of β-actin mRNA into axons
(Olink-Coux and Hollenbeck, 1996; Zhang et al., 2001) (M.
Olink-Coux and P.J.H., unpublished). However, the ADF
transcript, which has neither the β-actin zipcode nor any other
homology with the UTR or coding region of β-actin mRNA,
is nonetheless efficiently transported into neurons. This, along
with the sheer number of different transcripts present in axons
(Fig. 4) (Koenig, 1991; Eng et al., 1999; Zheng et al., 2001)
indicates that there are likely to be several different sequence
motifs that can mediate mRNA transport into the axon.
Furthermore, the rather small number of transcripts that seem
to be confined to the cell body raises the possibility that specific
sequence motifs could also prohibit entry into the axon.
Although some vertebrate axons contain abundant mRNA
but little or no protein synthesis (Wensley et al., 1995),
sympathetic neurons clearly contain mRNA that is actively
translated (Figs 3, 4). However, how large is the contribution
of axonal translation to the neuron as a whole? We found that
synthesis in the axons of our cultures ranged from 1.5% to
5.0% of the total for the neurons, a similar value to the 1.44.1% previously reported for goldfish Mauthner axons
(Alvarez and Benech, 1983) and a higher value than the 0.5%
for rat sympathetic neuronal culture (Eng et al., 1999),
probably because of differences in the lengths of the axons and
the proportions of the cell volume contained there. We further
calculated the specific translation activity per unit volume of
axonal cytoplasm to be 3-10% that of cell body cytoplasm.
Axons that contain just one-tenth of the protein synthetic
capacity per unit volume of the cell body must nonetheless
explain the fact that local protein synthesis is required to
support axonal growth and development, and synaptic
plasticity (Martin et al., 1997; Casadio et al., 1999; Campbell
and Holt, 2001; Zheng et al., 2001; Beaumont et al., 2001;
Ming et al., 2002; Schacher and Wu, 2002; Brittis et al., 2002;
Zhang and Poo, 2002). Furthermore, if total axonal
translational capacity scales up proportionally with axonal
volume then sympathetic axons with length much greater than
4.5 mm would carry out a larger proportion of the neuron’s
total protein synthesis. For example, for these neurons, the ratio
of axonal to non-nuclear cell body volume reaches 10:1 at an
axon length of ~50 mm. If linear scaling occurs, this is the axon
length at which the ratio of axonal to cell body translation
would reach 1:1. It is possible that, as axon length increases or
as neurons form synapses and mature, the specific translation
capacity of axonal cytoplasm declines. However, the discovery
of ribosomes and translation factors in adult vertebrate axons
(Koenig et al., 2000; Zheng et al., 2001) makes it unlikely that
axonal protein synthesis is a property only of developing or
regenerating neurons.
We thank J. R. Bamburg for his generous gift of antiserum against
ADF and S.-J. Kang for performing emulsion autoradiography
experiments. This work was supported by grant NS27073 from the
NIH.
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