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Volume 17, Number 5,
Issue of March 1, 1997
pp. 1539-1547
Copyright ©1997 Society for Neuroscience
Fragile X Mental Retardation Protein: Nucleocytoplasmic Shuttling
and Association with Somatodendritic Ribosomes
Yue Feng1, 2,
Claire-Anne Gutekunst4,
Derek E. Eberhart1, 2,
Hong Yi4,
Stephen T. Warren1, 2, 3, and
Steven M. Hersch4
1 Howard Hughes Medical Institute and Departments of
2 Biochemistry, 3 Pediatrics, and
4 Neurology, Emory University School of Medicine, Atlanta,
Georgia 30322
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Fragile X syndrome, a leading cause of inherited mental
retardation, is attributable to the unstable expansion of a CGG-repeat within the FMR1 gene that results in the absence of the encoded protein. The fragile X mental retardation protein (FMRP) is a ribosome-associated RNA-binding protein of uncertain function that
contains nuclear localization and export signals. We show here detailed
cellular localization studies using both biochemical and
immunocytochemical approaches. FMRP was highly expressed in neurons but
not glia throughout the rat brain, as detected by light microscopy.
Although certain structures, such as hippocampus, revealed a strong
signal, the regional variation in staining intensity appeared to be
related to neuron size and density. In human cell lines and mouse
brain, FMRP co-fractionated primarily with polysomes and rough
endoplasmic reticulum. Ultrastructural studies in rat brain revealed
high levels of FMRP immunoreactivity in neuronal perikarya, where it is
concentrated in regions rich in ribosomes, particularly near or between
rough endoplasmic reticulum cisternae. Immunogold studies also provided
evidence of nucleocytoplasmic shuttling of FMRP, which was
localized in neuronal nucleoplasm and within nuclear pores. Moreover,
labeling was observed in large- and small-caliber dendrites, in
dendritic branch points, at the origins of spine necks, and in spine
heads, all known locations of neuronal polysomes. Dendritic
localization, which was confirmed by co-fractionation of FMRP with
synaptosomal ribosomes, suggests a possible role of FMRP in the
translation of proteins involved in dendritic structure or function and
relevant for the mental retardation occurring in fragile X
syndrome.
Key words:
fragile X syndrome;
mental retardation;
ribosomes;
RNA
binding proteins;
FMRP;
fragile X mental retardation protein;
trinucleotide repeats;
dendritic protein synthesis
INTRODUCTION
Fragile X syndrome is the most frequent form of
inherited mental retardation in humans (Warren and Ashley, 1995 ) and
has a phenotype that commonly includes characteristic craniofacial
dysmorphisms and macro-orchidism. The mutational mechanism of fragile X
syndrome is the unstable expansion of a CGG trinucleotide repeat
within the 5 -untranslated region of the FMR1 gene (Fu et al., 1991; Oberle et al., 1991; Verkerk et al., 1991; Ashley et al., 1993). In
fragile X syndrome, the FMR1 repeat is massively expanded to an average
of 800 triplets, in contrast to the normal mode of 30 triplets (Brown
et al., 1993; Kunst and Warren, 1994; Rousseau et al., 1995). When
expanded beyond ~230 repeats, the FMR1 gene becomes aberrantly
methylated (Sutcliffe et al., 1992; Hornstra et al., 1993) and
transcriptionally silent (Pieretti et al., 1991). Thus, the FMR1 repeat
expansion results in the absence of the encoded protein, fragile X
mental retardation protein (FMRP), which appears to be responsible for
the phenotype (Devys et al., 1993). This has been confirmed by the
characterization of rare patients with the clinical phenotype of
fragile X syndrome, who harbor intragenic deletions or splice/missense
mutations (Meijer et al., 1994; Hirst et al., 1995; Lugenbeel et al.,
1995) rather than repeat expansions.
Attention has now focused on the normal function of FMRP. FMR1 mRNA
expression has been found to be widespread but not ubiquitous. Within
the brain, expression is limited to neurons and has been suggested to
be particularly prominent in the hippocampus, nucleus basalis, and
granular layer of the cerebellum (Abitbol et al., 1993; Hinds et al.,
1993; Hergersberg et al., 1995). FMRP contains amino acid domains that
are common among RNA-binding proteins and has been demonstrated to
interact with RNA homopolymers as well as with a subset of brain mRNAs,
including its own message (Ashley et al., 1993; Siomi et al., 1993).
Initial immunocytochemical studies suggested that FMRP localizes
predominantly to cytoplasm (Devys et al., 1993; Verheij et al., 1993).
Molecular studies, however, have demonstrated the presence of both
nuclear localization and nuclear export signals within FMRP, suggesting
the potential to shuttle between the nuclear and cytoplasmic
compartments (Eberhart et al., 1996). These data, and recent evidence
that FMRP co-fractionates with ribosomes (Eberhart et al., 1996;
Khandjian et al., 1996; Siomi et al., 1996), suggest that FMRP may be
involved in the transport of a subset of nuclear mRNAs, in their
subsequent association with ribosomes, and potentially in regulating
translation. In the current study, we have taken a combined biochemical
and morphological approach to examine whether the subcellular
localization of FMRP is consistent with this model and also to examine
how FMRP is distributed within neurons. We show that FMRP is
cytoplasmic, co-fractionating primarily with free polysomes. Using
immunogold electron microscopy (EM), we directly localize FMRP to
intraneuronal polysomes and provide the first direct visualization of
FMRP in nucleoplasm and nuclear pores. Finally, we find FMRP to be
associated with polysomes in dendrites and dendritic spines, suggesting
that FMRP may play a role in the translation of proteins related to dendritic function.
MATERIALS AND METHODS
Subcellular fractionation. All fractionation steps in
Figure 1 were carried out at 4°C. Cells were disrupted
by vacuum cavitation (200 psi for 10 min) in a buffer in 0.25 M sucrose, 50 mM Tris, pH 7.5, 25 mM KCl, 5 mM MgCl2, 1 mM PMSF, and 1 µg/ml each aprotinin, pepstatin, and
leupeptin (Sigma, St. Louis, MO). The fractionation scheme followed
that of Krajewski (1993). The lysate was subjected to 500 × g centrifugation for 5 min. The pellet was resuspended in
1.6 M sucrose and centrifuged through a 2.1 M
sucrose pad at 150,000 × g for 1 hr to isolate
cytoplasmic-free nuclei. The cytoplasmic supernatant was subjected to
10,000 × g centrifugation for 10 min to yield the
heavy membrane pellet and the postnuclear supernatant. The postnuclear
supernatant was then centrifuged for 1 hr at 130,000 × g. The resulting pellet contained light membrane and
polysomes, and the supernatant was centrifuged further at 180,000 × g for 3 hr to yield the insoluble and soluble cytoplasmic
fractions. Gradient 1 (Frangioni et al., 1992) contained sucrose layers
of 0.25 M, 1.35 M, 1.6 M, and 2.0 M. After centrifugation at 230,000 × g for
2 hr, the top layer containing cytosol, the low-density membrane at the
0.25/1.35 M interface, the high-density membrane at the
1.6/2.0 M interface, and the polysome pellet at the bottom were collected. Gradient 2 (Krajewski et al., 1993) contained sucrose
layers of 0.8 M, 1.23 M, 1.35 M,
and 2.1 M. Eighty microliters was taken from each
interface, which contained smooth endoplasmic reticulum (ER) (0.8/1.23
M interface), light rough ER (RER) (1.23/1.35 M
interface), and heavy RER (1.35/2.1 M interface). All the
fractions were lysed in 1× Laemmli buffer containing 8 M
urea (Feng et al., 1995a), and the protein concentration of each
fraction was determined by Bradford assay (Bio-Rad, Hercules, CA).
Fig. 1.
FMRP distribution in subcellular fractions of
human lymphoblastoid cells. A is a schematic
illustration of subcellular fractionation of EBV-transformed human
lymphoblastoid cells. The descriptor under key fractions
refers to the panels below
(B-D), with INT as interface. A detailed
description and protocol is provided in Materials and Methods. In
B, the left panel shows SDS-PAGE immunoblot analysis of FMRP and P0 in crude subcellular fractions. Total protein (3 µg) from each fraction of B1-B5 was loaded.
Densitometric analysis of immunoblot signals was used to calculate the
total yield of FMRP in each corresponding fraction, based on the total fraction volume. The relative percentage of FMRP in each fraction was
plotted as shown in the right panel. C
shows the SDS-PAGE immunoblot analysis of FMRP and P0 in separated
postnuclear supernatant fraction. C1-C4 in sucrose
gradient fractionation 1 represent cytosol; low-density membranes
(plasma membrane, Golgi, and smooth ER); high-density membranes (RER);
and free polysome pellet, respectively. Total protein (1.5 µg) from
each fraction was loaded. D shows the SDS-PAGE
immunoblot analysis of FMRP and P0 in various ER components.
D1-D3 in sucrose gradient fractionation 2 represent smooth ER, light RER, and heavy RER. Total protein (3 µg) from each
fraction was loaded.
[View Larger Version of this Image (29K GIF file)]
Fractionation of rat cortex homogenate and synaptosomal lysate.
Cerebral cortex from adult male Sprague Dawley rats was rapidly removed after decapitation and placed into 0.32 M sucrose
(10% w/v) containing 4 mM HEPES, pH 7.3, 5 mM
MgCl2, 200 µg/ml cycloheximide, 1 mM PMSF,
and 1 µg/ml each aprotinin, pepstatin, and leupeptin (Sigma). The
tissue was incubated for 20 min on ice to arrest polysome migration
before gentle homogenization (nine strokes) with a glass homogenizer.
The velocity centrifugation procedure was essentially as described by
(Huttner et al., 1983) to generate S1, S2, P2, S3, and P3, as
illustrated in Figure 2. Each fraction was then
subjected to lysis in 1× Laemmli buffer containing 8 M
urea. Bradford assay was carried out for each fraction to determine the
protein concentration, followed by SDS-PAGE analysis. To generate P2
lysate, the pellet was lysed in 600 µl 0.16 M sucrose
containing 2.5 mM MgCl2, 10 mM
Tris-HCl, pH 7.5, 50 mM KCl, and 0.5% NP40. For EDTA
lysis, P2 was lysed in the buffer described above, except that
MgCl2 was replaced with 30 mM EDTA. The P2
lysate was left on ice for 15 min, followed by microfuge centrifugation
to remove insoluble membrane components, and the corresponding
supernatant was loaded onto a 20-47% (w/w) sucrose gradient
containing 80 mM NaCl, 20 mM Tris, pH 7.5, and
5 mM MgCl2 or 30 mM EDTA,
correspondingly. After centrifugation in a Beckman SW41 rotor at 39,000 rpm for 100 min at 4°C, the entire gradient was fractionated by
upward displacement into twelve ~1 ml fractions using a gradient
fractionator (Isco, Lincoln, NE). The fractions were subjected to
Bradford assay to determine the corresponding protein concentration.
Because the top three fractions contained the highest total protein
levels, to avoid overloading, an aliquot from fractions 1 and 2 matching the amount of total protein in fraction 3 (70 µg) was
subjected to TCA precipitation, whereas for the rest of fractions, the
entire 1 ml was subjected to TCA precipitation. The precipitates were resuspended in 10 µl of 0.5 M Tris-HCl, pH 7.5, and 0.5 M NaCl, followed by the addition of 30 µl of 1× Laemmli
buffer containing 8 M urea before SDS-PAGE analysis.
Fig. 2.
FMRP immunocytochemistry in the rat brain.
A is a coronal section demonstrating widespread FMRP
labeling. The most intense labeling is in the cellular layers of the
hippocampus (h) and pyriform cortex
(p), which are regions with extremely high
neuronal densities. The deeper layers of the cerebral cortex
(c) are also well labeled. B demonstrates
FMRP immunoreactivity in the frontal cortex at higher magnification. It
appears that most neurons in each cortical layer are FMRP-positive.
C illustrates the cellular pattern of FMRP
immunoreactivity in layer V pyramidal cells from frontal cortex.
Staining is very dense in perikarya and proximal dendrites
(triangles). In contrast, nuclear staining
(arrows) is uncertain. D is an electron
micrograph of the soma of a cerebral cortical pyramidal cell. With
immunoperoxidase, dense cytoplasmic staining is evident. Although the
nucleus is somewhat dark, FMRP immunoreactivity is not clearly present.
Scale bars: A, 50 mm; B, 100 µm;
C, 50 µm; D, 1 µm.
[View Larger Version of this Image (175K GIF file)]
Immunoblot analysis and antibodies. Protein samples were
resolved on 12% SDS/polyacrylamide gels (Bio-Rad) along with
prestained molecular weight markers (Bio-Rad) and were subsequently
electroblotted at 30 V overnight onto nitrocellulose membranes
(Schleicher and Schuell, Keene, NH). Immunostaining and ECL detection
were performed at room temperature according to the manufacturer's
protocol (Amersham, Arlington Heights, IL) with the secondary antibody
incubation performed in a buffer containing 0.5× PBS, 0.5% milk, 10 mM Tris, pH 7.5, 75 mM NaCl, 0.5 mM
EDTA, 0.5% NP40, 0.25% deoxycholate, 0.05% SDS, and 2.5 mM NaF. The primary antibodies and the concentrations used
were as follows: the anti-FMRP monoclonal antibody mAb1a (Devys et al.,
1993) at 1:10,000; the anti-P polyclonal human autoantibody (Bonfa et
al., 1989) at 1:1000; the monoclonal antibody against the large subunit
of wheat germ RNA polymerase II (8WG16, Thompson et al., 1989) at
1:4000; the polyclonal antibody against rabbit mitochondrial branched
chain  -ketoacid dehydrogenase E2 (Heffelfinger et al., 1983) at
1:700; the monoclonal antibody against human LDH (Sigma) at 1:4000; and
the monoclonal antibody against mouse synaptophysin (Boehringer
Mannheim, Indianapolis, IN) at 1:150.
Immunocytochemistry. Four young adult male Sprague Dawley
(Harlan, Prattville, AL) rats were deeply anesthetized with chloral hydrate and perfused transcardially with 240 ml of 3% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer (PB).
Brains were removed and sectioned at 40 µm using a vibratome
(Technical Products International). Sections were collected in 100 mM PBS and rinsed in 50 mM TBS, pH 7.2. Some
sections were processed for immunoperoxidase using DAB as the
chromogen. Briefly, sections were incubated for 1 hr at 4°C in TBS
containing 4% normal goat serum (NGS) and avidin (Vector Research,
Burlingame, CA) at 10 µg/ml, rinsed in TBS, and incubated at 4°C on
a shaker for 48 hr in TBS containing 2% NGS, biotin (Vector Research)
at 50 µg/ml, and monoclonal mouse anti-FMRP antibodies mAb1a
(Devys et al., 1993) at 1/1000. Sections were then rinsed in TBS and
incubated overnight in biotinylated goat anti-mouse secondary antibody
(Vector Research) in TBS containing 2% NGS. After rinses in TBS,
sections were incubated in ABC Elite (Vector) for 4 hr followed by TBS rinses. Final development was done by incubation in 0.05% 3,3-DAB tetrahydrochloride (Sigma) and 0.01% hydrogen peroxide in 50 mM Tris buffer for 5-15 min. Sections were then rinsed
with TBS for another hour.
Immunogold labeling for EM was performed as follows. Rat brain sections
were preblocked in TBS containing 4% NGS and 0.05% Triton X-100 and
incubated in mouse monoclonal anti-FMRP antibody mAb1a (Devys et al.,
1993) at 1/1000 in 2% NGS-TBS for 60 hr at 4°C on a shaker
platform. Sections were then rinsed in TBS for a total of 1 hr and
incubated overnight in goat anti-mouse secondary antibody (1:50)
conjugated to 1.4 nm gold particles (Nanoprobes, Stony Brook, NY) in
TBS with 2% NGS. After rinsing in PB, the sections were fixed with 2%
glutaraldehyde. After several washes in PB, sections were
silver-intensified according to the silver intensification kit from
Nanoprobes (Stony Brook, NY). Finally, sections were post-fixed in 1%
OsO4 in PB, rinsed, and dehydrated in ascending
concentrations of ethanol and propylene oxide (1:1) and embedded in
Epon (Ted Pella, Redding, CA). Ultrathin sections (90 µm) were cut
using a Leica Ultracut S ultramicrotome. Lead staining was performed on
grids by 5 min incubation in 5% aqueous uranyl acetate followed by 10 min incubation in lead citrate. Ultrathin sections were examined using
a JEOL 100C electron microscope. Controls for both DAB and immunogold
included sections processed in parallel but without exposure to the
FMRP antibody and preabsorption controls in which the primary antibody
was first incubated with purified FMRP conjugated to CNBr-activated
Sepharose. EM was performed on the antibody deletion but not the
preabsorption controls, because the latter procedure usually leaves
trace residual specific staining at the EM level attributable to
absorption not being 100%.
RESULTS
FMRP co-fractionates with nonmembrane polysomes and the RER
To define the distribution of FMRP in various subcellular
compartments, normal EBV-transformed human lymphoblastoid cell lysate was fractionated, as illustrated in Figure 1A,
followed by immunoblot analysis of the fractions using an anti-FMRP
monoclonal antibody (Devys et al., 1993). The cell lysate was initially
fractionated by sequential velocity centrifugation, resulting in
fractions designated as nuclei, heavy membrane, light
membrane/polysome, insoluble cytoplasm, and soluble cytoplasm (Fig.
1A, fractions B1-B5). To verify the content of these
fractions, immunoblot analysis was performed using antibodies against
specific compartmentalized proteins (data not shown). RNA polymerase II
was found primarily in the nuclear fraction (B1);
mitochondrial branched chain -ketoacid dehydrogenase E2 subunit was
confined to the heavy membrane pellet (B2); ribosomal acidic
phosphoprotein P0 (Bonfa et al., 1989) was found primarily in the light
membrane/polysome pellet (B3); and lactate dehydrogenase was
found in the soluble cytoplasm (B5). These fractions were
then examined for the presence of FMRP. Densitometric quantitation of
FMRP signal on the immunoblot was used to calculate the percentage of
total cellular FMRP in each fraction based on the corresponding
fraction volume. Even with a conservative estimate, considering that
the most intensive signal in B3 may approach saturation, >80% of FMRP
was confined to the light membrane/polysome fraction (Fig. 1,
B3). The ~37 kDa P0 acidic phosphoprotein located on the
60S ribosomal subunit was also confined primarily to this fraction,
verifying the expected presence of RER and translating ribosomes. The
detection of both FMRP and P0 in the heavy membrane fraction
(B2) was most likely attributable to the presence of limited
RER in this fraction. Interestingly, a low level of FMRP (~4%) was
detected in the nuclear fraction with no P0 detected even after
prolonged exposure. Detection of FMRP in the insoluble cytoplasm
required prolonged exposure, and a negligible amount of FMRP was
detected in the soluble cytoplasm, indicating that FMRP is rarely
present as a free protein. Very similar subcellular distribution of
FMRP was observed in mouse brain in a parallel experiment (data not
shown).
To determine whether FMRP only co-fractionates with components that
carry ribosomes, i.e., RER, the postnuclear supernatant was
fractionated through a discontinuous sucrose gradient of 0.25, 1.35, 1.6, and 2.0 M sucrose to separate various membrane
components (Frangioni et al., 1992) as well as free polysomes (Fig.
1A, sucrose gradient fractionation 1).
Four fractions were obtained: free cytosol (C1, 0.25 M sucrose layer); low-density membrane containing plasma
membrane, Golgi, and smooth ER (C2, 0.25-1.35 M
sucrose interface); RER (C3, 1.6-2.0 M sucrose
interface); and nonmembrane associated polysomes (C4,
pellet). Examination of these fractions revealed that all the FMRP was
restricted to fractions containing RER and polysomes (Fig.
1C, C3, C4). P0 also localized
to these fractions, confirming the expected presence of 60S ribosomal
subunits. The exclusive fractionation of FMRP and P0 to C3 and C4 was
also observed when human fibroblasts, HeLa cells, and mouse brain were examined (data not shown).
The co-fractionation of FMRP with the RER was confirmed by a similar
fractionation (Krajewski et al., 1993) of the postnuclear supernatant
through a discontinuous sucrose gradient of 0.8, 1.23, 1.35, and 2.1 M sucrose (Fig. 1A, sucrose
gradient fractionation 2). This fractionation separated smooth ER
(D1, 0.8-1.23 M sucrose interface), light RER
(D2, 1.23-1.35 M sucrose interface), and heavy
RER (D3, 1.35-2.1 M sucrose interface). As
shown in Figure 1D, the majority of FMRP localized to
the heavy RER (D3), with little FMRP found in the light RER
(D2), and none detectable in the smooth ER fraction
(D1), even after prolonged film exposure. P0 fractionated to
both D2 and D3, as expected. The low level of FMRP in the light RER
fraction (D2) as compared with P0 suggests that FMRP is
associated with only a subset of ribosomes.
FMRP is highly expressed in neurons
FMRP expression was examined by light microscopy and
immunoperoxidase using the FMRP-specific monoclonal antibody, as
described above. As described previously in human (Devys et al., 1993), FMRP was highly expressed within neurons throughout the rat brain (Fig.
2A), whereas glial staining was minimal. As predicted
by in situ hybridization studies (Abitbol et al., 1993;
Hinds et al., 1993), hippocampus (Fig. 2A), nucleus basalis of Meynert, and cerebellum showed high levels of FMRP staining at low
magnification. When comparing the immunoreactivity of individual
neurons, however, many other neuronal types in a variety of forebrain
and hindbrain regions were just as intensely stained as the neurons in
these regions. Because the EM studies in this paper are in cerebral cortex, its FMRP immunoreactivity will be more fully described. Cerebral cortical pyramidal and nonpyramidal cells were filled with DAB
reaction product (Fig. 2B,C).
Reaction product densely filled perikarya and proximal dendrites (Fig.
2C). Some possible labeling was also apparent in nuclei but
always much lighter than in the cytoplasm. Smaller caliber elements
were also visible in the neuropil, but their identity could not be
resolved at the light microscopic level. All immunoreactivity was
abolished by preabsorption of primary antibodies with excess purified
FMRP conjugated to CNBr-activated Sepharose. Labeling was also absent when primary antibodies were omitted.
FMRP is present in nuclei and cytoplasm of neurons and co-localizes
with RER and somatodendritic polysomes
FMRP localization was examined at the ultrastructural level by
immunoperoxidase and immunogold EM. Immunoperoxidase provided greater
sensitivity for determining whether neuropil elements were labeled,
whereas immunogold provided sufficient spatial resolution to examine
the subcellular associations of FMRP. Most neuronal somata encountered
appeared to be labeled (Fig. 2D). Because the DAB
reaction product was difficult to distinguish from nucleoplasm, however, nuclear localization of FMRP could not be clearly determined in the immunoperoxidase material (Fig. 2D). The most
important finding with immunoperoxidase was that many dendrites of all
calibers were labeled, indicating that FMRP can be found in relatively distal dendrites.
The immunogold labeling provided much greater spatial resolution.
Immunogold particles were found within neuronal nuclei (Fig. 3A) but were rare in nuclei of any other cell
types including astrocytes (Fig. 3B), oligodendrocytes (Fig.
3C), and epithelial cells lining the blood vessels (data not
shown). Immunogold particles were visualized in nuclear pores within
neurons, most likely labeling FMRP molecules in transit between the
cytoplasm and the nucleus (Fig.
3D,E). In neuronal perikarya, most
immunogold particles were found free in the cytoplasm and appeared to
concentrate in regions rich in free ribosomes, particularly near or
between cisternae of the RER (Fig. 3F-H).
A small proportion of immunogold particles were also found directly in
contact with the membranes of RER (Fig.
3F,G). Immunogold particles were
rarely seen directly associated with the plasma membrane or with other
organelles, including mitochondria, transport vesicles, multivesicular
bodies, or Golgi apparatus (Fig. 3C). Even though the
highest concentrations of immunogold particles were seen in neuronal
perikarya, numerous collections of particles were also found in
dendrites at sites where ribosomes are customarily found. Specifically,
dendritic immunogold particles were frequently visualized near
cisternae of smooth ER (Fig.
4A,B), at dendritic
branch points, and at the origins of dendritic spines (Fig.
4C). Label was not confined to proximal dendrites but was also frequently found in relatively small-caliber dendrites. Many dendritic spines also contained immunogold particles that were either
free in the cytoplasm (Fig.
4E,F) or associated with the spine apparatus (Fig. 4D). In contrast to the intense
somatodendritic labeling, most axons and axon terminals were free of
immunogold particles. FMRP immunoreactive axon terminals contained only
one to three immunogold particles that were cytoplasmic in location (Fig. 4G). When using immunoperoxidase, which is more
sensitive than immunogold, more FMRP-immunoreactive axon terminals
could be identified (Fig. 4H).
Fig. 3.
A, Electron micrograph of the soma
of a labeled cerebral cortical pyramidal neuron. Immunogold particles
are present both in the nucleus (n) and perikaryon.
Little of the cytoplasmic label is associated with the plasma membrane,
mitochondria, or Golgi apparatus (arrow).
B, C, Few particles were found in the
nuclei or cytoplasm of astrocytes (a) or
oligodendrocytes (o). D, Tangential section through the nuclear envelope of a pyramidal cell showing three
nuclear pores (arrows), one of which (longer
arrow) contains an immunogold particle. E,
Cross-section through the nuclear envelope showing immunogold particles
within nuclear pores (arrows).
F-H, In perikarya, most immunogold
particles are clustered between the cisternae of RER. These regions are
especially rich in free ribosomes, visible here as fine electron-dense
particles (asterisks). A few immunogold particles are
also seen in direct contact with the cisternae (arrows).
Scale bars: A-C, 1 µm;
D-H, 500 µm.
[View Larger Version of this Image (185K GIF file)]
Fig. 4.
Electron micrographs demonstrating FMRP
localization in cellular processes in cerebral cortex.
A-C, Dendrites (d) in
cross-section and longitudinal section showing that immunogold
particles are either free in the cytoplasm or clustered around
cisternae of smooth ER (arrows) or at the origins of
dendritic spines (triangles). D-F, Dendritic spines (s)
containing immunogold particles, which are either free in the cytoplasm
or associated with the spine apparatus (arrows).
G, Rare axon terminals (a) contain
immunogold particles that are cytoplasmic in location.
H, FMRP-immunoreactive axon terminals (a)
are more easily identified using immunoperoxidase. Scale bars:
A-H, 500 nm.
[View Larger Version of this Image (85K GIF file)]
Background immunogold labeling was assessed by examining sections that
were processed in parallel but not exposed to FMRP antibody. In these
sections, scattered immunogold particles were diffusely present;
however, they were at a very low density, did not occur in clusters,
and did not show the selectivity described above for neurons, nuclear
pores, dendrites, or polysomes.
FMRP co-fractionates with brain polysomes, including those found
in synaptosomes
To confirm the microscopic finding that FMRP closely co-localizes
with somatodendritic polysomes and RER, rat cortex was gently homogenized and fractionated by velocity centrifugation to generate a
low-speed supernatant (Fig. 5, S1), a crude
synaptosomal pellet (P2) and corresponding supernatant
(S2), and finally a high-speed polysomal pellet
(P3) and corresponding supernatant (S3). Various marker antibodies were used in immunoblot analysis to verify contents in the above fractions. As shown in Figure 5A, the majority
of synaptophysin, a vesicle membrane protein broadly used as a
synaptosomal marker, was fractionated into the synaptosomal pellet.
Also as expected, the soluble cytoplasmic protein LDH was confined
primarily to the supernatant fractions. FMRP and P0 signals from the
cytoplasmic lysate were primarily confined to fraction P3, comparable
with what was observed in the light membrane/polysome fraction derived from lymphoblasts. The remaining FMRP and ribosomes fractionated into
the crude synaptosomal pellet (P2) and was not detected in the high-speed supernatant S3.
Fig. 5.
Association of FMRP with translating ribosomes in
rat cortex. A, Subcellular fractionation of rat cortex
by velocity centrifugation. The fractionation procedures are
illustrated on the left panel, with S
indicating supernatant and P indicating pellet. A
detailed description and protocol are provided in Materials and
Methods. The right panel shows SDS-PAGE immunoblot
analysis of FMRP and other marker proteins in the corresponding
fractions as indicated. Based on Bradford assay, 20 µg of total
protein from each fraction was used in this blot. B,
Association of FMRP with polysomes in synaptosomal lysate. SDS-PAGE
immunoblot analysis was performed using linear sucrose gradient
fractions containing synaptosomal lysates with the presence of
Mg2+ or EDTA, as described in Materials and Methods. The
signals of FMRP and P0 protein are indicated on the
right. The sedimentation of ribosomal components in
human lymphoblasts monitored at OD254 in a parallel gradient are
indicated on top of the corresponding fractions.
[View Larger Version of this Image (52K GIF file)]
To determine whether FMRP associates with polysomes in synaptosomes,
the majority of which originate from axospinous synapses, P2 was lysed
under mild conditions with a low level of nonionic detergent. After
removing the residual membrane component, the synaptosomal lysate was
fractionated through a linear sucrose gradient, and each fraction
collected was subjected to immunoblot analysis. FMRP co-fractionated
with the P0 protein in the fractions containing monosomes and polysomes
(Fig. 5B), similar to our previous observation in human
lymphoblasts (Eberhart et al., 1996). To provide additional evidence
for FMRP-ribosome association, P2 was lysed in the presence of 30 mM EDTA, a condition known to dissociate ribosomes into
subunits and release mRNP particles. The disappearance of P0 signals in
the polyribosomal fractions, together with the concomitant accumulation
of P0 signals in the top fractions (Fig. 5B, fractions
2 and 3), confirmed that EDTA caused ribosome
dissociation. FMRP shifted primarily to the top fractions containing
~60-100S particles (fractions 3 and
4). This result was consistent with our previous
observation in EDTA-treated lymphoblastoid lysate (Eberhart et al.,
1996), suggesting that FMRP associates with polysomes as an mRNP
component.
DISCUSSION
We have taken a combined biochemical and morphological approach to
study the cellular and subcellular localization and associations of
FMRP. Our primary goal was to seek evidence supporting the hypothesis
that FMRP shuttles between the nucleus and cytoplasm, carrying mRNAs to
ribosomes and perhaps playing a role in the regulation of translation.
Furthermore, because we have shown previously that FMRP binds only to a
subset of mRNAs (Ashley et al., 1993), our second major goal was to
determine whether this selectivity also extends to the ribosomes to
which the FMRP-mRNA complexes are presumed to bind. The results of
these experiments provide the first visualization of FMRP in neuronal
nuclei, in transit within nuclear pores, and in association with
polysomes in neuronal perikarya, but also distributed throughout the
dendritic tree. We hypothesize that some of the mRNAs carried by FMRP
code for proteins important for dendritic functions that are thus
compromised in fragile X syndrome, leading to mental retardation.
The cellular localization of FMRP
By immunocytochemistry, we have observed widespread and intense
FMRP immunoreactivity in neurons throughout the CNS. Previous studies
using in situ hybridization or immunocytochemistry have similarly detected abundant FMRP mRNA and protein and have suggested that it is especially abundant in hippocampal pyramidal cells, giant
cholinergic neurons of the nucleus basalis, and the cerebellum (Devys
et al., 1993; Hinds et al., 1993). It is our impression, however, that
the apparent enrichment of FMRP in these regions is attributable to the
high packing density of neurons they contain, because individual
neurons do not appear any more immunoreactive than do neurons in many
other regions. Immunoperoxidase, however, does not lend itself very
well to studying protein abundance of neurons, because the staining
process is nonlinear. In addition, neuronal size and packing density
are difficult to take into account. Thus, although FMRP expression in
the brain is strongly neuronal, it is not yet clear whether there are
meaningful differences in expression between neuronal populations.
Nucleocytoplasmic shuttling and ribosome association of FMRP
Several recent studies (Siomi et al., 1993; Burd and Dreyfuss,
1994) have suggested that FMRP may be a member of a new family of RNA
binding proteins (RNPs). Expression studies in vitro have shown that FMRP can bind to RNAs (Ashley et al., 1993; Siomi et al.,
1993) and that experimental mutations can disrupt RNA binding (Siomi et
al., 1994). We recently demonstrated a functional nuclear localization
signal and a functional nuclear export signal within FMRP (Eberhart et
al., 1996), suggesting that nascent FMRP may be imported into the
nucleus for RNP assembly, followed by export to the cytoplasm driven by
the FMRP nuclear export signal. Missing, however, has been direct
localization of native FMRP within the nucleus, perhaps because of the
predominant steady-state localization of FMRP to the cytoplasm.
Consistent with this, our cellular fractionation studies showed that
only ~4% of cellular FMRP co-fractionates with the nucleus. FMRP
immunocytochemistry using immunoperoxidase suggested nuclear labeling;
however, the reaction product was not definitively distinguishable from
the normal nuclear contents. Immunogold EM, however, unequivocally
localized FMRP within neuronal nucleoplasm. Furthermore, this method
was able to label FMRP within nuclear pores, presumably in transit
between the nucleus and cytoplasm. These results strongly support the
hypothesis that FMRP shuttles from cytoplasm to nucleus, where it
associates with RNA and perhaps other proteins before being exported as
an mRNP particle (Feng et al., 1995b; Eberhart et al., 1996) to the
cytoplasm.
Once in the cytoplasm, >85% of FMRP co-sediments with ribosomes
(Khandjian et al., 1996). In the present study, we have defined further
the association of FMRP and ribosomes using co-fractionation and
immunogold studies. We have determined that FMRP associates most
abundantly with a subset of free polysomes and also with some
RER-associated ribosomes. These associations were also directly visualized by immunogold EM in rat brain. FMRP was not found to associate with other organelles, including Golgi apparatus,
mitochondria, or plasma membrane, by either subcellular fractionation
or EM, indicating that FMRP is unlikely to play a role in the
corresponding cellular functions. These data directly confirm previous
biochemical suggestions that FMRP is chiefly associated with ribosomes
in mammalian cells.
FMRP in dendritic and axonal compartments
Neuronal ribosomes are not only present in the perikaryon but are
also distributed through their dendrites and dendritic spines (Steward
and Levy, 1982; Spacek, 1985; Steward and Reeves, 1988). There is
increasing evidence that particular mRNAs are targeted to distinct
intraneuronal compartments (Garner et al., 1988; Burgin et al., 1990;
Tiedge et al., 1991; Miyashiro et al., 1994; Mohr et al., 1995). This
targeting has been proposed as a mechanism to provide and regulate the
local synthesis of particular proteins (see Steward, 1994). Our first
indication that FMRP could be distributed to pre- or postsynaptic
compartments was provided by its co-fractionation with synaptosomal
ribosomes from rat cerebral cortex. When synaptosomal ribosomes were
dissociated with 30 mM EDTA, FMRP appeared to be present
primarily in complexes of 60-100S. This most likely represents FMRP
associated with the 60S subunit (Khandjian et al., 1996; Siomi et al.,
1996) and large mRNPs (Eberhart et al., 1996). These results also
suggest that synaptosomal FMRP is primarily postsynaptic, because
ribosomes are not found in axon terminals.
Using immunogold, we localized FMRP not only to neuronal perikarya, but
also to large- and small-caliber dendrites and dendritic spines.
Because individual immunogold particles are relatively large, direct
labeling of individual ribosomes could not be visualized. The
immunogold label, however, occurred in clusters at the exact intradendritic sites where ribosomes are usually found, including dendritic branch points, near cisternae of endoplasmic reticulum, at
the bases of dendritic spines, and within spine heads. Although FMRP
was found everywhere polysomes have been identified, this localization
of FMRP in dendrites and spines raises the possibility that FMRP may be
involved in regulating synthesis of proteins related to postsynaptic
function. Interestingly, synthesis of some postsynaptic proteins can be
upregulated by the actions of neurotransmitter receptors or by
increased ionic conductances (Weiler and Greenough, 1993). Because FMRP
is most abundant in tissues with high levels of protein synthesis, such
as CNS neurons, healing tissues, and cells which are induced for
proliferation (Devys et al., 1993; Khandjian et al., 1996), it has been
suggested that it may play a role in active translation.
Using immunoperoxidase and immunogold, FMRP was also localized in small
numbers of axons and axon terminals. Although axons do not contain
ribosomes, they do contain many mRNAs (Perrone-Capano et al., 1987).
Some mRNA species have been identified that appear to be specifically
targeted to axons, including those for  -actin (Olink-Coux and
Hollenbeck, 1996), vasopressin (Trembleau et al., 1994; Mohr et al.,
1995), oxytocin (Jirikowski et al., 1990), and BC1 (Tiedge et al.,
1993). The lack of axonal ribosomes has led to an assumption that
translation does not occur in axons, however, some evidence for axonal
protein synthesis is beginning to accumulate (reviewed in Van Minnen,
1994). It has also been hypothesized that axonal targeting of mRNA
down-regulates protein synthesis by removing these mRNAs from the
translating ribosomes (Jirikowski et al., 1990; Mohr et al., 1995).
Whether FMRP has any functional significance in the axon still remains
to be elucidated; however, it may play a role in the localization or
regulation of some axonal mRNAs.
Fragile X syndrome and FMRP
It is now clear that the phenotype associated with fragile X
syndrome, chiefly mental retardation, is the consequence of the absence
of FMRP. Evidence is now accumulating that the normal function of FMRP
is related to some aspect of mRNA transport and/or translation. FMRP
clearly does not assume a vital role in translation, given the
relatively subtle phenotype occurring in fragile X syndrome. This
possibility is also suggested by the general lack of major brain
pathology in the fmr1 knockout mouse (Dutch-Belgian Fragile X
Consortium, 1994). On the other hand, the general function FMRP serves
may be crucial, and its loss in fragile X syndrome may be partially
made up for by the presence of other members of its gene family, such
as FXR1 (Coy et al., 1995; Siomi et al., 1995) and FXR2 proteins (Zhang
et al., 1995).
The most notable neuropathology identified in postmortem brain tissue
from patients with fragile X syndrome is that cerebral cortical
dendritic spines are lengthened and possess enlarged heads (Rudelli et
al., 1985; Hinton et al., 1991). Our localization of FMRP to the spine
heads and bases raises the possibility that the spine dysmorphisms
occurring in fragile X are related to local alterations in protein
translation attributable to the loss of FMRP. It has been proposed that
postsynaptic mRNAs and ribosomes may serve to synthesize components
required for synapse development and plasticity (Steward and Levy,
1982; Steward, 1983; Steward and Falk, 1986) and for the induction and
maintenance of long-term potentiation (Frey et al., 1991; Fazeli et
al., 1993). Although direct evidence does not yet exist, we speculate
that altered protein synthesis in dendrites and spines occurs in
fragile X, leading to synaptic dysfunction and mental retardation.
FOOTNOTES
Received Aug. 23, 1996; revised Nov. 11, 1996; accepted Dec. 9, 1996.
This work was supported in part by National Institutes of Health Grants
HD20521 (S.T.W.) and NS01624 (S.H.). S.T.W. is an investigator of The
Howard Hughes Medical Institute. We thank Lisa Lakkis, Janelle Clark,
Ryan Berglund, and Priya Naik for assistance in preparation of the
manuscript and illustrations and Fuping Zhang for assistance and
discussion. We thank Drs. Jean-Louis Mandel, Keith Elkon, Daniel
Reines, and Dean Danner for providing antibodies.
Correspondence should be addressed to Dr. Steven M. Hersch, Department
of Neurology, Emory University School of Medicine, Woodruff Memorial
Building, Suite 6000, Atlanta, GA 30322.
Y.F. and C.-A.G. contributed equally to this work.
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