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The Journal of Neuroscience, September 1, 2000, 20(17):6385-6393
CaMKII 3' Untranslated Region-Directed mRNA Translocation in
Living Neurons: Visualization by GFP Linkage
Martha S.
Rook,
Mei
Lu, and
Kenneth S.
Kosik
Center for Neurological Diseases, Brigham and Women's Hospital,
Boston, Massachusetts 02115, and Department of Neurology, Harvard
Medical School, Boston, Massachusetts 02115
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ABSTRACT |
The CaMKII mRNA extends into distal hippocampal dendrites, and
the 3' untranslated region (3'UTR) is sufficient to mediate this
localization. We labeled the 3'UTR of the CaMKII mRNA in hippocampal
cultures by using a green fluorescent protein (GFP)/MS2 bacteriophage
tagging system. The CaMKII 3'UTR formed discrete granules throughout
the dendrites of transfected cells. The identity of the fluorescent
granules was verified by in situ hybridization. Over 30 min time periods these granules redistributed without a net increase in
granule number; with depolarization there is a tendency toward
increased numbers of granules in the dendrites. These observations
suggest that finer time resolution of granule motility might reveal
changes in the motility characteristics of granules after
depolarization. So that motile granules could be tracked, shorter
periods of observation were required. The movements of motile granules
can be categorized as oscillatory, unidirectional anterograde, or
unidirectional retrograde. Colocalization of CaMKII 3'UTR granules
and synapses suggested that oscillatory movements allowed the granules
to sample several local synapses. Neuronal depolarization increased the
number of granules in the anterograde motile pool. Based on the time
frame over which the granule number increased, the translocation of
granules may serve to prepare the dendrite for mounting an adequate
local translation response to future stimuli. Although the resident
pool of granules can respond to signals that induce local translation,
the number of granules in a dendrite might reflect its activation history.
Key words:
RNA localization; RNA translocation; CaMKII; 3'UTR; RNA granules; synaptic plasticity
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INTRODUCTION |
Local protein synthesis within
dendrites is an attractive mechanism potentially capable of explaining
many phenomena related to neuronal plasticity. In dendrites,
polyribosomes are positioned beneath the postsynaptic density and
therefore are situated ideally to perform protein synthesis in
response to neuronal activity (Steward and Levy, 1982 ; Steward and
Fass, 1983). Most mRNAs that reside in dendrites appear to be located
there constitutively (Miyashiro et al., 1994; Crino and Eberwine, 1996;
Steward, 1997; Kiebler and DesGroseillers, 2000), whereas others, such
as the Arc mRNA (Link et al., 1995; Lyford et al., 1995;
Steward et al., 1998; Guzowski et al., 1999), appear in dendrites only
after neuronal stimulation. In both cases the mechanisms that underlie
RNA motility, the signals that activate translocation and target mRNAs
to specific sites, and the nature of mRNA docking are understood only poorly.
mRNAs are present in dendrites along microtubules (Bassell et al.,
1994) in the form of granules that appear to be the active transport
unit and contain translational machinery (Ainger et al., 1993;
Ferrandon et al., 1994; Wang and Hazelrigg, 1994; Knowles et al.,
1996). Direct visualization in living cells demonstrates that
granules translocate along microtubules within dendrites (Knowles et
al., 1996) and oligodendrocytes (Ainger et al., 1993) and during
Drosophila oogenesis (Theurkauf and Hazelrigg, 1998) or
along actin filaments in the yeast bud (Bertrand et al., 1998). A few
proteins within these complexes, such as Staufen (Kiebler et al.,
1999), hnRNP A2 (Hoek et al., 1998), and the zipcode-binding protein
(Ross et al., 1997; Deshler et al., 1998), have been identified and
represent candidate regulators and effectors of RNA trafficking
Recently, green fluorescent protein (GFP) was linked to the ASH1 mRNA
in yeast, and translocation into the yeast bud was observed directly
(Bertrand et al., 1998; Beach et al., 1999). We have applied a similar
system to visualize the motility of the 3' untranslated region (3'UTR)
of calcium/calmodulin-dependent kinase II (CaMKII) in primary
hippocampal cultures. The mRNA for CaMKII is localized dendritically
(Steward, 1997), and its 3'UTR can mediate this localization (Mayford
et al., 1996). The function of CaMKII in the induction and
maintenance of long-term potentiation (Malenka and Nicoll, 1999) fits
well with many of the expected properties of a dendritic mRNA. After
induction of LTP the levels of CaMKII mRNA are increased in both the
cell body and the dendrites (Thomas et al., 1994; Roberts et al.,
1998), and strong tetanic stimulation of the Schaffer collateral
pathway leads to increased levels of CaMKII in dendrites earlier
than can be accounted for by transport from the cell bodies (Ouyang et
al., 1999). Thus the signals that activate CaMKII and initiate local
CaMKII translation may localize CaMKII mRNA-containing granules
at activated synapses also. Our dynamic observations demonstrate that
labeled CaMKII 3'UTR can assume the motility characteristics of RNA
granules and that neuronal depolarization increases labeled CaMKII
3'UTR granules in dendrites by driving oscillatory granules into an
anterograde motile pool.
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MATERIALS AND METHODS |
Plasmid construction. To make the RNA expression
vector RSV-lacZ-MS2bs-CaMKII3'UTR, we amplified the 3'UTR of
CaMKII from pBluescript containing the mouse CaMKII coding
sequence and 3'UTR (a gift from Dr. Mark Mayford, University of
California San Diego School of Medicine) with a 5' primer
containing two copies of the MS2-binding site and a
BglII site directly 3' of the MS2-binding site sequence,
5'-gcgatccaacatgaggatcacccatgtcagctggtcgactctagaaaacatgaggatcacccatg-tagatctggggcgccctccgtc-3', and a 3' primer complementary to the 3' end of CaMKII,
5'-ggattctttaaatttgtactatttat-3'. The resulting PCR fragment was
ligated into the PCRblunt vector (Invitrogen, San Diego, CA). The
resultant vector was cleaved with BamHI and ligated into
BamHI-cleaved RSV- -gal vector (a gift from Dr. Robert
Singer, Albert Einstein College of Medicine). To facilitate
subsequent subcloning, we deleted the 3' BamHI site by
amplifying the CaMKII 3'UTR with the same 5' primer and a 3' primer
replacing the 3' BamHI site with a NotI site,
5'-ataagaatgcggcccgctttaaatttgtagctatttattcc-3'. The resulting PCR
product was cleaved with BglII and NotI and ligated into BglII/NotI-cleaved
RSV--gal-MS2bs- CaMKII3'UTR. To generate plasmids with four
copies of the MS2-binding site, we cleaved the
RSV--gal-MS2bs-CaMKII3'UTR vector with BamHI and
NotI, generating a 3400 bp fragment containing two
MS2-binding sites and the 3'UTR. This fragment was ligated to the
RSV--gal-MS2bs-CaMKII3'UTR vector cleaved with BglII
and NotI; this process was repeated to generate plasmids
with eight copies of the MS2-binding site.
The GFP-MS2-nls vector was generated from a pEGFP-C1-MS2 coat protein
fusion (a gift from Dr. Jamie Williamson). The W83R mutation,
which makes the protein capsid assembly-deficient, was generated by
using the Stratagene Quick Change kit (La Jolla, CA). A nuclear
localization sequence was added to the mutant coat protein/GFP fusion
by amplifying the MS2 protein sequence with a 3' primer containing
three copies of a nuclear localization consensus sequence.
Hippocampal cell culture and transient transfection.
Pregnant embryonic day 18 (E18) Sprague Dawley rats were
killed by inhalation of CO2, and the
embryos were removed immediately by cesarean section. Hippocampi were
removed and digested in 0.25% trypsin in HEPES-buffered HBSS
without calcium or magnesium at 37°C for 15 min. The hippocampi were
washed three times with HBSS and manually dissociated with a fire-bored
Pasteur pipette. Cells were plated at a concentration of
25,000/cm2 on
poly-L-lysine-coated glass coverslips in plating medium
containing DMEM and 10% fetal bovine serum. After incubation overnight
the medium was changed to Neurobasal medium containing B27 supplement and 0.5 mM glutamine.
Primary hippocampal neurons were transiently transfected by using a
modified Ca2+-phosphate precipitation
method. The media of cultures varying from 7 to 11 d in
vitro (DIV) was changed to DMEM containing 1 mM sodium kynurenate and 10 mM MgCl2
(DMEM/Ky-Mg2+) and was incubated for 30 min at 37°C/5% CO2. During this incubation Ca2+-phosphate/DNA solution was prepared,
60 µl containing 4 µg of DNA (3 µg of the RNA vector; 1 µg of GFP vector if two plasmids were transfected); 250 mM CaCl2 was added
drop-wise to 60 µl of 2× HBS [containing (in
mM) 274 NaCl, 10 KCl, 1.4 Na2HPO4, 5 D-glucose, and 42 HEPES, pH 7.07]. Precipitate
was allowed to form for 20 min, and 60 µl was added drop-wise to 25 mm coverslips or 20 µl was added drop-wise to 12 mm coverslips. Cells
were incubated at 37°C/5% CO2 for from 30 to
90 min, washed once with DMEM/Ky-Mg2+,
once with Neurobasal containing B27 and glutamine, and returned to
conditioned Neurobasal medium. Coverslips were used for microscopy in
24-48 hr.
Riboprobe preparation. To determine the localization pattern
of transfected mRNA, we performed in situ hybridization
against the lacZ reporter RNA. Riboprobes were transcribed from
pBluescript containing the lacZ coding sequence. The plasmid was
linearized and transcribed in the presence of digoxygenin (DIG)-UTP
(Boehringer Mannheim, Indianapolis, IN) with either T7 RNA polymerase
or T3 RNA polymerase to generate sense or antisense DIG-labeled
riboprobe, respectively. To purify probes, we added 2.5 µl of 4 M LiCl and ethanol-precipitated the RNA. Probes
were resuspended in 50 µl of distilled deionized water and hydrolyzed
by the addition of 50 µl of carbonate buffer (40 mM NaHCO3 and 60 mM
Na2CO3); incubation occurred at 60°C for 5 min. The solution was neutralized by the addition of 100 µl of 0.2 M sodium acetate/1%
glacial acetic acid; 5 µl of 10 mg/ml glycogen was added as carrier,
and probes were ethanol-precipitated and resuspended in 50 µl of
distilled deionized water. Probes were checked for DIG incorporation by
dot blot.
Hybridization and immunocytochemistry. Cells were washed
three times with 1× PBS, fixed in 4% paraformaldehyde/5
mM MgCl2/PBS for 15 min at room
temperature, washed three more times in PBS, and incubated in 1× SSC
for 5 min at room temperature. Cells were permeabilized by incubation
in 1% Triton X-100/1× SSC for 30 min at room temperature, washed two
times with PBS for 5 min at room temperature, and incubated in 0.1 M glycine and 0.2 M Tris-Cl, pH 8, for 10 min
at room temperature. Cells were prehybridized in hybridization mix
(50% formamide, 2× SSC, 1% Denhardt's solution, 20% dextran
sulfate, 0.5 mg/ml of tRNA, and 0.25 mg/ml of sonicated salmon sperm
DNA) for 1 hr at 55°C; then the coverslips were placed cell-side down
on Parafilm containing 20 µl of hybridization mix plus 1 µl of
probe and hybridized in a humid chamber overnight at 55°C. After
hybridization the coverslips were washed three times with 50%
formamide/1× SSC for 20 min at 55°C, two times with 1× SSC for 20 min at room temperature, and two times with Tris-buffered saline, pH 8. Cells were blocked with blocking reagent from the Boehringer
Mannheim DIG detection kit for 30 min at room temperature.
Probes were detected with affinity-purified rhodamine-conjugated
anti-digoxygenin (Boehringer Mannheim). The anti-DIG antibody was
diluted 1:50 in blocking solution; at the same time the cells were
incubated with a 1:300 dilution of monoclonal anti--gal (Promega,
Madison, WI). The cells were incubated at room temperature for 1 hr and
washed in 3× PBS for 10 min. -Gal staining was detected with Alexa
488-conjugated goat anti-mouse secondary antibody (Molecular Probes,
Eugene, OR) at a 1:500 dilution in 1% neural goat serum, 0.1% BSA,
and PBS for 15 min at room temperature, washed three times in PBS for
10 min, and mounted on microscope slides with Antifade mounting medium.
For colocalization experiments elongation factor 1 (EF1) was
detected with mouse anti-EF1 (Upstate Biotechnology, Lake Placid,
NY). Coverslips were washed three times with PBS, fixed in 4%
paraformaldehyde/PBS, washed three more times with PBS, and blocked for
30 min at room temperature with 0.1% Triton X-100, 1% goat serum, and
PBS. Cells were incubated in anti-EF1 antibody diluted to 10 µg/ml
in blocking solution for 1 hr at room temperature and washed three
times with PBS. Anti-EF1 was detected with Alexa 594 goat anti-mouse
(Molecular Probes) secondary antibody. Synaptophysin staining was
detected with monoclonal anti-synaptophysin (Boehringer Mannheim) and
Alexa 594 goat anti-mouse (Molecular Probes) secondary antibody.
Fluorescence video microscopy. High-resolution fluorescence
video microscopy was performed on a Nikon microscope (Diaphot 300)
equipped with both a 40× oil immersion (1.0 numerical aperture) and a
100× oil immersion lens (1.4 numerical aperture). A specific GFP
filter set (Chroma Technology, Brattleboro, VT) was used for detecting
the fluorescence from the EGFP fusion protein, and a rhodamine filter
set was used for detecting anti-digoxygenin, anti-synaptophysin, and
anti-EF1. To monitor RNA granule motility, we transferred
transfected cells to a closed chamber maintained at 37°C on a heated
stage; the cells either were maintained in Neurobasal medium or were
changed into physiology buffer [containing (in mM) 119 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 25 HEPES, pH 7.5, and 30 glucose] or
physiology buffer with 10 mM KCl. Then fluorescence images
were captured with a highly sensitive back-thinned cooled CCD camera
(Princeton Instruments, Trenton, NJ) and processed with MetaMorph
software (Universal Imaging, Media, PA). To minimize photobleaching and
phototoxicity, especially for living cells, we used a computer-driven
automatic shutter to achieve the minimum illumination. For time-lapse
recordings 500-1000 msec exposures every 20 sec over a 5-10 min
period were used to capture images. Some exposures were taken every 10 sec for a shorter time period or every 30 sec over a longer time period.
Image analysis. For in situ hybridization
experiments MetaMorph software was used for all analyses. The length of
a particular dendrite was measured by tracing the distance from the
cell body to the end of the process highlighted by -gal staining.
The same region was overlaid onto the image containing the in
situ hybridization staining and divided into 20-µm-long
segments. Then the number of granules visualized in each segment was
counted. To calculate the percentage of length of dendrite-containing
RNA granules, we divided the distance to the most distal RNA granule by
the total length of the dendrite and multiplied it by 100. Granule density of GFP-labeled RNA granules was calculated by counting the
number of granules in dendrites ~20 µm long and 1 µm wide. In
all, 35 processes from 29 different control cells and 40 processes from
24 different treated cells were analyzed.
For colocalization experiments, images of the same region of a
coverslip were taken by using either the GFP or rhodamine filter set,
and a coordinate system was applied to the image by the MetaMorph software. To determine whether a particle labeled both by GFP and an
antibody colocalized, we compared the coordinates of the particle in
one image with those in the other; if the coordinates were the same,
the particles were considered to show colocalization.
To calculate the total distance a granule traveled, we called the
initial location of a granule "zero." The distances traveled (in
µm) in each 20 sec interval were summed with anterograde movements to
the previous value, and the retrograde movements were subtracted. For
histogram plots of distance from origin, the distance each granule
translocated was taken as the distance from the granule starting point
to the last position of the granule in a time-lapse series.
To measure rates, we measured the distance a particle traveled between
two adjacent time-lapse images and divided it by the time between
exposures. To calculate average rates and rate distributions for
untreated cells, we compared 10 oscillatory granules with 48 velocities
and 10 unidirectionally moving granules with 36 velocities. For
KCl-treated cells, four oscillatory granules with 30 velocities and 15 unidirectionally moving granules with 73 velocities were compared. A
granule was considered oscillatory if it reversed direction at least once.
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RESULTS |
The CaMKII 3'UTR is sufficient for RNA granule formation
and transport
The CaMKII mRNA is localized constitutively in high abundance
to the dendrites of hippocampal neurons (Burgin et al., 1990; Martone
et al., 1996; Steward, 1997; Malenka and Nicoll, 1999), and the 3'UTR
is sufficient to localize a reporter construct in the hippocampal
neurons of a transgenic mouse (Mayford et al., 1996). Therefore, this
mRNA is an excellent candidate for direct visualization of its
transport. To accomplish this goal, we designed two constructs that
would link GFP to a specific RNA. A construct (GFP-MS2-nls) was
assembled that included enhanced GFP, an RNA-binding protein, and a
nuclear localization signal under control of the CMV promotor (Fig.
1a). The RNA-binding protein
that was used was a capsid assembly-deficient MS2 coat protein, which
normally forms the capsid of the MS2 bacteriophage. Although deficient in capsid assembly, it binds tightly and specifically to a small RNA
hairpin. The second construct consisted of the 3248 bp mouse CaMKII
3'UTR fused to eight copies of the small RNA hairpin-binding element
and the lacZ mRNA as a reporter under control of the RSV promotor.
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Figure 1.
Dual constructs for GFP labeling of specific RNAs
in living cells. a, The GFP fusion expression is driven
by the strong CMV promotor. The MS2W82R capsid assembly-deficient
RNA-binding protein is expressed as a C-terminal fusion with GFP and
three copies of a consensus nuclear localization sequence. The RNA
construct is expressed by the strong RSV promotor and generates a
translationally competent RNA encoding the lacZ reporter gene, eight
copies of the 18 bp MS2-binding site RNA hairpin, and the RNA of
interest. After expression of both constructs the GFP fusion should
bind as a dimer to each MS2-binding site, leading to an amplified GFP
signal. b, Phase-contrast image of a transfected cell.
c, A live cell expressing both constructs with the
CaMKII 3'UTR mRNA as the RNA of interest. A higher-magnification
image of the boxed region shows many small GFP-labeled
granules in the dendrites. Arrows denote RNA-containing
granules. d, A live cell expressing the GFP construct
alone shows diffuse staining. A higher-magnification image of the
boxed region also shows smooth staining.
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To image the RNA fluorescently, we cotransfected the two plasmids
encoding the GFP construct and the CaMKII 3'UTR RNA construct into
rat primary hippocampal cultures. When the constructs are expressed,
the GFP-MS2-nls fusion protein should dimerize and bind to an RNA
hairpin labeling the RNA of interest (Fig. 1a). The presence
of eight hairpin-binding elements should increase the number of bound
GFP molecules and increase the signal-to-noise. The majority of the
unbound GFP fusion protein remains in the nucleus because of the
nuclear localization sequence. When the RNA construct was cotransfected
with the GFP-MS2-nls construct into hippocampal neurons, small bright
granules were seen in the dendrites, but not in the axons of
GFP-labeled neurons (Fig. 1c). Individual granules could not
be distinguished from background fluorescence in the cell body because
of the high nuclear GFP signal. When the GFP-MS2-nls construct was
transfected into hippocampal cultures alone, diffuse staining was seen
and no granules were detected in the processes (Fig.
1d).
Dendritically localized RNA-containing granules colocalized with the
GFP-labeled granules (Fig.
2e,f). The number of
granules detected by the GFP signal from the GFP-labeled CaMKII
3'UTR RNA-containing granules was only slightly lower than the number of RNA granules measured by in situ hybridization for lacZ
mRNA. This small difference is most likely attributable to the higher sensitivity of in situ hybridization. RNA-containing
granules in neurons generally are associated with ribosomes and
translational machinery. To determine whether the GFP-labeled granules
also were associated with the translational apparatus, we stained the cultures with an antibody against the translation elongation factor EF1. The immunostaining for EF1 colocalized with the GFP-labeled RNA-containing granules (Fig. 2g,h). RNA translation may be
important for the regulation of RNA transport. To show that the
transfected CaMKII 3'UTR could be translationally competent in the
presence of a coding sequence, we used the lacZ element included in the construct for the detection of -galactosidase. The cultures stained intensely with an antibody against -galactosidase in a diffuse (Fig.
2a,c), rather than a granular, pattern because
-galactosidase tends to diffuse throughout neuronal processes. These
findings suggest that the CaMKII 3'UTR can induce translationally
competent granules.
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Figure 2.
The CaMKII 3'UTR is necessary and sufficient to
localize RNA granules to distal dendrites. GFP-labeled granules contain
CaMKII 3'UTR RNA and components of translation. a, b,
A cell transfected with the lacZ-MS2-binding site empty vector.
a, -Gal staining shows that the RNA is
translationally competent and that -gal diffuses throughout the
processes. b, In situ hybridization with
probes for lacZ labels punctate RNA granules only in the cell body.
c, d, A cell transfected with the lacZ-MS2-binding
site-CaMKII 3'UTR fusion construct. Figures are a composite from
three separate images of the same cell. c, -Gal
staining is seen throughout the processes. d, In
situ hybridization labels punctate RNA granules in the cell
body and throughout the dendrites. e, f, CaMKII 3'UTR
containing GFP-labeled granules in dendrites colocalize with
lacZ-CaMKII 3'UTR RNA detected by in situ
hybridization. Arrows show colocalized granules. Images
were analyzed with MetaMorph software, coordinates were mapped onto
images that were taken by using GFP or a rhodamine filter set, and
granules were considered to colocalize when the coordinates of two
objects were the same. e, GFP-labeled RNA granules.
f, lacZ-CaMKII 3'UTR RNA detected by in
situ hybridization. g, h, GFP-labeled granules
colocalize with granules stained with anti-EF1.
Arrows show colocalized staining. g,
GFP-labeled granules. h, EF1 staining.
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To ensure that the CaMKII 3'UTR was sufficient and necessary to
direct localization of the reporter RNA to distal dendrites, we
performed fluorescence in situ hybridization against the
lacZ reporter RNA in cells that had been transfected with constructs either containing or lacking the CaMKII 3'UTR. LacZ was chosen as a
reporter to avoid background from endogenous CaMKII. -Gal immunostaining was used to determine the total length of processes, because -gal diffuses throughout the neuron. RNA containing lacZ mRNA and the MS2-binding sites was highly expressed in the cell body
and most proximal portion of the dendrite. RNA containing the CaMKII
3'UTR was detected at an average distance of 60 ± 23 µm from
the cell body and in some cases as far as 120 µm from the cell body
(Fig. 2c,d, Tables
1,
2). RNA containing only the
lacZ mRNA and MS2-binding sites was detected at an average distance of
23 ± 13 µm from the cell body (Fig. 2a,b).
Effect of KCl depolarization on granule density
Granule density was assessed both by in situ
hybridization and by GFP granule counts, both of which gave similar
results. The average granule density measured by in situ
hybridization was 10 ± 6 granules per 20 µm length of dendrite,
whereas the average granule density of GFP-labeled granules was 8 ± 3. Proximal dendrites tend to be thicker and have larger volumes;
therefore, this portion of the dendrite did have a greater granule
density. In these dendrites the granule density tended to decrease as
the dendrite tapered. However, when granule density in all of the processes was averaged, granule density was fairly uniform throughout the length of the dendrite (Table 2). The reason for this effect was
the large number of short thin processes ( 20 µm) with a low granule
density that masked the higher densities in the thicker proximal
segments of longer processes. This granule density was higher than that
observed when the total cellular RNA was labeled with the RNA-binding
dye SYTO 14 (Knowles et al., 1996); however, the RNA construct
monitored in our assay was overexpressed, and the cultures that were
used were older and had more complex dendritic arbors than those used
in the SYTO 14 experiments. Both methodologies SYTO 14 and
GFPrevealed increased numbers of granules at dendritic branch points
(Fig. 3a).
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Figure 3.
Proximity of GFP-labeled CaMKII 3'UTR RNA
granules to synapses. a, GFP-labeled CaMKII 3'UTR
mRNA-containing granules are localized at high density in junctions
(arrows) and often are seen at the surface of dendrites
and at the base of spines (arrowheads). b,
c, Some RNA-containing granules colocalize with
synaptophysin antibody. GFP-labeled RNA granules from a KCl-treated
cell are shown in b; the synaptophysin-labeled cell is
shown in c. The boxed regions are shown
at higher magnification below, and arrows
show colocalized granules. A region of dendrite 3 µm long is labeled
with the white bar and shows a GFP-labeled granule near
five synapses labeled with synaptophysin.
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In cells monitored over a 30 min period the position of many of the
granules changed, but there was no increase in their density (see Fig.
5c). In cells monitored over a 30 min period after KCl depolarization, there was both a change in the distribution of granules
and an increase in the total number of granules in the area of dendrite
that was imaged (see Fig. 5c); however, this difference did
not reach statistical significance. Given the large number of granules
present in the control cells and the variability of granule density
from cell to cell, a small increase might be masked. These observations
prompted a more detailed analysis of granule motility with finer time resolution.
RNA granules show two types of movement: Oscillatory
or unidirectional
To monitor RNA granule motility, we transferred transfected cells
to a chamber maintained at 37°C on a heated stage, and time-lapse images were taken of GFP-labeled neurons. Images generally were recorded every 20 sec over a 5-10 min time period; occasional exposures were taken every 10 or every 30 sec. Within this time interval the majority of granules was stationary during the imaging period; ~2-4% of the granules were motile. Motile granules
displayed two types of movement: oscillatory or unidirectional.
Oscillatory granules were bidirectional over short distances, often
traversing the same region of a dendrite multiple times. The distance
traveled was ~1 µm but occasionally was as large as 4 µm (Fig.
4a).
Unidirectionally moving granules traversed larger distances (2-8 µm)
and moved consistently in either the anterograde or retrograde
direction. Of the 23 motile granules that were analyzed, 52% were
oscillatory, 22% were anterograde unidirectional, and 26% were
retrograde unidirectional (see Fig. 7a). The movement of
both oscillatory and unidirectionally moving granules was
discontinuous; often granules would stop for one or two images and then
resume movement. This characteristic of the motility led to the
appearance of variable translocation rates, especially for oscillatory
granules. Rates were measured by measuring the distance a particular
granule moved in two consecutive frames of a time-lapse series and
dividing by the time interval of 20 sec. Hidden stops may occur during
the time-lapse recording. Therefore, the rates measured in this manner
are only the lower limit for translocation rates and show a broad
distribution (Fig. 6a). The
average rate for oscillatory granules was 0.04 ± 0.03 µm/sec
(which in general stop more frequently) and for unidirectionally moving
granules was 0.05 ± 0.03 µm/sec. The maximum rate was 0.1 µm/sec for oscillatory and 0.2 µm/sec for unidirectionally moving granules.
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Figure 4.
CaMKII 3'UTR RNA granules show both
oscillatory and unidirectional motility properties.
a, Time-lapse images of an oscillatory
granule. Frames are sequential images taken every 20 sec. The
arrowhead labels an oscillatory granule, whereas an
arrow labels a stationary granule. b,
Time-lapse of an anterograde-moving granule (arrowhead).
Total distance translocated was 5.85 µm. Average velocity over 160 sec was 0.04 ± 0.01 µm/sec. The granule was docked originally
at a junction, but after depolarization it moved in an anterograde
direction. QuickTime movies are available for these images.
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Figure 5.
Effects of KCl on RNA granule density.
a, KCl depolarization only slightly increases the
granule density in dendrites. The average granule density was
calculated from 35 processes from 29 different control cells and 40 processes from 24 different KCl-treated cells. b, KCl
depolarization increases granule density slightly over time, but this
difference does not reach statistical significance. Gray
bars represent the average granule density for
KCl-treated cells; black bars show the granule density
for control cells. c, Time-lapse images looking at an
individual process over long time periods show changes in granule
distribution. Under basal conditions the images taken at 0 min and
after 30 min show a redistribution of granules but no increase in
granule number, whereas cells treated with KCl show both a
redistribution of granules and an increase in the number of granules
present. Red dots represent stationary granules that
have not changed position in the 30 min recording interval, whereas
green dots represent new granules that have entered the
region of dendrite being imaged or granules that have changed position
in the dendrite during the 30 min recording interval. Granules were
assigned on the basis of having a well defined circular shape and a
pixel intensity above background levels in the process.
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Figure 6.
Effect of KCl depolarization on motility rates.
a, The distribution of rates for CaMKII
3'UTR RNA-containing granules under basal conditions. Oscillatory
granule rates are shown in black; unidirectional granule
rates are shown in gray. Rates were calculated by
measuring the change in position of a granule between two consecutive
time-lapse images and dividing this distance by the time between
images. b, The effect of KCl depolarization on the
distribution of motility rates for CaMKII 3'UTR RNA-containing
granules. After depolarization with KCl more granules moved
unidirectionally, and more granules exhibited rates higher than 0.1 µm/sec.
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Under basal conditions the distribution of granule displacements did
not indicate any tendency for net movement either toward or away from
the cell body. A histogram plot of the total distance traveled by 23 granules has a normal gaussian distribution (Fig. 7b). Most granules do not move
a significant distance from their starting points, and those that do
are divided equally between retrograde and anterograde movements
consistent with a steady-state RNA population.
View larger version (27K):
[in this window]
[in a new window]
|
Figure 7.
KCl depolarization shifts the motility
characteristics of granules from oscillatory to anterograde
unidirectional. a, Motile granules fall into three
directional categories: oscillatory, anterograde, and retrograde. The
plots show the distance each granule traveled during a time-lapse
recording at 20 sec intervals. Movements in the anterograde direction
are scored as positive, and movements in the retrograde direction are
scored as negative. Plots are shown for both control cells and cells
treated with 10 mM KCl. The addition of KCl shifts the
population of granules from oscillatory to anterograde.
b, The distance traveled by control granules displays a
gaussian histogram, whereas the histogram of the distance traveled by
granules in KCl-depolarized cells is shifted toward anterograde
movement.
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|
We questioned whether oscillatory movements could center on synapses.
First, to determine whether the granules were located in proximity to
synapses, we fixed and stained GFP/CaMKII 3'UTR-labeled cells with
anti-synaptophysin (see Fig. 3b,c). Although not all granules colocalized with synaptophysin antibody, many did, indicating that a subset of RNA-containing granules resides precisely at the
synapse. As the distance from the cell body increased, the RNA granules
were more likely to be found at the edge of the process, perhaps at the
base of spines (see Fig. 3a). As indicated in Figure 3, some
granules were present within a cluster of synapses that fall within the
range of oscillatory movements.
KCl depolarization of neurons leads to an increase in the
percentage of anterograde-moving granules
We sought to determine whether depolarization of the neurons
altered the steady-state distribution of the granules and perturbed their motility characteristics. KCl depolarization causes BDNF and TrkB
mRNAs to become localized to dendrites in hippocampal cultures and
slices (Tongiorgi et al., 1997), and induced seizures cause
Arc mRNA to become localized to dendrites in hippocampal slices (Roberts et al., 1998; Steward et al., 1998). To determine whether depolarization changes the motility characteristics of CaMKII 3'UTR mRNA-containing granules, we raised the KCl
concentration from 5 to 10 mM in cells that had
been transfected with the dual constructs. Living cells expressing the
GFP and CaMKII 3'UTR constructs were imaged from 10 to 90 min after
the KCl addition. The percentage of motile granules was not increased
significantly, but there was a shift in the population from oscillatory
to anterograde motile granules. Of 22 granules that were analyzed after
KCl, 18% were oscillatory, 55% moved in an anterograde direction, and 27% moved in a retrograde direction (Fig. 7a). The shift
from oscillatory to unidirectionally moving granules (compare the
baseline distribution of granules given above) raises the possibility
that KCl depolarization recruited granules from the oscillatory pool. Although depolarization did not change the average rate of granule movement (see Fig. 6b; oscillatory granules, 0.03 ± 0.01 µm/sec; unidirectional motile granules, 0.05 ± 0.04 µm/sec), a shift toward faster translocation rates (maximum rate,
0.25 µm/sec) did occur with depolarization. This shift probably
indicates that the granules experienced fewer pauses. A representative
time-lapse series of an anterograde-moving granule from KCl-treated
cells is shown in Figure 4b. Increased unidirectional
anterograde movements support the tendency toward increased numbers of
granules as observed above in the 30 min interval and suggest that even
longer intervals would show larger increases. In fact, even within the
5 min interval a subpopulation of cells appeared to have an increased
granule density in the presence of KCl.
| |
DISCUSSION |
Distribution of CaMKII 3'UTR mRNA granules
This distribution is similar to the distribution of the total
endogenous population of RNA granules as visualized with the RNA-binding fluorescent dye SYTO 14 (Knowles et al., 1996) and to
fluorescently labeled myelin basic protein (MBP) mRNA in microinjected oligodendrocytes (Ainger et al., 1993). Interestingly, the
density of the CaMKII 3'UTR-containing granules was higher than the
density of SYTO 14-labeled granules. In untreated cells a 40-µm-long
segment of dendrite contained 16 ± 6 GFP-labeled CaMKII 3'UTR
granules, whereas in SYTO 14-labeled cells there were 5.5 ± 0.5 RNA granules in an identical segment. The SYTO 14-labeled granules
presumably represent the total population of RNA granules, and those
granules containing endogenous CaMKII mRNA should reside in a subset
of them. It is likely that the introduction of CaMKII 3'UTR RNA induces the formation of granules. Indeed, in oligodendrocytes more
granules are induced when a larger amount of MBP mRNA is microinjected
(Ainger et al., 1993). Certainly, dendrites are densely packed with
ribosomes, but only a subset of these ribosomes assembles into
cohesively motile units termed RNA granules. One trigger for the
recruitment of ribosomes and the assembly of RNA granules is likely to
be the mRNA, and specifically signals in the 3'UTR. RNA transport often
is mediated by RNA-binding proteins such as Staufen (Ferrandon et al.,
1994), which associates with bicoid, oskar, and prospero mRNA in
Drosophila; the zipcode-binding protein that binds to
localization elements in -actin mRNA (Ross et al., 1997); and the
zipcode-binding protein homolog Vera, which binds to Vg1 in
Xenopus oocytes (Deshler et al., 1998). Currently, it is
unknown which signals in the 3'UTR direct granule formation and
interactions with RNA-binding proteins and whether the signals in the
RNA are also important for association with motor proteins and docking
to the cytoskeleton. The RNA labeling system that has been used here
will be ideal for determining which elements in the 3'UTR are
responsible for each of these functions.
Many of CaMKII 3'UTR RNA granules colocalize with synapses, as
detected with a synaptophysin marker. In the shaft of a representative dendrite we often observed a cluster of synapses within 3 µm of a
granule, thereby allowing granules to sample all of these sites (see
below). The CaMKII RNA granules are well positioned to contribute to
the synthesis of the CaMKII kinase, which concentrates in postsynaptic densities opposite glutamatergic terminals (Kennedy, 1998).
Basal motility and KCl depolarization-induced changes in RNA
granule motility
Assigning a motility rate to the CaMKII 3'UTR RNA granules was
complicated by the stop and go nature of granule translocation and the
time-lapse imaging techniques used in this study. Cessation of granule
movement for highly variable time intervals was frequent. Therefore,
the true rate of transport is greater than or equal to the fastest
rates measured. The fastest rate measured was 0.13 µm/sec and is
consistent with the rates measured for MBP RNA-containing granules in
oligodendrocytes (Ainger et al., 1993), SYTO 14-labeled granules
(Knowles et al., 1996), and RNA granules associated with GFP-Staufen
fusion proteins in hippocampal neurons (Kohrmann et al., 1999).
GFP-Staufen-containing granules also have discontinuous movements.
KCl depolarization leads to an increase in CaMKII activity (Bading et
al., 1993), and stimulation of hippocampal neurons increases the
transcription and localization of a number of RNAs (Mackler et al.,
1992; Miyashiro et al., 1994; Tongiorgi et al., 1997; Steward et al.,
1998; Schuman, 1999). If the translocation of RNAs in dendrites also
has physiological significance, particularly with regard to synaptic
plasticity, one might expect their motility properties to respond to
synaptic activity. According to the observations here, depolarization
of cultured neurons indeed does alter the motility properties of RNA
granules that transport the CaMKII 3'UTR. Under basal conditions
most of the labeled CaMKII 3'UTR granules were stationary. The
complete absence even of Brownian motion in these granules suggested
that they were docked. Triton X-100 extraction of the
doubly transfected cells indicated that the granules were resistant to
detergent (data not shown) and, therefore, were likely to be attached
to the cytoskeleton.
Within a brief observation time interval a small pool of the labeled
granules was motile, and one-half of the motile granules were
oscillatory. Granules that moved unidirectionally were equally likely
to move in the anterograde or retrograde directions. Depolarization induced anterograde granule movements, reduced the oscillatory movements, and reduced the distance that retrograde granules traveled (see Fig. 7). Presumably, depolarization sends a signal that causes CaMKII mRNA translocation to activated dendritic regions.
Unidirectionally moving granules may derive from an undocked
oscillatory pool via a depolarization signal, because the increase in
unidirectional anterograde granule movements is concomitant with the
decrease in oscillatory movements. Oscillatory granules may be
diffusing freely until they engage a motor and become unidirectional.
Unidirectionally moving granules also may form de novo,
emerge from the cell body, or dislodge from a stationary pool. When the
total movement of all granules was summed, their net displacements
peaked at zero (Fig. 7b), confirming that these movements
generally do not result in significant changes in granule localization
but, rather, may represent local adjustments in position. An intriguing
possibility is that oscillatory movements represent positional
adjustments in relation to a cluster of synapses that all are being
sampled by a single granule. The colocalization of many RNA granules
with synapses (see Fig. 3b) indirectly supports this idea.
A model for the function of RNA granule motility
It is well established that depolarization can activate multiple
"immediate-early" genes (Mackler et al., 1992; Roberts et al.,
1998; Steward et al., 1998; Schuman, 1999). The time scales over which
transcriptionally based mechanisms can deliver cargo to the dendrites
and the rates of RNA translocation we have observed are within similar
ranges. One confounder of previous studies was that the presence of new
endogenous RNA granules in dendrites after a stimulus could derive from
newly transcribed RNA. In the work that has been presented here, an
artificial mRNA is added exogenously; therefore, these experiments
definitively demonstrate that depolarization-induced translocation does
not represent newly transcribed mRNAs exiting from the nucleus.
Furthermore, these experiments suggest that CaMKII mRNA granules can
shift motility characteristics on the basis of signals in the 3'UTR
alone, because our constructs are under the control of a constitutive
promoter that is not affected by depolarization.
Figure 8 shows a model for the role that
the translocation of RNA granules to the dendrites plays in response to
synaptic activity. Under basal conditions the CaMKII 3'UTR is
sufficient to signal the formation of RNA granules containing
translation machinery and the localization of these granules to the
dendrites. Once in the dendrites these granules spend the majority of
their time docked to the cytoskeleton, but occasionally they are
released and show oscillatory movement, perhaps sampling a local
population of synapses. These oscillatory granules usually become
stationary again within a few minutes of undocking. When neurons are
stimulated, CaMKII transcription is activated and RNA transport
occurs. At the same time oscillatory granules already in the dendrites
move unidirectionally anterograde, perhaps traveling to specific
synapses or simply moving from local RNA reservoirs at junctions into
more distal dendrites. Although it has been established that local translation can occur in dendrites (Crino and Eberwine, 1996; Kang and
Schumann, 1996; Ouyang et al., 1999; Huber et al., 2000), what is the
function of an activity-related mechanism for delivering additional
mRNAs and translational machinery to dendritic sites? The rapidity of
local translational response to activity and the ability to exert local
control over translation offer functional advantages. However, these
rationales do not apply to the delivery of new mRNAs that arrive at
more distant synaptic sites within time frames comparable to the
arrival of proteins. Therefore, delivery of RNA granules probably does
not meet the immediate needs of an activated synapse. These needs are
supplied by translation of the RNAs already present at the site. One
possibility is that granule motility may provide fine local adjustments
in the number of granules within specific dendritic branches. The shift
from oscillatory to unidirectional granule motility suggests that
depolarization may affect the localization of subsets of RNA granules
already in the dendrites. More interestingly, the delivery of
additional mRNAs allows a neurite to reflect its experienceincreased
activity will draw in more RNA granules that will be available for
translation with persistent stimulation.
View larger version (28K):
[in this window]
[in a new window]
|
Figure 8.
A model for the function of RNA granule motility.
Under basal conditions RNA is transcribed and directed by the 3'UTR to
dendrites. Within the recording interval many granules appear
stationary and cluster at junctions. Some granules are motile and move
in an oscillatory manner, perhaps to sample a small number of synapses.
KCl depolarization activates transcription of endogenous (but not
GFP-labeled) CaMKII mRNA and initiates CaMKII translation at
synapses within localized RNA granules. RNA granules drawn from the
oscillatory pool translocate to activated synapses where they cluster
and the local translation occurs. GFP-labeled CaMKII 3'UTR
mRNA-containing granules are shown as circles.
Arrows denote moving granules; granules with
two-directional arrows are oscillatory. Microtubules are
shown as black bars. Arrows at synapses
represent activation. Tx, Transcription.
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FOOTNOTES |
Received April 5, 2000; revised June 6, 2000; accepted June 9, 2000.
This work was supported by National Institutes of Health Grant AG06601
and NASA Grant NAG2-964 to K.S.K. and a Lefler fellowship and National
Institutes of Health training grant to M.S.R. We thank Mark Mayford for
the CaMKII DNA, Robert Singer for the RSV-gal vector, James
Williamson and Regina Burris for the GFP-MS2 plasmid, and all for their
helpful discussions.
Correspondence should be addressed to Dr. Kenneth S. Kosik, Center for
Neurological Diseases, Brigham and Women's Hospital, HIM 760, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail:
kosik{at}cnd.bwh.harvard.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
