From the Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea
Received for publication, October 31, 2002
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ABSTRACT |
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KIF1A is a kinesin motor known to transport
synaptic vesicle precursors in neuronal axons, but little is known
about whether KIF1A mediates fast and processive axonal transport
in vivo. By monitoring movements of EGFP-labeled KIF1A in
living cultured hippocampal neurons, we determined the characteristics
of KIF1A movements. KIF1A particles moved anterogradely along the
neurites with an average velocity of 1.0 µm/s. The movements of KIF1A
were highly processive, with an average duration of persistent
anterograde movement of 11 s. Some KIF1A particles (17%)
exhibited retrograde movements of 0.72 µm/s, although overall
particle movement was in the anterograde direction. The anterograde
movement of KIF1A, however, did not lead to a detectable accumulation
of KIF1A in the periphery of neurons, suggesting that there are
mechanisms inhibiting the peripheral accumulation of KIF1A. These
results suggest that KIF1A mediates neuronal transport at a high
velocity and processivity in vivo.
KIF1A is a member of the KIF1/Unc104 family of plus-end-directed
kinesin motors (1, 2). Genetic deletion of Unc104, the
Caenorhabditis elegans homologue of KIF1A (3), leads to a
decrease in the number of axonal synaptic vesicles and an accumulation of similar vesicles in the cell body of neurons (4). Targeted deletion
of the KIF1A gene in mice causes a similar accumulation of clear small
vesicles in the cell body of neurons as well as marked neuronal death
(5). Biochemically, KIF1A associates with membrane organelles
containing synaptic vesicle proteins such as synaptophysin,
synaptotagmin, and Rab3A (1). These results suggest that KIF1A is
involved in the axonal transport of synaptic vesicle precursors.
A number of important questions on KIF1A, however, remain to be
addressed. Does KIF1A mediate a fast and processive transport in
vivo? EGFP-tagged synaptic vesicle and plasma membrane proteins including synaptophysin often show bidirectional movements in living
neurons (6-9). Does KIF1A also move bidirectionally? If so, what are
the underlying mechanisms? What happens to KIF1A when it reaches its destination?
A recent description of the movements of Unc104 in living C. elegans neurons provided direct evidence that Unc104 mediates a
fast and processive neuronal transport in vivo (10). Along with the previous measurement of kinesin-II movement in chemosensory neurons of living C. elegans (11), this is a significant
progress in that C. elegans is an important model system for
the study of intracellular sorting and transport (12). However, there seem to be additional complexities in mammalian homologues of Unc104.
There are four known mammalian homologues of Unc104: KIF1A, KIF1B,
KIF1C, and KIF1D (1, 13-18). Some of them have diverse splice
variants; for instance, KIF1B has two splice variants with different
tails (KIF1B In the present study, we characterized the movement of
EGFP1-tagged full-length
KIF1A, a well studied mammalian KIF1 family protein with a high overall
amino acid sequence identity (~55%) to Unc104, in living cultured
hippocampal neurons. Analysis of time-lapse images of the neurons
expressing EGFP-tagged KIF1A revealed that KIF1A exhibits fast and
processive anterograde movement with intermittent retrograde
(bidirectional) movement in living cultured hippocampal neurons.
Constructs--
To generate a mammalian expression construct
containing EGFP-tagged KIF1A (KIF1A-EGFP), an XbaI site was
created in GW1 KIF1A2 by
converting Ser-680 (TCC) and Arg-681 (AGG) of KIF1A into Ser (TCT) and
Arg (AGA) by the QuikChange kit (Stratagene). Subsequently, a
PCR-amplified EGFP (Clontech) was subcloned in
frame into GW1 KIF1A digested with XbaI. A KIF1A-EGFP mutant
with a point mutation (Thr312 into Met) in the motor domain
(KIF1A-EGFP-T312M) was generated using GW1 KIF1A-EGFP as a template.
KIF1A-EGFP- Antibodies--
The EGFP rabbit polyclonal antibody (1167) has
been described previously (21). Other antibodies used in the present
study are as follows: microtubule-associated protein 2 (MAP2; Sigma), neurofilament-H (Sigma), KIF1A (1131) (a rabbit polyclonal antibody raised against aa 657-937 of
KIF1A3) and liprin- Neuron Culture, Transfection, and Immunostaining--
Primary
hippocampal neuronal cultures were prepared from rat embryonic brain
(embryonic day 18) as described previously (22). Briefly, dissected
hippocampi were dissociated with trypsin and plated on coverslips (25 mm) coated with poly(L-lysine) (Mr
30,000-70,000) in neurobasal medium (Invitrogen) supplemented
with B27 (Invitrogen), 2 mM glutamine, and 25 µM glutamate (plating medium). After 2-3 h of
incubation, the plating medium was changed with a growth medium
(plating medium minus glutamate). Cultured hippocampal neurons (DIV
10-12) were transfected by the calcium phosphate method (23). For
immunofluorescence staining, cultured hippocampal neurons, after 2-3
days of transfection, were fixed in 4% paraformaldehyde, permeabilzed
in 0.1% Triton X-100 followed by incubation with EGFP (1167, 3 µg/ml), KIF1A (3 µg/ml), MAP2 (1:500), and neurofilament-H (1:500)
primary antibodies.
Image Acquisition from Living Neurons and Image
Analysis--
Transfected neurons on coverslips (25 mm diameter) were
set on a perfusion chamber with a thermostat (37 °C) and maintained in phenol red-free minimum essential medium supplemented with 1 mM pyruvate and 60 µM N-acetyl
L-cysteine during the observation as described previously
(8). Time-lapse images of moving KIF1A-EGFP were captured using a
63×/1.4 numerical aperture Plan-Apochromat objective and a confocal
laser scanning microscope (LSM510) under the following settings: 30%
intensity of argon laser to minimize bleaching and damage to neurons;
maximum pinhole opening (7.5 µm) to cover most of the thickness of
neurites; and 2-3× digital zoom for better viewing. One image of
512 × 512-pixel resolution was taken every 4.5 s (1.5 s of
scanning + 3-s interval). The interval of 3 s was used to minimize
laser-induced neuronal damages. Image analysis and generation of video
clips were performed using the MetaMorph software (Universal Imaging
Corp.). Statistical significance was assessed using the Student's
t test.
Coimmunoprecipitation--
COS7 cells were transfected with
combinations of GW1 KIF1A, GW1 KIF1A-EGFP, and pMT HA-liprin- MT-binding Assay--
The microtubule (MT)-binding assay was
performed as described previously (24). Briefly, human embryonic kidney
293T cells transfected for 48 h with various KIF1A-EGFP constructs
were solubilized in ice-cold lysis buffer (25 mM HEPES/KOH,
115 mM K+CH3COO Characterization of EGFP-tagged KIF1A--
To visualize the
movements of KIF1A in living neurons, we generated a construct
containing EGFP-tagged full-length KIF1A (KIF1A-EGFP). The EGFP domain
was inserted into the site (between aa 680 and 681) in the middle of
KIF1A around the N-terminal boundary of the liprin-
We next determined whether the subcellular distribution of KIF1A in
neurons is affected by the EGFP insertion. When expressed in cultured
hippocampal neurons, KIF1A-EGFP distributed to both MAP2-positive
dendrites (Fig. 1D) and neurofilament-H-positive axons (Fig.
1E), which is similar to the distribution pattern of
untagged exogenous KIF1A (data not shown) and endogenous KIF1A (Fig.
1F) in cultured neurons. The localization of endogenous KIF1A in both dendrites and axons was further confirmed by the electron
microscopic analysis of KIF1A distribution in rat brain neurons.3 Moreover, these results were consistent with the
movements of Unc104 observed in both dendrites and axons of living
C. elegans neurons (10). In further support of the minimal
effects of the EGFP insertion, EGFP-KIF1A moved processively along the
neurites of living neurons at the high velocity of 1.0 µm/s (see
below for more details), which is comparable with that of purified
KIF1A in the in vitro gliding assay (1.2 µm/s) (1) and the
in vivo movements of Unc104 in living C. elegans
neurons (1.02 µm/s) (10). Taken together, these results indicate that
KIF1A-EGFP possesses functional characteristics comparable with those
of untagged exogenous and endogenous KIF1A.
Movements of KIF1A-EGFP in Living Cultured Hippocampal
Neurons--
When expressed and visualized in living cultured
hippocampal neurons, KIF1A-EGFP was primarily associated with
fluorescent particles of various sizes and shapes (round or
tubulovesicular) (Fig. 2,
A-C). Because it was reported that overexpression of exogenous Unc104 in wild-type C. elegans neurons seems to
interfere with normal Unc104-driven transport (10), we obtained
time-lapse images of KIF1A movements only from neurons that expressed a
moderate amount of KIF1A, with KIF1A particles sparsely dispersed over the neuronal processes. Quantitative analysis on these neurons indicated that the expression levels of EGFP-KIF1A were ~40% of those of endogenous KIF1A; the average fluorescence intensity of
EGFP-KIF1A-transfected neurons revealed by KIF1A antibodies was
140 ± 16% (n = 10) that of neighboring
untransfected neurons (Fig. 2D). These results suggest that
the expression of EGFP-KIF1A is unlikely to exert dominant-negative
effects in transfected neurons.
When time-lapse images of transfected neurons were analyzed, prominent
KIF1A movements were mainly observed in thin and distal neurites (Figs.
3A and 4, A and
B), although KIF1A movements were also detected in thick and
proximal neurites (Fig. 3B), which may represent dendrites.
The KIF1A particles in thick neurites, however, were mostly stationary.
The stationary nature of KIF1A particles in proximal dendrites was
further confirmed by fluorescence recovery after photobleaching
analysis, in which we observed limited movements of KIF1A in bleached
regions (data not shown). The overall direction of KIF1A movements was
anterograde (moving away from the cell body) (Fig. 3, A and
B; supplementary movies 1 and 2), although retrograde
movements were observed in 17.1% of particles observed
(n = 293) (Fig. 4,
A and B). Some KIF1A vesicles changed their
direction of movement multiple times during observation (Fig.
4B; supplementary movie 3). Importantly, the fluorescence intensity of moving KIF1A particles did not change significantly (95%,
n = 20) during periods of retrograde movements,
compared with that during anterograde movements, suggesting that KIF1A particles change their direction of movement, whereas KIF1A proteins remain associated. The average velocity of KIF1A particles during their
persistent anterograde movements was 1.00 ± 0.61 µm/s
(mean ± S.D., n = 243) (Fig.
5A), whereas that of
retrograde movement was 0.72 ± 0.27 µm/s (n = 50) (Fig. 5B). The average durations of persistent
anterograde and retrograde movements were 11.0 ± 7.1 s
(n = 131) (Fig. 5C) and 8.17 ± 3.72 s (n = 56) (Fig. 5D), respectively.
To test whether the movement of KIF1A is driven by the motor domain
activity of KIF1A, we characterized the movements of two KIF1A-EGFP
mutants; deletion of the entire motor domain (aa 2-365) termed
KIF1A- In the present study, direct visualization of the movement of
KIF1A enabled us to analyze the characteristics of KIF1A movements in
living mammalian neurons.
Fast and Persistent Movements of KIF1A--
Several in
vitro and in vivo assays were employed to determine the
velocity and processivity (the distance that a motor protein moves
along MTs before detaching) of movements for the KIF1 family proteins.
In living C. elegans neurons, EGFP-tagged Unc104 moves at
the high speed of 1.02 µm/s over distances of 5 µm along MTs before
pausing (10, 26). In vitro reconstitution assays
demonstrated that DdUnc104 from Dictyostelium discoideum
mediates a processive (5-6 µm) transport of liposomes and native
vesicles at 2.0-2.1 µm/s (26, 27). In the same assay, truncated
Unc104 proteins (aa 1-653, termed CeU653) displayed minimal transport
activities, but fusion of the PH domain from D. discoideum
(PHDdUnc104) to the C terminus of CeU653 dramatically
stimulated cargo transports (processivity of 5-6 µm at 1.5-1.6
µm/s) (27). Intriguingly, it has been recently reported that CeU653
can form a dimer and show a fast and processive unidirectional movement
at high motor concentrations in the single-molecule fluorescence assay
(processivity of 1.47 µm at 1.73 µm/s), suggesting that Unc104 may
dimerize into a processive motor (28).
In the case of murine KIF1A, the single motor assay with a KIF1A
construct containing the catalytic core of the motor domain (aa 1-356)
moved along MTs by a biased one-dimensional diffusion at the much
slower speed of 0.14 µm/s, but with a high processivity (~0.85 µm
for 6.1 s) (29). A longer KIF1A construct (aa 1-693) that is
equivalent to CeU653 also exhibited fast (1.12 µm/s) and processive
movements at higher motor concentrations in the single-molecule fluorescence assay (28). In a conventional multi-motor gliding assay,
both truncated and full-length KIF1A proteins showed movements of high
velocity (1.2 µm/s) (1, 29). Although these results are highly
informative, the assays employed to measure KIF1A movements have some
limitations, in that they do not measure the transport of KIF1A in
association with cargoes and, more importantly, provide little
information on in vivo transport, where a number of
additional regulatory influences are integrated.
Our data demonstrate that KIF1A moves processively (11 µm for 11 s) along the neurites of living neurons at a high velocity of 1.0 µm/s. The velocity and processivity of moving KIF1A in living neurons
was comparable with that of Unc104 in living C. elegans
neurons (10), and that of KIF1A from in vitro assays (1, 28,
29). These results suggest that KIF1A mediates fast and processive
neuronal transport in vivo and that the fast and processive
in vivo movements are conserved in various KIF1/Unc104 family kinesin motors.
One of the most prominent differences between the movements of KIF1A
and Unc104 is that KIF1A showed limited movement in dendrites (Fig.
3B), whereas Unc104 exhibited fast and processive movement in dendrites (10). A possible source of the limited movements of KIF1A
in dendrites is the bidirectional nature of the MTs in the proximal
dendrites of hippocampal neurons, which is in contrast to the
unidirectional (plus-end-distal) polarity of axonal MTs (30). However,
because we occasionally observed fast and processive anterograde
movements in proximal dendrites (Fig. 3B), additional mechanisms seem to regulate the movements of KIF1A in this region. In
this regard, the active movements of Unc104 in C. elegans
dendrites may reflect a different polarity of MTs in this region, or
the presence of molecular mechanisms allowing fast and processive motor movements.
Bidirectional Movements of KIF1A--
KIF1A particles often move
bidirectionally along neurites. Along with the previously reported
bidirecitonal movements of Unc104 in living C. elegans
neurons (10), our results suggest that the KIF1/Unc104 family of
kinesin motors exhibit bidirectional movements in vivo. What
are the underlying molecular mechanisms? Because we measured KIF1A
movements mostly in thin neurites, which are probably axons, the
frequent retrograde movements of KIF1A are unlikely to be caused by the
bidirectional orientation of MTs, but rather by the intrinsic
properties of the KIF1A particles. It is generally accepted that moving
organelles or vesicles associate with antagonizing populations of
motors, the balance of which determines their direction of movement
(31). It is thus possible that KIF1A particles contain, in addition to
KIF1A, other functionally antagonizing motors. In support of this
hypothesis, KIF1A particles move bidirectionally at two different
velocities: anterograde (1.0 µm/s) and retrograde (0.72 µm/s).
Assuming that KIF1A particles contain antagonizing motors, a factor
that may regulate their balance would be the attachment or detachment
of antagonizing motors to and from their cargoes. Alternatively, the
activities of antagonizing motor proteins may be regulated, whereas
both motors remain associated with their cargoes, as suggested
previously (31). Our results indicate that KIF1A particles do not
significantly change their fluorescence during periods of retrograde
movements, suggesting that KIF1A particles may change their direction
of movement by mechanisms other than the attachment/detachment of KIF1A
proteins. A possible mechanism for the regulation of KIF1A activity is
phosphorylation, which has been shown to regulate the motor activity
and perhaps vesicle association of kinesin and dynein molecules (31,
32).
What is the fate of KIF1A at its destination? We demonstrated that the
overall direction of KIF1A movements was in the anterograde direction.
However, we did not observe any detectable accumulation of KIF1A in the
periphery of neurons. A possible explanation for these results is that
KIF1A is destroyed at its destination by the local machinery for
protein degradation (31). In support of this hypothesis, previous
studies in the peripheral nervous system have shown that
plus-end-directed motor proteins targeted to the synapse do not return
(33-36). However, we cannot exclude the possibility that KIF1A motors,
in a diffuse form, may move back to the cell body by retrograde
transport, as demonstrated previously for the cytoplasmic
dynein-dependent retrograde transport of the anterograde
kinesin-II motor in chemosensory neurons of C. elegans
(37).
In conclusion, our results, along with the previous report on Unc104
movement (10), support the hypothesis that the KIF1/Unc104 family
motors mediate fast and processive transport in living neurons of
various species. Identification of the molecular mechanisms that
underlie the intermittent retrograde movements of KIF1A and the minimal
peripheral accumulation of KIF1A would be interesting directions for
further study. Finally, the assay measuring motor movements in living
neurons may be used in the future for identifying the molecular
mechanisms that regulate the motor activity and cargo association of
KIF1A in vivo.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and KIF1B
) (13-16), which seems to contribute to
the binding of diverse cargoes (15, 16, 19). The environment within
mammalian neurons is likely to differ from that within invertebrate
neurons. Information from genomic sequences indicates that the
estimated number of kinesins in mammals (~45-50) exceeds that in
C. elegans (~20-25) (20). However, to our knowledge, no
direct visualization of any of the ~50 mammalian kinesins has been
made. Therefore, comparative analysis of the motor movements from
living C. elegans and mammalian neurons may help us
understand whether fast and processive in vivo movements of
kinesins are conserved across the species.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
MD (aa 366-1695) was generated by inverse PCR using
KIF1A-EGFP as a template.
(1127)
(a rabbit polyclonal antibody raised against aa 818-1202 of
liprin-
13).
1 using
the LipofectAMINE reagent (Invitrogen). Forty-eight hours after
transfection, COS cells were solubilized in phosphate-buffered saline
containing 1% Triton X-100 and protease inhibitors, incubated with
hemagglutinin (HA) monoclonal antibody (Hoffmann-La Roche) followed by
precipitation using the protein A-Sepharose resin (Amersham
Biosciences). Immunoprecipitates were analyzed by immunoblotting with
KIF1A (1131) and liprin-
(1127) antibodies.
, 5 mM Na+CH3COO
, 5 mM MgCl2, 0.5 mM EGTA, 1% Triton
X-100, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml
leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin A, 1 µg/ml
benzamidine, pH 7.2). After removing insoluble materials by
centrifugation of the lysates at 50,000 rpm in the TLA 100.2 rotor
(Beckman Coulter) at 4 °C for 30 min, the supernatant was added with
5'-adenylylimidodiphosphate (final concentration, 2.5 mM),
paclitaxel (Taxol; 20 µM), and paclitaxel-stabilized MTs (0.5-1 mg/ml). After 30 min of incubation at room temperature, the
mixture was overlaid on top of a lysis buffer bed (1/4 volume) containing 10% sucrose and 20 µM paclitaxel and
centrifuged at 30,000 rpm in the SW55Ti rotor at 22 °C for 30 min.
After centrifugation, the supernatant was carefully removed and mixed
with 2× SDS loading buffer. The precipitates were then washed with
lysis buffer and paclitaxel and resuspended in 1× SDS loading buffer.
The supernatants and precipitates were analyzed by immunoblotting with
EGFP (1167) antibodies.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-binding domain
(Fig. 1A), a putative domain
that binds to the multi-domain protein liprin-
.3
Insertion of the EGFP domain to the N- or C-terminal end of KIF1A was
not attempted because the insertion might have interfered with the
function of the motor domain or potential cargo binding. Because the
site of EGFP insertion in KIF1A was near the liprin-
-binding domain,
we tested whether this insertion affected the interaction between the
liprin-
-binding domain and liprin-
by coimmunoprecipitation experiments (Fig. 1B). In COS cells doubly transfected with
KIF1A (untagged or EGFP-tagged) and liprin-
1, both untagged and
tagged KIF1A showed a similar coimmunoprecipitation with liprin-
1.
We further tested whether the EGFP insertion affected the MT binding of
KIF1A. When human embryonic kidney 293T cell lysates singly transfected
with untagged KIF1A or KIF1A-EGFP were incubated with purified MTs, the
proteins showed similar MT binding (Fig. 1C). These results
indicate that the EGFP insertion does not affect the association of
KIF1A with liprin-
or MTs.
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Fig. 1.
Characterization of KIF1A-EGFP.
A, diagram of KIF1A-EGFP. The EGFP domain was inserted into
the site between aa 680 and 681 around the N terminus of the
liprin- -binding domain (LBD) of KIF1A. Motor,
motor domain; EGFP, enhanced green fluorescent protein;
PH, pleckstrin homology domain. B, similar
coimmunoprecipitation of untagged KIF1A or KIF1A-EGFP with liprin-
1.
COS cell lysates doubly transfected with KIF1A + HA-tagged liprin-
1
(HA-Lip), or KIF1A-EGFP + HA-tagged liprin-
1, were
immunoprecipitated with HA antibodies or mouse IgG (control) and
immunoblotted with KIF1A (1131) and liprin-
1 (1127) antibodies.
Transf, transfection; IP, immunoprecipitation.
Input, 2.5% of the lysates used for immunoprecipitation.
C, similar MT binding of KIF1A and KIF1A-EGFP. Lysates of
human embryonic kidney 293T cells singly transfected with KIF1A or
KIF1A-EGFP were incubated with purified MTs. MT binding of KIF1A-EGFP
is comparable with that of untagged KIF1A. S, supernatant;
P, pellet. D-E, insertion
of the EGFP domain does not change the subcellular distribution of
KIF1A in cultured neurons. Cultured hippocampal neurons (DIV 10-14)
were transfected with KIF1A-EGFP and visualized by double label
immunofluorescence staining with EGFP (1167) and MAP2 or EGFP and
neurofilament-H (NF-H) antibodies. KIF1A-EGFP
(green) distributed to both MAP2-positive (red)
dendrites and neurofilament-H-positive (red) axons.
Merge, merged images. Arrowheads indicate
examples of colocalization. Scale bar, 30 µm.
F, distribution of endogenous KIF1A to both dendrites and
axons in cultured neurons. Untransfected cultured hippocampal neurons
(DIV 10-14) were stained with KIF1A (1131) and MAP2 antibodies. KIF1A
(green) distributes to both MAP2-positive dendrites
(red, arrowhead) and MAP2-negative axons
(arrow). Scale bar, 30 µm.
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Fig. 2.
Expression and distribution of KIF1A-EGFP in
cultured hippocampal neurons. A-C, association of
KIF1A-EGFP with particulate structures. Cultured hippocampal neurons
(DIV 10-14) were transfected with KIF1A-EGFP. Subcellular distribution
of KIF1A-EGFP in living neurons was observed by confocal microscopy 3 to 5 days after transfection. EGFP-KIF1A fluorescence mainly associated
with round or slightly elongated structures (arrowheads).
B and C are enlarged images of the white
boxes in A and B, respectively. Scale
bars, 20 µm. D, comparison of the expression levels
of exogenous EGFP-KIF1A and endogenous KIF1A. Cultured hippocampal
neurons (DIV 10-14) were transfected with KIF1A-EGFP and visualized by
immunofluorescence staining with KIF1A antibodies (D2).
Green fluorescence from EGFP-KIF1A (D1) was preserved during
the staining process with KIF1A antibodies. The average fluorescence
intensity of EGFP-KIF1A in the cell body of transfected neurons was
140 ± 16% (n = 10) of that of neighboring
untransfected neurons. Scale bars, 20 µm.
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Fig. 3.
Movements of KIF1A-EGFP in living cultured
hippocampal neurons. A, movements of KIF1A-EGFP
particles in living neurons. A KIF1A particle (arrowheads)
moves anterogradely along the neurite away from the cell body (average
velocity, 1 µm/s). The numbers on the left indicate the
time points (seconds) at which the images were captured. Scale
bars, 10 µm. B, movements of KIF1A particles in a
proximal neurite, which may represent the dendrite. Scale
bars, 10 µm. Supplementary information: movies 1 and 2 for Fig.
3, A and B, respectively. Movie 1, a total of 10 frames corresponding to 45 s of imaging; movie 2, a total of 30 frames, corresponding to 135 s of imaging.
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Fig. 4.
Bidirectional movements of KIF1A.
A, retrograde movements of KIF1A (average velocity, 0.72 µm/s). Note that a KIF1A particle (arrowheads) is moving
retrogradely toward the cell body (not shown). Scale bar, 10 µm. B, multiple changes in the direction of KIF1A
movement. Scale bar, 10 µm. Supplementary information:
movie 3 for Fig. 4B, a total of 36 frames corresponding to
162 s of imaging.
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Fig. 5.
Velocity and duration of KIF1A
movements. A, histogram showing velocity distribution
of anterograde KIF1A movements. The number of KIF1A particles moving
within the indicated velocity ranges was counted. Velocity range,
0.34-3.8 µm/s; average velocity, 1.00 ± 0.61 µm/s (mean ± S.D., n = 243). Movements slower than 0.33 µm/s
were considered a pause in both anterograde and retrograde movements
and excluded from the calculation. B, velocity distribution
of retrograde KIF1A movements. Velocity range, 0.34-1.5 µm/s;
average velocity, 0.72 ± 0.27 µm/s (n = 50).
C, distribution of the duration of persistent anterograde
KIF1A movements. Duration range, 4.5-40.5 s; average duration,
11.0 ± 7.1 s (n = 131). D,
distribution of the duration of persistent retrograde KIF1A movements.
Duration range, 4.5-18 s; average duration, 8.17 ± 3.72 (n = 56).
MD and a point mutation (Thr-312 to Met) that impairs the
motor domain activity (KIF1A-T312M) (25) (Fig.
6A). When these mutant KIF1As
were expressed in cultured hippocampal neurons, most of the KIF1A
particles exhibited little or no movement and mainly distributed to the
region around the cell body (Fig. 6A). Quantitative analysis
showed that the number of moving KIF1A particles was significantly
reduced in both KIF1A mutants (0.20 ± 0.31, n = 5, p < 0.0001 in
MD; 1.11 ± 0.74, n = 4, p < 0.001 in T312M; Student's
t test) compared with the wild-type KIF1A (3.99 ± 1.16, n = 12) (Fig. 6B), indicating that the
observed movements of KIF1A are driven by the motor domain
activity.
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Fig. 6.
Movements of KIF1A is driven by the activity
of its motor domain. KIF1A mutants lacking the motor domain
( MD) or with a point mutation impairing the motor domain
activity (T312M) were expressed in cultured neurons.
A, the KIF1A mutants showed limited movements and associated
with distinct particles that are mainly confined to the regions around
the cell body of neurons. Full, full-length KIF1A.
Scale bar, 10 µm. B, quantitative analysis
showed that the number of moving KIF1A particles was significantly
reduced in both KIF1A mutants (0.20 ± 0.31, n = 5, Student's t test, *, p < 0.0001 for
MD; 1.11 ± 0.74, n = 4, *,
p < 0.001 for T312M) compared with the
wild-type full-length (Wt-full) KIF1A (3.99 ± 1.16, n = 12).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Michel Steruli at ImmunoGen,
Inc., for the pMT HA-liprin-1 construct.
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FOOTNOTES |
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* This work was supported by grants from the Korean Ministry of Science and Technology, the Korea Science and Engineering Foundation, and the Korea Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains three supplementary movies.
To whom correspondence should be addressed. Tel.: 42-869-2633;
Fax: 42-869-2610; E-mail: kime@mail.kaist.ac.kr.
Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M211152200
2 H. Shin and E. Kim, unpublished observations.
3 H. Shin and E. Kim, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: EGFP, enhanced green fluorescent protein; aa, amino acid(s); DIV, days in vitro; HA, hemagglutinin; MT, microtubule; MAP2, microtubule-associated protein 2.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Okada, Y., Yamazaki, H., Sekine-Aizawa, Y., and Hirokawa, N. (1995) Cell 81, 769-780[Medline] [Order article via Infotrieve] |
2. | Bloom, G. S. (2001) Curr. Opin. Cell Biol. 13, 36-40[CrossRef][Medline] [Order article via Infotrieve] |
3. | Otsuka, A. J., Jeyaprakash, A., Garcia-Anoveros, J., Tang, L. Z., Fisk, G., Hartshorne, T., Franco, R., and Born, T. (1991) Neuron 6, 113-122[Medline] [Order article via Infotrieve] |
4. | Hall, D. H., and Hedgecock, E. M. (1991) Cell 65, 837-847[Medline] [Order article via Infotrieve] |
5. |
Yonekawa, Y.,
Harada, A.,
Okada, Y.,
Funakoshi, T.,
Kanai, Y.,
Takei, Y.,
Terada, S.,
Noda, T.,
and Hirokawa, N.
(1998)
J. Cell Biol.
141,
431-441 |
6. |
Kaether, C.,
Skehel, P.,
and Dotti, C. G.
(2000)
Mol. Biol. Cell
11,
1213-1224 |
7. |
Nakata, T.,
Terada, S.,
and Hirokawa, N.
(1998)
J. Cell Biol.
140,
659-674 |
8. | Burack, M. A., Silverman, M. A., and Banker, G. (2000) Neuron 26, 465-472[Medline] [Order article via Infotrieve] |
9. |
Prekeris, R.,
Foletti, D. L.,
and Scheller, R. H.
(1999)
J. Neurosci
19,
10324-10337 |
10. |
Zhou, H. M.,
Brust-Mascher, I.,
and Scholey, J. M.
(2001)
J. Neurosci.
21,
3749-3755 |
11. | Orozco, J. T., Wedaman, K. P., Signor, D., Brown, H., Rose, L., and Scholey, J. M. (1999) Nature 398, 674[CrossRef][Medline] [Order article via Infotrieve] |
12. | Koushika, S. P., and Nonet, M. L. (2000) Curr. Opin. Cell Biol. 12, 517-523[CrossRef][Medline] [Order article via Infotrieve] |
13. | Conforti, L., Buckmaster, E. A., Tarlton, A., Brown, M. C., Lyon, M. F., Perry, V. H., and Coleman, M. P. (1999) Mamm. Genome 10, 617-622[CrossRef][Medline] [Order article via Infotrieve] |
14. | Gong, T. W., Winnicki, R. S., Kohrman, D. C., and Lomax, M. I. (1999) Gene 239, 117-127[CrossRef][Medline] [Order article via Infotrieve] |
15. | Zhao, C., Takita, J., Tanaka, Y., Setou, M., Nakagawa, T., Takeda, S., Yang, H. W., Terada, S., Nakata, T., Takei, Y., Saito, M., Tsuji, S., Hayashi, Y., and Hirokawa, N. (2001) Cell 105, 587-597[CrossRef][Medline] [Order article via Infotrieve] |
16. | Nangaku, M., Sato-Yoshitake, R., Okada, Y., Noda, Y., Takemura, R., Yamazaki, H., and Hirokawa, N. (1994) Cell 79, 1209-1220[Medline] [Order article via Infotrieve] |
17. | Rogers, K. R., Griffin, M., and Brophy, P. J. (1997) Brain Res. Mol. Brain Res. 51, 161-169[Medline] [Order article via Infotrieve] |
18. |
Dorner, C.,
Ciossek, T.,
Muller, S.,
Moller, P. H.,
Ullrich, A.,
and Lammers, R.
(1998)
J. Biol. Chem.
273,
20267-20275 |
19. |
Mok, H.,
Shin, H.,
Kim, S.,
Lee, J. R.,
Yoon, J.,
and Kim, E.
(2002)
J. Neurosci.
22,
5253-5258 |
20. | Goldstein, L. S. B. (2001) Trends Cell. Biol. 11, 477-482[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Choi, J., Ko, J.,
Park, E.,
Lee, J. R.,
Yoon, J.,
Lim, S.,
and Kim, E.
(2002)
J. Biol. Chem.
277,
12359-12363 |
22. | Banker, G., and Goslin, K. (1991) Culturing Nerve Cells , MIT Press, Cambridge, MA |
23. |
Xia, Z.,
Dudek, H.,
Miranti, C. K.,
and Greenberg, M. E.
(1996)
J. Neurosci.
16,
5425-5436 |
24. |
Verhey, K. J.,
Meyer, D.,
Deehan, R.,
Blenis, J.,
Schnapp, B. J.,
Rapoport, T. A.,
and Margolis, B.
(2001)
J. Cell Biol.
152,
959-970 |
25. |
Brendza, K. M.,
Rose, D. J.,
Gilbert, S. P.,
and Saxton, W. M.
(1999)
J. Biol. Chem.
274,
31506-31514 |
26. | Scholey, J. M. (2002) Dev. Cell 2, 515-516[Medline] [Order article via Infotrieve] |
27. | Klopfenstein, D. R., Tomishige, M., Stuurman, N., and Vale, R. D. (2002) Cell 109, 347-358[Medline] [Order article via Infotrieve] |
28. |
Tomishige, M.,
Klopfenstein, D. R.,
and Vale, R. D.
(2002)
Science
297,
2263-2267 |
29. |
Okada, Y.,
and Hirokawa, N.
(1999)
Science
283,
1152-1157 |
30. | Baas, P. W., Deitch, J. S., Black, M. M., and Banker, G. A. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8335-8339[Abstract] |
31. | Goldstein, L. S. B., and Yang, Z. (2000) Annu. Rev. Neurosci. 23, 39-72[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Sheetz, M. P.
(1999)
Eur. J. Biochem.
262,
19-25 |
33. |
Yang, Z.,
and Goldstein, L. S.
(1998)
Mol. Biol. Cell
9,
249-261 |
34. | Yamazaki, H., Nakata, T., Okada, Y., and Hirokawa, N. (1995) J. Cell Biol. 130, 1387-1399[Abstract] |
35. | Kondo, S., Sato-Yoshitake, R., Noda, Y., Aizawa, H., Nakata, T., Matsuura, Y., and Hirokawa, N. (1994) J. Cell Biol. 125, 1095-1107[Abstract] |
36. | Hirokawa, N., Sato-Yoshitake, R., Kobayashi, N., Pfister, K. K., Bloom, G. S., and Brady, S. T. (1991) J. Cell Biol. 114, 295-302[Abstract] |
37. |
Signor, D.,
Wedaman, K. P.,
Orozco, J. T.,
Dwyer, N. D.,
Bargmann, C. I.,
Rose, L. S.,
and Scholey, J. M.
(1999)
J. Cell Biol.
147,
519-530 |