From the Department of Biological Sciences, Korea
Advanced Institute of Science and Technology, Daejeon 305-701, Korea,
the § Center for Learning and Memory, RIKEN-MIT Neuroscience
Research Center and Howard Hughes Medical Institute, Massachusetts
Institute of Technology, Cambridge, Massachusetts 02139, the
Department of Cell Biology and Anatomy, University of North
Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, and the
** Department of Cancer Immunology and AIDS, Dana-Farber
Cancer Institute, and Department of Pathology, Harvard Medical School,
Boston, Massachusetts 02115
Received for publication, November 21, 2002
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ABSTRACT |
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Liprin- The liprin- Functionally, genetic deletion of syd-2 (for synaptic
defective-2), a Caenorhabditis elegans homolog of mammalian
liprin- The kinesin superfamily (KIF) of motor proteins transports cargo
vesicles or organelles on microtubule tracks (22, 23). KIF1A, a member
of the KIF1/Unc104 family of proteins (24), is a neuron-specific
kinesin motor known to transport synaptic vesicle precursors containing
synaptophysin, synaptotagmin, and Rab3A (24, 25). In support of this,
genetic deletion of unc-104, a C. elegans homolog
of KIF1A (26), results in the accumulation of clear vesicles in the
cell body (27). Mutation in the KIF1A gene in mice leads to a similar
accumulation of vesicles in the cell body and neuronal death (28).
Recently, fast and processive movements of Unc104/KIF1A were observed
in living C. elegans and mammalian neurons (29,
30), and molecular mechanisms underlying the processive movement of
Unc104/KIF1A have been extensively characterized (31-34). However,
relatively little is known about whether KIF1A transports cargoes other
than synaptic vesicle precursors and about the manner in which KIF1A
interacts with specific cargoes.
We report here a direct interaction between KIF1A and liprin- Yeast Two-hybrid Screen and Assay--
Liprin- Coimmunoprecipitation in Heterologous Cells--
For the KIF1A
expression construct, the full-length KIF1A cDNA was amplified by
reverse transcription-PCR, digested with HindIII and
EcoRI, and subcloned into GW1 (British Biotechnology).
HEK293T cells transfected with GW1-KIF1A and pMT2-HA-liprin- Antibodies--
To generate fusion protein immunogens, regions
of KIF1A (aa 657-937 for 1131 antibodies) and liprin- Immunohistochemistry and Immunoelectron Microscopy--
For
immunofluorescence staining of rat brain sections, adult rats were
perfused with 4% paraformaldehyde, and brain sections were cut using a
vibratome. Brain sections were permeabilized with phosphate-buffered
saline containing 50% ethanol at room temperature for 30 min,
incubated overnight with combinations of KIF1A (1131; 3 µg/ml),
liprin- Subcellular Fractionation--
Subcellular fractions of whole
rat brain were prepared as previously described (11). In brief, rat
brain homogenates (H in Fig. 6) were centrifuged at 900 × g to remove nuclei and other large debris
(P1). The supernatant was centrifuged at 12,000 × g to obtain a crude synaptosomal fraction (P2).
The supernatant (S2) was centrifuged at 250,000 × g to obtain light membranes (P3) and cytosolic
fraction (S3). In parallel, the P2 fraction was subjected to
hypotonic lysis and centrifuged at 25,000 × g to
precipitate synaptosomal membranes (LP1). The supernatant
(LS1) was further centrifuged at 250,000 × g to obtain a crude synaptic vesicle-enriched fraction
(LP2) and soluble fraction (LS2). For immunoblotting, nitrocellulose or polyvinylidene difluoride membranes were incubated with antibodies against KIF1A (1131; 1 µg/ml), liprin- Flotation Assay and Coimmunoprecipitation--
For the flotation
assay, brains of 6-week-old rats were homogenized in homogenization
buffer (20 mM HEPES, 100 mM potassium acetate,
40 mM KCl, 5 mM MgCl2, 320 mM sucrose, pH 7.4) supplemented with proteinase inhibitors
and 5 mM EGTA. Homogenate was centrifuged at 900 × g for 10 min, and the supernatant was centrifuged at 12,000 × g to obtain crude synaptosomes. Crude
synaptosomes were suspended in the homogenization buffer and
hypotonically lysed by adding nine volumes of H2O to
release synaptic vesicles. After centrifugation at 200,000 × g for 1 h, the membrane pellet was then adjusted to 2 M sucrose and loaded onto the bottom of a discontinuous sucrose gradient of 1.6, 1.0, and 0.3 M sucrose. The
sucrose gradient was centrifuged at 350,000 × g for
3 h. Fractions were taken from the top of the gradient to the
bottom. For coimmunoprecipitation, deoxycholate lysates (10) of the top
three fractions were incubated with antibodies against KIF1A (1131; 3 µg/ml), liprin- Nerve Ligation Assay--
Sciatic nerves of anesthetized adult
rats were ligated for 60 min followed by perfusion with 4%
paraformaldehyde. After a brief postfixation for 30 min, sections (20 µm) of sciatic nerves were cut using a cryotome and incubated
overnight with primary antibodies at 4 °C, followed by room
temperature incubation with Cy3- or fluorescein
isothiocyanate-conjugated secondary antibodies for 3 h. Primary
antibodies used include KIF1A (1131; 3 µg/ml), liprin- Neuron Culture and Immunostaining--
Low density hippocampal
primary cultures were prepared from E18 rat embryos as described
previously (36). Neurons were maintained in the neurobasal medium
supplemented with B27 (Invitrogen). For immunofluorescence
staining, neurons were fixed and permeabilized with cold methanol
( Characterization of the Interaction between KIF1A and Liprin-
We then tested the specificity of interactions between members of the
KIF1 and liprin-
To test whether full-length KIF1A and liprin- KIF1A and Liprin-
Since the yeast two-hybrid results indicated that KIF1A interacts with
liprin-
Similar to KIF1A, liprin- KIF1A Colocalizes with Liprin- Ultrastructural Localization of KIF1A in Rat Brain by
Immunoelectron Microscopy--
To further characterize the
distribution of KIF1A in central neurons, we determined the subcellular
localization of KIF1A by immunogold EM analysis of sections of rat
neocortex (Fig. 4). KIF1A immunogold
particles were observed in various subcellular sites of the neurons
including microtubules (Fig. 4A), consistent with the
function of KIF1A as a kinesin motor moving along microtubule tracks.
KIF1A particles were observed at both pre- and postsynaptic sites (Fig.
4B, example of an asymmetric spine synapse). Quantitative analysis revealed that KIF1A immunogold particles were concentrated close to the pre- and postsynaptic membranes (Fig. 4C,
left panel). The density of KIF1A particles was
constant along the lateral plane of the synaptic membrane (Fig.
4C, right panel). Similar to KIF1A,
liprin- KIF1A Coaccumulates with Liprin- KIF1A Cofractionates and Forms a Complex with Liprin-
To further characterize the association of KIF1A and liprin-
To determine whether KIF1A biochemically associates with liprin-
In further coimmunoprecipitation experiments in a reverse orientation,
liprin- Cargo-binding Domain in KIF1A--
We have shown that part of the
tail region of KIF1A, termed the LBD domain (aa 657-1105), interacts
with liprin-
It has been reported that the C-terminal pleckstrin homology domain of
Unc104 plays an important role in the recognition of phospholipids in
cargo vesicle membranes (45, 46). Our results demonstrate the LBD
domain of KIF1A that is located in the middle the molecule interacts
with liprin- KIF1A-mediated Transports in Dendrites and Axons--
Previous
studies on KIF1A were mainly focused on its transport in the axonal
compartment. However, several lines of evidence in our study indicate
that KIF1A exists in dendrites in addition to axons: 1) localization of
KIF1A in dendrites and axons of brain and cultured neurons revealed by
immunofluorescence staining (Fig. 2); 2) localization of KIF1A in both
pre- and postsynaptic sites revealed by immunogold EM analysis (Fig.
4); 3) biochemical association of KIF1A with both axonal
(synaptotagmin) and dendritic (AMPA receptors) proteins (Fig. 6). In
addition, movement of enhanced green fluorescent protein-tagged KIF1A
particles has been detected in proximal thick neurites (probably
dendrites) and axons of living cultured neurons (30). This is
consistent with the movement of enhanced green fluorescent
protein-tagged Unc104 particles observed in both dendrites and axons of
living C. elegans neurons (29). Collectively, these results suggest
that KIF1A/Unc104 proteins are involved in the transport of neuronal
proteins in both dendrites and axons.
Liprin-
It has been recently shown that Unc104 can exhibit a highly processive
movement through the formation of dimers at high motor concentrations
(34), which may occur in vivo through clustering of motor
proteins in phosphatidylinositol 4,5-bisphosphate-containing rafts on
the surface of cargo vesicles (45, 46). Similar to Unc104, KIF1A also
moves processively along the microtubule in the single molecule
motility assay, but some KIF1A molecules occasionally exhibit slow
movement (34) that is similar to the previously reported biased
diffusional movement (32). This suggests that KIF1A may form a
relatively unstable dimer, perhaps due to the weakness of the predicted
neck coiled-coil probability, and raises the possibility that KIF1A
dimers may be stabilized by additional mechanisms (34). Intriguingly,
liprin- GRIP-associated Proteins as KIF1A Cargoes--
GRIP and
GRIP-associated AMPA receptors comprise an important set of potential
KIF1A cargoes (Fig. 6, B-F). Several lines of evidence
indicate that GRIP is involved in neuronal transport. A significant
amount of GRIP immuno-EM labeling associates with vesicles that are
often very close to microtubules (10, 14). Biochemically, GRIP
distributes to small membrane- and vesicle-enriched fractions (11, 14),
similar to the subcellular distribution of liprin-
It has been reported that conventional kinesin heavy chain interacts
with GRIP1 and transports the AMPA receptor-GRIP complex (51). This
finding in conjunction with our results indicates that the AMPA
receptor-GRIP complex could be transported by more than one type of
kinesin motor, KIF1A and conventional kinesin. A similarly redundant
transport mechanism, which may exist for physiologically important
cargoes, has been identified for
N-methyl-D-aspartate glutamate receptors, which
associate with KIF17 through the LIN-2·LIN-7·LIN-10 complex
(49) and with KIF1B
Intriguingly, GRIP1 steers conventional kinesin to dendrites (51),
which raises the question of whether KIF1A is also steered to dendrites
by association with liprin- KIF1A, Liprin-
In conclusion, we have shown the first evidence for a protein
interaction of KIF1A with the multimodular protein liprin-/SYD-2 is a multimodular scaffolding
protein important for presynaptic differentiation and postsynaptic
targeting of
-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic
acid glutamate receptors. However, the molecular mechanisms
underlying these functions remain largely unknown. Here we report that
liprin-
interacts with the neuron-specific kinesin motor KIF1A.
KIF1A colocalizes with liprin-
in various subcellular regions of
neurons. KIF1A coaccumulates with liprin-
in ligated sciatic nerves.
KIF1A cofractionates and coimmunopreciptates with liprin-
and
various liprin-
-associated membrane, signaling, and scaffolding
proteins including
-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptors,
GRIP/ABP, RIM, GIT1, and
PIX. These results suggest that liprin-
functions as a KIF1A receptor, linking KIF1A to various
liprin-
-associated proteins for their transport in neurons.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/SYD-2 family of proteins was originally identified
as a cytosolic binding partner of the LAR family of receptor protein-tyrosine phosphatases (1). Liprin-
contains various domains
for protein interactions including a long coiled coil region in the
N-terminal half, three SAM domains in the middle, and a PDZ-binding
motif at the C terminus. The N-terminal coiled-coil region of
liprin-
mediates homomultimerization (1) and interacts with
GIT/Cat/p95-APP/PKL (2), a family of multidomain proteins with
GTPase-activating protein activity for ADP-ribosylation factor small
GTP-binding proteins (3-7), as well as RIM1 (Rab3-interacting molecule) (8), a scaffolding protein at the presynaptic active zone
regulating neurotransmitter release (8, 9). The SAM domains of
liprin-
interact with the intracellular domain of LAR (1). The C
terminus of liprin-
interacts with the GRIP/ABP family of multi-PDZ
proteins, which are known to bind various membrane, cell adhesion, and
signaling proteins including
AMPA1 glutamate receptors
(GluRs) (10-14), ephrin ligands, and receptors (15-17) and GRASP-1, a
neuronal Ras-specific guanine nucleotide exchange factor (18).
These results suggest that liprin-
may function as a multimodular
scaffolding protein.
, results in a diffuse distribution of presynaptic markers,
lengthening of the presynaptic active zone, and impairment of synaptic
transmission (19). Similarly, genetic deletion of
Dliprin-
, a Drosophila homolog of liprin-
, leads to an
alteration of the size and shape of active zones (20). In addition,
disruption of the interaction between liprin-
and GRIP eliminates
surface clustering of AMPA receptors in dendrites of neurons (21).
These results suggest that liprin-
/SYD-2 regulates presynaptic
differentiation of active zone as well as postsynaptic targeting of
AMPA receptors. However, it remains largely unknown how liprin-
regulates presynaptic differentiation and postsynaptic receptor
targeting. Importantly, liprin-
distributes to various nonsynaptic
structures in axons and dendrites (21), suggesting that liprin-
may
have novel functions at extrasynaptic sites in addition to its
suggested role as an organizer of synaptic multiprotein complexes.
, which
links KIF1A to various liprin-
-associated proteins including AMPA
receptors, GRIP, RIM, GIT1, and
PIX. Our results suggest that
liprin-
functions as a KIF1A "receptor" linking the KIF1A motor
to a cargo of liprin-
-associated proteins.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 (aa
351-1105) and liprin-
4 (aa 1-863) were amplified by PCR and
subcloned to pBHA bait vector (LexA DNA binding domain). The
following deletions of KIF1A were amplified by PCR and subcloned in
frame into the pGAD10 prey vector (GAL4 activation domain;
Clontech): 501-800, 501-937, 501-1105,
657-1105, 717-937, 717-1105, and 923-1105. Deletions of liprin-
1
were subcloned into pGAD10 vector: aa 1-221, 1-673, 1-848, 217-553,
221-673, 351-553, 351-673, 513-673, and 668-1202. KIF1B
(aa
666-1150) and KIF1B
(aa 697-1223) were amplified by reverse
transcription-PCR and subcloned into pGAD10. All yeast two-hybrid
constructs were confirmed by DNA sequencing.
1 were
extracted with Tris-buffered saline containing 1% Triton X-100 and
incubated with HA antibodies (mouse monoclonal; 4 µg/ml) or mouse IgG
(4 µg/ml), followed by incubation with protein A-Sepharose (Amersham Biosciences). Immunoprecipitates were immunoblotted with HA (rabbit polyclonal; 1 µg/ml) and KIF1A (1131; 1 µg/ml) antibodies.
1 (aa 351-673
for 1120 and 818-1202 for 1127 antibodies) were amplified by PCR and
subcloned into pRSETB (vector for H6 fusion protein; Invitrogen).
Fusion proteins were purified using Probond resin (Invitrogen).
Affinity purification of specific antibodies was performed using
immunogen immobilized on polyvinylidene difluoride membrane (Amersham
Biosciences). The specificity of KIF1A (1131) antibodies was determined
by Western blot analysis on GST-KIF1A (aa 657-937) and GST-KIF1B
(aa 697-1223). To examine the reactivity of liprin-
antibodies
(1120 and 1127) against the liprin-
family members, COS cell lysates
transfected with HA-liprin-
1 or HA-liprin-
2 were immunoblotted
with liprin-
(1120, 1127) and HA (mouse monoclonal; Roche Molecular
Biochemicals) antibodies. Other antibodies used include GRIP (C8399;
pan-GRIP antibody) (10, 11), GST (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), GluR1 (Chemicon), PSD-95 (K23/58; Upstate Biotechnology, Inc., Lake Placid, NY), GluR2/3 (Chemicon), synaptotagmin (Sigma), syntaxin (Sigma), MAP2 (Sigma), neurofilament-H (neurofilament 200;
Sigma), cortactin (Upstate Biotechnology), mouse monoclonal HA (Roche
Molecular Biochemicals), rabbit polyclonal HA (Santa Cruz
Biotechnology), GIT1 (2), and RIM (Transduction Laboratories) antibodies.
(1127; 3 µg/ml), MAP2 (1:200), neurofilament-H (1:100),
and GRIP (C8399; 3 µg/ml) antibodies, followed by incubation with
Cy3-, fluorescein isothiocyanate-, or Cy5-conjugated secondary
antibodies at dilutions of 1:1000, 1:300, and 1:500, respectively
(Jackson Immunoresearch). Images were captured using an LSM510 confocal
laser-scanning microscope (Zeiss). Postembedding immunogold electron
microscopy with KIF1A (1131; 3 µg/ml) antibodies was performed and
quantified as described previously (35).
(1120; 1 µg/ml), GRIP (C8399; 1 µg/ml), GluR1 (1 µg/ml), PSD-95 (1:1000), and synaptotagmin (1:500).
(1127; 3 µg/ml), GluR2/3 (Chemicon; 3 µg/ml),
or control IgG (rabbit or guinea pig; 3 µg/ml) followed by
precipitation with protein A-Sepharose (Amersham Biosciences).
Immunoprecipitates were analyzed by immunoblotting with KIF1A (1131; 1 µg/ml), liprin-
(1120; 1 µg/ml), GRIP (C8399; 1 µg/ml),
GluR2/3 (1 µg/ml), synaptotagmin (1 µg/ml), RIM (1 µg/ml), GIT1
(1 µg/ml),
PIX (1 µg/ml), syntaxin (1:400), and cortactin
(1:1000) antibodies.
(1127; 3 µg/ml), and syntaxin (1:200). Immunofluorescence images were captured
using an LSM510 confocal laser-scanning microscope (Zeiss).
70 °C) and incubated with KIF1A (1131; 3 µg/ml) and MAP2
(1:200) antibodies, followed by incubation with Cy3- or fluorescein
isothiocyanate-conjugated secondary antibodies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
by
the Yeast Two-hybrid Assay and Coimmunoprecipitation--
To better
understand the functions of liprin-
, we performed a yeast two-hybrid
screen of rat brain cDNA using liprin-
4, a member of the
liprin-
family, as bait. Out of ~1 × 106 yeast colonies, a cDNA fragment of KIF1A (aa
455-1105) containing roughly the middle third of the protein was
isolated. The minimal regions required for the interaction were
determined by characterizing deletion variants of KIF1A and
liprin-
1, a member of the liprin-
family for which a full-length
cDNA was available. The minimal liprin-
1-binding region in KIF1A
was narrowed down to aa 657-1105, which we termed the
liprin-
-binding domain (LBD) (Fig.
1A). The minimal KIF1A-binding
region in liprin-
1 was localized to a region (aa 351-673) largely
within the N-terminal coiled-coil region (aa 1-650; Fig.
1B).
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Fig. 1.
Characterization of the interaction between
KIF1A and liprin- by the yeast two-hybrid
assay and coimmunoprecipitation. A, minimal
liprin-
-binding region in KIF1A. Deletions of KIF1A were tested for
binding to liprin-
1 and liprin-
4 in the yeast two-hybrid assay.
The minimal liprin-
-binding region in KIF1A (aa 657-1105) is
indicated by the thicker line. The region that
the KIF1A (1131) antibodies were raised against is indicated by a
dotted line underneath the schematic of the
domain organization of KIF1A. Motor, motor domain;
LBD, liprin-
-binding domain; PH, pleckstrin
homology domain. HIS3 activity was as follows: +++ (>60%), ++
(30-60%), + (10-30%),
(no significant growth).
-Galactosidase
activity was as follows: +++ (<45 min), ++ (45-90 min), + (90-240
min),
(no significant
-galactosidase activity). B,
minimal KIF1A-binding region in liprin-
1. Deletions of liprin-
1
were tested for binding to KIF1A in the yeast two-hybrid assay. The
minimal KIF1A-binding region in liprin-
1 (aa 351-673) is indicated
by the thicker line. The regions that 1120 and
1127 liprin-
antibodies were raised against are indicated.
CC, coiled-coil domain; SAM, sterile
motif. A
PDZ domain-binding motif at the C terminus of liprin-
1 is indicated
by a vertical black line.
C, specificity of the interactions between members of the
KIF1 and liprin-
families. Both liprin-
1 and liprin-
4 interact
with KIF1A, whereas only KIF1A (not KIF1B
or KIF1B
) interacts
with liprin-
. 1'-863' in liprin-
4 (full aa sequence is not
available) is a region used as bait in the two-hybrid screen and
corresponds to aa 351-1202 in liprin-
1. D,
coimmunoprecipitation between KIF1A and liprin-
in HEK293T cells.
HEK293T cell lysates doubly transfected with KIF1A and HA-tagged
liprin-
1 (HA-liprin-
1) or singly transfected with KIF1A were
immunoprecipitated with mouse monoclonal HA antibodies and
immunoblotted with rabbit polyclonal HA and KIF1A (1131) antibodies.
KIF1A is specifically coimmunoprecipitated by HA antibodies
(lane 2) but not by control mouse IgG
(lane 3). Singly transfected KIF1A does not
cross-react with HA antibodies (lane 5).
Input, 5%.
families. In addition to liprin-
4, liprin-
1
(aa 351-1202, a region corresponding to the liprin-
4 bait) also
interacted with the KIF1A deletions that showed positive interaction
with liprin-
4 (Fig. 1A). In contrast, liprin-
1 and liprin-
4 did not interact with KIF1B (both KIF1B
and KIF1B
splice variants), another member of the KIF1 family (37-40) (Fig. 1C).
interact in a
mammalian cell environment, we performed coimmunoprecipitation experiments using HEK293T cells doubly transfected with KIF1A and
HA-tagged liprin-
1 (HA-liprin-
1) (Fig. 1D).
Immunoprecipitation with HA antibodies, but not control mouse IgG,
immunoprecipitated HA-liprin-
1 and coprecipitated KIF1A. HA
antibodies did not bring down KIF1A in the absence of
HA-liprin-
1.
Distribute to Both Dendrites and Axons in
Brain and Cultured Neurons--
To characterize the distribution of
KIF1A and liprin-
in vivo, we generated specific
antibodies against fusion proteins of KIF1A (aa 657-937; termed 1131 antibodies) and liprin-
1 (aa 351-673 for 1120 antibodies; aa
818-1202 for 1127 antibodies) (Fig. 1, A and B).
KIF1A (1131) antibodies specifically recognized KIF1A but not KIF1B
in immunoblot analysis (Fig.
2A). The two liprin-
(1120 and 1127) antibodies reacted equally with HA-liprin-
1 and HA-liprin-
2 (Fig. 2B). Liprin-
3 and liprin-
4 were
not tested because full-length cDNAs of these isoforms were not
available. However, since members of the liprin-
family share
similar aa sequences in the regions where the antibodies were raised,
it is likely that the liprin-
antibodies recognize all liprin-
isoforms. When tested against rat brain samples, the KIF1A and liprin-
antibodies recognized single bands with molecular masses of
~200 and ~160 kDa, respectively, which are comparable with those of
the same proteins transiently expressed in heterologous cells (Fig. 2,
C and D).
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Fig. 2.
KIF1A and liprin-
distribute to both dendrites and axons in rat brain and cultured
hippocampal neurons. A, specificity of KIF1A
antibodies. Equal amounts (100, 30, and 10 ng for lanes 1-3 and 4-6)
of GST fusion proteins containing KIF1A (aa 657-937) and KIF1B
(aa
697-1223, equivalent region) were immunoblotted with KIF1A (1131) and
GST antibodies. KIF1A antibodies specifically recognize KIF1A but not
the closely related KIF1B
. B, specificity of liprin-
(1120 and 1127) antibodies. HA-liprin-
1 and HA-liprin-
2 expressed
in COS cells were immunoblotted with HA and liprin (1120 and 1127)
antibodies. Liprin-
antibodies equally recognize liprin-
1 and
liprin-
2. HA-liprin-
proteins with a ratio of 10:3:1 were loaded
in lanes 1-3 and 4-6. C
and D, characterization of KIF1A (1131) and liprin-
(1120 and 1127) antibodies. Subcellular fractions of rat brain (S2 and P2
fractions) and lysates of COS cells transfected with KIF1A and
liprin-
-1, respectively, were immunoblotted with the KIF1A and
liprin-
antibodies indicated. P2, crude synaptosomes;
S2, supernatant after the removal of the P2;
Trans, transfected; Untrans, untransfected. A
small amount of endogenous liprin-
proteins is detected in the
untransfected lanes. E-M, immunodistribution of KIF1A and
liprin-
in vivo. Rat brain slices, spinal cord sections,
and cultured hippocampal neurons (DIV 21) were labeled by
immunofluorescence staining with combinations of KIF1A (1131),
liprin-
(1127), MAP2, and neurofilament H (NF-H) antibodies. KIF1A
colocalizes with MAP2, a dendritic marker, in cortex (E,
examples of colocalizations are indicated by arrowheads) and
hippocampus (F). KIF1A colocalizes with NF-H, an axonal
marker, in the white matter of cerebellum (G) and axon
bundles of spinal cord (H). Preincubation of KIF1A
antibodies with immunogen eliminates the staining (K,
hippocampal CA1 region). In cultured hippocampal neurons, KIF1A
distributes to MAP2-positive dendrites (M,
arrowhead) as well as MAP2-negative axons (M,
arrow). Similar to KIF1A, liprin-
distributes to
MAP2-positive dendrites (I, hippocampal CA1) and
NF-H-positive axons (J, cerebellar white matter).
Preincubation of liprin-
antibodies with immunogen eliminates the
staining (L, hippocampal CA1). P, pyramidal cell
layer; Sr, stratum radiatum. The arrowheads
indicate the sites of colocalization. Size bar,
30 µm (E-M).
, a protein that localizes to both dendrites and axons (21),
we first determined the subcellular distribution of KIF1A proteins in
rat brain and cultured neurons by immunofluorescence staining (Fig. 2,
E-H and M). Interestingly, we detected KIF1A in
both dendrites and axons. KIF1A overlapped with MAP2, a dendritic marker, in cortex (Fig. 2E) and hippocampus (Fig.
2F). Consistent with its known axonal localization (25),
KIF1A also colocalized with neurofilament H (NF-H), an axonal marker,
in the white matter region of cerebellum (Fig. 2G) and in
axon bundles of spinal cord (Fig. 2H). In cultured neurons,
KIF1A was found in MAP2-positive dendrites as well as MAP2-negative
axons (Fig. 2M). Preincubation of KIF1A antibodies with
immunogen eliminated the KIF1A staining (Fig. 2K, an example
from the CA1 region of hippocampus). Similar to endogenous KIF1A,
exogenous KIF1A was localized to both dendrites and axons of cultured
hippocampal neurons (data not shown). Consistently, KIF1A immunogold
particles distributed to both the pre- and postsynaptic sides in
electron microscopic (EM) analysis (Fig. 4; details described below).
Taken together, these results suggest that KIF1A plays a role in both
dendritic and axonal transport in neurons.
(1127 antibody) distributed to both
dendrites and axons as evidenced by colocalization with MAP2 (Fig.
2I, the CA1 region of hippocampus) and NF-H (Fig.
2J, the white matter of cerebellum). The other liprin-
(1120) antibodies showed essentially the same distribution pattern
(data not shown). Liprin-
staining was eliminated by preincubation
of the antibodies with immunogens (Fig. 2L, the CA1 region
of hippocampus). In cultured hippocampal neurons, both endogenous (21)
and exogenous (data not shown) liprin-
distribute to dendrites and
axons, similar to the subcellular distribution of liprin-
in brain.
and GRIP in Rat Brain--
We
tested colocalization between KIF1A, liprin-
, and GRIP (a
liprin-
-associated protein) by double or triple label
immunofluorescence staining on rat brain sections (Fig.
3). In rat brain, the distribution of
KIF1A overlapped that of both liprin-
(Fig. 3A, an
example from the CA1 dendrites of hippocampus) and GRIP (Fig.
3B, hippocampal CA1 dendrites). Triple labeling of KIF1A,
liprin-
, or GRIP and NF-H (axons) revealed that KIF1A colocalizes
with liprin-
(Fig. 3C) and GRIP (Fig. 3D) in
punctate subcellular structures in axons of cerebellar white matter.
These results indicate that KIF1A colocalizes with liprin-
and GRIP
in vivo.
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Fig. 3.
KIF1A colocalizes with
liprin- and GRIP in rat brain.
A and B, rat brain sections were doubly labeled
by immunofluorescence staining for KIF1A and liprin-a
(A), or KIF1A and GRIP (B). The
distribution of KIF1A largely overlaps that of liprin-
(A) and GRIP (B) (examples from the CA1 dendrites
of hippocampus). C and D, rat brain sections were
triply labeled for KIF1A (green), liprin-
(red) or GRIP (red), and NF-H (blue). KIF1A
colocalizes with liprin-
(C) or GRIP (D) in
punctate subcellular structures (arrowheads) of axons
(examples from axons of cerebellar white matter). Merge,
green plus red. Size bar,
80 µm (A-B) and 10 µm (C-D).
is distributed in pre- and postsynaptic sites of neurons at
the EM level (21). These results provide further evidence that KIF1A
distributes to dendritic and axonal sites.
View larger version (83K):
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Fig. 4.
Ultrastructural localization of KIF1A by
immunoelectron microscopy. Sections of rat cerebral cortex were
stained for KIF1A by immunogold EM. Some of the KIF1A immunogold
particles associate with microtubules (A,
arrows). KIF1A distributes to both pre- and postsynaptic
sites (B, example from an axospinous synapse). *,
presynaptic nerve terminal. Sp, postsynaptic spine. In a
quantitative analysis, KIF1A gold particles are concentrated at pre-
and postsynaptic membrane-proximal regions (C,
left panel; the right side
from the midline corresponds to the postsynaptic region).
The distribution of gold particles remains consistent along the lateral
plane of the synapse (C, right panel;
0 corresponds to the center, and 1.0 corresponds
to the edge of the synapse). Size bar, 0.25 µm.
in Ligated Sciatic
Nerves--
Okada et al. (25) showed that KIF1A accumulates
with synaptophysin but not with syntaxin in ligated sciatic nerve
fibers, suggesting that KIF1A selectively transports
synaptophysin-containing vesicles. Since liprin-
is detected in
sciatic nerve fibers by immunoblot analysis (data not shown), we tested
whether KIF1A comigrates with liprin-
in axons of motor neurons by
the nerve ligation assay (Fig. 5). In rat
sciatic nerves ligated for 60 min, KIF1A and liprin-
accumulated and
precisely colocalized on the proximal (cell body) side of the ligation
(Fig. 5A). Syntaxin also accumulated proximally but did not
colocalize with KIF1A (Fig. 5C), verifying the specificity
of KIF1A/liprin-
coaccumulation. KIF1A, liprin-
, and syntaxin did
not accumulate on the distal side of the ligation (Fig. 5, B
and D). These results suggest that KIF1A may anterogradely
transport liprin-
along axonal microtubules.
View larger version (37K):
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Fig. 5.
KIF1A and liprin-
coaccumulate in ligated sciatic nerve fibers. Rat sciatic
nerves were ligated for 60 min and labeled by double immunofluorescence
staining for KIF1A + liprin-
(A and B), or
KIF1A + syntaxin (C and D, negative control).
KIF1A specifically coaccumulates with liprin-
(A) but not
with syntaxin (C) in the proximal side of the ligation. The
site of nerve ligation is indicated by two
vertical arrows. Proximal (cell body)
(A and C) and distal (nerve terminal)
(B and D) sides of the ligation site are
indicated. Images on the proximal side of the ligation were
merged for better comparison. Size bar, 220 µm.
and
Liprin-
-associated Proteins in Brain--
If liprin-
is a KIF1A
receptor linking KIF1A to its vesicular cargoes, KIF1A and liprin-
should cofractionate into the subcellular fractions of neurons enriched
with light membranes and synaptic vesicles. To test this, we determined
fractionation patterns of KIF1A and liprin-
in subcellular fractions
of rat brain (Fig. 6A). Both
KIF1A and liprin-
were detected in the P3 (light membranes) and LP2
(synaptic vesicles) fractions. In addition, proteins associated with
liprin-
such as GRIP and GluR1 were also detected in the P3 and LP2
fractions.
View larger version (70K):
[in a new window]
Fig. 6.
KIF1A cofractionates and forms a complex with
liprin- and
liprin-
-associated proteins in brain.
A, KIF1A and liprin-
are detected in light membrane and
synaptic vesicle fractions of brain. Subcellular fractions of adult rat
brain were immunoblotted with the antibodies indicated. KIF1A and
liprin-
, along with GRIP and AMPA receptors (GluR1),
distribute to the light membrane (P3) and synaptic vesicle
(LP2) fractions. PSD-95 and synaptotagmin (a presynaptic
vesicle protein) were visualized for comparison. H, rat
brain homogenates; P1, nuclei and other large debris;
P2, crude synaptosomes; S2, supernatant after the
removal of the P2; S3, cytosolic fraction; P3,
light membranes; LP1, synaptic membrane-enriched fraction;
LS2, synaptic cytosol; LP2, synaptic
vesicle-enriched fraction. B, cofractionation of KIF1A and
liprin-
in the sucrose density flotation assay. Membrane-enriched
samples from rat brain (see "Experimental Procedures" for more
details) were loaded onto the bottom of a discontinuous sucrose density
gradient. KIF1A, along with liprin-
, GRIP, and AMPA receptors
(GluR2/3), but not cortactin, cofractionate in light fractions
(lanes 1-3, left panels).
The floating was eliminated (right panel) by the
addition of detergent to the samples before flotation (right
panels). C, coimmunoprecipitation of KIF1A with
liprin-
- and liprin-
-associated proteins in light membranes.
Light membrane fractions (1-3) from B were
solubilized with detergent, immunoprecipitated with KIF1A (1131)
antibodies or guinea pig IgG and immunoblotted with the antibodies
indicated. KIF1A coimmunoprecipitates with liprin-
, GRIP, GluR2/3,
and synaptotagmin but not syntaxin and cortactin. Input,
2%. D, in an independent coimmunoprecipitation experiment
similar to C, KIF1A selectively coimmunoprecipitates with
RIM, GIT1, and
PIX. Input, 1%. E and
F, detergent extracts of the floated light membranes were
also immunoprecipitated with liprin-
(1127), GluR2/3 (Chemicon)
antibodies, or rabbit IgG (negative control) and immunoblotted with the
antibodies indicated. Both liprin-
and GluR2/3 coimmunoprecipitate
with KIF1A.
with
membranes, we performed the sucrose density flotation assay (Fig.
6B). When samples enriched with membranes (see
"Experimental Procedures" for details) were loaded onto the bottom
of a discontinuous sucrose gradient, KIF1A and liprin-
floated and
cofractionated into the light fractions (lanes
1-3), along with GRIP, GluR2/3, and synaptotagmin, but not
with cortactin (Fig. 6B, left panel). Detergent treatment of the samples prior to centrifugation eliminated the floating (Fig. 6B, right panel),
suggesting that intact membranes are required for flotation.
in
the floated membranes, we performed coimmunoprecipitation experiments
on detergent extracts of the pooled light membranes (fractions 1-3).
KIF1A antibodies immunoprecipitated KIF1A and coprecipitated
liprin-
, GRIP, GluR2/3, and synaptotagmin, but not syntaxin and
cortactin (Fig. 6C). The liprin-
(1120) antibody recognizes both liprin-
1 and liprin-
2 (Fig. 2B), and
the GRIP (C8399) antibody recognizes both GRIP1 and GRIP2/ABP (10, 11). Thus, further study will be required to identify the specific isoforms
of liprin-
and GRIP that bind to KIF1A in vivo. The coimmunoprecipitation of GRIP and GluR2/3 is presumably due to their
association with liprin-
(21). The coimmunoprecipitation of
synaptotagmin suggests that KIF1A is biochemically associated with
synaptotagmin and is similar to the reported association between
synaptotagmin and the closely related KIF1B
(40). The lack of
coimmunoprecipitation of syntaxin that floated together with KIF1A in
the flotation assay (Fig. 6B) indicates the specific association of KIF1A with its cargoes, and the lack of
coimmunoprecipitation of cortactin with KIF1A is consistent with their
differential floating (Fig. 6B). Control immunoprecipitation
with guinea pig IgG did not bring down any of these proteins.
Interestingly, in an independent coimmunoprecipitation experiment on
detergent lysates of the floated samples, KIF1A antibodies
coimmunoprecipitated two additional liprin-
-binding proteins, RIM (a
scaffolding protein at active zones) and GIT1 (a multimodular
scaffolding protein with an ADP-ribosylation factor GTPase-activating
protein activity) (Fig. 6D). In addition, KIF1A antibodies
pulled down the
PIX/Cool-1 (Fig. 6D), a Rho-type guanine
nucleotide exchange factor that directly interacts with GIT1 (5, 6,
41).
antibodies immunoprecipitated liprin-
and coprecipitated
KIF1A and other liprin-
-associated proteins including GRIP and RIM
(Fig. 6E). In addition, GluR2/3 antibodies brought down
GluR2/3 and coprecipitated GRIP, liprin-
, and KIF1A (Fig. 6F), strongly suggesting that KIF1A and GluR2/3 are
biochemically associated in the floated membranes. Importantly, GluR2/3
antibodies did not bring down RIM (Fig. 6F), suggesting that
the KIF1A cargo vesicles containing postsynaptic proteins may not
contain presynaptic proteins. Taken together, these results indicate
that KIF1A biochemically associates with liprin-
and various
liprin-
-associated membrane, signaling, and scaffolding proteins in
the brain.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Fig. 1). The closely related KIF1B
(1770 aa long)
requires its tail region (aa 885-1770) for association with vesicles
containing synaptophysin, synaptotagmin, and SV2 (40). The C terminus
of KIF1B
, a shorter splice variant of KIF1B, interacts with the
PSD-95, SAP97, and S-SCAM PDZ domain-containing proteins (42).
KIF1C (1103 aa long), the third member of the KIF1 family, uses its
middle (aa 714-809) and C-terminal (last 60 aa residues) regions to
interact with protein-tyrosine phosphatase D1 and 14-3-3, respectively
(43, 44). Taken together, these results suggest that members of the KIF1 family of kinesin motors use various regions in their tails to
associate with specific cargoes.
, a multimodular protein that is linked to various
proteins including membrane proteins. Considering these results, it is
conceivable that the LBD and pleckstrin homology domains of KIF1A may
associate with cargo vesicles in a parallel fashion. In this model, the
pleckstrin homology domain of KIF1A may bind to the membrane of a cargo
vesicle, whereas liprin-
may associate with the proteins on the same
cargo vesicle. This parallel binding may help to determine the
specificity or affinity of the association of KIF1A with its cargoes.
as a KIF1A Receptor--
Recent studies have begun to
uncover the motor-binding "receptors" in cargoes (47, 48), which
include coat proteins, scaffolding proteins, small GTPases,
transmembrane proteins, and other motor proteins. Examples of motor
receptors similar to our results are the scaffolding proteins
LIN-2·LIN-7·LIN-10 and JIP-1·JIP-2·JIP-3 proteins, which
link KIF17 (49) and conventional kinesin (50), respectively, to their
specific cargoes. We propose that liprin-
functions as a cargo
receptor for KIF1A, since liprin-
directly interacts with KIF1A and
also associates with a variety of membrane proteins such as LAR and
AMPA receptors, thereby potentially linking KIF1A to cargo vesicles.
forms multimers (1), suggesting the possibility that
liprin-
may contribute to the processive movement of KIF1A through
the stabilization of KIF1A dimers. This would suggest a dual role for
liprin-
, that of both KIF1A receptor and a stabilizer of KIF1A dimers.
(Fig.
6A). It was reported that synaptic targeting of AMPA
receptors is eliminated by disrupting the liprin-
-GRIP interaction
by various dominant negative constructs (21). A possible explanation
for such results is that the disruption may prevent the AMPA
receptor-GRIP complex from associating with KIF1A through liprin-
.
Taken together, these results suggest that KIF1A, via liprin-
, may
transport GRIP, AMPA receptors, and possibly other GRIP-associated
membrane and signaling proteins including ephrin ligands, ephrin
receptors, and GRASP-1 (15-18).
through PSD-95 or S-SCAM (42). Similarly,
liprin-
could also be transported by both KIF1A and conventional
kinesin. The minimal effects of unc-104 mutations in C. elegans on the presynaptic targeting of SYD-2 (19) may support this
idea of a redundant mechanism for liprin-
/SYD-2 transport.
or GRIP. Our data indicate that
postsynaptic GluR2/3 coimmunoprecipitates with GRIP, liprin-
, and
KIF1A, but not with RIM, a presynaptic active zone protein (Fig.
6F), suggesting that pre- and postsynaptic cargo proteins partition into different KIF1A cargo vesicles. This suggests that further work is needed to identify the molecular determinants that
direct the polarized targeting of KIF1A cargo vesicles with pre- and
postsynaptic contents.
, and Presynaptic Differentiation--
Genetic
deletion of syd-2 in C. elegans and
Dliprin-
in Drosophila leads to abnormal
differentiation of the presynaptic active zone (19, 20). One
explanation for these results is that liprin-
functions as a
structural component of the active zone (52). An equally plausible
hypothesis based on our results is that defective liprin-
may limit
KIF1A-mediated axonal transport of various liprin-
-associated
proteins involved in presynaptic development. We demonstrated that
KIF1A associates with liprin-
and liprin-
-associated proteins
including RIM, GIT1, and
PIX (Fig. 6). RIM is a multimodular scaffolding protein of the active zone that is involved in the regulation of neurotransmitter release (8, 9). GIT1 distributes to both
pre- and postsynaptic sites at the EM level (2) and associates with
Piccolo/aczonin (53), a core component of active zones (54, 55).
Mutation in the dPix gene, a Drosophila homolog of mammalian
PIX, has been shown to modify synaptic structure and targeting of
various synaptic proteins (56). Taken together, these results suggest
that liprin-
may mediate the transport of these proteins to the
nerve terminal for presynaptic differentiation.
. Our
results suggest that liprin-
, as a KIF1A receptor, may link KIF1A to
various liprin-
-associated membrane, signaling, and cytoskeletal
proteins during their transport. It will be of use in the near future
to perform genetic analysis of the identified protein interactions and
determine the functional association of liprin-
with the
dimerization and polarized targeting of KIF1A.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. Dongeun Park (Seoul National
University) for the PIX antibody.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Korean Ministry of Science and Technology, the Korea Research Foundation, and the Korea Science and Engineering Foundation (to E. K.) and National Institutes of Health Grant NS-35527 (to R. J. W.).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.
¶ Present address: Amgen Inc., Thousand Oaks, CA 91320.
Present address: ImmunoGen, Inc., 128 Sidney St., Cambridge, MA 02139.
§§ Associate investigator of the Howard Hughes Medical Institute.
¶¶ To whom correspondence should be addressed: Dept. of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea. Tel.: 42-869-2633; Fax: 42-869-2610; E-mail: kime@mail.kaist.ac.kr.
Published, JBC Papers in Press, January 8, 2003, DOI 10.1074/jbc.M211874200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
AMPA, -amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid;
GluR, glutamate receptor;
aa, amino acid(s);
LBD, liprin-
-binding domain;
EM, electron microscopy;
GST, glutathione
S-transferase.
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