Characterization of the Movement of the Kinesin Motor KIF1A in Living Cultured Neurons*,

Jae-Ran Lee, Hyewon Shin, Jaewon Ko, Jeonghoon Choi, Hane Lee, and Eunjoon KimDagger

From the Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea

Received for publication, October 31, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (KIF1Balpha and KIF1Bbeta ) (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.

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-Delta MD (aa 366-1695) was generated by inverse PCR using KIF1A-EGFP as a template.

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-alpha (1127) (a rabbit polyclonal antibody raised against aa 818-1202 of liprin-alpha 13).

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-alpha 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-alpha (1127) antibodies.

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-, 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-alpha -binding domain (Fig. 1A), a putative domain that binds to the multi-domain protein liprin-alpha .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-alpha -binding domain, we tested whether this insertion affected the interaction between the liprin-alpha -binding domain and liprin-alpha by coimmunoprecipitation experiments (Fig. 1B). In COS cells doubly transfected with KIF1A (untagged or EGFP-tagged) and liprin-alpha 1, both untagged and tagged KIF1A showed a similar coimmunoprecipitation with liprin-alpha 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-alpha 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-alpha -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-alpha 1. COS cell lysates doubly transfected with KIF1A + HA-tagged liprin-alpha 1 (HA-Lip), or KIF1A-EGFP + HA-tagged liprin-alpha 1, were immunoprecipitated with HA antibodies or mouse IgG (control) and immunoblotted with KIF1A (1131) and liprin-alpha 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.

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.


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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.

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.


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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).

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-Delta 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 Delta 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 (Delta 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 Delta 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

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.

    ACKNOWLEDGEMENTS

We thank Dr. Michel Steruli at ImmunoGen, Inc., for the pMT HA-liprin-alpha 1 construct.

    FOOTNOTES

* 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.

Dagger 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.

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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[Abstract/Free Full Text]
6. Kaether, C., Skehel, P., and Dotti, C. G. (2000) Mol. Biol. Cell 11, 1213-1224[Abstract/Free Full Text]
7. Nakata, T., Terada, S., and Hirokawa, N. (1998) J. Cell Biol. 140, 659-674[Abstract/Free Full Text]
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[Abstract/Free Full Text]
10. Zhou, H. M., Brust-Mascher, I., and Scholey, J. M. (2001) J. Neurosci. 21, 3749-3755[Abstract/Free Full Text]
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[Abstract/Free Full Text]
19. Mok, H., Shin, H., Kim, S., Lee, J. R., Yoon, J., and Kim, E. (2002) J. Neurosci. 22, 5253-5258[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
25. Brendza, K. M., Rose, D. J., Gilbert, S. P., and Saxton, W. M. (1999) J. Biol. Chem. 274, 31506-31514[Abstract/Free Full Text]
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[Abstract/Free Full Text]
29. Okada, Y., and Hirokawa, N. (1999) Science 283, 1152-1157[Abstract/Free Full Text]
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[Abstract/Free Full Text]
33. Yang, Z., and Goldstein, L. S. (1998) Mol. Biol. Cell 9, 249-261[Abstract/Free Full Text]
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[Abstract/Free Full Text]


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