Characterization of Anti-KIF3B Antibody
As previously reported, our anti-KIF3B antibody revealed binding uniquely with the 85-kb polypeptide in SCG extracts ( D, lane 1), while preimmune sera did not ( D, lane 2). Immunofluorescence of cultured SCG neurons ( A) also revealed the localization of this protein, both in the cell body and the axons, the latter of which partly showed a punctate staining pattern ( C). With the previous ultrastructural finding that KIF3 is associated with vesicles isolated from cauda equina (Yamazaki et al. 1995
), this staining pattern is considered to represent the association of KIF3 with axonal membranous components. As shown in our previous study (Yamazaki et al. 1995
), this anti-KIF3B antibody blocks the in vitro motility of KIF3A/B heterodimers in a concentration-dependent manner.
Figure 1 Characterization of anti-KIF3B antibody. A, Fluorescence immunocytochemical staining of cultured SCG neurons. Staining with anti-KIF3B antibody can be observed throughout the neuron, and is especially evident in axons. When viewed at higher magnification (more ...)
Reduction of Fast Axonal Transport after Anti-KIF3B Fab Injection
To test the consequence of inhibiting KIF3 function, Fab fragments prepared from either anti-KIF3B antibody or normal mouse IgG (~3 mg/ml) were injected. To avoid possible cross-linking of vesicles to one another, resulting in a nonspecific traffic jam, Fab fragments were used.
About three hours after microinjection, we observed the SCG neurons emitting dim green fluorescence under the low-light level fluorescence microscope system equipped with the VEC–DIC system. Under the VEC–DIC, the ratio of the different categories of vesicles did not seem to have changed as a result of antibody injection (), but the number of vesicle passing through a certain point on the axon distant from the cell body (~200–400 mm) was significantly decreased (). Vesicle movements were quantified ( C), revealing that the microinjection process, per se, did not seem to have any deleterious effect on vesicle traffic, as injection of the control antibody gave almost the same results, as compared to uninjected samples (except for mitochondria, all groups show comparable vesicle traffic to the noninjected group). However, anti-KIF3B antibody injection dramatically diminished the total vesicle traffic along axons ( A and 3 B, control, 29.0 ± 0.3; anti-KIF3B Fab, 9.4 ± 0.1, mean ± SEM). In particular, the anterograde traffic (control, 13.7 ± 0.2; anti-KIF3B Fab, 3.8 ± 0.1; 27.5% of noninjected group) was more severely affected than that of retrogradely moving vesicles (control, 15.3 ± 0.2; anti-KIF3B Fab, 5.6 ± 0.2; 35.8% of noninjected control), the results reflecting the nature of KIF3 as an anterograde motor. Since mitochondria did not often move and were only infrequently observed in the SCG, it was difficult to judge whether mitochondrial traffic (control, 1.8 ± 0.1; anti-KIF3B Fab, 1.0 ± 0.1) was influenced or not. However, t test revealed no significant difference in mean values at the significance of P < 0.05, whereas other groups were significantly different at the level of P < 0.001. In addition, in COS cells or embryonic Reichert's membrane cells enriched in mitochondria, we could not observe any inhibitory effects of antibody injection on the mitochondrial motility (data not shown). Thus, we consider that this decreased traffic resulting from anti-KIF3B Fab injection was due to specific effects exerted on only vesicles transported by the KIF3 motor. Considering the resolution limit of the VEC–DIC system, it was almost impossible to discriminate and classify each vesicle by size. Therefore, we could categorize visually observed vesicles into only two groups: large and bright, or small and dark, which might represent endosomal vesicles and small membranous vesicles, respectively, as the former tend to move centripetally.
Figure 2 Examples of VEC–DIC images of SCG neuronal axons. SCG neurons grown on collagen type IV-treated coverslips were microinjected with either anti-KIF3B Fab or control normal mouse IgG Fab, and observed under VEC–DIC ~3 h after microinjection. (more ...)
Figure 3 Comparison of vesicle traffic before and after microinjection of Fabs. A, Bidirectional effect of anti-KIF3B Fab microinjection. Both antero- and retrograde traffic were inhibited by the microinjection of the Fab fragments, although the degree of inhibition (more ...)
Neurite Extension Was Inhibited by Anti-KIF3B Fab
Since anti-KIF3 Fab blocked axonal vesicle traffic as described above, kif3A−/−
embryos displayed neuronal abnormality (Nonaka et al. 1998
; Takeda et al. 1999
), and several lines of evidence implied the roles of KIF3 in fast axonal transport (Yamazaki et al. 1995
; Ray et al. 1999
), we speculated that the KIF3 motor possibly transports the components required for sprouting, construction, and maintenance of axons, which may be blocked by antibody microinjection, resulting in the failure of axonal elongation. To test this hypothesis, we carried out an experiment according to the following scheme.
Instead of microinjecting antibodies into SCG neurons with almost fully extended processes, we now injected cells (n = 60) harboring no neurites, ~12 h after plating. The concentrations of Fab fragment for both anti-KIF3B antibody and normal mouse IgG were adjusted to 3 mg/ml. In the case of control normal mouse IgG Fab (n = 50; and ), most of the neurons developed a single axonal structure with branching ~12 h after microinjection. In many cases, axons extended to >100 μm in length, exhibiting active growth cones, and some axons were >300-μm long (). However, microinjection of anti-KIF3B Fab blocked the sprouting of neurites ( and ), leaving most of the cells without neurites or with miniature sproutings. The median value () of the axonal sprout length 12 h after microinjection revealed significant differences between the two groups (median values for each group: control, 225 μm; anti-KIF3B Fab, 15 μm), implying the role of the KIF3 motor in neurite sprouting/elongation.
Figure 4 Neurite sprouting was affected by microinjection of anti-KIF3B Fab. Either anti-KIF3B Fab or normal mouse IgG Fab was injected to the cultured SCG neurons before neurite sprouting. Approximately 12 h after injection, the cells were fixed and viewed by (more ...)
KIF3 Motor Interacts with the Fodrin Molecule
We attempted to further characterize the properties of the vesicles transported by the KIF3 complex. However, the relatively weak and rather transient nature of the binding between the cargos and the motor protein complex (Cole et al. 1998
) prevented us from converting real-time dynamic phenomena into morphological evidence. To overcome this problem, we used the yeast two-hybrid screening. Because previous studies (Shimizu et al. 1996
; Yamazaki et al. 1996
) suggested the role of KAP3 as an associated protein of the KIF3 complex in intercalating the motor domain and the cargos, we used a part of the KAP3 sequence as a bait. Moreover, KAP3 has an Armadillo
-conserved sequence that is required for the protein–protein interaction (Gindhart and Goldstein 1996
; Shimizu et al. 1996
To identify the binding partner of KAP3, we generated a fusion construct of the DNA-binding domain and KAP3 coding sequence (aa 209–384). This fusion protein was used in yeast two-hybrid screening of mouse brain cDNA libraries. We selected several Leu+/LacZ+ colonies from the screening, and we isolated 17 independent positive clones. Out of these clones, three colonies were found to encode fragments of the fodrin gene (). Analysis of the other colonies is under way to determine the in vivo relevance of their affinity for KAP3.
Figure 6 Yeast two-hybrid assay of KAP3 with vesicle-associated proteins. After a yeast two-hybrid system screening, the interaction of α-fodrin with KAP3 was confirmed reproducibly by retransformation. Yeasts containing each combination of pLexA (no insert (more ...)
Furthermore, we constructed a series of deletion mutants of the KAP3 construct to determine the minimal binding domain to the fodrin molecule (). The minimal domain required for binding was located within the region where the Armadillo repeat is intercepted by a small insert (aa 209–292). In addition, we quantified the binding affinity of each candidate molecule to KAP3 by measuring β-galactosidase activity in liquid cultures of the yeast. The interaction of KAP3 with α-fodrin (αII-spectrin) was the strongest in the three positive clones (234 U for fodrin, 81–85 U for the other two clones, and 3 U for the control vector; U in an arbitrary scale). Therefore, we focused on fodrin in further experiments.
Figure 7 Schematic representation of the functional domain of KAP3 and the α-fodrin molecule obtained by the yeast two-hybrid system. This scheme depicts the entire amino acid sequence of the KAP3 molecule (1–793). We constructed bait vectors of (more ...)
KIF3 Interacts with Fodrin on the Vesicles that Travel Down Axons
To confirm whether the aforementioned results in the yeast two-hybrid assay reflected specific interaction in vivo, we performed the following two sets of experiments.
First, we prepared vesicles from the cauda equina, which is a thick bundle of axons, because they could be prepared without disrupting interactions of motor with vesicles (Okada et al. 1995b
). Moreover, this source and method enables us to isolate axonal vesicles with high purity by detaching them from MTs in the presence of 1 mM ATP. Therefore, we could avoid possible contaminations of plasma membrane fragments. Immunoprecipitation of these vesicles by using anti-KIF3B– or anti-KAP3–coated beads gave a band that was detected with antifodrin antibody in Western blotting ( C, lanes 1 and 3). This association was further confirmed by immunoprecipitation with antifodrin antibody-coated beads, which could pull down both KIF3B and KAP3 ( and , lane 5). Furthermore, as the results of the yeast two-hybrid experiment suggested, the binding of the KIF3 motor with fodrin may be direct, since detergent solubilization after immunoprecipitation did not destroy the interaction between the two molecules. In addition, immunoprecipitation by using antibodies against other KIFs, such as KIF5A and -B, did not reveal the association of the fodrin molecule to these motor proteins ( C, lanes 7 and 9), suggesting the specific interaction of KIF3 with the fodrin molecule.
Figure 8 Immunoprecipita-tion of the vesicle fraction of cauda equina by anti-KIF3B and KAP3 antibodies. Cauda equina vesicles were immunoprecipitated with either anti-KIF3B (lane 1), anti-KAP3 (lane 3), anti–αII-fodrin (lane 5), anti-KIF5A (lane (more ...)
Secondly, we carried out two immunoelectron microscopic experiments on the cauda equina vesicles to confirm morphologically the interaction between KIF3 and fodrin. As shown in F, 15.4% of the vesicles showed both KIF3 (10-nm gold particles) and fodrin (5-nm gold particles) labeling. In the case of single labeling for either KIF3B or fodrin, 37.2 and 29.3% of vesicles were labeled with the two antibodies, respectively. The size of the double-labeled vesicles ranged from 50 to 200 nm in this preparation (, A–D), which partially overlapped with diameters of those immunoprecipitated by anti-KIF3B immunobeads in the previous study (Yamazaki et al. 1995
). Colocalization of KIF3B and fodrin was also confirmed in the samples embedded in 2% agarose and epon ( and ), where relatively smaller vesicles (~30 nm) were found to be double-labeled ( H, arrow).
Figure 9 Immuno-colocalization of KIF3 and fodrin on cauda equina vesicles. A–D, Negatively stained cauda equina vesicles containing both KIF3B and fodrin. Vesicles ranging from 50–200 nm in diameter were double-labeled (arrowheads). 10-nm gold (more ...)
These data collectively suggest that the interface between the KIF3 motor and cargos might be mediated by fodrin, and those vesicles are transported as fast axonal flow en route to the axonal plasma membrane along MT tracks by KIF3.
Fodrin Travels Down the Axon with the KIF3 Motor
To probe the association of fodrin with the KIF3 motor in vivo, we conducted a radiolabeled pulse–chase experiment in the rat optic nerve. Although the same behavior of fodrin as the KIF3 motor within axons does not necessarily indicate a direct association between the two proteins, and further, with cargos by fodrin to KAP3 binding, it could be strong evidence in this situation, since several batteries of experiments revealed an association between the two proteins.
We injected radiolabeled amino acids ([35
S]methionine and [35
S]cysteine) into rat eyeballs and chased the newly synthesized proteins by immunoprecipitation. The radiolabeled KIF3 and fodrin moved into the optic nerve and tract toward the lateral geniculate ganglion. Four hours after administration, neither KIF3 nor fodrin was detected in the distal segment (10–15 mm). However, we could identify distinct bands in the proximal segment (5–10 mm); the velocity of fodrin transport was calculated to be between 46 and 51 mm/d (0.5–0.6 μm/sec; A). Since our previous in vitro motility assay yielded values of 0.53–0.59 μm/s for the KIF3 motor protein (Yamazaki et al. 1996
), the obtained value here for α-fodrin velocity shows good agreement with our previous report. The velocity of KIF3 was also in the same range as that of fodrin, whereas KIF1A (12–15 cm/d; 1.4–1.7 μm/sec) was much faster than that of KIF3, confirming the satisfactory resolution and reliability of this method. In this regard, it should be noted that the peaks of moving fodrin and KIF3 are not necessarily the same, even in the case when KIF3 transports vesicles associated with fodrin, because the dynamics of fodrin and KIF3 in the axoplasm, such as their dissociation and association with the vesicles, or their turnover, are possibly different.
Figure 10 Pulse-labeling study revealing the velocity of KIF3, KIF1A, and fodrin in the rat optic nerve. A, [35S]methionine was injected into the vitreous. 4, 6, and 8 h after administration, the optic nerve and tract were extirpated en bloc and further cut into (more ...)