Characterization of the Peptide Antibodies Against KIF2
The monospecificity of the affinity-purified rabbit polyclonal antibody (BKF2) raised against a peptide corresponding to amino acid residues 526–535 of mouse KIF2 is shown in Fig. A. This antibody recognizes a single band of ~97 kD in Western blots of whole cell homogenates from the cerebral cortex of developing rats (Fig. A, lane 1); an identical staining pattern is observed when equivalent blots are reacted with RKF2, a rabbit polyclonal antibody raised against a peptide corresponding to amino acid residues 114–123 of the KIF2 molecule (Fig. A, lane 3). The staining generated by these antibodies is completely abolished by neutralization with the corresponding purified peptides (Fig. A, lanes 2 and 4).
Fig. B shows that in the cerebral cortex the expression of the BKF2 immunoreactive protein species is higher at early postnatal days, declining gradually but considerably until adulthood, where the lowest levels are detected. In addition, Western blot analysis of subcellular fractions obtained from the cortex of 3-d-old rats revealed that the 97-kD protein is relatively concentrated in the microsomal fraction compared with the cytosol or the mitochondrial fraction (Fig. C). This means that a considerable amount of the protein recognized by the BKF2 antibody is associated with small membranous organelles, but only barely associated with larger ones such as mitochondria (Fig. C). In addition, microtubule binding experiments show that in the absence of ATP, the 97-kD protein cosediments with taxol-stabilized microtubules obtained from the cerebral cortex (Fig. D). This binding occurs in the presence (Fig. D) or absence (not shown) of AMP-PNP; on the other hand, the 97-kD protein is released from microtubules incubated with 10 mM ATP plus 100 mM NaCl, but not in the presence of ATP alone (Fig. D).
Since all the properties described above are identical to those previously reported for KIF2 (see Noda et al., 1995
), we conclude that our antibodies effectively recognize KIF2 and not a different protein having a similar molecular weight.
Subcellular Distribution of KIF2
To begin analyzing the type of cargo that KIF2 may transport, microsomal fractions from rat cerebral cortex were fractionated by isopicnic sucrose density gradient centrifugation and analyzed by immunoblotting with antibodies against KIF2, KHC, and several membrane proteins, including synaptic and nonsynaptic vesicle constituents. This type of approach has already proven useful to identify the type of membrane-bound organelle transported by KIF1A, another member of the kinesin superfamily (Okada et al., 1995
Approximately 70–80% of KIF2 and KHC were recovered in the P3 (high speed pellet) fraction, while <30% were present in the P2 (medium speed fraction) and S3 (high speed supernatant) fractions. In the P3 microsome fraction, KHC was recovered in a fraction that extends from 0.3 to 0.6 M sucrose (Fig. ). By contrast, KIF2 was recovered in a different fraction extending from 0.4 to 0.9 M sucrose, with a peak at 0.6–0.8 M sucrose (Fig. ). The distribution of KIF2 across the sucrose density gradient was then compared with that of synaptophysin and synapsin I, two well-characterized synaptic vesicle (SV) membrane proteins (Fletcher et al., 1991
). The results obtained showed that synaptophysin was recovered in lighter fractions (0.3–0.6 M) than those enriched in KIF2, while synapsin I was recovered in fractions extending from 0.6 to 1.0 M sucrose (Fig. ). The staining for synapsin I overlapped with that of KIF2, but the peak fractions were different.
Figure 2 The binding of KIF2 to membrane vesicles. Microsome fraction from developing rat cerebral cortex was fractionated by sucrose gradient centrifugation, and the same volume from each fraction was applied to SDS-PAGE, transferred to PVDF membranes, and (more ...)
Next we compared the distribution of KIF2 with that of three well-characterized growth cone membrane components. One of them, designated βgc
(a novel variant of the β-subunit of the IGF-1 receptor, which is highly enriched in growth cone membranes; Quiroga et al., 1995
; Mascotti et al., 1997), displayed a striking codistribution with KIF2, being highly enriched in the 0.6–0.8 M sucrose fractions (Fig. ). On the other hand, GAP-43 (Goslin et al., 1989) and APP (Ferreira et al., 1993
; Yamazaki et al., 1995b
) distributed across the sucrose gradient with a pattern clearly different from that of KIF2 (Fig. ). Thus, GAP-43 was recovered in all fractions of the sucrose gradient (0.3–1.6 M), while APP was enriched in either lighter (0.3–0.6 M) or heavier (1.0–1.6 M) fractions than those containing KIF2. The pattern of distribution of N-cadherin, a cell-adhesion molecule, was also compared with that of KIF2; this protein displayed a uniform distribution across the sucrose gradient, showing no enrichment in the fractions containing KIF2 or βgc
Taken collectively, these observations confirm previous studies suggesting the existence of at least two classes of organelles that contain SV membrane proteins: one contains synapsin I, and the other synaptophysin (Okada et al., 1995
). The former appears to be transported by KHC (Ferreira et al., 1992
), while the latter by KIF1A (Okada et al., 1995
). Our observations also suggest the existence of another type of organelle that appears to contain a nonsynaptic growth cone membrane component, namely βgc
, and KIF2. However, because other KIFs (KHC, KIF1A, and KIF3; Kondo et al., 1994
; Okada et al., 1995
) are also present in these fractions, definite conclusions cannot be drawn on the motor for the βgc
-containing organelles. Besides, the relationship between synaptophysin- and synapsin I–containing organelles with the ones enriched in KIF2 is also unclear, given that some degree of overlap exists among the fractions containing these proteins. Therefore, to clarify some of these points and directly determine the relationship between KIF2 and βgc
, immunoisolation of organelles from the microsomal fraction was performed with antibodies against KIF2. The remaining organelles were recovered by pelleting from the supernatant fraction. Fig. shows that with this method, the KIF2-containing organelles were quantitatively collected. In this immunoisolated organelle fraction βgc
was quantitatively recovered (Fig. A
). In contrast, KHC (Fig. B
), synaptophysin (Fig. C
), or synapsin (not shown) were not, or were only slightly detectable in this fraction. These proteins were quantitatively recovered in the remaining organelle fraction. They were not detected in the supernatant fraction after pelleting the remaining organelles, effectively ruling out the possibility that the lack of these proteins in the KIF2 organelle–containing fraction was due to dissociation during the immunoisolation procedure.
Figure 3 Immunoisolation of βgc-containing organelles with the BKF2 antibody (dilution 1:20). Lane 1, microsome fraction before immunoprecipitation. Lanes 2 and 3, microsome fraction incubated with BKF2 preimmune serum—pellet (lane 2) and remanent (more ...)
These results clearly and directly demonstrate that KIF2 is associated with a class of nonsynaptic, membranous organelle that contains βgc as one of its components. However, they do not provide evidence about the in vivo relationship between KIF2 expression and the transport of βgc-containing organelles, and/or the functional role of KIF2 during neuronal morphogenesis. Therefore, to obtain evidence about these aspects we decided to examine the pattern of expression and subcellular distribution of KIF2, as well as the consequences of KIF2 suppression on the distribution of βgc in PC12 cells. In this cell system, βgc expression is upregulated by NGF and highly correlated with neurite outgrowth. Even more importantly, in NGF-treated PC12 cells, βgc is selectively concentrated in the proximal growth cone region in vesicle-like structures, clearly different from those containing synaptophysin or synapsin I (Mascotti et al., 1997), a phenomenon that also suggests that βgc may be transported to the growth cone area by a motor different from KHC or KIF1A.
The Expression and Subcellular Localization of KIF2 in NGF-treated PC12 Cells
PC12 cells have proven to be an excellent model system for studying growth cone formation, neurite outgrowth, and the expression of structural and membrane proteins involved in nerve cell morphogenesis (Drubin et al., 1985
; Greene et al., 1987
; Bearer, 1992
; Ezmaeli-Azad et al., 1994; Mascotti et al., 1997). In the absence of NGF, PC12 cells have a round morphology with no neurites or growth cone-like structures. Upon stimulation with NGF, they extend several neurites tipped by well-defined growth cones, which are highly enriched with several SV and non-SV membrane markers, such as synaptophysin and βgc
(Mascotti et al., 1997).
Fig. shows that NGF induces the expression of KIF2 in PC12 cells (Fig. , lanes 1 and 2). Thus, only one band of about 100 kDa Mr is detected when whole cell extracts from PC12 cells, cultured for 72 h in the presence of NGF, are resolved in SDS-PAGE, blotted and immunostained with the anti-KIF2 antibodies (Fig. , lane 2). At equivalent, or even two to threefold higher protein loadings, KIF2 is barely detectable in cell extracts from PC12 cells cultured without NGF (Fig. 4, lane 1). In addition, if NGF-differentiated (4 d) PC12 cells are deprived of the neurotrophin for 6–12 h, neurite length decreases significantly (see Mascotti et al., 1997), as do KIF2 protein levels (Fig. , lane 3). These results showed that, in PC12 cells KIF2 expression (as in the case of βgc; Mascotti et al., 1997) is tightly controlled by NGF as well as differentiation.
Figure 4 KIF2 expression in PC12 cells. Immunoblot analysis of whole cell extracts from nontreated (lane 1) or NGF-treated (lane 2) PC12 cells reacted with the BKF2 antibody (dilution 1:50). NGF induces the expression of a single KIF2 immunoreactive protein (more ...)
In the next series of experiments the spatial distribution of KIF2 was studied by double immunolabeling with the BKF2 antibody and an mAb that recognizes tyrosinated α-tubulin (clone TUA 1.2). PC12 cells cultured in the absence of NGF have a round or polygonal morphology (Fig. A, tubulin antibody), and exhibit very weak immunofluorescence when incubated with the KIF2 antibody (Fig. B). As expected, a significant increase in KIF2 immunofluorescence becomes evident when PC12 are cultured in the presence of NGF. This phenomenon is detected ~48 h after the addition of NGF, when PC12 cells begin to acquire a neuron-like morphology. At this stage, the cells have several short neurites tipped with small growth cones. KIF2 immunofluorescence is preferentially localized to the perinuclear region and to the growth cones (Fig. D; compare with tubulin staining in Fig. C). An exception is the occasional short neurites that contain a continuous band of granular staining between the perinuclear region and the growth cones. After 72 h in the presence of NGF, PC12 cells have extended several long neurites that ended in prominent growth cones. At this stage KIF2 immunostaining has become very intense within the growth cone area, but has disappeared completely from neuritic shafts; a similar pattern is detected in PC12 cells cultured with NGF for longer periods of time (3–7 d). Observation of the growth cones at high magnification demonstrates that KIF2 staining labels a punctate organelle pattern (Fig. , E and F).
Figure 5 KIF2 becomes localized to growth cones in differentiated PC12 cells. Double immunofluorescence micrographs showing the distribution of tyrosinated α-tubulin (A, C, and E) and KIF2 (B, D, and F) in PC12 cells. PC12 cells, cultured in the (more ...)
Antisense Oligonucleotides Affect KIF2 Expression
Three phosphorothioate (S-modified) antisense oligonucleotides were tested for their ability to inhibit KIF2 expression. PC12 treated with NGF for 3 d and incubated for 24 h with each of the antisense oligonucleotides (5 μM dose) described in Materials and Methods show markedly reduced reactivity to the RKF2 antibody, as assessed by Western blotting of whole cell extracts (Fig. A; Table ). In contrast, cells treated with sense oligonucleotides are comparable in their immunoreactivity to untreated control cells (Fig. A). Exposure to the antisense oligonucleotides did not affect tubulin (Fig. A), KHC (Fig. B), dynein (not shown), βgc (Fig. C), or synaptophysin (Fig. D) immunoreactivity by the same assay. The presence of normal levels of these proteins in the KIF2-suppressed cells suggests that the effect of the antisense treatment is specific and that the regulation of the expression of other motor proteins (e.g., KHC or dynein) is independent of KIF2.
Figure 6 Effect of the KIF2 antisense oligonucleotide ASKF2a (5 μM) on KIF2 (A), tyrosinated α-tubulin (A), KHC (B), βgc (C), and synaptophysin (D) protein levels as revealed by Western blot analysis of whole cell homogenates obtained (more ...)
Effect of KIF2 Antisense Oligonucleotides on KIF2 and Tubulin Protein Levels in NGF-treated PC12 Cells
Distribution of βgc in Antisense-treated Neurons
Next we examined the distribution of βgc, synaptophysin, synapsin I, GAP-43, and APP in control and KIF2 antisense oligonucleotide-treated PC12 cells. As expected, KIF2 immunofluorescence is significantly reduced in differentiated PC12 cells treated with the ASKF2a or ASKF2b antisense oligonucleotides (not shown). A dramatic alteration in the distribution of βgc is also detected (Fig. ). Thus, while in nontreated or sense-treated differentiated control PC12 cells βgc is selectively and highly enriched at growth cones (Fig. , A and B), in the KIF2 antisense-treated cells, all of the labeling is present within the cell body, being completely absent from neuritic shafts including their tips (Fig. , C–F). By contrast, the distribution of synaptophysin, synapsin I, APP, and GAP-43 is unaltered in the KIF2 antisense-treated cells when compared with that observed in the control cells (nontreated or sense-treated); thus, all of these proteins are highly concentrated within the perinuclear region, presumably the Golgi complex, and the growth cones (Figs. , A–H, and 9, A and B).
Figure 7 KIF2 suppression alters the distribution of βgc in NGF-differentiated PC12 cells. (A and B) Double immunofluorescence micrographs showing tubulin (A) and βgc (B) staining in PC12 cells from a culture treated with a KIF2 sense oligonucleotide (more ...)
Figure 8 KIF2 suppression alters the distribution of βgc, but not of synaptophysin, GAP-43, or synapsin I in NGF-treated PC12 cells. (A–D) Double immunofluorescence micrographs showing the distribution of βgc (A and C), synaptophysin ( (more ...)
To determine if other motors may participate in βgc
transport, cells were treated with KHC antisense oligonucleotides. As shown in Fig. , KHC suppression does not alter the distribution of βgc
(Fig. , C
); however, and as previously described (see Ferreira et al., 1993
; Yamazaki et al., 1995b
) this treatment produces a dramatic reduction of APP immunolabeling at the growth cone and a concomitant accumulation within the cell body (Fig. , C
Figure 9 (A and B) Double immunofluorescence micrographs showing the distribution of βgc (A) and APP (B) in NGF-differentiated PC12 cells treated with the KIF2 antisense oligonucleotide ASKF2b (5 μM). Note that while βgc completely (more ...)
Effect of KIF2 Antisense Oligonucleotides on Neurite Extension
During the course of these experiments it became evident that one additional effect of KIF2 suppression was a reduction in the length of PC12 neurites. Therefore, to precisely examine this effect, neurite extension was measured in NGF-treated PC12 cells after either a 24- or a 36-h exposure to the KIF2 antisense oligonucleotides. When PC12-treated with NGF for 3 d are exposed to the ASKF2a antisense oligonucleotide (5 μM) and fixed 24 h later there is a slight decrease in neurite length. In the antisense-treated cultures, the mean total neurite length per cell is 225 ± 5 μm, a value lower than that observed in control (260 ± 8 μm) or sense-treated (265 ± 8 μm) cells. Neurite length in neurons exposed to KIF2 antisense oligonucleotides (5 μM) for 36 h is quantitated in Table . These cells exhibit a 60–70% decrease in neurite length. Our observations also show that the KIF2 antisense oligonucleotides affected neurite outgrowth in a dose-dependent manner; thus, at 2.5 μM the total neurite length was reduced 35% and at 1 μM it was only reduced 15% (Table ). While suppressing KIF2 expression, the KIF2 antisense oligonucleotides did not irreversibly damage the neurons. When, after 36 h in the presence of the antisense oligonucleotides, the cells are released from antisense inhibition by changing the medium, neurite extension resumed at a rate that paralleled that observed under control conditions (see below).
Effect of KIF2 Antisense Oligonucleotides on Neurite Length in NGF-treated PC12 Cells
In the final set of experiments, we used quantitative fluorescence and the morphometry of fixed cells to analyze expression of KIF2 and compare it with both the time course of βgc disappearance from the growth cone area, and the reduction of neurite length in PC12 cells treated with the ASKF2a antisense oligonucleotide for different periods of time. The results obtained show that the decrease in KIF2 immunofluorescence precedes the disappearance of βgc from the growth cone area, a phenomenon which in turn precedes the decrease in neurite length by several hours (Fig. , A and B). Similarly, when the cells are released from the antisense treatment the reexpression of KIF2 precedes the redistribution of βgc to the growth cone, a phenomenon which is then followed by an increase in neurite length (Fig. , A and B).
Figure 10 (A) Graph showing the effect of the KIF2 antisense oligonucleotide ASKF2a (closed symbols) on KIF2 (squares) and βgc (diamonds) immunofluorescence in NGF-differentiated PC12 cells. After 3 d in the presence of NGF (50 ng/ml), the cells were (more ...)