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Suppression of KIF2 in PC12 cells alters the distribution of a growth cone nonsynaptic membrane receptor and inhibits neurite extension.

Morfini G, Quiroga S, Rosa A, Kosik K, Cáceres A - J. Cell Biol. (1997)

Bottom Line: KIF2 suppression results in a dramatic accumulation of betagc within the cell body and in its complete disappearance from growth cones; no alterations in the distribution of synapsin, synaptophysin, GAP-43, or amyloid percursor protein are detected in KIF2-suppressed neurons.Instead, all of them remained highly enriched at nerve terminals.KIF2 suppression also produces a dramatic inhibition of neurite outgrowth; this phenomenon occurs after betagc has disappeared from growth cones.

View Article: PubMed Central - PubMed

Affiliation: Instituto Investigación Médica Mercedes y Martín Ferreyra, 5000 Córdoba, Argentina.

ABSTRACT
In the present study, we present evidence about the cellular functions of KIF2, a kinesin-like superfamily member having a unique structure in that its motor domain is localized at the center of the molecule (Noda Y., Y. Sato-Yoshitake, S. Kondo, M. Nangaku, and N. Hirokawa. 1995. J. Cell Biol. 129:157-167.). Using subcellular fractionation techniques, isopicnic sucrose density centrifugation of microsomal fractions from developing rat cerebral cortex, and immunoisolation with KIF2 antibodies, we have now identified a type of nonsynaptic vesicle that associates with KIF2. This type of organelle lacks synaptic vesicle markers (synapsin, synaptophysin), amyloid precursor protein, GAP-43, or N-cadherin. On the other hand, it contains betagc, which is a novel variant of the beta subunit of the IGF-1 receptor, which is highly enriched in growth cone membranes. Both betagc and KIF2 are upregulated by NGF in PC12 cells and highly concentrated in growth cones of developing neurons. We have also analyzed the consequences of KIF2 suppression by antisense oligonucleotide treatment on nerve cell morphogenesis and the distribution of synaptic and nonsynaptic vesicle markers. KIF2 suppression results in a dramatic accumulation of betagc within the cell body and in its complete disappearance from growth cones; no alterations in the distribution of synapsin, synaptophysin, GAP-43, or amyloid percursor protein are detected in KIF2-suppressed neurons. Instead, all of them remained highly enriched at nerve terminals. KIF2 suppression also produces a dramatic inhibition of neurite outgrowth; this phenomenon occurs after betagc has disappeared from growth cones. Taken collectively, our results suggest an important role for KIF2 in neurite extension, a phenomenon that may be related with the anterograde transport of a type of nonsynaptic vesicle that contains as one of its components a growth cone membrane receptor for IGF-1, a growth factor implicated in nerve cell development.

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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 (B), and GAP-43 (D) in  NGF-differentiated PC12 cells treated with a KIF2 antisense oligonucleotide (ASKF2a, 5 μM). Note that while βgc completely disappears from growth cones (arrows), synaptophysin and GAP-43 remain highly concentrated at neuritic tips. (E and F) Double immunofluorescence micrographs showing the distribution of tyrosinated α-tubulin (E) and synapsin I (F) in NGF-differentiated PC12 cells  treated with ASKF2a (5 μM). Note that synapsin I is highly concentrated at neuritic tips. For these experiments, cells were treated with  oligonucleotides as described in Fig. 7. Bar, 10 μm.
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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 (B), and GAP-43 (D) in NGF-differentiated PC12 cells treated with a KIF2 antisense oligonucleotide (ASKF2a, 5 μM). Note that while βgc completely disappears from growth cones (arrows), synaptophysin and GAP-43 remain highly concentrated at neuritic tips. (E and F) Double immunofluorescence micrographs showing the distribution of tyrosinated α-tubulin (E) and synapsin I (F) in NGF-differentiated PC12 cells treated with ASKF2a (5 μM). Note that synapsin I is highly concentrated at neuritic tips. For these experiments, cells were treated with oligonucleotides as described in Fig. 7. Bar, 10 μm.

Mentions: 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. 7). Thus, while in nontreated or sense-treated differentiated control PC12 cells βgc is selectively and highly enriched at growth cones (Fig. 7, 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. 7, 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. 8, A–H, and 9, A and B).


Suppression of KIF2 in PC12 cells alters the distribution of a growth cone nonsynaptic membrane receptor and inhibits neurite extension.

Morfini G, Quiroga S, Rosa A, Kosik K, Cáceres A - J. Cell Biol. (1997)

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 (B), and GAP-43 (D) in  NGF-differentiated PC12 cells treated with a KIF2 antisense oligonucleotide (ASKF2a, 5 μM). Note that while βgc completely disappears from growth cones (arrows), synaptophysin and GAP-43 remain highly concentrated at neuritic tips. (E and F) Double immunofluorescence micrographs showing the distribution of tyrosinated α-tubulin (E) and synapsin I (F) in NGF-differentiated PC12 cells  treated with ASKF2a (5 μM). Note that synapsin I is highly concentrated at neuritic tips. For these experiments, cells were treated with  oligonucleotides as described in Fig. 7. Bar, 10 μm.
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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 (B), and GAP-43 (D) in NGF-differentiated PC12 cells treated with a KIF2 antisense oligonucleotide (ASKF2a, 5 μM). Note that while βgc completely disappears from growth cones (arrows), synaptophysin and GAP-43 remain highly concentrated at neuritic tips. (E and F) Double immunofluorescence micrographs showing the distribution of tyrosinated α-tubulin (E) and synapsin I (F) in NGF-differentiated PC12 cells treated with ASKF2a (5 μM). Note that synapsin I is highly concentrated at neuritic tips. For these experiments, cells were treated with oligonucleotides as described in Fig. 7. Bar, 10 μm.
Mentions: 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. 7). Thus, while in nontreated or sense-treated differentiated control PC12 cells βgc is selectively and highly enriched at growth cones (Fig. 7, 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. 7, 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. 8, A–H, and 9, A and B).

Bottom Line: KIF2 suppression results in a dramatic accumulation of betagc within the cell body and in its complete disappearance from growth cones; no alterations in the distribution of synapsin, synaptophysin, GAP-43, or amyloid percursor protein are detected in KIF2-suppressed neurons.Instead, all of them remained highly enriched at nerve terminals.KIF2 suppression also produces a dramatic inhibition of neurite outgrowth; this phenomenon occurs after betagc has disappeared from growth cones.

View Article: PubMed Central - PubMed

Affiliation: Instituto Investigación Médica Mercedes y Martín Ferreyra, 5000 Córdoba, Argentina.

ABSTRACT
In the present study, we present evidence about the cellular functions of KIF2, a kinesin-like superfamily member having a unique structure in that its motor domain is localized at the center of the molecule (Noda Y., Y. Sato-Yoshitake, S. Kondo, M. Nangaku, and N. Hirokawa. 1995. J. Cell Biol. 129:157-167.). Using subcellular fractionation techniques, isopicnic sucrose density centrifugation of microsomal fractions from developing rat cerebral cortex, and immunoisolation with KIF2 antibodies, we have now identified a type of nonsynaptic vesicle that associates with KIF2. This type of organelle lacks synaptic vesicle markers (synapsin, synaptophysin), amyloid precursor protein, GAP-43, or N-cadherin. On the other hand, it contains betagc, which is a novel variant of the beta subunit of the IGF-1 receptor, which is highly enriched in growth cone membranes. Both betagc and KIF2 are upregulated by NGF in PC12 cells and highly concentrated in growth cones of developing neurons. We have also analyzed the consequences of KIF2 suppression by antisense oligonucleotide treatment on nerve cell morphogenesis and the distribution of synaptic and nonsynaptic vesicle markers. KIF2 suppression results in a dramatic accumulation of betagc within the cell body and in its complete disappearance from growth cones; no alterations in the distribution of synapsin, synaptophysin, GAP-43, or amyloid percursor protein are detected in KIF2-suppressed neurons. Instead, all of them remained highly enriched at nerve terminals. KIF2 suppression also produces a dramatic inhibition of neurite outgrowth; this phenomenon occurs after betagc has disappeared from growth cones. Taken collectively, our results suggest an important role for KIF2 in neurite extension, a phenomenon that may be related with the anterograde transport of a type of nonsynaptic vesicle that contains as one of its components a growth cone membrane receptor for IGF-1, a growth factor implicated in nerve cell development.

Show MeSH
Related in: MedlinePlus