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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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(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 disappears from growth cones (arrowheads) APP remains  highly concentrated at neuritic tips. (C and D) Double  immunofluorescence micrographs showing the distribution of βgc (C) and APP (D)  in NGF-differentiated PC12  cells treated with the KHC  antisense oligonucleotide -11/ 14 hkin (50 μM). Note that  while this treatment does  not affect the growth cone localization of βgc, it dramatically decreases APP immunofluorescence at neuritic tips.  For these experiments, cells  were treated with antisense  oligonucleotides as described  in Fig. 7. Bar, 10 μm.
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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 disappears from growth cones (arrowheads) APP remains highly concentrated at neuritic tips. (C and D) Double immunofluorescence micrographs showing the distribution of βgc (C) and APP (D) in NGF-differentiated PC12 cells treated with the KHC antisense oligonucleotide -11/ 14 hkin (50 μM). Note that while this treatment does not affect the growth cone localization of βgc, it dramatically decreases APP immunofluorescence at neuritic tips. For these experiments, cells were treated with antisense oligonucleotides as described in Fig. 7. Bar, 10 μm.

Mentions: To determine if other motors may participate in βgc transport, cells were treated with KHC antisense oligonucleotides. As shown in Fig. 9, KHC suppression does not alter the distribution of βgc (Fig. 9, C and D); 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. 9, C and D).


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)

(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 disappears from growth cones (arrowheads) APP remains  highly concentrated at neuritic tips. (C and D) Double  immunofluorescence micrographs showing the distribution of βgc (C) and APP (D)  in NGF-differentiated PC12  cells treated with the KHC  antisense oligonucleotide -11/ 14 hkin (50 μM). Note that  while this treatment does  not affect the growth cone localization of βgc, it dramatically decreases APP immunofluorescence at neuritic tips.  For these experiments, cells  were treated with antisense  oligonucleotides as described  in Fig. 7. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2141628&req=5

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 disappears from growth cones (arrowheads) APP remains highly concentrated at neuritic tips. (C and D) Double immunofluorescence micrographs showing the distribution of βgc (C) and APP (D) in NGF-differentiated PC12 cells treated with the KHC antisense oligonucleotide -11/ 14 hkin (50 μM). Note that while this treatment does not affect the growth cone localization of βgc, it dramatically decreases APP immunofluorescence at neuritic tips. For these experiments, cells were treated with antisense oligonucleotides as described in Fig. 7. Bar, 10 μm.
Mentions: To determine if other motors may participate in βgc transport, cells were treated with KHC antisense oligonucleotides. As shown in Fig. 9, KHC suppression does not alter the distribution of βgc (Fig. 9, C and D); 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. 9, C and D).

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