Limits...
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.

Show MeSH

Related in: MedlinePlus

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 from PC12  cells. For this experiment PC12 cells cultured in the presence of  NGF were treated for 24 h with the ASKF2a oligonucleotide.  Antisense treatment was initiated 3 d after the addition of NGF  (50 ng/ml). Control cultures were treated with equivalent doses  of sense oligonucleotides. (A) Blot reacted with antibodies  against KIF2 (RKF2 diluted 1:50) and tyrosinated α-tubulin  (mAb TUB-1A2), or with mAb SUK 4 (B), or with an affinity-purified polyclonal antibody against βgc (C), or with mAb SY38  (D). 30 μg of total cellular protein were loaded in each lane.  Blots were revealed using the ProtoBlot staining kit (Promega).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2141628&req=5

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 from PC12 cells. For this experiment PC12 cells cultured in the presence of NGF were treated for 24 h with the ASKF2a oligonucleotide. Antisense treatment was initiated 3 d after the addition of NGF (50 ng/ml). Control cultures were treated with equivalent doses of sense oligonucleotides. (A) Blot reacted with antibodies against KIF2 (RKF2 diluted 1:50) and tyrosinated α-tubulin (mAb TUB-1A2), or with mAb SUK 4 (B), or with an affinity-purified polyclonal antibody against βgc (C), or with mAb SY38 (D). 30 μg of total cellular protein were loaded in each lane. Blots were revealed using the ProtoBlot staining kit (Promega).

Mentions: 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. 6 A; Table I). In contrast, cells treated with sense oligonucleotides are comparable in their immunoreactivity to untreated control cells (Fig. 6 A). Exposure to the antisense oligonucleotides did not affect tubulin (Fig. 6 A), KHC (Fig. 6 B), dynein (not shown), βgc (Fig. 6 C), or synaptophysin (Fig. 6 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.


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)

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 from PC12  cells. For this experiment PC12 cells cultured in the presence of  NGF were treated for 24 h with the ASKF2a oligonucleotide.  Antisense treatment was initiated 3 d after the addition of NGF  (50 ng/ml). Control cultures were treated with equivalent doses  of sense oligonucleotides. (A) Blot reacted with antibodies  against KIF2 (RKF2 diluted 1:50) and tyrosinated α-tubulin  (mAb TUB-1A2), or with mAb SUK 4 (B), or with an affinity-purified polyclonal antibody against βgc (C), or with mAb SY38  (D). 30 μg of total cellular protein were loaded in each lane.  Blots were revealed using the ProtoBlot staining kit (Promega).
© Copyright Policy
Related In: Results  -  Collection

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

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 from PC12 cells. For this experiment PC12 cells cultured in the presence of NGF were treated for 24 h with the ASKF2a oligonucleotide. Antisense treatment was initiated 3 d after the addition of NGF (50 ng/ml). Control cultures were treated with equivalent doses of sense oligonucleotides. (A) Blot reacted with antibodies against KIF2 (RKF2 diluted 1:50) and tyrosinated α-tubulin (mAb TUB-1A2), or with mAb SUK 4 (B), or with an affinity-purified polyclonal antibody against βgc (C), or with mAb SY38 (D). 30 μg of total cellular protein were loaded in each lane. Blots were revealed using the ProtoBlot staining kit (Promega).
Mentions: 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. 6 A; Table I). In contrast, cells treated with sense oligonucleotides are comparable in their immunoreactivity to untreated control cells (Fig. 6 A). Exposure to the antisense oligonucleotides did not affect tubulin (Fig. 6 A), KHC (Fig. 6 B), dynein (not shown), βgc (Fig. 6 C), or synaptophysin (Fig. 6 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.

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