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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 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  absence of NGF, display low  positive immunofluorescence  for the RKF2 antibody (A  and B). A dramatic increase  in KIF2 immunofluorescence  is detected in PC12 cells  treated with NGF for 2 d. In  these cells, KIF2 is localized  to the perinuclear region and  highly enriched in growth  cones (arrowheads). High  power micrographs of neuritic tips reveal a granular  (vesicle-like) appearance of  the KIF2 immunostaining  (F). Bars: 10 μm.
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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 absence of NGF, display low positive immunofluorescence for the RKF2 antibody (A and B). A dramatic increase in KIF2 immunofluorescence is detected in PC12 cells treated with NGF for 2 d. In these cells, KIF2 is localized to the perinuclear region and highly enriched in growth cones (arrowheads). High power micrographs of neuritic tips reveal a granular (vesicle-like) appearance of the KIF2 immunostaining (F). Bars: 10 μm.

Mentions: 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. 5 A, tubulin antibody), and exhibit very weak immunofluorescence when incubated with the KIF2 antibody (Fig. 5 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. 5 D; compare with tubulin staining in Fig. 5 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. 5, E and F).


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 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  absence of NGF, display low  positive immunofluorescence  for the RKF2 antibody (A  and B). A dramatic increase  in KIF2 immunofluorescence  is detected in PC12 cells  treated with NGF for 2 d. In  these cells, KIF2 is localized  to the perinuclear region and  highly enriched in growth  cones (arrowheads). High  power micrographs of neuritic tips reveal a granular  (vesicle-like) appearance of  the KIF2 immunostaining  (F). Bars: 10 μm.
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Related In: Results  -  Collection

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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 absence of NGF, display low positive immunofluorescence for the RKF2 antibody (A and B). A dramatic increase in KIF2 immunofluorescence is detected in PC12 cells treated with NGF for 2 d. In these cells, KIF2 is localized to the perinuclear region and highly enriched in growth cones (arrowheads). High power micrographs of neuritic tips reveal a granular (vesicle-like) appearance of the KIF2 immunostaining (F). Bars: 10 μm.
Mentions: 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. 5 A, tubulin antibody), and exhibit very weak immunofluorescence when incubated with the KIF2 antibody (Fig. 5 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. 5 D; compare with tubulin staining in Fig. 5 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. 5, E and F).

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