Axonal guidance protein FEZ1 associates with tubulin and kinesin motor protein to transport mitochondria in neurites of NGF-stimulated PC12 cells

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Abstract

Fasciculation and elongation protein zeta-1 (FEZ1) promotes efficiently the neurite elongation of rat phaeochromocytoma PC12 cells. We here characterized FEZ1 in PC12 cells. Nerve growth factor (NGF) stimulation induces significant expression of endogenous FEZ1 in PC12 cells. Upon NGF stimulation FEZ1 localizes in both cytoplasm and neuritis, co-localizing with mitochondria. Silencing of FEZ1 by RNA interference efficiently reduces NGF-induced neurite elongation and the anterograde motility of mitochondria in PC12 cells. Immunoprecipitation and pulldown assay shows that FEZ1 interacts with kinesin superfamily protein 5 (KIF5) and tubulin. Thus, our results suggest that the FEZ1/kinesin complex functions for the transport of mitochondria along microtubules toward the extending neurites in differentiating PC12 cells.

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Materials and methods

Cell culture. PC12 cells were grown at 37 °C in Dulbecco’s modified Eagle’s medium with high glucose (Sigma, St. Louis, MO) containing 10% (v/v) fetal bovine serum (FBS) and 4 mM l-glutamine in a humidified atmosphere of 5% CO2. Neuronal differentiation of PC12 cells was induced by incubation with NGF at 100 ng/ml (2.5S, Roche Diagnostics, Indianapolis, IN) in the absence of FBS for 0–120 h.

Immunocytochemical observation. PC12 cells (approximately 5 × 104 cells) grown on a 35-mm glass bottom dish

NGF-induced expression of FEZ1 protein and mRNA in PC12 cells

Firstly, we confirmed the expression of endogenous FEZ1 protein in PC12 cells. Western blotting of total cell lysates with an anti-FEZ1 antibody showed that the expression of FEZ1 protein was induced by NGF stimulation and sustained for at least 72 h (Fig. 1A). Immunocytochemical staining of PC12 cells using the same antibody confirmed the induction of endogenous FEZ1 protein (Fig. 1B). From 24 to 72 h after NGF stimulation, the expression of FEZ1 protein was enhanced along with the neurite

Discussion

We showed herein that the synthesis of FEZ1 mRNA and FEZ1 protein is induced upon NGF stimulation in PC12 cells (Fig. 1). Silencing of FEZ1 by RNAi strongly inhibits the neurites elongation and the velocity of mitochondria transport in the anterograde direction in NGF-stimulated PC12 cells (Fig. 2). We found that FEZ1 not only interacts with kinesin motor protein KIF5B but also tubulin in PC12 cells (Fig. 4). Therefore, our results clearly show that the interaction of FEZ1 with kinesin and

Acknowledgments

We thank Profs. Kozo Kaibuchi, Yuko Fukata, Yasuo Uchiyama, and Kyoko Isahara for their helpful advice. We thank Profs. Nicolas Demaurex and Tullio Pozzan for providing DsREDmit. We thank Prof. Werner Schlegel for critical reading of the manuscript. This work was supported by Grants-in-Aid for the 21st Century Center of Excellence Program “Towards Creating New Industries Based on Inter-Nanoscience”, “Structural and Functional Proteomics Consortium for Research on the Proteins Working in Brain

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1

Present address: University of Oslo, Faculty Division: The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway.

2

These authors have contributed equally to the work.

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