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Research Article |
1 Department of Physics, Lewis and Clark College, Portland, OR 97219, USA
2 CNR-Cellular and Molecular Pharmacology Center, Department of Pharmacology,
Via Vanvitelli 32, Milan, Italy
3 Photon Medical Research Center, Hamamatsu University School of Medicine,
Hamamatsu 431-3192, Japan
4 Division of Pharmacology, National Institute of Health Sciences, Tokyo
158-8501, Japan
5 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, M5G
1x5 Canada
* Author for correspondence (e-mail: bethe{at}lclark.edu )
Accepted 5 March 2002
Neuronal calcium sensor-1 (NCS-1) and its non-mammalian homologue, frequenin, have been implicated in a spectrum of cellular processes, including regulation of stimulated exocytosis of synaptic vesicles and secretory granules (SGs) in neurons and neuroendocrine cells and regulation of phosphatidylinositol 4-kinase beta activity in yeast. However, apart from these intriguing putative functions, NCS-1 and frequenin are relatively poorly understood. Here, the distribution, dynamics and function of NCS-1 were studied using PC12 cells that stably express NCS-1-EYFP (NCS-1 fused to enhanced yellow fluorescent protein) or that stably overexpress NCS-1. Fluorescence and electron microscopies show that NCS-1-EYFP is absent from SGs but is present on small clear organelles, some of which are just below the plasma membrane. Total internal reflection fluorescence microscopy shows that NCS-1-EYFP is associated with synaptic-like microvesicles (SLMVs) in growth cones. Overexpression studies show that NCS-1 enhances exocytosis of synaptotagmin-labeled regulated secretory organelles (RSOs) under basal conditions and during stimulation by UTP. Significantly, these studies implicate NCS-1 in the enhancement of both basal and stimulated phosphoinositide-dependent exocytosis of RSOs in PC12 cells, and they show that NCS-1 is distributed strategically to interact with putative targets on the plasma membrane and on SLMVs. These studies also reveal that SLMVs undergo both fast directed motion and highly hindered diffusive motion in growth cones, suggesting that cytoskeletal constituents can both facilitate and hinder SLMV motion. These results also reveal interesting similarities and differences between transport organelles in differentiated neuroendocrine cells and neurons.
Key words: GFP, PtdIns(4,5)P2, Synaptophysin, Frequenin, Synaptotagmin
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