Neuronal calcium sensor-1 binds to regulated secretory organelles and functions in basal and stimulated exocytosis in PC12 cells.
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.
Scalettar
Bethe A
BA
Department of Physics, Lewis and Clark College, Portland, OR 97219, USA. bethe@lclark.edu
Rosa
Patrizia
P
Taverna
Elena
E
Francolini
Maura
M
Tsuboi
Takashi
T
Terakawa
Susumu
S
Koizumi
Schuichi
S
Roder
John
J
Jeromin
Andreas
A
D000818:Animals; D001370:Axonal Transport / Q000502:physiology; D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005089:Exocytosis / Q000502:physiology*; D020439:Growth Cones / Q000378:metabolism / Q000648:ultrastructure; D007150:Immunohistochemistry; D008164:Luminescent Proteins; D008854:Microscopy, Electron; D009420:Nervous System / Q000378:metabolism* / Q000648:ultrastructure; D051580:Neuronal Calcium-Sensor Proteins; D009474:Neurons / Q000378:metabolism* / Q000648:ultrastructure; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D009490:Neurosecretory Systems / Q000378:metabolism / Q000648:ultrastructure; D015388:Organelles / Q000378:metabolism* / Q000648:ultrastructure; D016716:PC12 Cells; D011485:Protein Binding / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D011993:Recombinant Fusion Proteins; D022142:Secretory Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D013572:Synaptic Vesicles / Q000378:metabolism / Q000648:ultrastructure; D016708:Synaptophysin
D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; D008164:Luminescent Proteins; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides; D011993:Recombinant Fusion Proteins; D016708:Synaptophysin; C101464:frequenin calcium sensor proteins; C054562:yellow fluorescent protein, Bacteria