title: Secretagogue stimulation of neurosecretory cells elicits filopodial extensions uncovering new functional release sites.
abstract: Regulated exocytosis in neurosecretory cells relies on the timely fusion of secretory granules (SGs) with the plasma membrane. Secretagogue stimulation leads to an enlargement of the cell footprint (surface area in contact with the coverslip), an effect previously attributed to exocytic fusion of SGs with the plasma membrane. Using total internal reflection fluorescence microscopy, we reveal the formation of filopodia-like structures in bovine chromaffin and PC12 cells driving the footprint expansion, suggesting the involvement of cortical actin network remodeling in this process. Using exocytosis-incompetent PC12 cells, we demonstrate that footprint enlargement is largely independent of SG fusion, suggesting that vesicular exocytic fusion plays a relatively minor role in filopodial expansion. The footprint periphery, including filopodia, undergoes extensive F-actin remodeling, an effect abolished by the actomyosin inhibitors cytochalasin D and blebbistatin. Imaging of both Lifeact-GFP and the SG marker protein neuropeptide Y-mCherry reveals that SGs actively translocate along newly forming actin tracks before undergoing fusion. Together, these data demonstrate that neurosecretory cells regulate the number of SGs undergoing exocytosis during sustained stimulation by controlling vesicular mobilization and translocation to the plasma membrane through actin remodeling. Such remodeling facilitates the de novo formation of fusion sites.
authors.affiliation: The Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, St Lucia, Queensland 4072, Australia.
authors.forename: Andreas
authors.initials: A
authors.lastname: Papadopulos
authors.forename: Sally
authors.initials: S
authors.lastname: Martin
authors.forename: Vanesa M
authors.initials: VM
authors.lastname: Tomatis
authors.forename: Rachel S
authors.initials: RS
authors.lastname: Gormal
authors.forename: Frederic A
authors.initials: FA
authors.lastname: Meunier
mesh_terms: D000199:Actins / Q000378:metabolism; D000205:Actomyosin / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002459:Cell Fusion; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000502:physiology / Q000648:ultrastructure; D022162:Cytoplasmic Vesicles / Q000502:physiology / Q000648:ultrastructure; D003599:Cytoskeleton / Q000502:physiology; D005089:Exocytosis / Q000502:physiology; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D024462:Myosin Type II / Q000502:physiology; D009473:Neuronal Plasticity / Q000502:physiology; D009490:Neurosecretory Systems / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D058105:Polymerization; D011554:Pseudopodia / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D022142:Secretory Vesicles / Q000502:physiology / Q000648:ultrastructure
chemical_list: D000199:Actins; D000205:Actomyosin; D024462:Myosin Type II