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.

Activity-driven relaxation of the cortical actomyosin II network synchronizes Munc18-1-dependent neurosecretory vesicle docking

“…Recent evidence suggests that SVs can organize their own release sites on reaching the plasma membrane 32,43 . Indeed, syntaxin-1 molecules have been shown to concentrate under docked vesicles before fusion, suggesting that a pool of plasma membrane syntaxin-1 can be recruited by an unknown mechanism under SVs 43 .…”

“…Fusion events were counted within the first 30 s of stimulation. Fusion events were detected as an increase in fluorescence intensity in TIR-illumination followed by the disappearance of a vesicle 31,32 (Fig. 5a).…”

Annexin A2–dependent actin bundling promotes secretory granule docking to the plasma membrane and exocytosis

“…However, such a housekeeping function for actin is inconsistent with several functional studies suggesting an active role for actin in the exocytotic machinery (for reviews see Malacombe et al, 2006 ; Gutiérrez, 2012 ). For instance, actin filaments in association with myosins participate in the transport and guiding of secretory granules to their fusion sites ( Papadopulos et al, 2013a ,b ; Tomatis et al, 2013 ). Actin has also been proposed to play a positive role in late stages of exocytosis by controlling the fusion pore and specifying kiss and run versus full exocytosis ( Gutiérrez, 2012 ) and/or by directly expelling secretory content ( Miklavc et al, 2012 ).…”

F‐actin cytoskeleton and the fate of organelles in chromaffin cells

“…Furthermore, F‐actin also plays a plastic role during cell stimulation to promote filopodial formation, therefore, creating new and fully functional releasing sites (Papadopulos et al . ).…”