Mapping Organelle Motion Reveals a Vesicular Conveyor Belt Spatially Replenishing Secretory Vesicles in Stimulated Chromaffin Cells

Abstract

How neurosecretory cells spatially adjust their secretory vesicle pools to replenish those that have fused and released their hormonal content is currently unknown. Here we designed a novel set of image analyses to map the probability of tracked organelles undergoing a specific type of movement (free, caged or directed). We then applied our analysis to time-lapse z-stack confocal imaging of secretory vesicles from bovine Chromaffin cells to map the global changes in vesicle motion and directionality occurring upon secretagogue stimulation. We report a defined region abutting the cortical actin network that actively transports secretory vesicles and is dissipated by actin and microtubule depolymerizing drugs. The directionality of this “conveyor belt” towards the cell surface is activated by stimulation. Actin and microtubule networks therefore cooperatively probe the microenvironment to transport secretory vesicles to the periphery, providing a mechanism whereby cells globally adjust their vesicle pools in response to secretagogue stimulation.

The Munc18-1 domain 3a hinge-loop controls syntaxin-1A nanodomain assembly and engagement with the SNARE complex during secretory vesicle priming

“…We noticed that the cage radius at 120 s was significantly larger than that of the prestimulation condition. This could be caused by vesicle replenishment, a process that requires active movement of SVs toward the plasma membrane ( Maucort et al, 2014 ). Importantly, expression of Munc18-1 Δ317-333 resulted in a complete rescue of SV docking at 30 s, but remarkably, the undocking of SVs was impaired, as their mobility remained restricted after 120 s. The rescue of a docking phenotype through expression of either Munc18-1 WT or Munc18-1 Δ317-333 was confirmed by electron microscopy (Fig.…”

Control of Autophagosome Axonal Retrograde Flux by Presynaptic Activity Unveiled Using Botulinum Neurotoxin Type A

“…Time-lapse movies were captured at 20 frames/s through an EM-CCD (Evolve512 delta; Photometrics) using MetaMorph software (Molecular Devices) for the indicated period and analyzed using custom-made single-molecule analysis software (Kechkar et al, 2013; Nair et al, 2013). To isolate trajectories of carriers that underwent directed motion from nonspecific noise and immobile objects, we selected the trajectories based on their duration (>25 consecutive frames) and with a mean square displacement (MSD) (Steyer and Almers, 1999; Papadopulos et al, 2013) fitting a polynomial equation of the type: ⟨ r 2 ⟩( t ) = at 2 + b with R 2 > 0.99 (Maucort et al, 2014). Using this fit, we also extracted trajectory speeds using italica=v.…”

Multiple Mechanisms Driving F-actin-Dependent Transport of Organelles to and From Secretory Sites in Bovine Chromaffin Cells

“…In addition to the possible transport of chromaffin vesicles along actin tracks or fibers, F-actin could also transport vesicles that appear to be trapped inside cages formed in the peripheral cytoskeleton (Giner et al, 2007 ), thereby following the dynamics of the overall F-actin structure. Similarly, other studies support the idea that entire regions of the cytosol could transport vesicles as if on a conveyor belt, therefore vectoring the overall transport of vesicles during exocytosis toward the cell periphery (Maucort et al, 2014 ). More recently, a new potential transport mechanism was described based on the coordinated displacement of F-actin structures embedding chromaffin granules, a process defined as a “cast” system (Papadopulos et al, 2015 ).…”