The biogenesis of adrenal chromaffin granules

The cortical cytoskeleton is a dense network of filamentous actin (F-actin) that participates in the events associated with secretion from neuroendocrine cells. This filamentous web traps secretory vesicles, acting as a retention system that blocks the access of vesicles to secretory sites during the resting state, and it mediates their active directional transport during stimulation. The changes in the cortical cytoskeleton that drive this functional transformation have been well documented, particularly in cultured chromaffin cells. At the biochemical level, alterations in F-actin are governed by the activity of molecular motors like myosins II and V and by other calcium-dependent proteins that influence the polymerization and cross-linking of F-actin structures. In addition to modulating vesicle transport, the F-actin cortical network and its associated motor proteins also influence the late phases of the secretory process, including membrane fusion and the release of active substances through the exocytotic fusion pore. Here, we discuss the potential interactions between the F-actin cortical web and proteins such as SNAREs during secretion. We also discuss the role of the cytoskeleton in organizing the molecular elements required to sustain regulated exocytosis, forming a molecular structure that foments the efficient release of neurotransmitters and hormones.

Amperometry is a simple and powerful technique to study exocytosis at the single cell level. By positioning and polarizing (at an appropriate potential at which the molecules released by the cell can be oxidized) a carbon fiber microelectrode at the top of the cell, each exocytotic event is detected as an amperometric spike. More particularly, a portion of these spikes has previously been shown to present a foot, i.e. a small pedestal of current that precedes the spike itself. Among the important number of works dealing with the monitoring of exocytosis by amperometry under different conditions, only a few studies focus on amperometric spikes with a foot. In this work, by coupling our previous and recent experiments on chromaffin cells (that release catecholamines after stimulation) with literature data, we bring more light on what an amperometric foot and particularly its features, represents.

Dense vesicles can be observed in live bovine chromaffin cells using fluorescent reflection confocal microscopy. These vesicles display a similar distribution, cytoplasmic density and average size as the chromaffin granules visualized by electron microscopy. In addition, the acidic vesicles labeled with Lysotracker Red comprised a subpopulation of the vesicles that are visualized by reflection fluorescence. A combination of fluorescence reflection and transmitted light images permitted the movements of vesicles in relation to the cortical cytoskeleton to be studied. The movement of vesicles located on the outside of this structure was restricted, with an apparent diffusion coefficient of 1.0±0.4×10⁻⁴ μm²/s. In contrast, vesicles located in the interior moved much more freely and escaped from the visual confocal plane. Lysotracker labeling was more appropriate to study the movement of the faster moving vesicles, whose diffusion coefficient was five times higher. Using this type of labeling we confirmed the restriction on cortical movement and showed a clear relationship between vesicle mobility and the kinetics of cytoskeletal movement on both sides of the cortical cytoskeleton. This relationship was further emphasized by studying cytoskeletal organization and kinetics. Indeed, an estimate of the size of the cytoskeletal polygonal cages present in the cortical region and in the cell interior agreed well with the calculation of the theoretical radius of the cages imprisoning vesicle movement. Therefore, these data suggest that the structure and kinetics of the cytoskeleton governs vesicle movements in different regions of chromaffin cells.

Annexin 2 is a member of the annexin family which has been implicated in calcium-regulated exocytosis. This contention is largely based on Ca²⁺-dependent binding of the protein to anionic phospholipids. However, annexin 2 was shown to be associated with chromaffin granules in the presence of EGTA. A fraction of this bound annexin 2 was released by methyl-β-cyclodextrin, a reagent which depletes cholesterol from membranes. Restoration of the cholesterol content of chromaffin granule membranes with cholesterol/methyl-β-cyclodextrin complexes restored the Ca²⁺-independent binding of annexin 2. The binding of both, monomeric and tetrameric forms of annexin 2 was also tested on liposomes of different composition. In the absence of Ca²⁺, annexin 2, especially in its tetrameric form, bound to liposomes containing phosphatidylserine, and the addition of cholesterol to these liposomes increased the binding. Consistent with this observation, liposomes containing phosphatidylserine and cholesterol were aggregated by the tetrameric form of annexin 2 at submicromolar Ca²⁺ concentrations. These results indicate that the lipid composition of membranes, and especially their cholesterol content, is important in the control of the subcellular localization of annexin 2 in resting cells, at low Ca²⁺ concentration. Annexin 2 might be associated with membrane domains enriched in phosphatidylserine and cholesterol.

In ciliates, a variety of processes are regulated by Ca²⁺, e.g., exocytosis, endocytosis, ciliary beat, cell contraction, and nuclear migration. Differential microdomain regulation may occur by activation of specific channels in different cell regions (e.g., voltage- dependent Ca²⁺ channels in cilia), by local, nonpropagated activation of subplasmalemmal Ca stores (alveolar sacs), by different sensitivity thresholds, and eventually by interplay with additional second messengers (cilia). During stimulus-secretion coupling, Ca²⁺ as the only known second messenger operates at ∼5 μM, whereby mobilization from alveolar sacs is superimposed by “store-operated Ca²⁺ influx”' (SOC), to drive exocytotic and endocytotic membrane fusion. (Content discharge requires binding of extracellular Ca²⁺ to some secretory proteins.) Ca²⁺ homeostasis is reestablished by binding to cytosolic Ca²⁺-binding proteins (e.g., calmodulin), by sequestration into mitochondria (perhaps by Ca²⁺ uniporter) and into endoplasmic reticulum and alveolar sacs (with a SERCA-type pump), and by extrusion via a plasmalemmal Ca²⁺ pump and a Na⁺/Ca²⁺ exchanger. Comparison of free vs total concentration, [Ca²⁺] vs [Ca], during activation, using time-resolved fluorochrome analysis and X-ray microanalysis, respectively, reveals that altogether activation requires a calcium flux that is orders of magnitude larger than that expected from the [Ca²⁺] actually required for local activation.

This is a detailed characterization of a secretory mutant incapable of releasing secretory contents despite normal exocytotic membrane fusion performance. Trichocyst non-discharge strain tndl of Paramecium caudatum and its wildtype (wt) both show a transient cortical [Ca ²⁺]_i increase and exocytotic membrane fusion in response to the polyamine secretagogue, aminoethyldextran (AED), or to caffeine. tndl cells frequently display spontaneous Ca²⁺ signals parallelled by spontaneous exocytotic membrane fusion. This remains undetected, unless the trichocyst matrix is shown to be freely accessible to the inert, non-membrane permeable fluorochrome, F₂FITC, from the outside. In these tndl cells, spontaneous and AED- or caffeine-induced membrane fusion, always without contents expulsion by decondensation (i.e. several-fold stretching), is ascertained by electron microscopy. Exocytotic openings, with condensed trichocysts retained, may persist for hours without impairing cells. Trichocyst decondensation normally requires micromolar [Ca²⁺]_e, but an increase to 10 mM has no effect on tndl trichocyst expansion in vivo or in vitro (when isolated and exposed to ionophore A23187 + Ca²⁺). Paracrystalline packing of the major secretory components (trichynins) does occur, despite incomplete proteolytic precursor processing (according to SDS-PAGE). However, ⁴⁵Ca²⁺-binding by secretory components is considerably reduced — the likely cause of the non-discharge phenotype. Our findings imply significant untriggered membrane fusion in a system normally following the triggered pathway and clear separation of exocytotic membrane fusion from any later Ca²⁺-dependent steps of the secretory cycle.