A 20-nm step toward the cell membrane preceding exocytosis may correspond to docking of tethered granules.

In endocrine cells, plasma membrane (PM)-bound secretory granules must undergo a number of maturation stages (i.e., priming) to become fusion-competent. Despite identification of several molecules involved in binding granules to the PM and priming them, the exact nature of events occurring at the PM still largely remains a mystery. In stimulated BON cells, we used evanescent wave microscopy to study trajectories of granules shortly before their exocytoses, which provided a physical description of vesicle-PM interactions at an unprecedented level of detail, and directly lead to an original mechanistic model. In these cells, tethered (T), nonfusogenic, vesicles are prevented from converting to fusogenic, docked (D) ones in resting conditions. Upon elevation of calcium, T-vesicles perform a 21-nm step toward the PM to become D, and fuse approximately 3 s thereafter. Our ability to directly visualize different modes of PM-attachment paves the way for clarifying the exact role of various molecules implicated in attachment and priming of granules in future studies.

Characterization of sequential exocytosis in a human neuroendocrine cell line using evanescent wave microscopy and "virtual trajectory" analysis.

Secretion of hormones and other bioactive substances is a fundamental process for virtually all multicellular organisms. Using total internal reflection fluorescence microscopy (TIRFM), we have studied the calcium-triggered exocytosis of single, fluorescently labeled large, dense core vesicles in the human neuroendocrine BON cell line. Three types of exocytotic events were observed: (1) simple fusions (disappearance of a fluorescent spot by rapid diffusion of the dye released to the extracellular space), (2) "orphan" fusions for which only rapid dye diffusion, but not the parent vesicle, could be detected, and (3) events with incomplete or multi-step disappearance of a fluorescent spot. Although all three types were reported previously, only the first case is clearly understood. Here, thanks to a combination of two-color imaging, variable angle TIRFM, and novel statistical analyses, we show that the latter two types of events are generated by the same basic mechanism, namely shape retention of fused vesicle ghosts which become targets for sequential fusions with deeper lying vesicles. Overall, approximately 25% of all exocytotic events occur via sequential fusion. Secondary vesicles, located 200-300 nm away from the cell membrane are as fusion ready as primary vesicles located very near the cell membrane. These findings call for a fundamental shift in current models of regulated secretion in endocrine cells. Previously, sequential fusion had been studied mainly using two-photon imaging. To the best of our knowledge, this work constitutes the first quantitative report on sequential fusion using TIRFM, despite its long running and widespread use in studies of secretory mechanisms.

Analysis of transient behavior in complex trajectories: application to secretory vesicle dynamics.

Analysis of trajectories of dynamical biological objects, such as breeding ants or cell organelles, is essential to reveal the interactions they develop with their environments. Many previous works used a global characterization based on parameters calculated for entire trajectories. In cases where transient behavior was detected, this usually concerned only a particular type, such as confinement or directed motion. However, these approaches are not appropriate in situations in which the tracked objects may display many different types of transient motion. We have developed a method to exhaustively analyze different kinds of transient behavior that the tracked objects may exhibit. The method discriminates stalled periods, constrained and directed motions from random dynamics by evaluating the diffusion coefficient, the mean-square displacement curvature, and the trajectory asymmetry along individual trajectories. To detect transient motions of various durations, these parameters are calculated along trajectories using a rolling analysis window whose width is variable. The method was applied to the study of secretory vesicle dynamics in the subplasmalemmal region of human carcinoid BON cells. Analysis of transitions between transient motion periods, combined with plausible assumptions about the origin of each motion type, leads to a model of dynamical subplasmalemmal organization.

Serotonin secretion by human carcinoid BON cells.

BON cells are human carcinoid cells that secrete serotonin (5-HT) and various peptides. Secretion of [(3)H]5-HT by cell cultures was investigated. Acetylcholine (Ach) stimulated secretion through a somatostatin-sensitive muscarinic pathway, whereas isoproterenol was inefficient. [(3)H]5-HT secretion also was induced by Ca(2+) in the presence of the ionophore A-23187 or after digitonin permeabilization. These two processes were insensitive to stomatostatin. Ba(2+) induced an efficient somatostatin-sensitive [(3)H]5-HT secretory response. Secretion also was analyzed at the single-cell level, using carbon fiber amperometry and evanescent-field fluorescence microscopy, after labeling the secretory vesicles by transfection of the cells with a NPY-GFP construct. Both techniques revealed slow kinetics of secretory responses, suggesting that ready-to-fuse vesicles do not accumulate in these cells. Single secretory vesicles were imaged either in resting conditions or after addition of Ca(2+) ions to digitonin-permeabilized cells. The three-dimensional movements of the vesicles before exocytosis were analyzed. The mean velocity of vesicles that released their content was lower than that of silent ones. Even in the case of mobile vesicles, exocytosis often was preceded by a period of arrest lasting at least 15 seconds, consistent with a docking/priming step.

Rab27A and its effector MyRIP link secretory granules to F-actin and control their motion towards release sites.

The GTPase Rab27A interacts with myosin-VIIa and myosin-Va via MyRIP or melanophilin and mediates melanosome binding to actin. Here we show that Rab27A and MyRIP are associated with secretory granules (SGs) in adrenal chromaffin cells and PC12 cells. Overexpression of Rab27A, GTPase-deficient Rab27A-Q78L, or MyRIP reduced secretory responses of PC12 cells. Amperometric recordings of single adrenal chromaffin cells revealed that Rab27A-Q78L and MyRIP reduced the sustained component of release. Moreover, these effects on secretion were partly suppressed by the actin-depolymerizing drug latrunculin but strengthened by jasplakinolide, which stabilizes the actin cortex. Finally, MyRIP and Rab27A-Q78L restricted the motion of SGs in the subplasmalemmal region of PC12 cells, as measured by evanescent-wave fluorescence microscopy. In contrast, the Rab27A-binding domain of MyRIP and a MyRIP construct that interacts with myosin-Va but not with actin increased the mobility of SGs. We propose that Rab27A and MyRIP link SGs to F-actin and control their motion toward release sites through the actin cortex.