Giant unilamellar vesicles with diameters ranging from 10 to 60 microns were obtained by the swelling of phospholipid bilayers in water in the presence of an AC electric field. This technique leads to a homogeneous population of perfectly spherical and unilamellar vesicles, as revealed by phase-contrast optical microscopy and freeze-fracture electron microscopy. Freshly prepared vesicles had a high surface tension with no visible surface undulations. Undulations started spontaneously after several hours of incubation or were triggered by the application of a small osmotic pressure. Partially deflated giant vesicles could undergo further shape change if asymmetrical bilayers were formed by adding lyso compounds to the external leaflet or by imposing a transmembrane pH gradient that selectively accumulates on one leaflet phosphatidylglycerol. Fluorescence photobleaching with 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labeled phospholipids or labeled dextran trapped within the vesicles enabled the measurement of the membrane continuity in the dumbbell-shaped vesicles. In all instances phospholipids diffused from one lobe to the other, but soluble dextran sometimes was unable to traverse the neck. This suggests that the diameter of the connecting neck may be variable.
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.
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.
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