Dynamics of Immature Secretory Granules: Role of Cytoskeletal Elements during Transport, Cortical Restriction, and F-Actin-dependent Tethering

Secretory granules store neuropeptides and hormones and exhibit regulated exocytosis upon appropriate cellular stimulation. They are generated in the trans-Golgi network as immature secretory granules, short-lived vesicular intermediates, which undergo a complex and poorly understood maturation process. Due to their short half-life and low abundance, real-time studies of immature secretory granules have not been previously possible. We describe here a pulse/chase-like system based on the expression of a human chromogranin B-GFP fusion protein in neuroendocrine PC12 cells, which permits direct visualization of the budding of immature secretory granules and their dynamics during maturation. Live cell imaging revealed that newly formed immature secretory granules are transported in a direct and microtubule-dependent manner within a few seconds to the cell periphery. Our data suggest that the cooperative action of microtubules and actin filaments restricts immature secretory granules to the F-actin-rich cell cortex, where they move randomly and mature completely within a few hours. During this maturation period, secretory granules segregate into pools of different motility. In a late phase of maturation, 60% of secretory granules were found to be immobile and about half of these underwent F-actin-dependent tethering.

Dynamical hierarchy in transition states: Why and how does a system climb over the mountain?

How a reacting system climbs through a transition state during the course of a reaction has been an intriguing subject for decades. Here we present and quantify a technique to identify and characterize local invariances about the transition state of an N-particle Hamiltonian system, using Lie canonical perturbation theory combined with microcanonical molecular dynamics simulation. We show that at least three distinct energy regimes of dynamical behavior occur in the region of the transition state, distinguished by the extent of their local dynamical invariance and regularity. Isomerization of a six-atom Lennard–Jones cluster illustrates this: up to energies high enough to make the system manifestly chaotic, approximate invariants of motion associated with a reaction coordinate in phase space imply a many-body dividing hypersurface in phase space that is free of recrossings even in a sea of chaos. The method makes it possible to visualize the stable and unstable invariant manifolds leading to and from the transition state, i.e., the reaction path in phase space, and how this regularity turns to chaos with increasing total energy of the system. This, in turn, illuminates a new type of phase space bottleneck in the region of a transition state that emerges as the total energy and mode coupling increase, which keeps a reacting system increasingly trapped in that region.