Chromaffin granule motion near the plasma membrane.

摘要

The majority of studies concerning regulated protein secretion in neuroendocrine cells measure only the final events of fusion and release of secretory granule contents. Kinetic analysis of the secretory response has revealed that granules undergo priming reactions prior to fusion triggered by Ca2+. However, the relationship of priming events to movement of granules to the plasma membrane, the association of granules and their eventual molecular interaction with the membrane are not understood. It was the goal of this thesis to characterize the movement of granules, manipulate proteins necessary for exocytosis and to determine their effects on granule motion near the plasma membrane.;To quantitatively study motion and distribution of secretory granules near the plasma membrane of living bovine chromaffin cells, total internal reflection fluorescence microscopy (TIRFM) was employed. TIRFM selectively illuminates subcellular features close to the plasma membrane at cell-substrate contact regions. A novel TIRFM analysis was performed on the basal state motions of granules in the direction normal to the membrane ("z" direction). This z-motion of granules is much slower than expected from free Brownian motion, and strongly restricted over tens of nanometer distances. Diffusion coefficients decrease by two orders of magnitude within less than 150 nm of the plasma membrane. The analyses indicate that granule motion is increasingly restricted upon approach to the membrane, suggesting that structures or processes severely limit granule motion in the immediate vicinity of the membrane.;Three proteins, synaptobrevin (VAMP), SNAP-25 (synaptosomal associated protein of 25 kDa) and syntaxin, form the "SNARE" complex between the granule and plasma membranes, which is necessary for exocytosis. The effects of this complex on granule z-motion were investigated. Overall, z-motion was unaffected by disruption of the SNARE complex, indicating it is not responsible for the restricted nature of the granules. A subpopulation of granules was identified which spent less time very close to the plasma membrane than granules from control cells. These granules may be sampling the plasma membrane, transiently forming SNARE complexes. Disruption of the SNARE complex prevents them from forming these interactions and thus they spend less time closely associated with the plasma membrane.