Restriction of secretory granule motion near the plasma membrane of chromaffin cells

We used total internal reflection fluorescence microscopy to study quantita tively the motion and distribution of secretory granules near the plasma me mbrane (PM) of living bovine chromaffin cells. Within the similar to 300-nm region measurably illuminated by the evanescent field resulting from total internal reflection, granules are preferentially concentrated close to the PM. Granule motion normal to the substrate (the z direction) is much slowe r than would be expected from free Brownian motion, is strongly restricted over tens of nanometer distances, and tends to reverse directions within 0. 5 s. The z-direction diffusion coefficients of granules decrease continuous ly by two orders of magnitude within less than a granule diameter of the PM as granules approach the PM. These analyses suggest that a system of tethe rs or a heterogeneous matrix severely limits granule motion in the immediat e vicinity of the PM. Transient expression of the light chains of tetanus t oxin and botulinum toxin A did not disrupt the restricted motion of granule s near the PM. indicating that SNARE proteins SNAP-25 and VAMP are not nece ssary for the decreased mobility. However. the lack of functional SNAREs on the plasma or granule membranes in such cells reduces the time that some g ranules spend immediately adjacent to the PM.