There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.
Abstract
The trajectory of secretory vesicles to their fusion sites at the plasma membrane
is expected to give insight into the mechanisms that underlie vesicle transport, maturation
and the initiation of membrane fusion. Evanescent-wave (EW) microscopy allows the
tracking of fluorescently labeled granules and vesicles prior to fusion with nanometer
precision in xy-direction. At the same time, the exponential sensitivity of granular
fluorescence to experimental parameters can preclude quantitative estimates of the
granule's approach to the plasma membrane. Thus, it has remained controversial to
which extent axial distance can be obtained from simple intensity measurements. We
used the information contained in a stack of images acquired at 80-125 nm penetration
depth of the EW field to estimate individual granule diameter and axial distance.
A population analysis on 90 granules revealed an average diameter of 305 +/- 47 nm,
below the diffraction-limited 352 +/- 31 nm obtained from xy measurements at fixed
depth penetration. Stimulation of exocytosis by potassium depolarization resulted
in the selective loss of the 18 +/- 5% of granules located closest to the plasma membrane,
while a second population of granules located 60 nm deeper within the cytoplasm increased
by recruitment of granules previously located at > or = 120 nm depth. These measurements
extend and corroborate previous observations at fixed penetration depth of functionally
distinct granule populations. Parameters influencing the accuracy of the parameter
estimation are evaluated in the appendix.