Direct Measurement of Secretory Vesicle-Plasma Membrane Tethering Interactions by Correlated AFM Force-Clamp and TIRF Microscopy

Abstract

The molecular mechanisms behind the tethering of secretory vesicles to the plasma membrane are not well understood. To study these mechanisms, we use a combined AFM/TIRF method applying forces to GFP-tagged secretory granules tethered to PC-12 cell membrane sheets. In the experiment shown in the figure, the AFM tip captured a granule, and was then lowered onto the sheet (a-b). Pulling forces were applied, ranging from ∼100pN to ∼700pN (c-e). Extension and rupture events are apparent as transient variations in the pulling force, as well as increases in the scanner z position sensor. Change in sensor position is consistent with changes in vesicle height as observed in the TIRF, based on the calibrated excitation decay constant (c-d). Rapid transients with small amplitude reveal partial tether extensions by 10-20nm, while large transients with slow decay reveal more gradual extensions, exceeding 1μm, that appear to be composed of multiple steps. These observations suggest that the dynamics of vesicle tether extensions are much more complex than previously thought. A large data set of recordings like that in the figure is being analyzed. Supported by NIH grant R21NS072577.View Large Image | View Hi-Res Image | Download PowerPoint Slide