AFM/TIRF force clamp measurements of neurosecretory vesicle tethers reveal characteristic unfolding steps

Abstract

Although several proteins have been implicated in secretory vesicle tethering, the identity and mechanical properties of the components forming the physical vesicle-plasma membrane link remain unknown. Here we present the first experimental measurements of nanomechanical properties of secretory vesicle-plasma membrane tethers using combined AFM force clamp and TIRF microscopy on membrane sheets from PC12 cells expressing the vesicle marker ANF-eGFP. Application of pulling forces generated tether extensions composed of multiple steps with variable length. The frequency of short (<10 nm) tether extension events was markedly higher when a fluorescent vesicle was present at the cantilever tip and increased in the presence of GTPγS, indicating that these events reflect specifically the properties of vesicle-plasma membrane tethers. The magnitude of the short tether extension events is consistent with extension lengths expected from progressive unfolding of individual helices of the exocyst complex, supporting its direct role in forming the physical vesicle-plasma membrane link.

How did correlative atomic force microscopy and super-resolution microscopy evolve in the quest for unravelling enigmas in biology?

“…More recently, mechanisms of intercellular tension involving cytoskeletal proteins, like catenin and vinculin, have been observed in real time by combining AFM and TIRFM. 174 The authors visualized α-catenin stretched using AFM and the simultaneous recruitment of fluorescently labeled vinculins. The work highlights the potential of the hybrid instrument in the field of mechanobiology (Fig.…”

Advanced Imaging Approaches to Reveal Molecular Mechanisms Governing Neuroendocrine Secretion

“…Other modifications of TIRFM that have gained prominence over the last decade are the result of its merger with super-resolution techniques. Harris et al [86] combined TIRFM and AFM force clamp to identify the nanomechanical properties of the secretory granule-plasma membrane tether by force clamp measurements. They prepared plasma membrane sheets from PC12 cells to locate and track the movement of secretory granules attached to the AFM cantilever tip.…”