AFM/TIRF force clamp measurements of neurosecretory vesicle tethers reveal characteristic unfolding steps.
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.
School of Applied and Engineering Physics, Engineering, Cornell University, Ithaca, NY, United States of America.
Mark C
MC
Harris
School of Applied and Engineering Physics, Engineering, Cornell University, Ithaca, NY, United States of America.
Dillon
D
Cislo
School of Applied and Engineering Physics, Engineering, Cornell University, Ithaca, NY, United States of America.
Joan S
JS
Lenz
Department of Materials Science and Engineering, Cornell University, Ithaca, NY, United States of America.
Christopher
C
Umbach
School of Applied and Engineering Physics, Engineering, Cornell University, Ithaca, NY, United States of America.|Laboratory for Nanoscale Cell Biology, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany.
Manfred
http://orcid.org/0000-0002-5722-9557
M
Lindau
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D022162:Cytoplasmic Vesicles / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D055595:Mechanical Phenomena; D018625:Microscopy, Atomic Force / Q000379:methods*; D008857:Microscopy, Interference / Q000379:methods*; D016716:PC12 Cells; D051381:Rats; D022142:Secretory Vesicles / Q000378:metabolism*
D005456:Fluorescent Dyes; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D009320:Atrial Natriuretic Factor