A Combined TIRF/AFM Approach to Investigate Nanomechanical Features at the Cytoplasmic Face of a Plasma Membrane

Abstract

The cytoplasmic face of cell membranes has multiple features, such as cortical actin and tethered vesicles, the nanomechanical properties of which are largely unknown. To study these properties, we prepared plasma membrane sheets from a PC12 cell clone (Lang 2008 Meth.Mol.Biology, 440: 51-9) constitutively expressing ANF-EGFP, GFP-tagged proANF (atrial natriuretic factor) (Han et al 1999 PNAS 96:14577-82) as a secretory vesicle marker. The membrane sheets are firmly attached to the coverslip with their extracellular face while the cytoplasmic face is exposed. We combined a Nikon Ti-E/B total internal reflection fluorescence (TIRF) microscope with an Agilent PicoPlus atomic force microscope (AFM). Soft AFM cantilevers (Olympus Biolevers) were brought into contact with the exposed membrane face, binding spontaneously to the surface and to secretory vesicles. The membrane sheets and the motion of the cantilever tips were imaged by TIRF microscopy while forces of approximately 50pN or more were applied, pulling the tip away from the surface. At 50 ms time resolution, the noise level of the mechanical measurements was ∼1 pN and 0.5 nm (r.m.s.). Dynamic interactions associated with displacements in the 10-40 nm range were recorded. The role of secretory vesicles in this behavior will be investigated.