NANOPHYSIOLOGY OF CELLS, CHANNELS AND NUCLEAR PORES - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 117

The ideal sample for high-resolution AFM imaging is hard and lat.
Hardness reduces vertical and lateral movement of sample structures
as well as a lat sample minimizes tip convolution artefacts. The plasma
membrane of eucaryotic cells is generally anything but lat. The curvature of
a cell is formed by lamellopodia, cell body and cell nucleus. The membrane
shows major structures like membrane rufles, microvilli and cilia and also
submembranous structures like the cytoskeleton. A huge variety of proteins
are heterogeneously distributed within the membrane, and many membrane
proteins are equipped with highly branched sugars forming the glycocalyx.
The glycocalyx, a network of polysaccharides that protrudes up to 100 nm
from cellular surfaces, limits the tip access to the membrane surface, thus
reducing the resolution. Therefore, we isolated cell membranes on a solid
support in such a way that the intracellular face of the membrane is accessible
for the AFM tip (“inside-out” orientation). An example of images obtained for
human lung epithelial cells (cell line 16HBE14o

) is shown in Fig. 6.1 .
The extracellular face of the cell membrane shows a dense distribution
of microvilli maintaining the mucocilliary clearance. These microvilli are up
to 1 μm in length with diameters of several hundred nanometres ( Fig. 6.1a ).
This microvilli layer generates an extreme roughness of the surface, which
causes a serious tip convolution and therefore a reduction of resolution.
Protein structures are hardly detectable even in smaller scan areas with
increased resolution ( Fig. 6.1b ) . Contrarily the intracellular faces of theses
membranes are rather lat with height differences below 50 nm ( Fig. 6.1c,d ) .
Protein structures are clearly detectable, enabling AFM studies on single-
molecule level.
Together, isolated, “inside-out” oriented membranes offer several
advantages: (i) the membrane is lat because no curvature is imposed by
underlying structures (e.g. nucleus, cytoskeleton), (ii) the membrane is hard
because it lies on a hard support instead of a soft cytosol, (iii) the intracellular
face of the membrane can be imaged by AFM, which means that no glycocalyx
disturbs high-resolution imaging. Furthermore, the majority of membrane
proteins are located intracellularly and therefore accessible by the AFM tip in
an inside-out coniguration.

6.1.2 The Cysc Fibrosis Transmembrane Conductance

Regulator

A membrane protein of clinical importance is the cystic ibrosis
transmembrane conductance regulator (CFTR). 1 CFTR is a plasma membrane
cyclic AMP-activated Cl

channel that is expressed in several functionally
diverse tissues, including the kidney, pancreas, intestine, heart, vas deferens,