NANOPHYSIOLOGY OF CELLS, CHANNELS AND NUCLEAR PORES - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 118

sweat duct and lung. It is a protein of the ATP-binding cassette transporter
superfamily, known to play a crucial role in maintaining the salt and water
balance on the epithelium. Stimulating CFTR by cAMP increases channel
activity and increases the total number of CFTR channels in the membrane,
which is achieved by the insertion and removal of CFTR channels from the
plasma membrane. Mutations in CFTR affect the number of channels in the
plasma membrane, channel activity and the intracellular traficking of CFTR.
A mutation in the gene encoding for CFTR results in cystic ibrosis (CF), a
very common lethal genetic disease.
One of the goals of our AFM studies on CFTR in isolated cell membranes
was to quantify this protein in its native environment and to elucidate
membrane traficking of CFTR.
The most predominant mutation, ∆F508, results in a defective protein
traficking, which manifests in organ pathology. 2 To perform its task, CFTR has
to be correctly incorporated into the cell membrane in a suficient number.
The issue regarding the number of CFTR within the cellular membrane is
gaining increasing interest for developing ∆F508-CFTR-rescuing strategies
and gene therapies for CF. Although a wealth of information has been
gathered using different quantiication approaches, the conclusions obtained
so far regarding the CFTR number were indirectly drown. Identiication of
CFTR within the cell membrane at single-molecule level makes it feasible to
visualize the distribution and organization of CFTR proteins within the cell
membrane of healthy individuals and CF patients.

6.1.3 Visualisaon and Quanficaon of Plasma Membrane

Dynamics

We used oocytes as expression system for human CFTR. We

examined the expression of CFTR after injection of CFTR-cRNA with voltage-
clamp experiments and isolated the membranes of oocytes exhibiting cAMP-
inducible currents in voltage-clamp analysis. This experimental procedure
ensures that the plasma membrane investigated by AFM contains functional
CFTR. We used the AFM to image the cytoplasmic surface of native plasma
membranes of CFTR-expressing

Xenopus l.

Xenopus l. oocytes before and after cAMP

stimulation.
AFM revealed large patches of inside-out oriented plasma membrane and
areas without membrane. Edges of membrane were used to determine total
height of plasma membrane and its protruding structures. Figure 6.2 shows
a 3D colour-coded view of a 9 μm 2 scan area containing plasma membrane
fragments attached to the poly-L-lysine-coated glass surface. Poly-L-lysine-
coated glass is shown in “blue”, the lipid bilayer membrane is shown in