NANOPHYSIOLOGY OF CELLS, CHANNELS AND NUCLEAR PORES - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 121

distribution shows two peak values, at 9 nm and at 14 nm, corresponding to
molecular masses of 275 kDa and 750 kDa, respectively ( Fig. 6.3c , red hatched
area). The data obtained in CFTR-expressing oocytes indicate that the protein
covered area increases in response to cAMP by about 110%. This observation
strongly suggests protein insertion into the plasma membrane. CFTR-
expressing oocytes exhibited upon cAMP stimulation two new peaks at 275
kDa and 750 kDa. Since both peaks do not appear in CFTR-negative oocytes
in response to cAMP stimulation, we conclude that the two peaks are caused
by CFTR. Considering the molecular mass of 180 kDa for a CFTR monomer,
the peak at 275 kDa and 750 kDa could be multimeric CFTR or CFTR forming
clusters with other proteins. Together, upon stimulation with cAMP, CFTR is
inserted into the plasma membrane, indicated by a shift in protein density
and protein distribution. Insertion of CFTR in the plasma membrane leads
to the formation of clusters, heteromeric structures composed of CFTR and
other proteins with yet unknown stoichiometry.
These data show that the dynamics of plasma membrane protein
distribution could be visualized and quantiied with AFM.

6.1.4 Quanficaon of CFTR in Human Red Blood Cells

CFTR is distributed in various cell types, and it is also shown for red
blood cells (RBCs).

Interestingly, CF patients do not show hematological
disorders; therefore, the meaning of CFTR on RBCs is unclear. We performed a
quantiication study of the CFTR copies in RBC membranes at single-molecule
level and compared the difference between healthy donors and CF patients
with the homozygous ΔF508 mutation. For this purpose, two different AFM
techniques were used: (1) immunostaining with quantum dot (Qdot)-labelled
antibodies and (2) topography and recognition imaging.
The membrane isolation approach was used not only to achieve high
resolution but also to have the intracellular portion of CFTR freely accessible
for antibodies. RBCs are non-adherent cells, and therefore we glued them
onto poly-L-lysine-coated glass. These attached RBCs were sheared open
with a jet stream of isotonic phosphate buffered saline (with 0.2 mM EGTA).

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The quality of membrane preparations was assessed, with AFM revealing
large areas of freely accessible intracellular plasma membrane surfaces ( Fig.
6.4 ) . Membranes appear as lat round structures, with protrusions up to 25
nm in height. A high density of erythrocytes during preparation causes an
overlapping of membrane edges, resulting in multilayered membrane areas
clearly visible in the AFM images ( Fig. 6.4a-c ) .

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