TOPOGRAPHY AND RECOGNITION IMAGING OF CELLS - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 154

distribution of microdomains (in green) on the cellular surface with domain
sizes from ~30 up to ~350 nm, with a mean ± SD of 99 ± 81 nm (

= 25) on
the long domain axis. During several subsequent rescans, recognition maps
of hERG channels remained unchanged. Next, ErgTx1 was very slowly (~50
M

n

L/min) injected in the luid cell while scanning the same sample. After the
irst and second injection of ErgTx1 (concentration of ~400 nM), no visual
changes in the recognition maps were observed. However, the recognition
clusters disappeared, only in part, when the concentration of ErgTx1 reached
1

M

M ( Fig. 7.7d ) , whereas no change in the topography image was observed
( Fig. 7.7d ) . The speciic binding between anti-Kv11.1 and the cellular surface
was abolished when free ErgTx1 molecules bound to the hERG channels
and thus blocked the antibody access to interact with epitope tags on hERG
subunits. The topography of a scanned cell surface area showed a complex
picture of linear and branched ilamentous structures with some globular
features. Most domains were found to be located near and between ilaments
( Fig. 7.7b ) . TREC results suggest that ErgTx1 does not only interact with the
extracellular surface of the pore domain (S5-S6) but might also interact with
the voltage-sensing domains (S1-S4) of the hERG K + channel.

(b)

(a)

Figure 7.8. Detection of hERG K + channels on live cells with anti-Kv11.1-
functionalized AFM tip. (a) Quantitative comparison of binding probabilities
obtained on living HEK-293 cells expressing hERG K + channels in the absence ( left

light gray ) and presence of either free anti-Kv11.1 antibodies ( middle light gray ) or
free antigen peptides ( right light gray ); binding probably on parent HEK-293 cells is
shown in black . Consecutive injection of ErgTx-1 (300 nM, 1 μM) reduces the binding
probability ( white ). Values are mean ± SEM, n = 2000-4000. (b) Force distributions
(pdf ) observed in the absence of ErgTx1 (

) and in the presence of
ErgTx1 ( dot [300 nM] and short dashed-dot lines [1 μM]). Areas under the curves are
scaled to the corresponding binding probabilities.

solid blue line

To extend TREC measurements, AFM force-distance cycles with a tip
carrying an epitope-speciic antibody (anti-Kv11.1) were collected on
living and gently ixed hERG HEK-293 cells. Both studies conducted on