SAMPLE PREPARATION

Immediately after cataract surgery,the membranes were extracted
from cataract debris, and pelleted byultracentrifugation. The membrane
solution was injected into a droplet ofadsorption buffer (10 mM Tris-HCl pH
7.4, 150 mM KCl, 25 mM MgCl2) on topof a freshly cleaved mica sheet. Afterincubation, the sample was rinsed
using recording buffer (10 mM Tris-HCl
pH 7.4, 150 mM KCl).13
Imaging was performed on healthy12
and cataract13 lens cell membranes
using a customized Veeco NanoScope™
E AFM equipped with a 130 μm
J-scanner and Olympus Si3N4 (length =100 μm; k = 0.09 N/m). The loading force
was ~100 pN and the scan rate was
4–7 Hz. See references 12 and 13 forfurther sample preparation details.
RESULTS

AFM images of cataract membranes
revealed lipid bilayer cell membranes
adsorbed to the mica support. Thesemembranes contained protein domains
identified as junctional microdomainsthat connect adjacent lens cells. The
microdomains were significantly

larger in cataract membranes thanthose observed in membranes fromhealthy cells. The cataract membrane
junctional microdomains were foundto be composed exclusively of AQP0transmembrane channel proteins.
Image resolution was sufficient toallow identification of individual helixconnecting loops, which protrude
from the membrane surface, of aboutfour amino acids in length; and thesedata coincide closely with predicted
models. The sub-nanometer resolutionof these features was extracted from
topographical images and compared
to previously published data on healthy
sheep lens cell membranes.12
A systematic structural comparison
between healthy and cataract lensmembranes revealed that, in healthylens cells, AQP0 molecules are wellorganized in small dense patchessurrounded and confined by connexons.
In stark contrast, the cataract lensmembranes did not contain connexons(see figure 1). As a consequence,
junction arrays appeared significantly
enlarged and malformed in themembranes of cataract lens cells.

Junctional microdomain in healthy lens membranes

It would seem that the connexons
are progressively degraded duringcataract development, ultimately
leading to a breakdown of lens cellnutrition. From a physiological pointof view, in a healthy lens cell thesupramolecular assembly of AQP0
and connexons is required for celladhesion through junction formation,
as well as normal ion, metabolite, andwater flow between adjacent cells
through gap junctions. Moreover, thehomogeneous distribution of smaller
junctional microdomains allows abetter connection between neighborcells, decreasing the probability of nonadhering membrane areas. In contrast,
the absence of connexons from themembranes of cataract lens cells
results in a heterogeneous distributionof the adhering/non-adhering
membrane areas. Finally, nutrientsand ions are not delivered to cells deep
inside the lens and waste products
accumulate (see figure 2). These cells
will become unhealthy and will not
be able to maintain transparency,
ultimately leading to blindness.

Junctional microdomain in cataract lens membranes

Figure 1: Contact mode high-resolution AFM topography images showing substructure on individualtransmembrane channels in healthy sheep (left) and human cataract (right) lens cell membranes. In thehealthy case, AQPO molecules (cross-shaped tetrameric proteins with a diameter of 6nm) form small
and regular patches edged by connexons (flower-shaped hexameric proteins with a diameter of 8nm) thatdelimit the AQPO microdomains. In the pathological case, connexons are lacking.

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Figure 2: Representation of the structural differences between healthy (a) andcataract (b) membranes. In healthy membranes, themomogeneous distribution ofcontact areas ensures normal communication between neighboring cells (c) whereas
in cataract membranes, the lack of connexons results in abnormal cell-to-celladhesion (d). Furthermore, the absence of connexons in the unhealthy tissue resultsin cellular starvation and waste accumulation.

CONCLUSION

High-resolution AFM imaging provides
an ideal means to investigate thestructural differences between healthy
and cataract lens cell membranes.
This is a very promising result in theongoing push to utilize SPM technology
in the investigation of disease causes
at the molecular level. AFM has anestablished capability to analyze
individual molecules. Since it is now
well accepted that many pathologies
originate from molecular disorders, it
can be expected that the AFM techniquewill become increasingly important inmedical imaging in the near future.

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