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Biophysics of Proteins at Surfaces: Assembly, Activation, Signaling
Thursday Speaker Abstracts
Spatial Organization of Na
+
/K
+
-ATPase and Lipid Domains in Free-standing Membranes
Captured on a Solid Support
Tripta Bhatia
1
, John Hjort Ipsen
1
, Flemming Cornelius
2
, Ole G. Mouritsen
1
,
Adam Cohen
Simonsen
1
.
1
University of Southern Denmark, Odense M, Denmark,
2
Aarhus University, Aarhus C,
Denmark.
Imaging the lateral organization of membrane proteins and coexisting lipid phases at the
nanoscale holds the key to establish a link between membrane structure and function. A dilemma
has been that available high-resolution imaging techniques are often only applicable to supported
membranes while information on the free-standing analog is wanted. As a possible solution, we
demonstrate a general methodology to fixate and image giant unilamellar vesicles (GUVs) at
sub-optical length scales. Individual GUVs are rapidly transferred to a solid support forming
planar bilayer patches. These are taken to represent a fixated state of the free-standing
membrane, where in-plane spatial structures are kinetically trapped.
High-resolution images of domain patterns in the liquid-ordered (lo) and liquid-disordered (ld)
co-existence region in the phase-diagram of ternary lipid mixtures are revealed by Atomic Force
Microscopy (AFM) scans of the patches. Macroscopic phase separation as known from
fluorescence images is found, but with superimposed fluctuations in the form of nanoscale
domains of the lo and ld phases. The size of the fluctuating domains increases as the composition
approaches the critical point, but with the enhanced spatial resolution, such fluctuations are
detected even deep in the coexistence region.
Likewise, we demonstrate the reconstitution of the Na
+
/K
+
-ATPase pump in GUVs and the
subsequent collapse to planar patches. AFM imaging reveals the spatial organization of pumps
with single molecule resolution both in host-membranes with one fluid phase and in membranes
containing two coexisting fluid phases. We comment on the localization of pumps with respect
to the domain border and the influence of pump activity on the stability of the patches.