Download Imaging Membrane Lipid Domains by Scanning Transmission X

Imaging Membrane Lipid Domains by Scanning Transmission X-Ray Microscopy
P. Greimel, Y. Senoh, T. Kobayashi
背景と目的
The cell membrane is the most essential border in the biological world. This frontier acts not only as a
barrier, but also plays a crucial role during metabolic processes. The emerging key themes indicate that
membranes are patchy, with lipid regions varying in thickness and composition. It is envisioned, that
segregated regions rich in saturated lipids and cholesterol float in a sea of unsaturated lipids poor in
cholesterol. While both regions are considered to be in the liquid phase, the region high in cholesterol –
which tends to order the lipids - is referred to as a liquid-ordered phase. The coexistence of two liquid
phases in the membrane causes phase separation, leading to a lateral variation in lipid composition. It is
postulated, that these lateral variations play a key role in many cellular events. Unfortunately, it has been
difficult to probe membrane composition at sufficient resolution in the region of tens or hundreds of
nanometers. This work aimed at the direct observation of the lipid distribution within a phase separated
membrane with a lateral resolution of less than 100 nanometers by scanning transmission X-ray
microscopy (STXM).
成 果
In order to gain a better understanding of phase separation processes in membranes, we prepared artificial
liposomes consisting of an equimolar mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)
and 1',3'-bis[1,2-dimyristoyl-sn-glycero-3-phospho]-sn-glycerol (TMCL). While DMPC and TMCL are
fully miscible in membranes at room temperature, phase separation was induced by the addition of
calcium, a divalent ion.
Figure 1: STXM image of dry DMPC/TMCL liposomes after calcium treatment at the (A) calcium, (B)
nitrogen and (C) oxygen K edges. (D) The merged image indicates the presence of distinct regions of DMPC
(contains nitrogen) and TMCL (co-localized with calcium).
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Dried liposomes were deposited on silicon nitride membranes, while liposomes suspended within a water
layer were detained between silicon nitride membranes. Imaging by STXM was conducted at the ‘PolLux’
beamline of the swiss light source (SLS) near Zuerich, Switzerland. The samples were visualized at the
calcium (~340 eV), nitrogen (~400 eV) and oxygen (~540 eV) K edges. The acquired elemental maps (see
figure 1) revealed the presence of distinct regions with a high concentration of nitrogen, only present in
DMPC as well as regions with a high concentration of calcium predominantly co-localized with TMCL.
This indicates a successful visualization of phase separation of DMPC and TMCL in the presence of
calcium at a resolution of 50 nm.
In a separate set of experiments at the SLS (‘PolLux’ beamline), we conducted near edge x-ray absorption
fine
structure
(NEXAFS)
analysis
(see
figure
2)
at
the
carbon
K-edge
of
dried
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
(DPPC) liposomes.. The DPPC spectrum exhibited the characteristic absorption maxima at about 287.2 eV
and 287.8 eV of aliphatic carbonyls and alkyl chains, respectively. DOPC exhibited an additional
absorption maximum at around 285 eV due to the presence of an unconjugated double bond from its oleic
acid residue. This distinct absorption difference of soft X-rays at 285 eV will allow a clear discrimination
between DOPC and DPPC. Furthermore, imaging of DPPC liposomes in water solution, a more natural
environment, at the carbon, nitrogen and oxygen K-edges was achieved. In general, the combination of the
revealed NEXAFS difference between DPPC and DOPC as well as the demonstration of liposome
imaging in solution represent an important prerequisite to enable the visualization of temperature
dependant phase separation in the binary system of DOPC and DPPC in the future.
Figure 2: NEXAFS of DOPC (orange line) and DPPC (blue line). Distinct absorption difference at ~285 eV
between DOPC and DPPC (green box) revealed.
参考文献
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