Biology Reference
In-Depth Information
Incorporation of new lipid esters to existing LDs was shown by polarizing flow
cytometry of isolated LDs (
Kellner-Weibel, McHendry-Rinde, Haynes, & Adelman,
2001
), fluorescence microscopy using a polyene-lipid (
Kuerschner, Moessinger, &
Thiele, 2008
), and multiplex coherent anti-Stokes Raman scattering microscopy
(
Rinia, Burger, Bonn, & Muller, 2008
). The EM method described here has an ad-
vantage over the other methods in that it can observe much smaller LDs than other
methods and determine whether newly synthesized lipid esters make LDs on
their own.
13.2
OBSERVATION OF THE LD SURFACE
13.2.1
Rationale
The absence of a distinct membrane structure in the periphery of LDs indicates that
LDs are not covered by the conventional phospholipid bilayer membrane. Instead,
the LD surface is thought to be made of a phospholipid monolayer, which should
form a stable interface by making hydrophilic head groups and hydrophobic acyl
chains to face the cytoplasm and lipid esters, respectively.
The presence of a phospholipid monolayer in the LD surface was proved by TEM
utilizing the presence of a single layer of phosphorus atoms (
Z
ΒΌ
15) (
Tauchi-Sato,
Ozeki, Houjou, Taguchi, & Fujimoto, 2002
). For this purpose, isolated LDs were
directly observed without any fixation, embedding, or staining, so that the electron
density of the phosphorus atom stood out among elements of small atomic numbers
(e.g., H, C, N, O). Observation of such biological materials requires the use of cryoe-
lectron microscopy that keeps the specimen in a frozen state to minimize the damage
from the electron bombardment.
13.2.2
Methods
13.2.2.1
Isolation of LDs
All the procedures are done at 4
C. Culture cells are detached from the substrate by
incubating in PBS containing 5 mM EDTA. The cells suspended in a homogeniza-
tion buffer (HB: 25 mM Tris, 100 mMKCl, 1 mM EDTA, 1 mM EGTA, pH 7.4) are
kept in a nitrogen bomb at 400 psi for 15 min and disrupted by a slow release from
the bomb. The homogenate is centrifuged for 5 min at 300
g
to pellet down undis-
rupted cells and nuclei. The supernatant is mixed with an equal volume of 1.08 M
sucrose in HB to make the final sucrose concentration at 0.54 M and placed at the
bottom of the ultracentrifugation tube. It is overlayed with 0.27 M sucrose in HB,
0.135 M sucrose in HB, and a top solution (25 mMTris, 1 mM EDTA, 1 mM EGTA,
pH 7.4), and centrifuged at 30,000 rpm for 1 h by using a Beckman SW41 rotor. LDs
are obtained as a white layer on the top surface.
