Biology Reference
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size, we proposed that the internal pressure difference due to size difference of the
contacting LDs may provide the driving force for directional lipid transfer. Both the
LD surface tension and the fusion pore/channel in LDCS may influence the LD fu-
sion rate (
Gong et al., 2011
). We labeled the LD surface and LDCS with Fsp27-GFP
and documented the process of Fsp27-mediated LD fusion under spinning disk mi-
croscope. The neutral lipid transfer rate in cultured adherent cells is then quantified
by assessing the LD size change in live cells.
14.3.1
Materials and experimental procedures
14.3.1.1
Materials
3T3-L1 preadipocytes
35 mm Labtek live cell imaging culture chamber
10 mM oleic acid coupled to BSA (10 mg/ml)
Lipofectamine 2000 (Life Technologies)
Plasmids encoding GFP-tagged LD proteins
Spinning disk microscope
14.3.1.2
Experimental procedures
1.
Procedures for 3T3-L1 preadipocytes culture, transfection, and visualization of
LD morphology by fluorescent microscopy are similar as described above. After
overnight culture, cells are transfected with Fsp27-GFP using Lipofectamine
2000 according to the manufacturer's instruction.
2.
4-6 h after transfection, cells are washed with DMEM and incubated in DMEM
containing 10% FBS and 200
m oleic acids for another 18 h.
3.
Cells are incubated in the live cell imaging chamber on the microscope for at least
15 min at 37
C with 5% CO
2
.
4.
Set 60
m
oil immersion objective.
5.
Select contacted LD pair that has intense green fluorescent signal at the LDCS in
Fsp27-GFP transfected cells. Note that the size difference between contacted
LDs has to be at least 20% or higher in order to observe efficient lipid transfer.
6.
To compare the lipid transfer rate between different conditions, we
recommend choosing similarly sized LD pairs. We usually select acceptor LD
with a diameter around 5
m(5
0.5
m) and the donor LD with a diameter
m
m
m).
7.
Set the laser power that can clearly visualize both the bright Fsp27-GFP at LDCS
and the faint GFP signal on LD surface.
8.
Take live cell imaging for 1-2 h with 30 s intervals.
around 3
m(3
0.5
m
m
14.3.2
Data analysis and representative results
According to the proposed model of Fsp27-meidated LD fusion (
Gong et al., 2011
),
the lipid transfer rate will most likely change in a nonlinear manner even in a single
fusion event due to the dynamic changes of LD sizes and LD surface tension.
