Biology Reference
In-Depth Information
differential equation (for detailed mathematical deduction please see (
Gong et al.,
2011
)), we express the neutral lipid exchange rate per LDCS,
'
as follows:
d
I
1
dln
a
ð
bI
1
Þ
' ¼
d
t
¼
ð
a
bI
1
Þ
b
d
t
I
1
is the mean optical intensity of LD1 at different time points. Since we assume
'
and
b
as constant, ln(
a
bI
1
) and
t
are linearly correlated.
We next performed linear fitting of ln(
a
bI
1
) against
t
to calculate the lipid
exchange rates. At least four time points after photobleaching should be used for lin-
ear fittings.
R
2
(correlation coefficient square) of all linear fittings should be above
0.95. Note that
a
can be calculated from each time point and will not fluctuate sig-
nificantly. Using time-adapted constant
a
will further increase the linearity of fitting.
14.2.2.2
Representative results
Using the approach described above, we have identified Plin1 as a specific activator
of Fsp27. When coexpressed with Fsp27-GFP in 3T3-L1 preadipocytes, Plin1 can
drastically increase Fsp27-mediated neutral lipid exchange by 10-fold (
Fig. 14.2
).
Since the rate of neutral lipid exchange is positively correlated with size of the fusion
pore/channel, Plin1 may help expand the pore or lipid transfer channel to enhance
Fsp27-mediated lipid exchange (
Sun et al., 2013
). Same approach has been used
to measure inter-LD neutral lipid exchange in 3T3-L1 adipocytes and MEF-derived
adipocytes (
Gong et al., 2011
).
It is important to note that in our algorithm, the LD size has been taken into
account and normalized in the calculation. Although LD size affects the fluorescence
recovery curve, we observed that LD pairs with various size combinations show sim-
ilar lipid exchange rates in cells expressing Fsp27-GFP. In addition, we observed that
lipid exchange rates are similar between LD pairs with unequal sizes, regardless
which LD (the large or small one) is photobleached. Therefore, the size of LD within
the contacted LD pairs does not affect the lipid exchange rate. We have previously
reported that Fsp27-meidated lipid exchange was observed in paraformaldehyde-
fixed cells, suggesting that the structure of lipid-diffusion pore can be preserved after
fixation. However, cautions need to be taken in this experiment as the duration and
effectiveness of fixation may affect the pore structure and the lipid exchange rate.
FRAP assay performed in live cells is more reliable.
14.3
MEASURING LD FUSION RATE
The final step of Fsp27-mediated LD fusion involves the transfer of bulk neutral
lipids from the smaller to the larger LD within the contacting pair, resulting in
the fusion of two LDs into a larger one. Thus, the fusion rate could be expressed
as the net lipid transfer rate from the donor LD to the acceptor LD within the fusion
pair. Since Fsp27-dependent LD fusion did not occur between LDs with the same
