Biology Reference
In-Depth Information
4.1.5.1
Class III Arf1/COPI complex regulates LD breakdown
ADP-ribosylation factor 1 (Arf1) and coatomer protein complex I (COPI) regulate
Golgi to endoplasmic reticulum (ER) retrograde vesicular transport (
D'Souza-
Schorey & Chavrier, 2006
). We were surprised to observe knocking down eight
subunits of the Arf1/COPI complex specifically caused slightly larger and more dis-
persed LDs in S2 cells (
Guo et al., 2008
). We referred to this striking phenotype as
“marbles on the table” during our visual scoring. This phenotype is evolutionary con-
served in mammalian cells and yeast. We also discovered that the ARF-GDP form is
enriched on LD surfaces and the ARF-GTP form promotes LD breakdown. The
COPI complex has also been identified as a lipid homeostasis regulator from an in-
dependent RNAi screen in
Drosophila
Kc
167
cells using a different RNAi library
(
Beller et al., 2008
). Although this phenotypic class has been independently
confirmed by several groups (
Soni et al., 2009; Takashima et al., 2011
), it remains
controversial at this point whether the LD phenotype is through Arf1/COPI complex
dependent transport of adipose triglyceride lipase (ATGL) to LD surfaces.
4.1.5.2
Class V phosphatidylcholine biosynthesis genes regulate LD size
Class V is the most visually appealing phenotype identified in our screen. Knocking
down genes in this class, we observed most cells contained a few giant LDs com-
pared to the LD clusters in the control condition (
Fig. 4.1
). Genes in this class reg-
ulate phosphatidylcholine (PC) biosynthesis in
Drosophila
.
The most abundant phospholipid on S2 cell LD surfaces was PE (phosphatidyleth-
anolamine), followed by PC, then PS (phosphatidylserine) and PI (phosphatidylinosi-
tol). Only the knockdown of PC biosynthesis enzymes specifically caused LD fusion to
form the giant LDs (
Krahmer et al., 2011
). In subsequent studies, we identified the rate
limiting step enzyme in PC biosynthesis, CTP:phosphocholine cytidylyltransferase
(CCT), which targeted LD surfaces and mediated LD expansion (
Krahmer et al.,
2011
). PC is uniquely crucial among phospholipids to stabilize growing LDs and pre-
vent their coalescence. This might be attributed to its unique biophysical property
of having a larger head group and thus being more cylindrical compared to other phos-
pholipids. This characteristic leads to better shielding of the neutral lipid stored within
the LD core and might make the negatively curved fusion intermediate less energet-
ically favorable to prevent LD fusion events (
Krahmer et al., 2011
).
Genome-wide RNAi screens provide an unbiased approach to identify novel reg-
ulators for biological processes. Our experience with RNAi screens in
Drosophila
S2
cells shows that by combining thoughtful design with robust readout and stringent
validation, biological processes can be studied on a genome-wide scale within a rea-
sonable time frame and at affordable cost in a standard academic laboratory setting.
Several screens focused on LD biology in the past few years provide the initial road-
map to understanding this fascinating cellular organelle.
We also acknowledge that there are general limitations in both the cell-based
screens and the organism-level screens. For the two cell-based LD screens (
Beller
et al., 2008; Guo et al., 2008
), both S2 and Kc
167
cells are cultured
Drosophila
cell
lines derived from late stage
Drosophila
embryos (
Bourouis & Jarry, 1983;
