In metazoa, the nuclear envelope (NE) is comprised of a double
membrane and an underlying nuclear lamina. The double
membrane is peppered with nuclear pores that enable movement
of cellular components between the nucleus and the cytoplasm,
whereas the nuclear lamina forms contacts with chromosomes and
contributes to overall nuclear architecture (reviewed by Crisp and
Burke, 2008). During cell division, the NE undergoes dramatic
changes (reviewed by Hetzer et al., 2005): the NE breaks down
early in mitosis, allowing spindle microtubules to gain access to
the chromosomes, and it reassembles around the segregated DNA
masses during later stages of mitosis. The endoplasmic reticulum
(ER) has an integral role in this cycle of NE breakdown (NEBD)
and reassembly (Prunuske and Ullman, 2006): during NEBD,
components of the NE, such as nuclear pore components and inner
nuclear membrane proteins, are absorbed by the ER, and the ER
itself becomes enriched in membrane tubules (Ellenberg et al.,
1997; Puhka et al., 2007). During the initial stages of NE
reassembly, ER tubules form contacts with chromosomes. These
tubules then flatten to form an intact nuclear double membrane
(Anderson and Hetzer, 2007). Given that the NE is an extensive
membrane sheet, the formation of the NE requires a change in
the ER topology, from tubules to sheets. Indeed, Anderson and
Hetzer showed recently that the levels of reticulons, conserved
proteins that stabilize ER tubules (Voeltz et al., 2006), affect the
timing of NE formation: high levels of reticulons, which favor
tubule formation, delay NE formation whereas low levels of
reticulons, which increase the abundance of ER sheets, accelerate
NE formation (Anderson and Hetzer, 2008).
Reticulons are not the only cellular component that affects ER
shape. Studies in yeast have shown that deletion of the gene encoding
the phosphatidic acid phophohydrolase Pah1p (also known as Smp2p
in budding yeast and Ned1p in fission yeast), which converts
phosphatidic acid to diacylglycerol, leads to the appearance of ER
sheets (Campbell et al., 2006; Han et al., 2006; Santos-Rosa et al.,
2005; Siniossoglou et al., 1998; Tange et al., 2002). Budding yeast
pah1? mutants exhibit elevated levels of phosphatidic acid,
phosphatidylinositol and phosphatidylethanolamine and reduced
levels of diacylglycerol and triacyglycerol. In addition, pah1?
mutants and mutations in genes that regulate Pah1p (e.g. spo7?,
nem1?) exhibit an abnormal nuclear morphology (Campbell et al.,
2006; Han et al., 2006; Santos-Rosa et al., 2005; Siniossoglou et al.,
1998; Tange et al., 2002). The effect of Pah1p inactivation on both
ER and nuclear morphology is intriguing, because it further suggests
a functional link between ER organization and NE dynamics.
However, because both budding and fission yeasts undergo closed
mitosis (i.e. the NE does not break down during mitosis), these model
organisms are not suitable for examining whether Pah1p-dependent
ER perturbations affect NEBD and reassembly.
PAH1 is the yeast homolog of the mammalian lipin (Reue and
Brindley, 2008); mammals have three lipin homologs: LPIN1,
LPIN2 and LPIN3. In mice, inactivation of lipin-1 causes reduced
adipose tissue mass, insulin resistance and lipid storage defects
(Peterfy et al., 2001; Reue, 2007). The latter is consistent with a
The nuclear envelope (NE) is a dynamic structure, undergoing
periods of growth, breakdown and reassembly during the cell
cycle. In yeast, altering lipid synthesis by inactivating the yeast
homolog of lipin, a phosphatidic acid phosphohydrolase, leads
to disorganization of the peripheral ER and abnormal nuclear
shape. These results suggest that lipid metabolism contributes
to NE dynamics; however, since yeast undergo closed mitosis,
the relevance of these observations to higher eukaryotes is
unclear. In mammals, lipin has been implicated in adipose tissue
differentiation, insulin resistance, lipid storage and obesity, but
the underlying cellular defects caused by altering lipin levels
are not known. Here, we identify the Caenorhabditis elegans
lipin homolog (LPIN-1) and examine its affect on NE dynamics.
We find that downregulating LPIN-1 by RNAi results in the
appearance of membrane sheets and other abnormal structures
in the peripheral ER. Moreover, lpin-1 RNAi causes defects in
NE breakdown, abnormal chromosome segregation and
irregular nuclear morphology. These results uncover cellular
processes affected by lipin in metazoa, and suggest that lipid
synthesis has a role in NE dynamics.
Supplementary material available online at
Key words: Peripheral ER, Lamin, Nuclear shape, Nuclear
architecture, Lipid droplets, Diabetes, Insulin resistance, Majeed
Inactivation of the C. elegans lipin homolog leads to
ER disorganization and to defects in the breakdown
and reassembly of the nuclear envelope
Andy Golden1, Jun Liu2and Orna Cohen-Fix3,*
1The Laboratory of Biochemistry and Genetics and National Institute of Diabetes and Digestive and Kidney Diseases and 3The Laboratory of
Molecular and Cellular Biology, National Institutes of Health, 8 Center Drive, Bethesda, MD 20892, USA
2Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
*Author for correspondence (e-mail: email@example.com)
Accepted 9 March 2009
Journal of Cell Science 122, 1970-1978 Published by The Company of Biologists 2009
Journal of Cell Science
Lipin affects ER and nuclear morphology
defect in the synthesis of triacylglycerol, the major component of
stored lipid. In addition, lipin-1 overexpression leads to obesity
(Phan and Reue, 2005). In humans, mutations in LPIN2 cause
Majeed syndrome (Ferguson et al., 2005), an autoinflammatory
disorder, and mutations in either LPIN1 or LPIN2 are associated
with metabolic syndrome and type-2 diabetes (Reue and Brindley,
2008). Little is known about the function of lipin-3. Although the
physiological consequences of mammalian lipin inactivation and
overexpression have been examined, the effect of mammalian lipin
inactivation on ER structure and NE dynamics has not been
explored. In this study, we took advantage of the nematode C.
elegans, which has a single lipin gene, to examine what effect, if
any, lipin has on ER organization and NE breakdown and
C. elegans lipin is needed for lipid storage and development
The sequence of the C. elegans genome revealed that unlike
mammals, which have three lipin genes, C. elegans has only one
putative lipin homolog, H37A05.1 (Lykidis, 2007). Since mice
lacking lipin-1 have no adipose tissue, we wished to determine
whether the C. elegans lipin homolog, which we named lpin-1,
affects the ability to accumulate and store fat. Unlike mammals,
which store fat in adipose cells, worms accumulate fat in lipid
droplets that form in intestinal and hypodermal cells. These droplets
can be visualized by the vital dye Nile red (Ashrafi et al., 2003).
To examine whether downregulation of LPIN-1 affected the
accumulation of lipid droplets, embryos of wild-type (N2) worms
were laid on bacteria expressing either control double-stranded RNA
(dsRNA) or dsRNA against lpin-1 [henceforth lpin-1(RNAi)]. The
bacterial cultures were mixed with Nile red, which was taken up
by the worms during feeding. During the first 4 days after the
embryos were laid, worms that hatched on lpin-1(RNAi) bacteria
were indistinguishable from the control worms in both size and Nile
red accumulation (Fig. 1A; and data not shown). However,
differences between control and lpin-1(RNAi) worms became
apparent as worms reached adulthood: at days 5 and 6, the lpin-
1(RNAi) worms were smaller (Fig. 1A) and accumulated
significantly less Nile red (Fig. 1B) than the control RNAi-treated
worms (see also supplementary material Fig. S1). The reduced body
size and decreased lipid droplet accumulation in the lpin-1(RNAi)
worms was not a result of decreased food intake, as the pumping
rate of lpin-1(RNAi) and control animals was similar (5.8±0.45
pumps/second and 4.96±0.01 pumps/second, respectively). The
effect of lpin-1(RNAi)on lipid droplet accumulation was comparable
to that of sbp-1(RNAi), which was found by Ashrafi and colleagues
(Ashrafi et al., 2003) to induce a significant reduction in lipid droplet
accumulation (supplementary material Fig. S1). These results
suggest that, as in mammalian cells, downregulating the activity of
the C. elegans LPIN-1 reduces the amount of neutral lipids in the
worm and leads to a lipid-storage defect. Consistent with its role
in lipid storage, a GFP::LPIN-1 fusion (see Materials and Methods)
was present in the gut, where it localizes to the cytoplasm and the
nucleus, as well as in other tissues such as head neurons, hypodermis
and vulva muscles (Fig. 1C; and data not shown). Our gfp::lpin-1
construct was not expressed in the germline, but genetic evidence
presented below suggests that LPIN-1 is present in the germline as
Despite their smaller size, lpin-1(RNAi)worms produced and laid
embryos during the same time interval as control worms (4-6 days
post-hatching), indicating that their smaller size was not due to a
slower developmental program. However, lpin-1(RNAi) worms did
have a smaller brood size (66.33±11.75 per worm) compared with
controls (307.67±10.01 per worm). Moreover, although less than
1% of embryos laid by control worms failed to hatch (0.91±0.79%
of total brood), nearly 50% of embryos from lpin-1(RNAi) worms
were dead (46.67±15.90% of total brood), and embryonic lethality
exceeded 80% for embryos laid on day 6 (82.78±13.37% of
embryos laid on day 6 post-hatching). To determine whether the
embryonic lethality stems from the downregulation of LPIN-1
expressed in the soma or the germline, hatching experiments were
repeated with strains carrying ppw-1(pk1425) or rrf-1(pk1417),
leading to defects in RNAi in the germline or soma, respectively
(Tijsterman et al., 2002; Grishok et al., 2005). L4 larvae of these
two strains, as well as an N2 wild-type control, were placed on
bacteria expressing lpin-1 dsRNA or control dsRNA, and hatching
of embryos laid 24-48 hours later was determined. As expected,
hatching of control worms was nearly 100% for all three strains
(n>300 for each of the three strains). By contrast, lpin-1(RNAi)
induced embryonic lethality in N2 and the rrf-1(pk1417) strain
[lethality, 91.18±6.04% (n=453) and 71.71±15.56% (n=365),
respectively], whereas no lethality was observed for the lpin-
1(RNAi)ppw-1(pk1425) strain (100% hatching in three independent
experiments, n=358). Since rrf-1(pk1417) strains are defective in
RNAi only in the soma, and ppw-1(pk1425) strains are defective
in RNAi only in the germline, these results suggest that the
embryonic lethality induced by lpin-1(RNAi) is probably due to
downregulation of LPIN-1 present in the germline.
We also examined the phenotype of a small deletion in the lpin-
1 gene, lpin-1(ok2761), which removed 518 bp near the 5? end of
the lpin-1 gene coding region, deleting most of the second exon
and part of third exon. Worms homozygous for lpin-1(ok2761)
hatched normally, but failed to progress past the L1 stage and died
2-3 days after hatching (see Materials and Methods). Given the
RNAi results, it is likely that maternal LPIN-1 is required for
embryogenesis, and that lpin-1(ok2761) homozygous worms, which
are derived from heterozygous mothers, can develop until the
maternal stores of LPIN-1 are depleted. Taken together, these results
show that C. elegans lpin-1 is needed for the formation of lipid
droplet and is important for development.
Downregulation of LPIN-1 results in disorganization of the
Thus far, the effect of lipin inactivation on ER structure has only
been examined in yeast (Campbell et al., 2006; Han et al., 2006;
Santos-Rosa et al., 2005; Siniossoglou et al., 1998; Tange et al.,
2002). To determine whether lipin of a metazoan organism has an
analogous function, L4-staged C. elegans larvae expressing the ER
marker SP12::GFP (Poteryaev et al., 2005) and histone H2B fused
to Cherry Red (henceforth H2B::CR) were grown on bacteria
expressing either control dsRNA or lpin-1 dsRNA. After 48 hours,
ER structure was examined in young embryos at two focal planes:
a central plane that traverses the nuclei and a peripheral plane that
includes mostly peripheral ER. In control worms, the peripheral
ER appears as a network of fine tubules with occasional small
patches (Fig. 2A) (Poteryaev et al., 2005). lpin-1(RNAi) led to a
disruption of ER structure, with the appearance of membrane sheets,
patches and ring or vesicle-like structures (Fig. 2B; Fig. 3B).
Although the morphology of the ER is known to change during the
cell cycle (Poteryaev et al., 2005), the ER structures seen following
lpin-1 dsRNA did not resemble an ER organization seen at any
stage during a normal cell cycle. The abrogation of ER structure
Journal of Cell Science
Keren Witkin and Lynn Boyd for comments on the manuscript and
helpful discussions, Herong Shi and Rachel Fairbank for help in
generating the npp-1::gfp strains, and members of the Golden and
Cohen-Fix laboratories for technical support and encouragement.
Finally, we thank Mátyás Gorjánácz and Iain Mattaj for sharing their
results prior to publication. This work was funded by intramural NIDDK
grants to A.G. and O.C.F., and NIH R01 GM066953 and a grant from
the Muscular Dystrophy Association to J.L. Deposited in PMC for
release after 12 months.
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Journal of Cell Science