Polarity is critical for axis specification and for generating cell
diversity during development. In metazoans, cell polarity is
mediated in part by a conserved set of regulatory proteins, known
collectively as the PAR (partitioning-defective) proteins. The one-
cell C. elegans embryo establishes an anterior-posterior (A-P) axis
shortly after fertilization and serves as a model for studying
polarity (Goldstein and Macara, 2007; Schneider and Bowerman,
2003). The PAR proteins include the PDZ domain-containing PAR-
3 and PAR-6, the atypical protein kinase PKC-3, the serine/
threonine kinase PAR-1 and, in nematodes, the putative ubiquitin
E3 ligase PAR-2.
In the C. elegans embryo, a cue associated with the sperm
centrosome signals a local downregulation of contractile forces in
the posterior (Motegi and Sugimoto, 2006), triggering a myosin II-
dependent contraction towards the future anterior pole (Munro et
al., 2004). The contraction generates cortical flows away from the
paternal pronucleus, and serves to restrict PAR-3, PAR-6 and PKC-
3 to the anterior of the one-cell embryo (Cheeks et al., 2004;
Munro et al., 2004). As PAR-3, PAR-6 and PKC-3 become
enriched on the anterior cortex, PAR-2 and PAR-1 are recruited to
the posterior cortex (Cuenca et al., 2003). The asymmetric
distribution of the actomyosin cytoskeleton toward the anterior
gives rise to a transient cortical invagination called the
pseudocleavage furrow that marks the boundary between the
contractile anterior cortex and the smooth posterior cortex (Munro
et al., 2004).
Additionally, pseudocleavage represents the completion of
polarity ‘establishment’ and marks the beginning of the
‘maintenance’ phase (Cuenca et al., 2003). During polarity
maintenance, the anterior and posterior PAR proteins act in a
mutually antagonistic manner to perpetuate the cortical
asymmetries generated by the cortical flows (Cheeks et al., 2004;
Cuenca et al., 2003; Munro et al., 2004). PAR-2 prevents the return
of the anterior PAR proteins to the posterior cortex by inhibiting a
flow of cortical cytoplasm directed towards the posterior (Cheeks
et al., 2004; Munro et al., 2004). In turn, phosphorylation by PKC-
3 antagonizes the cortical localization of PAR-2, preventing the
posterior PAR-2 domain from extending into the anterior (Hao et
Although most of the PAR proteins have been shown to be crucial
polarity components in a variety of animal systems (Goldstein and
Macara, 2007), PAR-2 is puzzling because it appears to be nematode
specific. A possible answer to this puzzle is that PAR-2 has taken on
a function in nematodes that is more commonly carried out by
another protein, or proteins, in other polarity systems. In many
systems, the PAR proteins interact with a number of other polarity
modules, one of which includes the conserved tumor-suppressor
protein Lethal giant larvae [Lgl; also known as L(2)gl] (Betschinger
et al., 2003; Plant et al., 2003; Vasioukhin, 2006; Wirtz-Peitz and
Knoblich, 2006; Yamanaka et al., 2003). The overall sequence and
domain structure of Lgl are well conserved in metazoans. Lgl family
members contain a characteristic C-terminal ‘Lgl domain’ that is not
predicted to have a catalytic function. The protein includes a highly
conserved series of aPKC phosphorylation consensus sequences
(Vasioukhin, 2006). The N-terminal domain of Lgl homologs
typically contains a series of WD-40 repeats that are predicted to
form consecutive seven-bladed b-propeller structures. b-propeller
structures are often involved in scaffolding protein-protein
interactions (Vasioukhin, 2006).
Development 137, 3995-4004 (2010) doi:10.1242/dev.056028
© 2010. Published by The Company of Biologists Ltd
Department of Molecular Biology and Genetics, Cornell University, 433
Biotechnology Building, Ithaca, NY 14850, USA.
*Author for correspondence (firstname.lastname@example.org)
Accepted 27 September 2010
Polarity is essential for generating cell diversity. The one-cell C. elegans embryo serves as a model for studying the establishment
and maintenance of polarity. In the early embryo, a myosin II-dependent contraction of the cortical meshwork asymmetrically
distributes the highly conserved PDZ proteins PAR-3 and PAR-6, as well as an atypical protein kinase C (PKC-3), to the anterior.
The RING-finger protein PAR-2 becomes enriched on the posterior cortex and prevents these three proteins from returning to the
posterior. In addition to the PAR proteins, other proteins are required for polarity in many metazoans. One example is the
conserved Drosophila tumor-suppressor protein Lethal giant larvae (Lgl). In Drosophila and mammals, Lgl contributes to the
maintenance of cell polarity and plays a role in asymmetric cell division. We have found that the C. elegans homolog of Lgl, LGL-
1, has a role in polarity but is not essential. It localizes asymmetrically to the posterior of the early embryo in a PKC-3-dependent
manner, and functions redundantly with PAR-2 to maintain polarity. Furthermore, overexpression of LGL-1 is sufficient to rescue
loss of PAR-2 function. LGL-1 negatively regulates the accumulation of myosin (NMY-2) on the posterior cortex, representing a
possible mechanism by which LGL-1 might contribute to polarity maintenance.
KEY WORDS: PAR proteins, Asymmetric cell division, Polarity, Caenorhabditis elegans
The C. elegans homolog of Drosophila Lethal giant larvae
functions redundantly with PAR-2 to maintain polarity in the
Alexander Beatty, Diane Morton and Kenneth Kemphues*
Lgl is required in a number of polarized cell types (Vasioukhin,
2006; Wirtz-Peitz and Knoblich, 2006). In Drosophila, Lgl is
involved in the maintenance of polarity in epithelial cells (Bilder
et al., 2000; Hutterer et al., 2004). In Drosophila embryonic
epithelial cells, Lgl is primarily localized to the basolateral
membrane, where it contributes to the maintenance of polarity by
restricting apical proteins to the appropriate cortical domain
(Hutterer et al., 2004). Lgl acts by antagonizing the activity of
apical protein complexes that consist of Par-6–Bazooka(Par-3)-
aPKC and Crumbs-Stardust-Patj (Hutterer et al., 2004; Tanentzapf
and Tepass, 2003). Similarly, the apical complexes act to inhibit
Lgl function on the apical membrane (Hutterer et al., 2004). This
antagonistic relationship results in the maintenance of distinct
cortical domains and is reminiscent of the mutual exclusion
feedback loop that facilitates polarity maintenance in the C. elegans
Drosophila Lgl also plays a role in asymmetric cell division
(Ohshiro et al., 2000; Peng et al., 2000; Wirtz-Peitz et al., 2008).
In neuroblasts, Lgl is required for the basal targeting of fate
determinants prior to mitotic division and, in this context, plays a
role in the formation of polarity early in mitosis (Ohshiro et al.,
2000; Peng et al., 2000; Wirtz-Peitz and Knoblich, 2006). Lgl may
be involved in spindle positioning in neuroblasts (Albertson and
Doe, 2003) and is also involved in the asymmetric cell divisions of
sensory organ precursors, where the protein is required for
asymmetric localization of the cell-fate determinant Numb (Wirtz-
Peitz et al., 2008).
Recent studies have shown that Lgl is involved in the
polarization of the A-P axis in the Drosophila oocyte (Doerflinger
et al., 2010; Fichelson et al., 2010; Li et al., 2008; Tian and Deng,
2008). In the early oocyte, Lgl is required for the proper posterior
translocation of cell fate determinants as well as the centrosomes
(Fichelson et al., 2010; Tian and Deng, 2008). At mid-oogenesis,
phosphorylation by aPKC restricts Lgl to the posterior of the
oocyte, along with Par-1 (Tian and Deng, 2008). After the oocyte
has been polarized into distinct cortical domains, Lgl is likely to
stabilize the cortical localization of Par-1, and these proteins act to
reciprocally inhibit the anterior Bazooka complex. As in other
contexts, the mutual antagonism between proteins in opposing
cortical domains serves to maintain polarity (Doerflinger et al.,
Despite being a fundamental polarity component in a number of
polarized cell types, the role of an Lgl homolog in C. elegans has
not yet been determined. Here, we show that a C. elegans homolog
of Lgl, LGL-1, functions redundantly with PAR-2 to maintain
polarity, and provide evidence that LGL-1 acts by preventing the
cortical accumulation of NMY-2 in the posterior cortex of the early
MATERIALS AND METHODS
Nematodes were cultured using standard conditions (Brenner, 1974). N2
(Bristol) was used as wild type. Mutations used in this analysis include par-
2(e2030), par-2(it5) (Kemphues et al., 1988), par-2(lw32), par-2(it87)
(Cheng et al., 1995), nmy-2(ne1490), nmy-2(ne3401) (Liu et al., 2010),
unc-119(ed4) (Maduro and Pilgrim, 1995), lgl-1(tm2616), provided by the
National Bioresource Project at Tokyo Women’s Medical College, and lgl-
1(it31) (this study). We confirmed that the tm2616 allele of lgl-1 is a 211
bp deletion with a 9 bp insertion that begins in the sixth intron and ends in
the seventh intron. We determined the transcript produced by the tm2616
mutant using RT-PCR (First-Strand cDNA Synthesis Kit, Amersham)
followed by sequencing. tm2616 was out-crossed to N2 six times.
We also used the transgene zuIs45[nmy-2::NMY-2::GFP] (Nance et al.,
2003). lgl-1 transgenic strains created for this study are listed in Table 1.
RNA interference (RNAi)
RNAi was performed by feeding (Timmons and Fire, 1998), with the
exception of lgl-1(RNAi) in par-2(it5), which was performed either by
feeding or by injection (Fire et al., 1998). All RNAi feeding experiments
involving par-2 mutants were performed at 16°C, and the worms were
allowed to feed for 48-60 hours. All other RNAi experiments were
performed at 25°C, and worms were allowed to feed for 36 hours prior to
imaging or immunostaining.
Images of live embryos using either differential interference contrast (DIC)
or wide-field fluorescence microscopy were captured using a Leica DM
RA2 microscope with a 63? Leica HCX PL APO oil-immersion lens, a
Hamamatsu ORCA-ER digital camera, and Openlab software
(Improvision). Blastomere cross-sectional areas were measured using
Openlab. Fixed embryos were imaged using a Leica TCS SP2 system with
a Leica DMRE-7 microscope and an HCX PL APO 63? oil-immersion
lens. Images were processed using Leica Confocal Software and Adobe
Photoshop CS4. The images in Fig. 8 were captured with a PerkinElmer
UltraVIEW LCI confocal scanner with a Nikon Eclipse TE2000-U
microscope using UltraVIEW Imaging Suite v5.5. The step size was 1 mM
with 10-14 sections per stack. The sections were stacked and processed
using ImageJ and Adobe Photoshop CS4.
LGL-1 polyclonal antibody production
A C-terminal fragment of LGL-1 (amino acids 490-941) fused to GST was
used to generate a polyclonal antibody in guinea pigs. Antibody production
was performed by Pocono Rabbit Farm & Laboratory (Canadensis, PA,
USA). Crude serum was blot-affinity purified using GST-LGL-1(490-941)
prior to use.
Immunostaining of PAR-2, GFP, LGL-1 and PKC-3 in embryos was
performed using methanol fixation as described (Guo and Kemphues,
1995). Primary antibodies used include: anti-PAR-2 rabbit polyclonal
(Boyd et al., 1996); anti-GFP
Immunochemicals); anti-PKC-3 rat polyclonal (Hung and Kemphues,
1999) and anti-LGL-1 guinea pig (this study). Secondary antibodies used
goat polyclonal (Rockland
Development 137 (23)
Table 1. Transgenic strains used in this study
unc-119(ed3); zuIs45[nmy-2::NMY-2::GFP + unc-119(+)]; lgl-1(tm2616)
itIs256[plgl-1::lgl-1::gfp + unc-119(+)]; unc-119(ed4); lgl-1(tm2616)
itIs279[plgl-1::lgl-1::mCherry + unc-119(+)]; unc-119(ed4); lgl-1(tm2616)
itIs281[plgl-1::lgl-1S661A;S665A;T669A::mCherry+ unc-119(+)]; unc-119(ed4); lgl-1(tm2616)
itIs282[plgl-1::lgl-1S661E;S665E;T669E::mCherry+ unc-119(+)]; unc-119(ed4); lgl-1(tm2616)
itIs256[plgl-1::lgl-1::gfp + unc-119(+)]; unc-119(ed4); par-2(it5); lgl-1(tm2616)
itIs285[plgl-1::lgl-1S877N::gfp + unc-119(+)]; unc-119(ed4); par-2(it5)/sC1 [dpy-1(s2171)]; lgl-1(tm2616)
itIs285[plgl-1::lgl-1::gfpS877N+ unc-119(+)]; unc-119(ed4); lgl-1(tm2616)
itIs281[plgl-1::lgl-1::mCherryS661A;S665A;T669A+ unc-119(+)]; unc-119(ed4); par-2(it5)/sC1 [dpy-1(s2171)], lgl-1(tm2616)
itIs282[plgl-1::lgl-1::mCherryS661E;S665E;T669E+ unc-119(+)]; unc-119(ed4); par-2(it5)/sC1 [dpy-1(s2171)]; lgl-1(tm2616)
itIs279[plgl-1::lgl-1::mCherry + unc-119(+)]; unc-119(ed4); par-2(it5); lgl-1(tm2616)
par-2(lw32), unc-45(e286ts), itIs256[plgl-1::lgl-1::gfp + unc-119(+)]; unc-119(ed4)
include: donkey anti-rat Cy3, donkey anti-rabbit Cy3, donkey anti-goat
Cy3, goat anti-guinea pig Cy3 (Jackson Laboratories, West Grove, PA,
USA) and donkey anti-goat Alexa 488 (Invitrogen). Samples were
mounted using VectaSheild with DAPI (Vector Laboratories).
lgl-1 transgenic constructs were generated by fosmid recombineering using
a galK-positive/counterselection cassette (Warming et al., 2005). To begin,
the galK cassette (Warming et al., 2005) was amplified using primers that
include 75 bp arms on the 5? ends that are homologous to the regions
flanking the region of genomic DNA to be modified. The purified PCR
product was then transformed into SW102 cells containing a fosmid that
included lgl-1 (WRM065bB11). Homologous recombination was induced
and recombinants were selected as described previously (Warming et al.,
2005). Next, another cassette containing the region to be inserted flanked by
the 75 bp homology arms was generated either using PCR (as was the case
for fluorophores) or by annealing two homologous single-stranded primers
(as was the case for mutations). The purified cassette was transformed,
homologous recombination was induced, and recombinants were selected as
previously (Warming et al., 2005). After making the desired modification to
lgl-1 in the fosmid, the gene as well as the upstream and downstream
intergenic regions was recombineered into pJKL702 (unc-119 in pBSIISK+),
kindly provided by Kelly Liu, Cornell University. Approximately 500 bp
homology regions, corresponding to the regions directly downstream of the
gene upstream of lgl-1 (X:872011-872531) and the region directly upstream
of the gene downstream of lgl-1 (X:863852-864392), were cloned into
pJKL702 adjacent to one another in the same orientation. The vector was
then linearized by cutting between the homology regions and used as a
cassette for recombineering. Constructs were transformed using microparticle
bombardment (Praitis et al., 2001). At least two independent integrated
transgenic lines were examined for each construct. For the KK1080 strain
(see Table 1), we sequenced the transgene expressed in the line after
bombardment to ensure that the correct construct was transformed. One line
for each construct was used to test for rescue.
Loss of lgl-1 function enhances the maternal-
effect embryonic lethality of weak par-2 mutants
The C. elegans gene F56F10.4 (lgl-1) is predicted to encode a
protein homologous to Lgl (Vasioukhin, 2006). Although clearly a
member of the Lgl family, lgl-1 is more diverged at the primary
sequence level than other family members [13.7% identical to
Drosophila lgl and 14.1% identical to mouse Llgl1 (Mgl1)]. To
determine whether lgl-1 is required for polarity in the early
embryo, we examined nematodes homozygous for a deletion/
insertion allele of lgl-1, tm2616. tm2616 is predicted to result in a
frameshift after codon 342, causing a premature stop codon after
amino acid 350 (Fig. 1). Embryos from lgl-1(tm2616) mutants
were 99.2±0.4% embryonic viable (Fig. 2A, n675) and did not
exhibit early polarity defects (Fig. 2C). In both wild-type and
lgl-1(tm2616) embryos the anterior blastomere (AB) accounted for
similar proportions of the total area of the two-cell zygote
[56.8±2.2% in wild type (N2) and 56.5±2.1% in lgl-1(tm2616);
P0.74, n15]. Furthermore, as in wild type, the second cleavages
in lgl-1(tm2616) embryos were asynchronous, and the mitotic
spindle was oriented transversely in the AB cell and longitudinally
in the P1 cell (Fig. 2C, n15). Thus, in contrast to its essential role
in other polarity systems (Vasioukhin, 2006; Wirtz-Peitz and
Knoblich, 2006), LGL-1 function is dispensable for polarity in the
C. elegans embryo.
In C. elegans, the putative ubiquitin E3 ligase PAR-2 is required
to maintain polarity in the early embryo by restricting the
distribution of the anterior polarity proteins PAR-3, PAR-6 and
PKC-3 (Cuenca et al., 2003; Hao et al., 2006; Kemphues et al.,
1988). Of the PAR proteins, only PAR-2 is not conserved outside
of nematodes (Goldstein and Macara, 2007). Because the role of
Lgl proteins in polarity in other animals is analogous to that of
PAR-2 in C. elegans, we hypothesized that C. elegans LGL-1
functions redundantly with PAR-2. To test this hypothesis, we used
RNAi to deplete LGL-1 in par-2(it5) at the permissive temperature
of 16°C. We noted a dramatic enhancement of embryonic lethality.
Embryos from homozygous par-2(it5) are predominantly viable at
the permissive temperature, but the mutants exhibit strong
temperature sensitivity (Cheng et al., 1995). At permissive
temperature, RNAi control par-2(it5) worms exhibited 15.4±4%
embryonic lethality (n878). By contrast, the embryonic lethality
of par-2(it5ts) treated with lgl-1(RNAi) was 97.0±0.5% (Fig. 2A,
n1252). Furthermore, par-2(it5); lgl-1(tm2616) double mutants
were 100% maternal-effect embryonic lethal at the permissive
temperature (Fig. 2A, n1000). This enhancement was not allele
specific: lgl-1(tm2616) in double mutant combination with the
maternal-effect sterile par-2 alleles e2030 or it87 (Cheng et al.,
1995; Kemphues et al., 1988) gave over 99% maternal-effect
embryonic lethality (Fig. 2B, n765 or 857, respectively).
In addition, we determined that depleting another C. elegans
homolog of Lgl, tom-1 (Vasioukhin, 2006), could not enhance par-
2(it5) lethality. RNAi control par-2(it5) worms gave 10.3±12%
embryonic lethality (n715), whereas par-2(it5) worms treated
with tom-1(RNAi) showed 14.9±10.3% lethality (P0.50, n839).
Mutation of lgl-1 enhances par-2 polarity defects
in the early embryo
Although loss of lgl-1 function enhanced the maternal-effect
embryonic lethality of hypomorphic par-2 mutants, it was unclear
whether the embryonic lethality was a result of enhanced early
embryonic polarity defects or whether it revealed a cryptic role for
the proteins in later embryogenesis. We used DIC microscopy to
examine the first two mitotic divisions of embryos from par-2(it5);
lgl-1(tm2616) mutants at permissive temperature. At this
temperature, most embryos from par-2(it5) mothers divided
asymmetrically (n18/19) and exhibited an asynchronous second
mitotic division with spindles oriented transversely in AB and
longitudinally in P1 (Fig. 2C, n10/13). By contrast, all embryos
from par-2(it5); lgl-1(tm2616) exhibited a strong par-2 mutant
phenotype (Fig. 2C) (Cheng et al., 1995; Hao et al., 2006;
Kemphues et al., 1988); the double mutant embryos exhibited a
symmetrical first cleavage (the AB blastomere accounted for
49.9±1.9% of the total area of the two-cell embryo) and a
synchronous second cleavage (Fig. 2C, n12/12). Additionally, the
mitotic spindles in both the AB and P1 cells were of transverse
orientation with respect to the longitudinal axis of the embryo (Fig.
1C, n12/12). Therefore, compromising lgl-1 function enhances the
polarity defects associated with par-2 in the early embryo.
LGL-1 and C. elegans polarity maintenance
Fig. 1. The C. elegans Lgl protein (LGL-1). The conserved Lgl domain
[Pfam LLGL2 (Katoh, 2004)] spans amino acids 273-368. Conserved
serines 661 and 665 and threonine 669 (asterisks) are the sites of the
LGL 3A and 3E mutations. The tm2616 deletion/insertion removes
codons for amino acids 343 to 404 and results in a frameshift (dashed
red line) and early stop to produce a 350 amino acid truncated protein.
The location of the it31 mutation is indicated.
LGL-1 is asymmetrically localized to the posterior
of the one-cell embryo and to the basolateral
cortex in epithelial cells
To determine the subcellular localization of LGL-1 in the early
embryo, we generated transgenic lines that express gfp- and
mCherry-tagged lgl-1 under the control of its endogenous promoter
(see Materials and methods). Similar to PAR-2 (Boyd et al., 1996),
LGL-1::GFP and LGL-1:mCherry localized asymmetrically to the
posterior cortex of the one-cell embryo (Fig. 3A; see Movie 1 in
the supplementary material; see below). Unlike PAR-2, however,
low levels of LGL-1::GFP were present throughout the cortex just
prior to polarization, and the anterior localization of the protein
persisted even as it became enriched at the posterior (see Movie 1
in the supplementary material).
The fluorescently tagged transgenes rescued the enhanced
maternal-effect embryonic lethality of par-2(it5) by lgl-1(tm2616) at
the permissive temperature. For example, expression of LGL-1::GFP
in par-2(it5); lgl-1(tm2616) mutants resulted in 98% viable embryos
(Fig. 3B, n927). Immunostaining fixed embryos with a polyclonal
antibody raised against the N-terminus of LGL-1 confirmed the
localization pattern of LGL-1 (see Fig. S1 in the supplementary
material). The subcellular localization of LGL-1 to the posterior
cortex in the early embryo is consistent with the hypothesis that
LGL-1 acts redundantly with PAR-2 to maintain polarity.
We also observed that LGL-1::GFP localized asymmetrically in
differentiated epithelial cells. In the elongating embryo, LGL-
1::GFP localized to the basolateral cortex of gut and epidermal cells
(Fig. 4). This subcellular distribution suggests that LGL-1 has a
role in polarity in differentiated epithelial cells in addition to its role
in the early embryo. Because the embryos from lgl-1(tm2616) are
viable, the function of lgl-1 in these epithelial tissues, if any, is
likely to be redundant.
PKC-3 is required for the asymmetric cortical
localization of LGL-1
In asymmetrically dividing Drosophila neuroblasts, migrating
fibroblasts and polarized mammalian epithelial cells, Lgl is a
substrate for aPKC (Betschinger et al., 2003; Plant et al., 2003;
Yamanaka et al., 2003). In Drosophila, phosphorylation of Lgl on
a series of conserved aPKC consensus sites results in an
intramolecular association between the N-terminal and C-terminal
domains of the protein, resulting in cortical disassociation and,
presumably, inactivation (Betschinger et al., 2005).
C. elegans LGL-1 includes a highly conserved motif that
contains three putative aPKC phosphorylation sites (S661,
S665 and T669), suggesting that LGL-1 might be similarly
regulated by the aPKC homolog PKC-3 in the early embryo
(Fig. 1) (Vasioukhin, 2006). To test this hypothesis, we used
RNAi to deplete PKC-3 and monitored the localization of LGL-
1::GFP. After depleting PKC-3, LGL-1::GFP was no longer
restricted to the posterior cortex following polarity establishment
(Fig. 5A; see Movie 2 in the supplementary material). Thus,
PKC-3 is required for the asymmetric localization of LGL-
To determine whether the putative PKC-3 phosphorylation
sites are required for LGL-1 asymmetry, we generated transgenic
lines expressing a mutant form of LGL-1::mCherry (LGL-
13A::mCherry) in which the three putative PKC-3 phosphorylation
sites are mutated to alanine. Consistent with the expected role of
these conserved serines, LGL-13A::mCherry was strongly cortical
prior to, and failed to become asymmetric during, polarity
establishment. Instead, the mutant protein remained uniformly
distributed throughout the cortex (Fig. 5B). Additionally, the
cortical signal of LGL-13A::mCherry appeared more intense
relative to the cytoplasmic signal when compared with wild type
(Fig. 5B). LGL-13A::mCherry failed to rescue the enhancement
of par-2(it5) by lgl-1(tm2616) at the permissive temperature: both
par-2(it5); lgl-1(tm2616)and par-2(it5); lgl-1(tm2616)
expressing LGL-13A::mCherry were 100% maternal-effect
embryonic lethal (n1000 for each genotype). Furthermore, par-
2(it5)/sC1 [dpy-1(s2171)]; lgl-1(tm2616) worms expressing
LGL-13A::mCherry were 91±4.0% viable at the permissive
temperature, suggesting that ectopic localization of LGL-
13A::mCherry at the anterior of the one-cell embryo did not
substantially affect embryonic viability in a dominant-negative
Development 137 (23)
Fig. 2. Loss of lgl-1 function enhances weak
par-2 mutants. (A)The percentage of C. elegans
embryos of the indicated genotypes that failed to
complete embryogenesis. Error bars indicate s.d.
(B)Percentage embryonic lethality of weak par-2
alleles compared with the respective par-2; lgl-1
double mutant. The lgl-1 mutation used is
tm2616. (C)DIC images of two-cell embryos
during interphase (left column) and prior to the
second mitotic division (right column). The black
lines mark the orientation of the mitotic spindles.
Anterior is to the left in all figures.
We also generated a phosphomimetic mutant (LGL-
1S661E;S665E;T669E, or LGL-13E) by mutating the potential PKC-3
phosphorylation sites to glutamic acid. We expected this mutant to
be cytoplasmic. However, although cytoplasmic levels were clearly
higher than for wild-type LGL-1::mCherry, LGL-13E::mCherry was
still detectable at the cortex (Fig. 5B). Expression of LGL-
13E::mCherry failed to rescue the enhancement of par-2(it5) by
lgl-1(tm2616): embryos from par-2(it5); lgl-1(tm2616); lgl-
13E::mCherry exhibited 100% lethality (n1000).
We conclude that one or more of the three putative PKC-3
phosphorylation sites is required for LGL-1 asymmetry, consistent
with the hypothesis that phosphorylation of LGL-1 by PKC-3
negatively regulates the cortical accumulation of LGL-1 in the
Overexpression of LGL-1 is sufficient to rescue
PAR-2 loss of function
par-2(lw32) is a strong allele that produces a truncated PAR-2
protein of a predicted 233 amino acids (Levitan et al., 1994) that
lacks the domain required for cortical localization (Hao et al.,
2006). If LGL-1 and PAR-2 function redundantly, we hypothesized
that overexpression of LGL-1 might be sufficient to rescue the
lethality of par-2(lw32). To test this, we crossed the lgl-1::gfp
transgene into par-2(lw32) and quantified embryonic lethality of
par-2(lw32) expressing LGL-1::GFP. Embryos from par-2(lw32)
exhibited 98.0±0.4% lethality (n2216). By contrast, the
embryonic lethality of par-2(lw32); lgl-1::gfp was 5.7±0.6%,
suggesting that overexpression of LGL-1 robustly rescued par-2
loss of function (Fig. 6A, n1128).
par-2(lw32) produces a truncated protein and might not be a true
functional null. To confirm that expression of the lgl-1::gfp
transgene bypassed the need for PAR-2, rather than acting through
residual PAR-2, we treated par-2(lw32); lgl-1::gfp worms with
par-2(RNAi). As expected, if par-2(lw32) were a functional null,
par-2(RNAi) did not enhance the embryonic lethality of par-
2(lw32) (Fig. 6A, n2473). Furthermore, par-2(lw32); lgl-1::gfp
treated with par-2(RNAi) had similar levels of embryonic lethality
to those fed bacteria containing empty vector (Fig. 6A, n2091).
Assuming that par-2 RNAi removes any residual active PAR-2 in
the lw32 background, we conclude that expression of the lgl-1::gfp
transgene can bypass the need for PAR-2.
Finally, to confirm that the viability of par-2(lw32); lgl-1::gfp
resulted from LGL-1 overexpression, we depleted LGL-1 in par-
2(lw32); lgl-1::gfp using RNAi and scored embryonic lethality.
Embryos from par-2(lw32); lgl-1::gfp; lgl-1(RNAi) worms
exhibited 99.4±0.8% lethality, indicating that the rescue of par-
2(lw32) was the result of LGL-1 overexpression (Fig. 6A, n696).
Embryos from par-2(lw32) worms treated with lgl-1(RNAi)
exhibited 100% lethality, suggesting that the small percentage of
viable embryos produced by the par-2(lw32) mutant can be
attributed to LGL-1 function (Fig. 6A, n917).
We also compared the cortical polarization of early embryos
from par-2(lw32), par-2(lw32); lgl-1(tm2616) and par-2(lw32);
lgl-1::gfp by immunostaining endogenous PKC-3. In one-cell wild-
type embryos during anaphase, PKC-3 occupied 63.3±3.8% of the
cortex (Fig. 6B, n10). In par-2(lw32), PKC-3 extended
significantly further into the posterior (84.2±9.8%, Fig. 6B;
P1.7?10–4, n10). Furthermore, PKC-3 was found throughout the
entire cortex in par-2(lw32); lgl-1(tm21616) embryos (Fig. 6B;
P6.7?10–4, n8), consistent with the hypothesis that both PAR-2
and LGL-1 contribute to polarity maintenance. By contrast, the
extent of cortex occupied by PKC-3 in par-2(lw32); lgl-1::gfp
embryos was not significantly different than in wild type
(65.5±7.0%, Fig. 6B; P0.40, n10), indicating that overexpression
of LGL-1 can rescue the loss of par-2 function.
In wild-type two-cell embryos, PKC-3 is enriched on the anterior
cortex of the AB blastomere and in the most anterior portion of P1
(Fig. 6C) (Tabuse et al., 1998). By contrast, PKC-3 was cortically
LGL-1 and C. elegans polarity maintenance
Fig. 3. LGL-1 localizes to the posterior
cortex of the early C. elegans embryo.
(A)Confocal mid-sections of fixed embryos
immunostained for LGL-1::GFP (green) and
PAR-2 (red). Establishment refers to the time
following pronuclear decondensation but prior
to pronuclear meeting. The embryos were
dissected from mothers of genotype lgl-1::gfp;
lgl-1(tm2616). Transgenes were crossed or
transformed into the lgl-1(tm2616) mutant
background because the cortical signal of the
transgene was stronger in the absence of
functional endogenous protein. (B)Percentage
embryonic lethality for par-2(it5); lgl-
1(tm2616) and par-2(it5); lgl-1(tm2616); lgl-
1::gfp. Error bars indicate s.d.
Fig. 4. LGL-1 is asymmetrically localized to the basolateral cortex
of differentiated epithelial cells. (A)Confocal immunofluorescence
image of an elongating C. elegans embryo stained for GFP (LGL-1::GFP,
green) and PKC-3 (red). (B-G)High magnification of epidermal cells (B-
D, left-hand box in A) and gut cells (E-G, right-hand box in A).
localized in both AB and P1 in par-2(lw32) embryos, although the
intensity of the PKC-3 signal was notably weaker in the posterior
than in the anterior (Fig. 6C). Furthermore, in embryos from par-
2(lw32); lgl-1(tm2616), PKC-3 was also localized to the cortex of
both the anterior and posterior blastomeres during the two-cell
stage, but the difference in signal intensity was less substantial (Fig.
6C). We quantified the difference and found that in par-2(lw32)
two-cell embryos, the posterior to anterior signal ratio was
0.65±0.22 (n8), whereas the ratio in par-2(lw32); lgl-1(tm2616)
embryos was 0.97±0.32 (n8), indicating that the distribution of
PKC-3 is significantly more symmetrical in par-2(lw32); lgl-
1(tm2616) than in par-2(lw32) (P0.03). These data are consistent
with the idea that PAR-2 and LGL-1 function redundantly to
maintain polarity. Finally, in embryos from par-2(lw32); lgl-1::gfp,
the asymmetrical cortical localization of PKC-3 was restored and
PKC-3 was again enriched in the AB blastomere and in the most
anterior portion of P1 (Fig. 6C). Embryos from par-2(lw32); lgl-
1::gfp worms exhibited asymmetric first cell divisions (n17/19),
and the mitotic spindles of the second cell division were oriented
in transverse to the A-P axis in the AB cell and along the A-P axis
in the P1 cell (n16/18). LGL-1::GFP was also asymmetrically
localized in these embryos (see Movie 3 in the supplementary
We conclude that overexpression of LGL-1 is sufficient to
restore viability in the absence of functional PAR-2 by rescuing the
early embryonic failure in polarity maintenance of par-2 mutants.
it31 is a hypomorphic allele of lgl-1
par-2(e2030) was initially isolated in a strain with a maternal-effect
embryonic lethal phenotype; however, the embryonic lethality of
the strain was dependent on an additional locus linked to the X
chromosome (K.K., unpublished). When separated from the X-
linked locus, par-2(e2030) exhibits a maternal-effect sterile
phenotype (Kemphues et al., 1988). The X-linked mutation it31
also enhanced the maternal-effect embryonic lethality of par-2(it5).
At the permissive temperature, par-2(it5) single mutants and par-
2(it5); (it31) double mutants displayed 4.9±2.8% (n509) and
61.6±14.9% (n1077) maternal-effect embryonic lethality,
respectively (Fig. 7A). As a single mutant, it31 was viable
(2.5±1.9% embryonic lethality, Fig. 7A, n440) and did not display
Development 137 (23)
Fig. 5. PKC-3 is required for the asymmetric localization of LGL-1.
(A)Confocal mid-sections of fixed C. elegans embryos at the indicated
stage of development immunostained for LGL-1::GFP (red). The top
row shows control embryos from lgl-1::gfp; lgl-1(tm2616) worms
treated with the L4440 vector alone, and the bottom row shows
embryos from lgl-1::gfp; lgl-1(tm2616); pkc-3(RNAi). (B)Wide-field
fluorescence images from time-lapse movies of embryos progressing
through the first mitotic cell cycle. Embryos expressing LGL-1::mCherry
(top), LGL-13A::mCherry (middle) or LGL-13E::mCherry (bottom) prior to
establishment (between meiosis II and pronuclear decondensation), at
pseudocleavage, nuclear envelope breakdown (NEB), and after the first
Fig. 6. Overexpression of LGL-1 rescues par-2 loss of function.
(A)The percentage embryonic lethality in par-2(lw32) and par-2(lw32);
lgl-1::gfp C. elegans when fed L4440 vector alone (left), par-2(RNAi)
(middle) or lgl-1(RNAi) (right). Error bars indicate s.d. (B,C)Confocal
mid-sections of anaphase one-cell embryos (B) and two-cell embryos
(C) immunostained for PKC-3 (red). The centrosomal staining (B) is non-
any detectable mutant phenotype. Genetic mapping placed it31 at
the left end of linkage group X at approximately –20 cM, very near
lgl-1 at –19.5 cM, raising the possibility that it31 is a mutation
in lgl-1. Sequencing revealed a missense mutation, S877N.
Furthermore, it31 failed to complement lgl-1(tm2616) for the
ability to enhance par-2(it5). An average of 99.8±0.3% of embryos
from six par-2(it5); lgl-1(tm2616)/it31 mothers failed to hatch
(n636). Thus, it31 is an allele of lgl-1.
To determine the effect of the it31 S877N mutation on the
subcellular localization of LGL-1 we generated transgenic lines
expressing LGL-1S877N::GFP. In early embryos, LGL-1S877N::GFP
localized very weakly to the posterior cortex when compared with
LGL-1::GFP (Fig. 7B). Additionally, we stained endogenous LGL-
1 in it31 embryos and observed that the cortical signal of LGL-1
was notably weaker than in the wild type (see Fig. S1 in the
supplementary material). Taken together, these data suggest that the
serine at position 877 is required for the normal cortical
localization of LGL-1 or for stability of the protein. As expected
for a hypomorphic mutation, LGL-1S877N::GFP suppressed the
lethality of lgl-1(tm2616);par-2(it5ts) embryos less well than did
LGL-1::GFP (Fig. 7A, n970; compare with Fig. 3B).
Mutation of lgl-1 affects the cortical accumulation
of NMY-2 during polarity maintenance
During the maintenance phase of polarity, PAR-2 is required to
prevent the recruitment of NMY-2 to the posterior cortex (Cuenca
et al., 2003; Munro et al., 2004). Because LGL-1 functions
redundantly with PAR-2 during polarity maintenance, we
hypothesized that LGL-1 might also affect the posterior cortical
accumulation of NMY-2. To test this, we compared the localization
of NMY-2::GFP in par-2(RNAi) and par-2(RNAi); lgl-1(tm2616)
embryos during the first mitotic division. In wild-type embryos,
NMY-2::GFP foci became asymmetrically distributed to the anterior
half of the embryo during the establishment phase (Fig. 8A; see
Movie 4 in the supplementary material). Around the time of
pronuclear meeting, the NMY-2 foci were reorganized into finer
filaments, which remained enriched in the anterior of the embryo
(Fig. 8B) (Munro et al., 2004). During metaphase, NMY-2::GFP
was mostly restricted to the anterior half of the embryo, although
there was a patch of NMY-2::GFP that appeared at the posterior
pole (Fig. 8C). In lgl-1(tm2616), the dynamics of NMY-2::GFP
were similar to wild type (Fig. 8E,F; see Movie 5 in the
supplementary material). In par-2(RNAi) embryos, establishment of
NMY-2 asymmetry occurred relatively normally, although the cap
of NMY-2::GFP foci extended further into the posterior than in wild
type (Fig. 8G; see Movie 6 in the supplementary material) (Munro
et al., 2004). The asymmetry failed to be maintained, and shortly
after pseudocleavage NMY-2::GFP asymmetry was lost (Fig. 8H)
(Munro et al., 2004). Around the time of nuclear envelope
breakdown, however, NMY-2::GFP partially cleared from the
posterior (Fig. 8I, n10/12). NMY-2::GFP filaments extended into
the posterior, but little NMY-2::GFP was observable on the posterior
pole. In par-2(RNAi); lgl-1(tm2616) embryos, the dynamics of
NMY-2::GFP were similar to that of par-2(RNAi) embryos until
nuclear envelope breakdown (Fig. 8J,K; see Movie 7 in the
supplementary material); thereafter, in contrast to par-2(RNAi)
LGL-1 and C. elegans polarity maintenance
Fig. 7. The it31 S877N mutation compromises the ability of LGL-1
to accumulate on the posterior cortex. (A)The percentage of C.
elegans embryos of the indicated genotypes that failed to complete
embryogenesis. Error bars indicate s.d. (B)Confocal mid-sections of
fixed two-cell embryos immunostained for GFP (green).
Fig. 8. LGL-1 negatively regulates the accumulation of NMY-2 in
the posterior in the absence of PAR-2. (A-L)Confocal projections of
cortical NMY-2::GFP in single C. elegans embryos of the indicated
genotypes at pseudocleavage, during pronuclear (PN) migration, and at
nuclear envelope breakdown (NEB
embryos, the NMY-2::GFP filaments remained almost uniformly
distributed around the cortex in par-2(RNAi); lgl-1(tm2616)
embryos and, in most cases, failed to clear from the posterior (Fig.
8L, n11/12). We quantified the extent of NMY-2::GFP clearing
based on total embryo length in par-2(RNAi) and par-2(RNAi); lgl-
1(tm2616). Although NMY-2 failed to clear in most par-2(RNAi);
lgl-1(tm2616) embryos, if we included the few embryos that showed
some clearing, the average clearing was 5.3±7.1%, compared with
21.8±11.1% in par-2(RNAi) (n10, P0.001). These data suggest
that LGL-1 functions redundantly with PAR-2 to negatively regulate
the accumulation of NMY-2 in the posterior of the one-cell embryo.
Because LGL-1 inhibits the accumulation of NMY-2::GFP in the
posterior in par-2(RNAi) embryos, we hypothesized that decreasing
the dose of NMY-2 in the early embryo might be sufficient to
partially rescue par-2(it5); lgl-1(tm2616). To test this hypothesis,
we used partial RNAi to reduce NMY-2 levels in par-2(it5); lgl-
1(tm2616). NMY-2 was depleted such that a low level of lethality
was observed in N2 (15.1±13.1%). Similar to par-2(it5); lgl-
1(tm2616) controls, par-2(it5); lgl-1(tm2616); nmy-2(RNAi) was
100% embryonic lethal. Results were similar when we
compromised NMY-2 function using either of two conditional nmy-
2 alleles (Liu et al., 2010) at semi-restrictive temperature,
suggesting that reduced NMY-2 function is insufficient to partially
rescue par-2(it5); lgl-1(tm2616).
Having determined that LGL-1 influences the cortical
accumulation of NMY-2, we screened genes identified in a previous
study as affecting the cortical dynamics of the early embryo
(Sonnichsen et al., 2005) to determine whether RNAi depletion of
any of the genes blocked the ability of LGL-1::GFP to rescue par-
2(lw32) mutants. We found several genes that caused higher levels
of embryonic lethality in par-2(lw32); lgl-1::gfp relative to wild
type. One of these, let-502, a homolog of the Rho-associated kinase
ROCK (Piekny and Mains, 2002; Wissmann et al., 1997), also
compromised early embryonic polarity in par-2(lw32); lgl-1::gfp
(see Fig. S2 in the supplementary material). Specifically, five out of
12 par-2(lw32); lgl-1::gfp; let-502(RNAi) embryos had symmetrical
first cleavages, and the mitotic spindles of the second cell division
were oriented transversely to the A-P axis in both the AB and P1
cells (see Fig. S2 in the supplementary material, n13/13). When
LET-502 was depleted in N2, similar polarity defects were not
observed (see Fig. S2 in the supplementary material; n15/15) (see
also Sonnichsen et al., 2005).
Having identified a requirement for let-502 for rescue of par-
2(lw32) by overexpression of lgl-1, we also tested the possible
requirement for myotonic dystrophy-related Cdc42-binding kinase
homolog (MRCK-1). MRCK-1 is a potential downstream effector
of CDC-42, and both LET-502 and MRCK-1 are involved in the
cortical recruitment of NMY-2 in the one-cell embryo (Kumfer et
al., 2010). In embryos from par-2(lw32); lgl-1::gfp; mrck-1(RNAi)
we observed an increased frequency of symmetrical first cleavages
(n6/12) and the mitotic spindles of the second cell division were
oriented transverse to the A-P axis in both the AB and P1 cell
(n12/12), but we did not observe similar defects in mrck-1(RNAi)
embryos (see Fig. S2 in the supplementary material; n15/15) (see
also Sonnichsen et al., 2005).
PAR-2 and LGL-1 function redundantly in polarity
We have shown that LGL-1, the C. elegans homolog of Lgl,
functions redundantly with PAR-2 to maintain polarity in the
early embryo. Loss of LGL-1 function robustly enhances both
the embryonic lethality and early polarity phenotypes of
hypomorphic par-2 mutants. We also found that LGL-1 and PAR-
2 colocalize in the early embryo, and overexpressing LGL-1 in a
putative par-2 null was sufficient to restore embryonic viability
and rescue the early polarity defects associated with par-2 loss of
function. These results indicate that LGL-1 and PAR-2 function
redundantly and suggest that the respective pathways to which the
proteins belong must ultimately converge on a common target or
set of targets.
We noted that LGL-1, in addition to posterior cortical
localization in the early embryo, is strongly expressed in C. elegans
epithelial cells and is localized basolaterally. However, lgl-
1(tm2616) worms are viable and fertile with no obvious defects in
epithelial function. Since we do not detect PAR-2 in epithelial cells
and lgl-1(tm2616); par-2(lw32) worms exhibit only maternal-effect
lethality, we speculate that a polarity protein other than PAR-2
functions redundantly with LGL-1 in epithelial cells or that LGL-
1 has no function in these cells.
The cortical asymmetry of LGL-1 is regulated by
Lgl proteins in flies and mammals are regulated via
phosphorylation by aPKC (Betschinger et al., 2003; Plant et al.,
2003; Tian and Deng, 2008; Yamanaka et al., 2003). Our results are
consistent with conservation of this regulation in C. elegans. The
asymmetric localization of LGL-1 depends on the aPKC homolog
PKC-3, and mutating three conserved putative PKC-3 target sites
in LGL-1 to alanines blocks asymmetry. However, mutating these
three phosphorylation sites to glutamic acid yielded unexpected
results. We hypothesized that the phosphomimetic mutant would
fail to localize to the cortex. Instead, LGL-13E::mCherry localized
cortically and symmetrically, in addition to being distributed to the
cytoplasm. There are several possible explanations for this
unexpected result. Perhaps glutamic acid insufficiently mimics a
phosphate group to completely block the cortical localization of
LGL-1. Another possibility is that residual cortical LGL-1 is the
result of overexpression of the transgene relative to wild type lgl-
1. Alternatively, regulation of the cortical localization of LGL-1
might depend on additional sites in the protein. Finally, it is
possible that the conserved sites do not serve as phosphorylation
sites in C. elegans.
Two potential modes of LGL-1 action in C. elegans
Currently, the molecular mechanism by which Lgl participates in
polarity is not well understood. Results from Drosophila and
mammalian cells suggest three non-mutually exclusive hypotheses
to explain how LGL-1 could function (Vasioukhin, 2006; Wirtz-
Peitz and Knoblich, 2006). One hypothesis, based initially on work
on the LGL-1 homologs Sro7/77 in yeast (Hattendorf et al., 2007;
Vasioukhin, 2006; Wirtz-Peitz and Knoblich, 2006), is that LGL
could regulate polarized vesicular trafficking. Additional evidence
from metazoans supports this role: Mlgl binds a component of the
exocytic machinery, syntaxin 4, in mammalian epithelial cells
(Musch et al., 2002). The second hypothesis proposes that Lgl
could negatively regulate the activity of non-muscle myosin II.
Drosophila and human Lgl proteins bind non-muscle myosin II
(Strand et al., 1994; Strand et al., 1995). Additionally, in
Drosophila neuroblasts, reducing the dosage of the myosin II gene
zipper suppresses the loss of basal protein targeting associated with
the lgl mutation (Ohshiro et al., 2000; Peng et al., 2000).
Furthermore, Lgl may function in neuroblasts to restrict myosin to
the apical cortex (Barros et al., 2003), although this may be
Development 137 (23)
facilitated indirectly by inhibition of aPKC activity at the basal
cortex (Atwood and Prehoda, 2009). Finally, in asymmetrically
dividing cells in the Drosophila nervous system (Atwood and
Prehoda, 2009; Betschinger et al., 2003; Wirtz-Peitz et al., 2008),
Lgl appears to function by regulating the activity of aPKC, either
by inhibiting its activity (Atwood and Prehoda, 2009) or by altering
its target specificity (Wirtz-Peitz et al., 2008). It accomplishes this,
at least in part, by exchanging with PAR-3 in the PAR-6–aPKC
complex (Wirtz-Peitz et al., 2008). A similar exchange with PAR3
also occurs in mammalian epithelial cells (Plant et al., 2003;
Yamanaka et al., 2003).
The mode of action of LGL-1 in the early C. elegans embryo is
unclear. Our evidence argues strongly that the major role of LGL-
1 in the early embryo is in the maintenance, rather than
establishment, of polarity. Thus, at the time that LGL-1 acts, it is
not in a complex with PKC-3 and PAR-6 but acts to exclude these
proteins from the posterior cortex. It is possible that LGL-1
prevents binding of PAR-6 and PKC-3 at the cortex by forming
PAR-6–LGL-1–PKC-3 complexes that, in contrast to the situation
in Drosophila and mammalian cells, can no longer bind cortically.
In this model, the observed accumulation of myosin in the posterior
of lgl-1(tm2616); par-2(RNAi) embryos is a consequence, rather
than a cause, of the abnormal presence of the PAR-6–PKC-3–PAR-
3 complex. However, if LGL-1 acted by promoting dissociation of
PAR complexes from the cortex, we would expect to see dominant
effects of mislocalizing LGL-1 to the anterior and we do not.
Alternatively, LGL-1 might have an activity that is independent
of complex formation with PAR-6 and PKC-3, such as regulating
membrane trafficking or myosin activity. Of these, regulating the
recruitment of myosin or its activity at the cortex is most consistent
with our data. Inhibition of myosin activity by Lgl was previously
proposed in Drosophila embryonic neuroblasts (Peng et al., 2000).
In Drosophila neuroblasts, lgl mutations can be suppressed by
compromising myosin activity (Peng et al., 2000). We carried out
similar experiments in C. elegans to no effect, however: reducing
myosin activity using either temperature-sensitive nmy-2 mutations
or weak nmy-2(RNAi) failed to suppress the enhancing effects of
loss of LGL-1 on par-2 mutants. Evidence that LGL-1 does act at
least indirectly through myosin comes from our discovery that
rescue of par-2 mutants via LGL-1 overexpression is dependent
upon the activities of Rho kinase (let-502) and mrck-1, a
downstream effector of CDC-42. However, in contrast to the
proposed role as an inhibitor of myosin contractility, the
requirement for LET-502 and MRCK-1 argues that LGL-1
promotes myosin contractility. Perhaps, by blocking myosin
accumulation in the posterior, LGL-1 indirectly promotes increased
myosin accumulation and hence contractility in the anterior.
The discovery of a role for Lgl in polarity in C. elegans
underscores the degree to which cell polarity mechanisms are
conserved. The creation of a C. elegans strain that is completely
dependent upon LGL-1 provides a new opportunity to explore the
precise mode of action of this key protein.
Note added in proof
While this paper was under review, a similar analysis was
published by Hoege and colleagues (Hoege et al., 2010).
We thank Wendy Hoose and Mona Hassab for technical support; Sylvia Lee for
helpful comments on the manuscript; the National Bioresource Project for the
experimental animal C. elegans and for lgl-1(tm2716); the Biological Resources
Branch at NCI for recombineering reagents; the Caenorhabditis Genetics
Center for providing worm strains; and Jun Kelly Liu for providing pJKL702.
This research was supported by National Institutes of Health grants HD27689
and GM079112 to K.K. Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material for this article is available at
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Embryonic Lethality (%)