Regulation of Cell Polarity and Protrusion Formation by Targeting RhoA for Degradation

Abstract

The Rho family of small guanosine triphosphatases regulates actin cytoskeleton dynamics that underlie cellular functions such
as cell shape changes, migration, and polarity. We found that Smurf1, a HECT domain E3 ubiquitin ligase, regulated cell polarity
and protrusive activity and was required to maintain the transformed morphology and motility of a tumor cell. Atypical protein
kinase C zeta (PKCζ), an effector of the Cdc42/Rac1-PAR6 polarity complex, recruited Smurf1 to cellular protrusions, where
it controlled the local level of RhoA. Smurf1 thus links the polarity complex to degradation of RhoA in lamellipodia and filopodia
to prevent RhoA signaling during dynamic membrane movements.

Full text

normal ␣Syn function and its misfunction
in synucleinopathies. We established that
the ability of the protein to inhibit PLD,
promote the accumulation of lipids, influ-
ence the balance of vesicular pools, asso-
ciate with membranes in a highly selective
manner, induce ubiquitin accumulation,
and inhibit the proteasome when misfolded
are all intrinsic and biologically relevant
properties of the protein that can be uncou-
pled from each other by the effects of ␣Syn
mutations. Constructs expressing mutant
Q103 Htt did not produce similar biological
effects (supporting online material), and
unbiased genetic screens confirmed that
distinct pathways are involved in ␣Syn and
Htt toxicities (30). Notably, membrane-
bound ␣Syn is in dynamic equilibrium with
cytoplasmic forms. Just a twofold differ-
ence in expression was sufficient to cause a
catastrophic change in its behavior, induc-
ing nucleated polymerization and recruiting
protein previously associated with mem-
branes to cytoplasmic inclusions. This nu-
cleated polymerization process suggests a
mechanism by which even small changes in
the QC balance of aging neurons could
produce a toxic gain of ␣Syn function con-
comitantly with a loss of normal function.
Thus, two hypotheses [gain of toxic func-
tion or loss of normal function (31)] put
forward to explain PD can be reconciled by
a single molecular mechanism.
Note added in proof: Very recently Sin-
gleton et al.(32) reported that a triplication of
the ␣Syn locus on one chromosome (presum-
ably doubling the expression of wild-type
␣Syn) causes premature onset of PD, strong-
ly supporting our model that a small change
in the expression of ␣Syn relative to the cell’s
quality-control systems causes disease-relat-
ed toxicity.
References and Notes
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33. This work was funded by NIH/National Institute of
Neurological Disorders and Stroke (grant NS044829-
01). T.F.O. was partially supported by Programa Prax-
is XXI, Fundacao para a Ciencia e Tecnologia, Portu-
gal. We thank P. Lansbury, R. Esposito, J. Engebrecht,
H. Chang, and S. Henry for plasmids and strains and
members of the Lindquist laboratory for critical read-
ing of this manuscript.
Supporting Online Material
www.sciencemag.org/cgi/content/full/302/5651/1772/
DC1
Materials and Methods
Figs. S1 to S4
Tables S1 and S2
References
14 August 2003; accepted 13 October 2003
Regulation of Cell Polarity and
Protrusion Formation by
Targeting RhoA for Degradation
Hong-Rui Wang,*
1
Yue Zhang,*
1
Barish Ozdamar,
1,2
Abiodun A. Ogunjimi,
1
Evguenia Alexandrova,
3
Gerald H. Thomsen,
3
Jeffrey L. Wrana
1,2
†
The Rho family of small guanosine triphosphatases regulates actin cytoskeleton
dynamics that underlie cellular functions such as cell shape changes, migration,
and polarity. We found that Smurf1, a HECT domain E3 ubiquitin ligase, reg-
ulated cell polarity and protrusive activity and was required to maintain the
transformed morphology and motility of a tumor cell. Atypical protein kinase
C zeta (PKC␨), an effector of the Cdc42/Rac1-PAR6 polarity complex, recruited
Smurf1 to cellular protrusions, where it controlled the local level of RhoA.
Smurf1 thus links the polarity complex to degradation of RhoA in lamellipodia
and filopodia to prevent RhoA signaling during dynamic membrane movements.
The Rho family of small guanosine triphos-
phatases (GTPases) cycle between an active
guanosine 5⬘-triphosphate (GTP)– bound and
inactive guanosine 5⬘-diphosphate (GDP)–
bound state to control cell shape, motility,
polarity, and behavior (1–5). At the leading
edge of motile cells, Cdc42 and Rac1 regulate
the actin cytoskeleton to form fingerlike
filopodia and sheetlike lamellipodia, respec-
tively, whereas in the cell body RhoA induc-
es assembly of focal adhesions and contrac-
tile actin-myosin stress fibers. Active Rho
GTPases signal through effector complexes
(5, 6), one of which is the PAR (for parti-
tioning defective) polarity complex (7).
PAR6 is a key component of this complex
that binds atypical protein kinase C zeta
(PKC␨)(8–10), recruits it to active Cdc42
(8–10), and is important for cell transforma-
tion (10), polarity (11, 12), and epithelial
tight junctions (9, 13–15). One effector path-
way of this complex involves GSK-3␤ and
APC, which links PAR6 to microtubules and
astrocyte polarity (16); another, involving
Lgl, affects asymmetric cell divisions (17)
and polarization of migrating cells (18). A
direct link between the polarity complex and
regulation of actin cytoskeleton dynamics has
not been defined.
Conjugation of polyubiquitin chains to
protein targets triggers their degradation and
is mediated by E3 enzymes, which include
the Smurf family of C2-WW-HECT ubiquitin
ligases (19, 20) that regulate transforming
growth factor–␤ (TGF-␤) signal transduction
(21–23). Ubiquitin ligases are not known to
regulate cell shape, motility, and polarity.
1
Program in Molecular Biology and Cancer, Samuel
Lunenfeld Research Institute, Mount Sinai Hospital,
Toronto M56 1⫻5, Canada.
2
Department of Medical
Genetics and Microbiology, University of Toronto,
Toronto M5S 1A8, Canada.
3
Department of Biochem-
istry and Cell Biology, Center for Developmental Ge-
netics, CMM 348, Stony Brook University, Stony
Brook, NY 11794 –5215, USA.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-
mail: wrana@mshri.on.ca
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However, when Smurf1 or yellow fluorescent
protein (YFP)–tagged Smurf1 was over-
expressed in Mv1Lu epithelial cells, we ob-
served large numbers of dynamic protrusions
in 95% of expressing cells (24). These were
not present in the controls, nor in cells ex-
pressing a catalytic mutant of Smurf1, YFP-
Smurf1(C699A) (Movies S1 and S2) (Fig.
1A). Furthermore, movement of YFP-Smurf1
to the tips of processes often preceded active
extension (Fig. 1A), whereas movement to
the cell body presaged retraction (Movie S1).
Smurf1 expression also strongly reduced
stress fiber formation (Movie S1). In contrast,
Smurf2 rarely induced protrusive activity
(15% of Smurf2-expressing cells). Next, we
examined how increased expression of
Smurf1 affected the behavior of NIH3T3 fi-
broblasts in a wounding assay in which cells
are induced to polarize and migrate into a
wound that is created by scratching the
monolayer with a pipette tip. In controls, the
leading-edge cells displayed organized mi-
gration (fig. S1), whereas Smurf1-expressing
cells were highly disorganized and extended
more protrusions at the leading edge (2.4
protrusions per cell in Smurf1-expressing
populations versus 1.3 in the controls) (Fig.
1B). This suggested that Smurf1 disrupted
polarity, which we examined directly by an-
alyzing the localization of pericentrin, a com-
ponent of the microtubule organization center
(MTOC) (25) (fig. S1). In controls, 76 ⫾ 8%
of cells at the wound edge exhibited polarized
MTOC localization, compared with 30 ⫾ 6%
in wild-type Smurf1-expressing cells, indi-
cating nearly random (25%) polarity (Fig.
1B). In contrast, Smurf1(C699A) had only
slight effects on protrusive activity and
MTOC polarity. Thus, elevated Smurf1 ex-
pression induces protrusive activity and dis-
rupts fibroblast polarity in a manner that is
dependent on the catalytic activity of its
HECT domain.
To determine the importance of endoge-
nous Smurf1 on protrusive activity, we de-
signed small interfering RNA (siRNA) to
Smurf1 that reduced Smurf1 protein by 60%
compared with controls (fig. S2A). We used
the human embryonic kidney (HEK) 293T
tumor cell line because these cells are highly
transfectable, express Smurf1, extend many
cellular protrusions, and display a disorga-
nized actin cytoskeleton typical of a trans-
formed phenotype (Fig. 1C). Examination of
the morphology of Smurf1 siRNA-
transfected cells revealed a marked change at
40 hours after transfection. The cells lost their
protrusions, the actin cytoskeleton was re-
arranged into a cortical F-actin staining pat-
tern, and the cells assumed a cuboidal mor-
phology (Fig. 1C). Similar effects were ob-
served with a second siRNA directed to
Smurf1. In addition, HEK293T motility
through modified Boyden chambers was re-
duced sixfold (fig. S3). These data indicate
that Smurf1 plays an important role in regu-
lating protrusive activity and the transformed
phenotype of HEK293T cells.
Elevated expression of the Rho family
GTPases Cdc42 or Rac1 induces high levels
of protrusive activity, disrupts polarity, and
contributes to cell transformation (26, 27).
Previous studies have established that the
PAR6-PKC␨ complex is a key effector of
Cdc42 and Rac1 that controls polarity, is
localized at lamellipodia and filopodia, and is
required for the oncogenic activities of Cdc42
and Rac1 (8–11). PKC␨ activity also medi-
ates the loss of stress fibers caused by acti-
vated Cdc42 (26), and in HEK293T cells
PKC␨ inhibitors caused morphological
changes that were similar to those in Smurf1
siRNA-treated cells (28). All of this suggest-
ed that Smurf1 might function as an effector
of this complex. In support of this notion, in
Mv1Lu and NIH3T3 cells, Smurf1 was local-
ized to both lamellipodial- and filopodial-like
protrusions (Fig. 2, A and B), where it colo-
calized extensively with PKC␨ (Fig. 2B).
Furthermore, both the expression of Smurf1
and the kinase activity of PKC␨ were re-
quired for the localization of both proteins to
Fig. 1. Smurf1 is localized to lamellipodia and filopodia and regulates protrusive activity, polarity,
and motility. (A) Mv1Lu cells were transfected with YFP control or YFP fused to wild-type Smurf1
(WT) or Smurf1(C699A) (CA) and imaged by time lapse fluorescence microscopy. Shown are frames
extracted from the supplemental movies at the indicated times (in minutes). The start position of
the tip of a protrusion in a YFP-Smurf1– expressing cell at the beginning of data collection (time
0) is marked (red bar), and movement of the YFP-Smurf1 puncta is shown highlighted (yellow
arrowhead). Note that extension of the tip coincides with the arrival of Smurf1 at the tip. (B)
Regulation of protrusive activity and polarity in NIH3T3 cells. (Top) Protrusions formed per cell in
a NIH3T3 wounding assay were quantitated (mean ⫾ SEM) and are plotted for each of the
indicated cell lines. (Bottom) Polarity was measured by counting the percentage (⫾SEM) of cells in
the front row that oriented their MTOC in the forward-facing quadrant. (C) Loss of protrusions and
actin cytoskeleton rearrangements induced by Smurf1 siRNA. HEK293T cells were transfected with
control or Smurf1 siRNA and visualized 40 hours later by phase contrast microscopy (top) or Texas
red–phalloidin staining (red; bottom).
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membrane protrusions (fig. S4, A and B).
This is consistent with the requirement for
atypical PKC activity in the assembly and
function of PAR complexes in Caenorhabdi-
tis elegans and during tight junction forma-
tion in epithelial cells (13, 14, 29, 30). Next,
we demonstrated that endogenous PKC␨
bound Smurf1 and that this was unaffected by
treatment with PKC␨ kinase inhibitors (Fig.
2C). Similar kinase-independent interactions
were observed between bacterially produced
proteins (fig. S4C) and in transiently trans-
fected HEK293T cells, where the steady-state
level of PKC␨ was unaffected by Smurf1 (fig.
S4D). This indicates that PKC␨ is not a sub-
strate of Smurf1. Thus, PKC␨ binds directly
to Smurf1 independent of its kinase activity,
but both the expression of Smurf1 and PKC␨
kinase activity are required to localize the
complex to filopodia and lamellipodia.
We hypothesized that Smurf1 might be an
effector of the Cdc42-PAR6-PKC␨ pola-
rity complex that mediates the ubiquitin-
dependent degradation of RhoA. In support
of this, wild-type Smurf1, but neither Smurf2
nor catalytically inactive Smurf1, decreased
the steady-state level of RhoA, but had no
effect on Cdc42 or Rac1 (Fig. 3A); this result
was confirmed with the UPR assay (31) (fig.
S5). Two proteasome inhibitors, LLnL and
lactacystin, reversed this effect (Fig. 3B)
(28). Furthermore, Smurf1 expression mark-
edly increased ubiquitin-conjugated RhoA,
whereas Smurf1(C699A) blocked all detect-
able ubiquitination of RhoA (Fig. 3C). Next,
we examined RhoA interaction with Smurf1,
using catalytically inactive Smurf1 to trap the
ligase substrate (21, 32, 33). Smurf1(C699A)
was found to interact with the dominant in-
active form of RhoA, RhoA
N19
(Fig. 3D),
which binds constitutively to guanine nucle-
otide exchange factors (GEFs) (34). Smurf1
also interacted in vitro with either nucleotide-
free or GDP-bound RhoA, whereas loading
with GTP␥S inhibited the interaction (Fig.
3E). Because most GDP-bound RhoA in cells
is associated with guanine dissociation inhib-
itor (GDI) (35), these results suggest that GDI
may inhibit binding of Smurf1 to GDP-bound
RhoA in mammalian cells. Finally, Smurf1
directly catalyzed ubiquitination of RhoA in
an in vitro ubiquitination assay (Fig. 3F).
Thus, RhoA is a direct substrate of Smurf1,
and degradation of RhoA in mammalian cells
may require nucleotide exchange and be de-
pendent on a Rho GEF.
The PKC␨-dependent recruitment of
Smurf1 to active membrane protrusions sug-
gested that at endogenous levels of expres-
sion, Smurf1 activity toward RhoA was re-
stricted to sites where the Smurf1-PKC␨
complex assembled with Cdc42-PAR6 to in-
duce membrane protrusions. Consistent with
this notion, we did not see marked changes in
total RhoA protein levels upon reducing
Smurf1 expression (28). Thus, we considered
that the morphological change in HEK293T
cells treated with Smurf1 siRNA might be
due to ectopic accumulation of RhoA in pro-
trusions. To test this hypothesis, we exam-
ined endogenous RhoA localization at 18
hours after Smurf1 siRNA treatment. At this
early time point— before morphological
changes have occurred—we observed strong
accumulations of RhoA and colocalized F-
actin in the tips of most cellular protrusions.
We also observed the initiation of RhoA ac-
cumulation at cell-cell junctions (Fig. 4A).
Thus, the maintenance of low local levels of
RhoA in protrusions was dependent on en-
dogenous Smurf1. To demonstrate that RhoA
degradation was a key target for Smurf1-
dependent regulation of the transformed phe-
notype of HEK293T cells, we next tested the
epistatic relation between Smurf1 and RhoA
using siRNA. siRNA directed to RhoA
blocked expression of the protein (fig. S2B
and Fig. 4B), but had no effect on the mor-
phology (fig. S6A) or F-actin distribution in
HEK293T cells (Fig. 4B). This result indi-
cates that there was little RhoA-dependent
regulation of the actin cytoskeleton in these
cells, consistent with the absence of a dis-
cernible, well-organized stress fiber network.
Notably, cotransfection with RhoA siRNA
strongly suppressed the effect of Smurf1
siRNA (fig. S6A and Fig. 4B). Thus, reduc-
ing RhoA expression blocks the morpholog-
ical transformation and actin cytoskeleton re-
arrangements caused by reducing Smurf1
levels. We also observed that siRNA to RhoA
severely impaired MTOC polarity in NIH3T3
cells (fig. S6B), supporting the notion that
Smurf1 targeting of RhoA is important for
regulating polarity. Our results point to a key
role for Smurf1 as an effector of PKC␨ that
regulates actin cytoskeleton dynamics during
protrusion formation by targeting the local-
ized degradation of RhoA via a ubiquitin-
dependent mechanism.
Regulation of cell polarity and shape by
RhoGTPase-dependent regulation of the actin
cytoskeleton is a key biological pathway that
governs diverse cell functions such as local-
ization of embryonic determinants, establish-
ment of tissue and organ architecture, and cell
motility. The precise temporal and spatial
coordination of Rho family GTPase activity
is important in a broad range of cellular
activities (2–5). Our findings strongly sug-
gest that Smurf1 is a key effector of the
Cdc42/Rac1-PAR6-PKC␨ pathway that an-
tagonizes RhoA through ubiquitin-dependent
degradation. In cells in which Smurf1 expres-
sion is knocked down by siRNA, RhoA and
associated F-actin accumulate in cellular pro-
trusions, indicating that the activity of
Fig. 2. PKC␨ colocalizes with and binds
Smurf1. (A) Localization of endogenous
Smurf1 was visualized by immunostaining
Mv1Lu cells with a Smurf1 monoclonal anti-
body followed by fluorescein isothiocyanate–
conjugated secondary antibody (green); the
actin cytoskeleton was visualized with Texas
red–phalloidin staining (red). Smurf1 in protru-
sions is marked (yellow arrowheads). Controls
conducted in the absence of primary antibody
showed no detectable signal (27). (B) Colocal-
ization of Smurf1 and PKC␨. Endogenous PKC␨
(i) and Smurf1 CA (ii) were visualized in
NIH3T3-Smurf1 CA cells, and colocalization
(iii) of PKC␨ and Flag-tagged Smurf1 to pro-
trusions and lamellipodial-like structures is in-
dicated (yellow arrowheads). (C) Interaction of
endogenous Smurf1 and PKC␨ in Mv1Lu cells. Lysates prepared from cells incubated in the absence
or presence of the indicated inhibitors were subjected to immunoprecipitation with rabbit antibody
to Smurf1 (anti-Smurf1) followed by immunoblotting with anti-PKC␨.
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Fig. 3. Smurf1 targets RhoA for ubiquitination and degradation through
ubiquitin-dependent proteasome pathway. (A) Expression of Smurf1 de-
creases the steady-state level of RhoA. HEK293T cells were transiently
transfected with the indicated combinations of wild-type (WT) or cata-
lytically inactive (CA) Flag-tagged Smurf1 (F/Smurf1) or Flag-tagged
Smurf2 (F/Smurf2) and Flag-tagged RhoA (F/RhoA), Cdc42 (F/Cdc42), or
Rac1 (F/Rac1). Steady-state protein levels were determined by immuno-
blotting (IB) total cell lysates with anti-Flag. (B) Proteasome inhibitors
decrease RhoA down-regulation by Smurf1. HEK293T cells transfected
with hemagglutinin (HA)–tagged RhoA (HA/RhoA) and F/Smurf1 were
treated for 4 hours with or without 40 ␮M LLnL, and RhoA steady-state
levels determined. (C) Ubiquitination of RhoA in HEK293T cells. After
overnight treatment with 20 ␮M LLnL, lysates from cells transfected with
HA-tagged ubiquitin (HA/Ub), F/RhoA, or Myc-tagged Smurf1 (M/
Smurf1) (WT or CA) were subjected to anti-Flag immunoprecipitation,
eluted by boiling in 1% SDS, and then reprecipitated with anti-Flag (2X
IP). Ubiquitin-conjugated RhoA [(Ub)
n
-RhoA] and free RhoA were detected
by immunoblotting with the appropriate antibodies. M/Smurf1 expression
was confirmed by anti-Myc immunoblotting samples of total lysates. (D)
Interaction of Smurf1 with RhoA. HEK293T cells were transfected with
F/Smurf1(CA) and various versions of HA/RhoA (WT, V14, or N19). Cell
lysates were subjected to anti-Flag immunoprecipitation followed by immu-
noblotting (IB) with rat anti-HA to detect associated RhoA. Total protein
expression was confirmed by immunoblotting as shown. (E) Interaction
between Smurf1 and RhoA in vitro. Samples of bacterially produced RhoA
treated with or without 0.25 mM GDP or GTP␥S, as indicated, were
incubated with glutathione beads coupled to glutathione S-transferase
(GST ) or GST/Smurf1(CA). Associated RhoA (top) and input RhoA (bottom)
were determined by immunoblotting (IB) with the indicated antibody. GST
was detected by Ponceau S staining. (F) Direct ubiquitination of RhoA by
Smurf1. RhoA and WT or C699A Smurf1 were purified from bacteria and
subjected to an in vitro ubiquitination assay.
Fig. 4. Endogenous RhoA is targeted by Smurf1 in protrusions. (A)
Reduction in Smurf1 leads to appearance of endogenous RhoA in pro-
trusions of HEK293T cells. HEK293T cells transfected with luciferase
control or Smurf1 siRNA as indicated were fixed 18 hours after trans-
fection. Endogenous RhoA staining was visualized with an anti-RhoA
primary antibody (green), and actin was detected with Texas red–
conjugated phalloidin (red). Superimposition of the images (merge)
reveals colocalization of RhoA and F-actin in the protrusions of Smurf1
siRNA-treated HEK293T cells (yellow arrowheads). (B) Reduction of
endogenous RhoA by RhoA siRNA rescues the morphological change
caused by Smurf1 siRNA. HEK293T cells were transfected with Smurf1,
RhoA, or control siRNA, as indicated. Cells were fixed 40 hours after
transfection, and RhoA and F actin were visualized by immunofluores-
cence microscopy as described in (A).
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Smurf1 toward RhoA is locally restricted to
sites of active protrusion. This is likely to be
achieved through PKC␨-dependent recruit-
ment of Smurf1 to filopodia and lamellipo-
dia, which is in agreement with a key role for
this kinase in controlling the activity of the
polarity complex both in C. elegans (29, 30)
and in mammals (13, 14). Furthermore, we
showed that Smurf1 binds RhoA in a GEF-
dependent manner, suggesting that Smurf1
activity is further restricted by a RhoA GEF
that colocalizes with the polarity complex in
filopodia and lamellipodia. Localizing the deg-
radation of RhoA to protrusive regions likely
acts to prevent inappropriate stress fiber forma-
tion during dynamic actin cytoskeletal remod-
eling that is required to drive rapid filopodial
and lamellipodial membrane extensions in re-
sponse to Cdc42 and Rac1 activation. More-
over, our observation that knocking down
Smurf1 expression suppresses the tumorigenic
morphology and motility of HEK293T cells
suggests that this pathway plays a key role in
maintaining the protrusive activity and trans-
formed phenotype of cancer cells.
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36. We thank L. Attisano and members of the Wrana
laboratory for discussion and comments on the
manuscript; S. Elowe, D. Lin, T. Pawson, I. B. Wein-
stein, D. Bohmann, J. Sheng, and A. Varshavsky for
providing reagents; S. Kulkarni for help with decon-
volution microscopy and time-lapse imaging; and L.
Locke for advice on the motility assay. We are in-
debted to J. Trimmer and L. Buckwalder for help in
preparation of monoclonal antibodies. This work was
supported by grants from the Canadian Cancer Soci-
ety and the Canadian Institutes of Health Research
(J.L.W.) and NIH grant HD32429 and Carol M. Bald-
win Breast Cancer Foundation (G.H.T.). Y.Z. and B.O.
are supported by a Canadian Institutes of Health
Research (CIHR) Postdoctoral Fellowship and Doctor-
al Studentship awards, respectively, and E.A. is the
recipient of an Institute for Cell and Developmental
Biology Predoctoral Fellowship from Stony Brook
University. J.L.W. is a CIHR Investigator and an Inter-
national Scholar of the Howard Hughes Medical
Institute.
Supporting Online Material
www.sciencemag.org/cgi/content/full/302/5651/1775/
DC1
Materials and Methods
Figs. S1 to S6
Movies S1 and S2
25 August 2003; accepted 16 October 2003
Redox Regulation of Germline
and Vulval Development in
Caenorhabditis elegans
Yukimasa Shibata,* Robyn Branicky, Irene Oviedo Landaverde,
Siegfried Hekimi†
In vitro studies have indicated that reactive oxygen species (ROS) and the
oxidation of signaling molecules are important mediators of signal transduc-
tion. We have identified two pathways by which the altered redox chemistry
of the clk-1 mutants of Caenorhabditis elegans acts in vivo on germline de-
velopment. One pathway depends on the oxidation of an analog of vertebrate
low density lipoprotein (LDL) and acts on the germline through the Ack-related
tyrosine kinase (ARK-1) kinase and inositol trisphosphate (IP
3
) signaling. The
other pathway is the oncogenic ras signaling pathway, whose action on germ-
line as well as vulval development appears to be modulated by cytoplasmic ROS.
Reactive oxygen species (ROS) are short-
lived reactive molecules that can modify
cellular components including nucleic ac-
ids, proteins, and lipids. For example, the
oxidation of LDLs by ROS is one of the
causative factors of atherosclerosis (1).
ROS are toxic but the oxidation of macro-
molecules can also serve as a signaling
device (2). Moreover, in vitro studies have
shown that ROS act as intracellular mes-
sengers in signal transduction pathways,
such as ras signaling (3, 4). However, little
is known about the effect of ROS on signal
transduction in intact animals (5).
Ubiquinone (UQ or coenzyme Q) is a
redox-active lipid that has numerous bio-
chemical roles and is involved in the
production of ROS. However, UQ is also an
antioxidant that prevents the initiation and/
or propagation of lipid peroxidation in cellu-
lar membranes (6). The Caenorhabditis el-
egans clk-1 gene encodes a conserved en-
zyme that is necessary for UQ biosynthesis
(7). In the absence of CLK-1, mutants accu-
mulate demethoxyubiquinone (DMQ) (8, 9),
which can partially replace UQ as an electron
carrier (8). However, clk-1 mutants require
dietary UQ for their survival (9, 10).
clk-1 mutants show a highly pleiotropic
phenotype that includes an average slowing
down and deregulation of a number of phys-
iological processes, including aging (11).
Presumably, given that clk-1 mutants obtain
significant amounts of UQ from their diet
(12), these phenotypes result from the pres-
ence of DMQ, which might be a better anti-
oxidant than UQ (13). Thus, the clk-1 mutant
phenotypes could be the consequence of al-
tered redox signaling.
In addition to the previously described
phenotypes, we find that somatic and germ-
line development are desynchronized in clk-1
mutants. In wild-type hermaphrodites, prima-
ry spermatocytes and sperm are observed at
the late fourth larval stage (L4), and oogen-
esis commences shortly after the adult molt
(Fig. 1). However, the majority of clk-1 mu-
tants are either before or in the process of
spermatogenesis at the adult molt (fig. S1A),
and only 3% of the anterior gonads have
initiated oogenesis at 6 hours after the adult
molt (Fig. 1, B and C). Although the devel-
Department of Biology, McGill University, 1205 Ave-
nue Docteur Penfield, Montre´al, Que´bec, Canada, H3A
1B1.
*Present address: RIKEN Center for Developmental
Biology, 2-2-3 Minatojima Minamimachi, Chuo-ku,
Kobe 650-0047, Japan.
†To whom correspondence may be addressed. E-mail:
siegfried.hekimi@mcgill.ca
R EPORTS
www.sciencemag.org SCIENCE VOL 302 5 DECEMBER 2003 1779