Revisiting the role of microtubules in C. elegans polarity.
ABSTRACT Cells must break symmetry to acquire polarity. Microtubules have been implicated in the induction of asymmetry in several cell types, but their role in the Caenorhabditis elegans zygote, a classic polarity model, has remained uncertain. One study (see Tsai and Ahringer on p. 397 of this issue) brings new light to this problem by demonstrating that severe loss of microtubules impairs polarity onset in C. elegans.
- [show abstract] [hide abstract]
ABSTRACT: Polarity establishment requires a symmetry-breaking event, resulting in an axis along which determinants are segregated. In Caenorhabditis elegans, oocytes are apolar and are triggered to polarize rapidly along one axis after fertilization. The establishment of this first polarity axis is revealed by the asymmetric distribution of PAR proteins and cortical activity in the one-celled embryo. Current evidence suggests that the centrosome-pronucleus complex contributed by the sperm is involved in defining the polarization axis. Here we directly assess the contribution of the centrosome to polarity establishment by laser ablating the centrosome before and during polarization. We find that the centrosome is required to initiate polarity but not to maintain it. Initiation of polarity coincides with the proximity of the centrosome to the cortex and the assembly of pericentriolar material on the immature sperm centrosome. Depletion of microtubules or the microtubule nucleator gamma-tubulin did not affect polarity establishment. These results demonstrate that the centrosome provides an initiating signal that polarizes C. elegans embryos and indicate that this signalling event might be independent of the role of the centrosome as a microtubule nucleator.Nature 10/2004; 431(7004):92-6. · 38.60 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The par genes participate in the process of establishing cellular asymmetries during the first cell cycle of Caenorhabditis elegans development. The par-2 gene is required for the unequal first cleavage and for asymmetries in cell cycle length and spindle orientation in the two resulting daughter cells. We have found that the PAR-2 protein is present in adult gonads and early embryos. In gonads, the protein is uniformly distributed at the cell cortex, and this subcellular localization depends on microfilaments. In the one-cell embryo, PAR-2 is localized to the posterior cortex and is partitioned into the posterior daughter, P1, at the first cleavage. PAR-2 exhibits a similar asymmetric cortical localization in P1, P2, and P3, the asymmetrically dividing blastomeres of germ line lineage. This distribution in embryos is very similar to that of PAR-1 protein. By analyzing the distribution of the PAR-2 protein in various par mutant backgrounds we found that proper asymmetric distribution of PAR-2 depends upon par-3 activity but not upon par-1 or par-4. par-2 activity is required for proper cortical localization of PAR-1 and this effect requires wild-type par-3 gene activity. We also find that, although par-2 activity is not required for posterior localization of P granules at the one-cell stage, it is required for proper cortical association of P granules in P1.Development 11/1996; 122(10):3075-84. · 6.21 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: It has long been appreciated that spermiogenesis, the cellular transformation of sessile spermatids into motile spermatozoa, occurs in the absence of new DNA transcription. However, few studies have addressed whether the physical presence of a sperm nucleus is required either during spermiogenesis or for subsequent sperm functions during egg activation and early zygotic development. To determine the role of the sperm nucleus in these processes, we analyzed two C. elegans mutants whose spermatids lack DNA. Here we show that these anucleate sperm not only differentiate into mature functional spermatozoa, but they also crawl toward and fertilize oocytes. Furthermore, we show that these anucleate sperm induce both normal egg activation and anterior-posterior polarity in the 1-cell C. elegans embryo. The latter finding demonstrates for the first time that although the anterior-posterior embryonic axis in C. elegans is specified by sperm, the sperm pronucleus itself is not required. Also unaffected is the completion of oocyte meiosis, formation of an impermeable eggshell, migration of the oocyte pronucleus, and the separation and expansion of the sperm-contributed centrosomes. Our investigation of these mutants confirms that, in C. elegans, neither the sperm chromatin mass nor a sperm pronucleus is required for spermiogenesis, proper egg activation, or the induction of anterior-posterior polarity.Development 02/2000; 127(2):355-66. · 6.21 Impact Factor
T H E J O U R N A L O F C E L L B I O L O G Y
© The Rockefeller University Press $30.00
The Journal of Cell Biology, Vol. 179, No. 3, November 5, 2007 367–369
Most cells become polarized during development to divide
asymmetrically, to migrate, or to organize in tissues. Local-
ized changes in the actin-rich cortex are essential to establish
and maintain polarity in many cell types (Siegrist and Doe,
2007), but what initially triggers these changes is not always
fully understood. Because polarization involves long-term re-
organization throughout the cell, the initial cue must be accu-
rate and self-reinforcing. Accumulating evidence suggest that
microtubules often serve as an internal source of asymmetry
(Siegrist and Doe, 2007). Now, a new study implicates micro-
tubules in polarization of the C. elegans zygote (Tsai and
Microtubules are polar fi laments with stable minus ends,
which are typically pointed toward the centrosome near the nu-
cleus, and unstable plus ends, which extend outward toward the
cell cortex. This organization makes them ideally suited for the
local delivery of regulators of cortical actomyosin. For example,
in newly divided fi ssion yeast, microtubules deliver Tea4p to
the new cell tip. Tea4p interacts with the actin nucleator formin/
For3p, stimulating local actin cable assembly and growth
(Martin et al., 2005). Similarly, in migrating fi broblasts, growing
microtubules at the leading edge activate the small GTPase
Rac1. Activated Rac1 stimulates actin fi lament assembly and
lamellipodial protrusions, which, in turn, accelerates micro-
tubule growth (Wittmann and Waterman-Storer, 2001). In princi-
ple, positive feedback loops between the microtubule and actin
systems could reinforce even small initial differences and lead
to robust symmetry-breaking signals.
The C. elegans zygote may, at fi rst glance, look like an-
other example of microtubule-induced polarity, but the evidence
so far has been contradictory (Siegrist and Doe, 2007). The zy-
gote becomes polarized shortly after fertilization under the in-
fl uence of the sperm and its associated centrosome (Goldstein
and Hird, 1996; Sadler and Shakes, 2000). The sperm–centrosome
complex remains near the cortex for several minutes after fer-
tilization and, therefore, is in an ideal position to deliver a
symmetry-breaking cue to the overlying actin cytoskeleton.
The actin cytoskeleton initially is under dynamic tension
throughout the cortex but becomes destabilized near the sperm–
centrosome complex, coincident with the accumulation of peri-
centriolar material (PCM) and microtubule nucleation (Fig. 1;
Munro et al., 2004). This local disruption leads to a fl ow of cor-
tical actomyosin away from the sperm–centrosome complex,
which transports polarity regulators PAR-3 (partitioning defec-
tive 3), PAR-6, and PKC-3 to the opposite pole (Munro et al.,
2004). Reciprocal inhibitory interactions between PAR-6/PKC-3/
PAR-3 in the anterior and PAR-1/PAR-2 in the posterior eventu-
ally lead to the formation of two nonoverlapping PAR domains
(Kemphues, 2000; Cuenca et al., 2003).
How does the sperm–centrosome complex trigger polar-
ity? Mutants that block PCM assembly (e.g., spd-5 and spd-2)
and laser ablation of the centrosome delay or prevent polarity
initiation (O’Connell et al., 2000; Wallenfang and Seydoux,
2000; Hamill et al., 2002; Cowan and Hyman, 2004). Although
all of the available evidence points to the centrosome as the
source of polarity, the specifi c component involved has been
diffi cult to pin down. Obvious candidates are the microtubules,
which appear around the centrosome coincident with the onset
of polarity. Indirect evidence for the involvement of micro-
tubules fi rst came from analyzing zygotes arrested in the fi rst
meiotic division (Wallenfang and Seydoux, 2000). These zy-
gotes fail to nucleate microtubules around the centrosome and
instead become polarized by the acentriolar meiotic spindle.
The meiotic spindle typically is located at the other end of the
zygote, causing an apparent reversed polarity (Fig. 1). This re-
versed polarity is sensitive to nocodazole, implicating micro-
tubules. Direct evidence for a role for microtubules in “normal”
polarity, however, has been diffi cult to obtain. Two studies using
α- and β-tubulin RNAi and/or nocodazole treatment to disrupt
microtubule assembly in wild-type zygotes failed to uncover
polarity defects (Cowan and Hyman, 2004; Sonneville and
Gonczy, 2004). In both studies, low levels of tubulin remained
Revisiting the role of microtubules in C. elegans
Fumio Motegi and Geraldine Seydoux
Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Center for Cell Dynamics, Johns Hopkins School of Medicine, Baltimore, MD 21205
Cells must break symmetry to acquire polarity. Micro-
tubules have been implicated in the induction of asymmetry
in several cell types, but their role in the Caenorhabditis
elegans zygote, a classic polarity model, has remained
uncertain. One study (see Tsai and Ahringer on p. 397
of this issue) brings new light to this problem by demon-
strating that severe loss of microtubules impairs polarity
onset in C. elegans.
Correspondence to Fumio Motegi: firstname.lastname@example.org; or Geraldine Seydoux:
JCB • VOLUME 179 • NUMBER 3 • 2007 368
around the centrosome. However, these levels appeared lower
than those observed in spd-2 mutants that fail to initiate polar-
ity, suggesting that microtubule nucleation and polarity estab-
lishment are not correlated.
This issue has been taken up again in a new study by Tsai
and Ahringer (2007), who used RNAi to achieve severe deple-
tion of α/β tubulin by RNAi in gravid hermaphrodites. Sus-
tained depletion of tubulin eventually leads to sterility, so the
authors analyzed the last embryos produced before the onset of
sterility. Zygotes that experienced the most severe loss of α/β
tubulin assembled microtubules around the sperm centrosome
later than in wild type. In these zygotes, PAR-2 localization to
the cortex was also delayed, suggesting a correlation between
microtubules and polarity. Although nonspecifi c effects caused
by the severe tubulin depletion could not be excluded, the accu-
mulation of two centrosomal markers appeared unaffected. Tsai
and Ahringer also found that a mutant that fails in PCM assem-
bly (spd-5) often develops a reversed PAR-2 domain during mi-
tosis, indicating that a centrosome-independent mechanism of
polarity exists even in embryos that are not blocked in meiosis
(Fig. 1). The spd-5 reversed polarity was highly sensitive to tu-
bulin depletion and appeared later than wild type, suggesting a
microtubule-dependent cue that is less effi cient when uncoupled
from the centrosome. Interestingly, in wild-type embryos, PAR-2
occasionally appears transiently at both poles, but the PAR-2
domain nearest the centrosome always eventually wins out
(Boyd et al., 1996; Cuenca et al., 2003). Together, these obser-
vations suggest that the symmetry-breaking cue involves
microtubules as well as a centrosome-dependent mechanism
to in crease robustness.
How could microtubules affect polarity? Actomyosin con-
tractions in the cortex require the small GTPase RHO-1, its
activator the RhoGEF (guanine nucleotide exchange factor)
ECT-2, and its negative regulator the RhoGAP (GTPase-activating
protein) CYK-4 (Jenkins et al., 2006; Motegi and Sugimoto,
2006; Schonegg and Hyman, 2006). Local inactivation of RHO-1
by the down-regulation of ECT-2 or up-regulation of CYK-4
in principle could weaken the actomyosin network, causing
an asymmetric contraction and cortical fl ows away from the
centrosome. In fact, ECT-2 and CYK-4 have been shown to be
excluded and enriched, respectively, from the cortex overlying
the centrosome (Jenkins et al., 2006; Motegi and Sugimoto,
2006). Whether microtubules contribute to these distributions
remains to be determined. RhoGEFs and RhoGAPs have been
reported to interact with proteins that associate with micro-
tubule plus ends in several organisms (Siegrist and Doe, 2007).
Figure 1. Working model for the initiation of
cortical polarity in C. elegans zygotes. Wild
type: fertilization results in a zygote with a
sperm pronucleus (light blue; right) at one end
of the zygote and a maternal pronucleus (light
blue; left) undergoing meiosis at the other end.
After meiosis (polar bodies are extruded
outside the zygote [small gray circle]), the
actomyosin-rich cortex undergoes dynamic
contractions (arrows). Upon entry into mitosis,
microtubules (green) and PCM (light orange)
are assembled around the sperm-donated cen-
trosome (orange) at one end of the zygote.
Microtubules and/or PCM-associated proteins
stimulate local disassembly of the actomyosin
network (gray) and cortical fl ows that clear an-
terior PARs, allowing PAR-2 (red) to associate
with the cortex. At the other end of the zygote,
a hypothetical meiotic spindle remnant also nu-
cleates microtubules, but without the help of a
centrosome. This weaker microtubule nucle-
ation site induces a transient PAR-2 domain
(light red), which quickly is overtaken by the
cortical fl ows coming from the opposite end.
mat-1 mutant: mat-1 mutants are arrested dur-
ing the fi rst meiotic division and never assem-
ble microtubules or PCM around the sperm
centrosome. Microtubules from the acentriolar
meiotic spindle induce an unopposed reversed
PAR-2 domain. spd-5 mutant: spd-5 mutants
progress through meiosis normally but fail to
nucleate microtubules and PCM around the
sperm centrosome in mitosis. Microtubules
from the hypothetical meiotic spindle remnant
induce an unopposed reverse PAR-2 domain.
MICROTUBULES IN C. ELEGANS POLARITY • MOTEGI AND SEYDOUX369
In C. elegans, CYK-4 is known to bind the kinesin-like protein
ZEN-4 (Mishima et al., 2002). In Drosophila melanogaster
neuroblasts, the kinesin Khc-73 is required to link the mi-
totic spindle axis to cortical polarity (Siegrist and Doe, 2005).
Clearly, it will be important to investigate whether microtubule-
associated proteins affect polarity in C. elegans. The role of the
centrosome also remains to be investigated. One possibility is
that the centrosome increases the robustness of the microtubule-
dependent cue simply by stimulating microtubule polymerization.
Alternatively, the centrosome may act independently of micro-
tubules, as suggested by the lack of correlation between polar-
ity and microtubule nucleation in spd-2 mutants (Cowan and
The results of Tsai and Ahringer (2007) provide a new
impetus for investigating the role of microtubule and centro-
some-associated proteins in C. elegans polarity. Their results
also are an important reminder of the challenges associated
with investigating the role of essential cytoskeletal components,
which are diffi cult to deplete while avoiding catastrophic effects.
Hopefully, identifi cation of the molecules that make up the
symmetry-breaking cues will clarify the role of microtubules in
C. elegans zygotes and perhaps uncover new unifying princi-
ples for how cells break symmetry.
Submitted: 9 October 2007
Accepted: 16 October 2007
Boyd, L., S. Guo, D. Levitan, D.T. Stinchcomb, and K.J. Kemphues. 1996.
PAR-2 is asymmetrically distributed and promotes association of P gran-
ules and PAR-1 with the cortex in C. elegans embryos. Development.
Cowan, C.R., and A.A. Hyman. 2004. Centrosomes direct cell polarity inde-
pendently of microtubule assembly in C. elegans embryos. Nature.
Cuenca, A.A., A. Schetter, D. Aceto, K. Kemphues, and G. Seydoux. 2003.
Polarization of the C. elegans zygote proceeds via distinct establishment
and maintenance phases. Development. 130:1255–1265.
Goldstein, B., and S.N. Hird. 1996. Specifi cation of the anteroposterior axis in
Caenorhabditis elegans. Development. 122:1467–1474.
Hamill, D.R., A.F. Severson, J.C. Carter, and B. Bowerman. 2002. Centrosome
maturation and mitotic spindle assembly in C. elegans require SPD-5, a
protein with multiple coiled-coil domains. Dev. Cell. 3:673–684.
Jenkins, N., J.R. Saam, and S.E. Mango. 2006. CYK-4/GAP provides a local-
ized cue to initiate anteroposterior polarity upon fertilization. Science.
Kemphues, K. 2000. PARsing embryonic polarity. Cell. 101:345–348.
Martin, S.G., W.H. McDonald, J.R. Yates III, and F. Chang. 2005. Tea4p links
microtubule plus ends with the formin for3p in the establishment of cell
polarity. Dev. Cell. 8:479–491.
Mishima, M., S. Kaitna, and M. Glotzer. 2002. Central spindle assembly and
cytokinesis require a kinesin-like protein/RhoGAP complex with micro-
tubule bundling activity. Dev. Cell. 2:41–54.
Motegi, F., and A. Sugimoto. 2006. Sequential functioning of the ECT-2 RhoGEF,
RHO-1 and CDC-42 establishes cell polarity in Caenorhabditis elegans
embryos. Nat. Cell Biol. 8:978–985.
Munro, E., J. Nance, and J.R. Priess. 2004. Cortical fl ows powered by asym-
metrical contraction transport PAR proteins to establish and maintain
anterior-posterior polarity in the early C. elegans embryo. Dev. Cell.
O’Connell, K.F., K.N. Maxwell, and J.G. White. 2000. The spd-2 gene is required
for polarization of the anteroposterior axis and formation of the sperm
asters in the Caenorhabditis elegans zygote. Dev. Biol. 222:55–70.
Sadler, P.L., and D.C. Shakes. 2000. Anucleate Caenorhabditis elegans sperm
can crawl, fertilize oocytes and direct anterior-posterior polarization of
the 1-cell embryo. Development. 127:355–366.
Schonegg, S., and A.A. Hyman. 2006. CDC-42 and RHO-1 coordinate acto-my-
osin contractility and PAR protein localization during polarity establish-
ment in C. elegans embryos. Development. 133:3507–3516.
Siegrist, S.E., and C.Q. Doe. 2005. Microtubule-induced Pins/Galphai cortical
polarity in Drosophila neuroblasts. Cell. 123:1323–1335.
Siegrist, S.E., and C.Q. Doe. 2007. Microtubule-induced cortical cell polarity.
Genes Dev. 21:483–496.
Sonneville, R., and P. Gonczy. 2004. zyg-11 and cul-2 regulate progression
through meiosis II and polarity establishment in C. elegans. Development.
Tsai, M.-C., and J. Ahringer. 2007. Microtubules are involved in anterior-posterior
axis formation in C. elegans embryos. J. Cell Biol. 179:397–402.
Wallenfang, M.R., and G. Seydoux. 2000. Polarization of the anterior-posterior
axis of C. elegans is a microtubule-directed process. Nature. 408:89–92.
Wittmann, T., and C.M. Waterman-Storer. 2001. Cell motility: can Rho GTPases
and microtubules point the way? J. Cell Sci. 114:3795–3803.