The TAO kinase KIN-18 regulates contractility and establishment of polarity in the C. elegans embryo

Cell polarity is important in many developmental systems, and cytoskeleton remodeling is an essential mechanism for establishing cell polarity. In the C. elegans one-cell embryo, the actomyocin cytoskeleton is required for asymmetric localization of the Par proteins. Anterior PAR proteins exert feedback on contractility of the early embryo. Understanding the roll of different proteins in these cytoskeletal dependent mechanisms is important in understanding how embryos develop, particularly in the very early stages. Through this deeper understanding of developmental systems, we can hope to improve our ability to prevent and cure developmental problems in other biological systems.


In many systems, including C. elegans, acto-myosin cytoskeleton remodeling is coordinated by RhoGTPases. The small GTPase Rho is important in regulating contractility of the acto-myosin cytoskeleton by controlling actin polymerization and myosin contractility. In C. elegans, RHO-1 is an important protein establishing polarity in the zygote. Shortly after meiosis, the PAR-3 complex is localized around the entire cortex and the cortex is uniformly contractile. A signal from sperm during fertilization triggers posterior cortical smoothening and breaks the initial symmetry of the embryo. Asymmetric cortical ruffling results in anterior-directed cortical flows and the PAR-3 complex is localized to the anterior of the embryo and PAR-1 and 2 localize to the posterior cortex. Once the polarity is establish, contractions stop and the polarity is maintained by the antagonism of anterior and posterior PAR proteins. The establishment of this PAR localization is dependent on the cortical contractions driven by a Rho cytoskeleton remodeling process. The absence of RHO-1 in early-embryonic development causes cortical ruffling to be abolished and the asymmetric contraction that localized the PAR-3 complex does not occur.

The authors of this paper identify a new gene, kin-18, required to regulate early contractility of the C. elegans embryo, and try to elucidate its role in the establishment of the contractility and polarization of the embryo.

C. elegans Development Resources:

KIN-18, Cell-Cycle Progression, and Pseudocleavage Relaxation

The authors use C. elegans embryos depleted of KIN-18 by RNA interference to analyze the phenotype of KIN-18 depleted embryos. By comparing time-lapse differential interference contrast microscopy of wild-type and KIN-18 depleted embryos, the authors find that several key events in early development of KIN-18 depleted embryos occur at delayed intervals relative to wild-type.

Fig. 1. KIN-18 depletion delays pseudocleavage relaxation and cell cycle progression. (A) Stills from DIC time-lapse recordings, Pro-Nuclear Meeting (PNM) time is set to 0 s. White arrowheads indicate the site of pseudocleavage ingression. (B) Quantification of phenotypes observed in (A) (see text for details). ⁎p<0.01 with respect to wild type (wild type n=23, kin-18(RNAi) n=18). Averages are shown, error bars represent s.e.m., scale bar 10 μm.

These differences can be seen in Fig 1 above. The time to relaxation of the pseudocleavage of KIN-18 depleted embryos and time from pronuclei meeting (PNM) to the time the nuclear envelope breaks down (NEBD) are geratly increased, while the time from NEBD to cytokinesis remains the same (Fig 1b).

Since this suggests that KIN-18 leads to cell-cycle delay, the persistence of the pseudocleavage may be an effect of the delayed cell-cycle progression. Inactivation of div-1, a component of DNA replication, creates cell-cycle delay in C. elegans embryos and leads to persistence of the pseudocleavage much like the KIN-18 depleted embryos. This div-1 delay is caused by activation of the alt-1 DNA damage checkpoint. The authors create alt-1 mutant embryos to rescue the cell-cycle delay and check for continued prolonged pseudocleavage relaxation in KIN-18 depleted embryos versus wild-type and div-1 RNA interference depleted embryos.

Fig. 2. kin-18(RNAi) delay in pseudocleavage relaxation does not depend on the cell cycle delay. (A) Stills from DIC time-lapse recordings of the indicated genotypes, PNM time is set to 0 s. White arrowheads indicate the site of pseudocleavage ingression. (B) Quantification of pseudocleavage persistence and PNM to NEBD timing in the indicated genotypes. *p<0.005 with respect to control RNAi (control RNAi n=8, kin-18(RNAi) n=9, div-1(RNAi) n=11, atl-1(tm853) n=11, kin-18(RNAi); atl-1(tm853) n=11, div-1(RNAi); atl-1(tm853) n=8). Averages are shown, error bars represent s.e.m., scale bars 10 μm. The difference in time of pseudocleavage persistence between KIN-18 depletion in WT and in the atl-1(tm853) mutant is not statistically significant (p=0.174).

The alt-1 mutant KIN-18 depleted embryos rescued cell-cycle delay, but did not rescue pesuducleave persistence, as can be seen in alt-1 mutant pseudocleavage persistence (Fig 2B). In combination, these two findings suggest that KIN-18 plays two roles, one in promoting cell-cycle progression and one in triggering psuedocleavage relaxation.

KIN-18 and Contractility

In order to answer whether KIN-18 depletion increased cortical contractions in conjunction with persistence of the pseudocleavage, the authors use a strain of C. elegans with a GFP membrane marker and count the number of invaginations in both RNA interference control and KIN-18 depleted embryos. KIN-18 depleted embryos showed significantly more invaginations indicating a positive regulatory effect of KIN-18 on cortical contractions.

Fig. 4. KIN-18 acts via RHO-1 and is negatively regulated by PAR-3. (A) Stills from DIC time-lapse recordings of the genotypes indicated, PNM time is set to 0 s. White arrowheads indicate the site of pseudocleavage ingression. Control RNAi is pde-5(RNAi) (see Materials and methods for details). (B) Quantification of pseudocleavage persistence in the indicated genotypes. *p<0.01 with respect to kin-18(RNAi). °p<0.01 with respect to control RNAi; par-3(it71), note that the difference in pseudocleavage persistence between kin-18(RNAi) and par-3(it71); kin-18(RNAi) is not significant (p=0.51), and both are significantly different from wild type (p25, control RNAi;kin-18(RNAi) n=20, control RNAi;rho-1(RNAi) n=17, rho-1(RNAi);kin-18(RNAi) n=16). Averages are shown, error bars represent s.e.m., scale bars 10 μm.

Since the anterior PAR proteins exert positive feedback on contractions and loss-of-function mutations of PAR-3 result in strongly reduced contractility, the authors compare  the pseudocleavage persistence of wild-type, KIN-18 depleted, PAR-3 mutants, and KIN-18 depleted/PAR-3 mutant embryos. The PAR-3 mutant embryos showed significantly lower pseudocleavage persistence than wild-type, while both the KIN-18 and KIN-18/PAR-3 mutant embryos showed significantly higher pseudocleavage persistence and the difference between KIN-18 and KIN-18/PAR-3 embryos was not significant (Fig4B-C). This indicates that PAR-3 may negatively regulate KIN-18. To further probe this possibility, the authors test whether KIN-18 was able to bind to PAR-3 in yeast two hybrid experiments. The results indicated that KIN-18 interacts with the C-terminal of PAR-3.

KIN-18 and RHO-1

In order to probe whether the contractions observed in KIN-18 depleted embryos required RHO-1, the authors observed the percentage of embryos with pseudocleavage in wild-type, KIN-18 depleted, RHO-1 depleted, and RHO-1/KIN-18 depleted embryos. Wild-type and KIN-18 depleted embryos showed near 100% pseudocleavage, while RHO-1 depleted and RHO-1/KIN-18 depleted embryos showed little to no pseudocleavage. KIN-18 antibody staining of RHO-1 depleted embryos showed a depletion of KIN-18. This means that observed KIN-18 depleted embryos’ increase in contractions is RHO-1 dependent, and KIN-18 may negatively regulate RHO-1 dependent contractility.

To observe localization of RHO-1 in KIN-18 depleted embryos, embryos expressing GFP-tagged RHO-1 were observed in control and KIN-18 depleted embryos. Specificity of RHO-1 localization to KIN-18 was checked by localization of RHO-1 in rga-3/4 depleted embryos, another positive regulator of contractility.

Fig. 6. KIN-18 negatively regulates cortical localization of RHO-1. (A) Fluorescence pictures of embryos from control RNAi and kin-18(RNAi) in a GFP::RHO-1 expressing strain. (B) Quantification of relative cortical intensities in one cell embryos (from PNM to the appearance of the cytokinesis furrow) of the indicated genotypes. RHO-1 is enriched in the anterior in control embryos (*p<0.01 between anterior and posterior corteces). In KIN-18 depleted embryos, the posterior cortical levels of RHO-1 are significantly increased compared to the posterior in control embryos (°p<0.01). Anterior and posterior levels of GFP::RHO-1 in kin-18(RNAi) are not significantly different (p=0.38, control RNAi n=24, kin-18(RNAi) n=18). (C) Fluorescence pictures of embryos from control RNAi and rga-3/4(RNAi) of a GFP::RHO-1 expressing strain. (D) Quantification of relative cortical intensities in one cell embryos (from PNM to the appearance of the cytokinesis furrow) of the indicated genotypes. Anterior levels are significantly different from posterior levels in both control RNAi (**p<0.01) and rga-3/4(RNAi) embryos (p<0.02). The increase in levels observed at the anterior in rga-3/4(RNAi) compared to the anterior in control RNAi embryos is not significant (°°p=0.18) (control RNAi n=35, rga-3/4(RNAi) n=23). Averages are shown, error bars represent s.e.m., scale bars 10 μm

A significant difference in anterior localization of RHO-1 was observed in KIN-18 depleted embryos and not rga-3/4 depleted embryos (Fig6 B,D). This indicates that KIN-18 may play a roll in RHO-1 anterior localization and cell polarity in the embryo.

Fig. 7. Localization of KIN-­18 in the early embryo. (A) Staining of representative embryos of the indicated stage, and of a kin-­18(RNAi) embryo as control. Arrowheads indicate cortical KIN-­18, blue is DNA, white is KIN-­18 staining. (B) Fluorescence pictures of representative gfp::kin-­18 expressing embryos at one and two cells stage. (C) Cortical quantification of GFP::KIN-18 levels in, from the left, control, par-3(RNAi), par-2(RNAi), control and rho-1(RNAi) embryos. See materials and methods for the experimental settings. GFP::KIN-18 cortical levels are higher at the anterior cortex in both experiments (*p<0.05 and **p<0.001). In par mutants this asymmetry is lost. In par-2(RNAi) embryos, the increase in levels at anterior is not significantly different from the anterior in control embryos (p=0.39), while the increase in the posterior is significant (°p<0.02). The reduced KIN-18 anterior cortical levels in rho-1(RNAi) embryos is significant compared to anterior in control embryos (°°p<0.001), but anterior and posterior are still significantly different (+p<0.001) (PAR protein experiment: control RNAi n=21, par-3(RNAi) n=13 and par-2(RNAi) n=8; RHO-1 experiment: control RNAi n=14, rho-1(RNAi) n=19). Averages are shown, error bars represent s.e.m., scale bar 10 μm.

GFP-tagged KIN-18 embryos are consistent with these findings, showing anterior localization of KIN-18 in control and RHO-1 depleted embryos, but no anterior localization in PAR-3 and PAR-2 depleted embryos (Fig 7 C). This indicates that localization of KIN-18 is dependent on establishment of polarization and PAR-3 and is consistent with the association of KIN-18 with PAR-3 indicated previously. This indicates that PAR-3 may contribute to the cortical localization of KIN-18.

KIN-18 plays a role in polarity-establishment

Since PAR-2 mutants establish polarity, but cannot maintain the established polarity, the authors tested the effect KIN-18 depletion on PAR-2 mutants. The authors measured the embryonic viability of both wild-type and PAR-2 mutants for PAR-6, KIN-18, and DIV-1 depleted embryos.

Fig. 10. KIN-18 plays a role in polarity establishment. (A) Schematic representation of polarity establishment (before PNM) and polarity maintenance phases (after PNM, and after pseudocleavage relaxation for kin-­18(RNAi)) in the indicated genotypes. PAR-­6 is depicted in blue, PAR-­2 in red, cortex devoid of both PAR-­6 and PAR-­2 is in black. Thickness and darkness of the lines reflect abundance or activity of the indicated protein. (B)­par-­2(it5ts) embryonic lethality suppression by RNAi depletion of the indicated genes. Depletion of PAR-6, KIN-18 or DIV-1 all significantly suppress par-2(it5ts) lethality (*p<0.001 with respect to par-­2(it5ts) in control RNAi). In these conditions, par-6(RNAi) results in weak lethality in wild type embryos (°p500 embryos were counted. Note that the levels of suppression of par-2(it5ts) lethality vary between the different RNAi. This might depend on the mechanism exerted by the gene in the system, but as well as on the specific RNAi penetrance and the balance between toxic/favorable effects of each combination. For instance par-3(RNAi) or pkc-3(RNAi) is less efficient than par-6(RNAi) to rescue par-2(it5ts) lethality, although they should in principle impinge on the same pathway.

The viability of KIN-18 depleted PAR-2 mutants was significantly increased from that of the control (Fig 10B). The authors propose this may have been due to a farther anterior pseudocleavage restricting the dispersion of PAR-3 to the anterior and destroying the established polarity.


The authors establish that KIN-18 plays a roll in RHO-1 dependent cortical contractility, a delay in cortical relaxation, and a cell-cycle delay, and that these effects are likely independent of one another. They also establish that KIN-18 can associate with PAR-3 and plays some roll in maintaining the polarity of the embryo. The authors propose that these findings may suggest a new role for Kin-18 is setting the boundaries between the PAR proteins during polarity establishment via RHO-1, providing a link between cytoskeleton remodeling and cell polarity establishment.

This study represents an initial probing into the roll of Kin-18 in C. elegans’ embryo polarity establishment. The authors begin by establishing the phenotype of Kin-18 depleted embryos to establish a starting point for further inspection of Kin-18’s role. This leads them to further narrow the scope of Kin-18’s role by a progression of experiments, which each reinforce eachothers’ findings and establish new dependencies and rolls for Kin-18. This methodical progression provides not only a good basis for further study, but establishes a solid footing for the understanding of Kin-18’s intricate role in contractility.

Additionally, the authors use very carefully designed experiments, which implement RNA interference controls, functionally related genes, and confirmation of localization and interaction. These controls and reinforcements bring increased certainty to the authors’ conclusions. The only weak points are a small amount of uncertainty in establishing the 0 point in time that the initial phenotype conclusions are based on and the use of vagination count, which are both based on a definition chosen by the authors and introduces a small amount of bias. Despite, this small weakness, the paper comes to conclusions with a high degree of certainty and establishes an excellent framework for further exploration of Kin-18 and contractility.


Fabio M. Spiga, Manoel Prouteau, Monica Gotta, The TAO kinase KIN-18
regulates contractility and establishment of polarity in the C.
elegans embryo, Developmental Biology, Volume 373, Issue 1, 1
January 2013, Pages 26-38

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