Context and Background:
Asymmetric cell division is universally used in development to ensure cell fate diversity and to orchestrate axes formation and embryonic body plan patterning. The early C. elegans embryo is one of the most widely used systems to gain genetic and molecular insights into the processes that underlie symmetry breaking (Rose L. et al., 2014). In this model organism, asymmetric cell division is evident from the very first cleavage of the zygote and goes on for the next 4 rounds, during which the major body axes are established. The A-P axis rises upon fertilisation and the position of the paternal centrosome marks the future posterior side of the embryo. At the molecular level, symmetry breaking derives from a change in the uniform contraction of the acto-myosin cortex, involving the small GTPase RHO-1 and its activator ECT-2. At the onset of polarity establishment, upon a still unknown signal, ECT-2 is displaced from the future posterior cortex and Myosin-II is disassembled, leading to cortex relaxation and increased acto-myosin flow towards the anterior (Motegi F. et al., 2006; Zonies S. et al., 2010). This unbalance favours the recruitment of PAR-2 at the posterior pole while pushing the anterior PAR complex (PAR-3/PAR-6/PKC-3) to the opposite end.
Whether and how centrosomes dictate symmetry breaking and how they signal the cell cortex to recruit polarity complexes is still unclear and under investigation. Two recent preprints from the Gönczy and the Kotak labs look at the centrosome as a signalling hub for the establishment of polarity and identify the Aurora A kinase (AIR-1 in C. elegans) as a central player in polarity establishment.
Both studies start with the observation that inhibition of AIR-1, either by RNAi or using a dead kinase variant, results in the formation of two PAR-2 domains, one at each pole of the zygote. This unusual distribution of PAR-2 is also accompanied by a delocalisation of the anterior PAR-3/PAR-6/PKC-3 complex in the middle, suggesting that, under normal conditions, Aurora A controls the spatial distribution of the PAR complexes within the embryo.
As the cortical acto-myosin flow is critical for the asymmetric distribution of the PAR components, the two groups analyse this variable by image velocimetry (PIV) analysis and found that AIR-1 knockdown results in reduced cortical flow. Specifically, they registered two flows of small intensity that start from both poles and propagate towards the centre. The reduction of cortical flow nicely correlates with no changes in the localisation of Myosin-II at the posterior cortex upon polarity establishment.
Next, both preprints address whether centrosomes play a role in symmetry breaking. Previous studies found these organelles indispensible for setting up polarity in the C. elegans zygote. The work from the Gönczy lab now questions this assumption given that in the AIR-1 RNAi background, in which centrosomes are not functional, polarity is altered but yet established. They observed that eggs fertilised with sperm from such-1 (t1668) mutants (which do not harbour centrioles) still form a PAR-2 crescent either at the anterior or at both poles, indicating that centrioles are not required for symmetry breaking. Nevertheless, the data suggest that localisation of AIR-1 at the centrosome provides a spatial cue to organise a posterior PAR domain. To test this idea, Klinkert et al. force the localisation of a RNAi-resistant AIR-1 to an immature, non-functional centriole (SPD-2 RNAi) to test whether it would rescue the defective asymmetry upon RNAi of endogenous AIR-1. Surprisingly, centrioles lacking SPD-2 establish a unique, posterior PAR2 domain, rescuing the AIR-1 RNAi phenotype. This set of experiments suggests therefore that the C. elegans one-cell embryo has an intrinsic ability to polarise; however, AIR-1 at the centrosome provides the spatial information for proper PAR-2 localisation at the posterior pole, ensuring the uniqueness of symmetry breaking. Kapoor et al. reach a similar conclusion using a different approach. They explore symmetry breaking in zyg-12 mutants, in which centrosomes do not keep their association to the nucleus and move freely in the cytoplasm towards the anterior pole, resulting in an anterior PAR-2 domain. Down-regulation of the kinase leads to anterior and posterior cortical PAR-2 localisation, no matter the position of the centrosome. These results reinforce the notion that centrosomal Aurora A instructs the posterior cortex to allocate PAR-2.
Given that in the absence of centrosomes the zygote is able to self-organise its polarisation, is there any physical property intrinsic to the system that might drive PAR-2 localisation to the cortex? Klinkert et al. set out to answer this question by using triangular PDMS chambers into which squeeze the embryos and developing an integrated physical model for symmetry breaking. Combining these methods, they reach the conclusion that PAR-2 tends to localise to region of high membrane curvature.
Finally to get at the mechanism of how PAR-2 posterior localisation is spatially controlled by Aurora A, Kapoor et al. investigate whether the kinase works upstream of ECT-2 and thus acts as master regulator of the acto-myosin flow. They find that simultaneous depletion of AIR-1 and ECT-2 rescues the AIR-1 RNAi phenotype restoring the formation of a single PAR-2 domain. Moreover, by monitoring ECT-2 localisation upon polarity establishment, they find that AIR-1 RNAi results in persistent localisation of ECT-2 at the posterior cortex. These data support the idea that Aurora A spatially controls symmetry breaking through the displacement of ECT-2 from the posterior cortex.
Importance: a new working model for Aurora A activity in symmetry breaking:
The data from the preprints advance our knowledge of symmetry breaking and polarity establishment in the C. elegans zygote. This new research suggests that the system is able to self-organise polarity in the absence of functional centrosomes by exploiting areas of high membrane curvature. Nevertheless centrosomes contribute to the process: by controlling ECT-2 localisation, centrosomal AIR-1 provides spatial uniqueness to symmetry breaking by redirecting PAR-2 to only one side of the embryo. AIR-1 acts upstream of the small RHO-GEF ECT-2 and promotes its displacement from the posterior cortex (Figure 1B, B’). This in turn leads to local cortical relaxation and initiates a robust actin flow towards the anterior, pushing the anterior PAR complex to the opposite side, thereby ensuring the formation of a single A/P axis.
Figure 1: Representation of the sequential steps that lead to a unique A/P axis in the C. elegans zygote. A) Polarised C. elegans one-cell embryo. Upon fertilisation, a dramatic reorganisation results in the formation of a unique A/P axis characterised by the establishment of robust anterior and posterior PAR domains (in red and green, respectively). B, B’) Zoomed-in view of the posterior pole at the time of symmetry breaking. Centrosomal AIR-1 (in yellow) signals the posterior cortex (blue arrows) where ECT-2 (in orange) is uniformly distributed. Following AIR-1 activity, ECT-2 is displaced from the cortex and Myosin-II patches disassemble in the area, leading to cortical relaxation and formation of a region of high membrane curvature. Par-2 localises to this region (in green). Actin flow (black arrows) moves anteriorly, pushing the anterior PAR components in this direction.
Future directions and Questions to the authors:
- Previous work suggested that a pool of AIR-1 is present at the cell cortex. The Gönczy lab exploited the GFP-GBP system to address whether the kinase has any non-centrosomal function and suggested that AIR-1 at the cortex prevents unregulated symmetry breaking events from happening. How does the kinase localise at the cortex? Does it used the same binding mode/domain to localise at the centrosome? What are the targets of AIR-1 at the cortex? Presumably, cortical AIR-1 needs to be cleared solely from the posterior cortex to allow symmetry breaking. Can you speculate on how AIR-1 cortical localisation is fine-tuned?
- PAR-2 has high affinity for regions of high membrane curvature. Does PAR-2 bind these regions directly? From a structural point of view, does it have BAR domains? Do you think this is a feature shared with other PAR proteins?
- Is ECT-2 a direct target of AIR-1?
- Both preprints look at the role of the microtubule cytoskeleton in the establishment of polarity. Reduced number of astral microtubules (TBG-1 RNAi) or complete loss of microtubules (Nocadozole treatment) do not impair PAR-2 localisation to the cortex upon AIR-1 RNAi, reinforcing the idea that the system is able to self-organise polarity. However mutation of the PAR-2 microtubule binding motif completely abrogates its binding to the cortex upon AIR-1 RNAi, suggesting that in this context the presence of microtubules is way more detrimental for polarity establishment. To what extent does the microtubule cytoskeleton contribute to symmetry breaking? And how are the actin and microtubule networks integrated to ensure the formation of a single A/P axis?
- The role of the acto-myosin cytoskeleton in regulating polarity establishment and positioning of polarity markers has been tested and proved in other systems, such as the Drosophila neuroblast (Broadus and Doe, 1997; Hannaford et al., 2018). How conserved is the role of Aurora A in dictating symmetry breaking in other developmental contexts? And how relevant is it to physiopathological conditions?
- Rose L. and Gönczy P. – Polarity establishment, asymmetric division and segregation of fate determinants in early elegans embryos- December 30, 2014), WormBook, doi/10.1895/wormbook.1.30.2, http://www.wormbook.org.
- Motegi F. and Sugimoto A. – Sequential functioning of the ECT-2 RhoGEF, RHO-1 and CDC-42 establishes cell polarity in Caenorhabditis elegans – Nature cell biology 8, 978 (Sep, 2006).
- Zonies S. et al.- Symmetry breaking and polarization of the elegans zygote by the polarity protein PAR-2- Development 137, 1669 (May, 2010).
- Broadus and Doe- Extrinsic cues, intrinsic cues and microfilaments regulate asymmetric protein localization in Drosophila neuroblasts- Current Biology 1997 (7)-11: 827-35
- Hannaford MR et al.- aPKC-mediated displacement and actomyosin-mediated retention polarize Miranda in Drosophila neuroblasts- Elife, eLife. 2018; 7:e29939. doi:10.7554/eLife.29939.
Posted on: 9th September 2018