| dc.description.abstract | Polarity establishment is critical for the development and stem cell lineages. The one-cell stage of the Caenorhabditis elegans embryo polarizes soon after fertilization. As a result, the first division of the embryo is asymmetric. The parent P0 cell divides into a large AB and a smaller P1 cell. The AB daughter cell specifies the somatic lineage, while the P1 cell lineage forms the germline. Failure to accurately establish polarity results in embryonic lethality. It is well-established that centrosomes are responsible for determining the axis of polarity in the one-cell embryo; however, the identity of the centrosome-associated polarity cue and the precise mechanism of polarity establishment remained unknown.
After polarity establishment, the one-cell C. elegans embryo enters in mitotic phase, where the male and female pronuclei expand their size and condense their chromatin. The two pronuclei migrate towards each other, followed by the nuclear envelope breakdown (NEBD) that allows the mixing of the maternal and paternal genomes. Subsequently, the mitotic spindle is assembled, and parental genomes are aligned on the metaphase plate. At the onset of anaphase, the differential cortical pulling forces position the mitotic spindle towards the embryo posterior. Since the position of the mitotic spindle dictates the site for cleavage furrow/cytokinesis, this leads to unequal cell division, producing a larger anterior AB cell and posterior smaller P1 cell. The process of NEBD is conserved in all metazoans that undergo 'open' mitosis and is vital for the accurate segregation of the chromosomes. However, the precise mechanism by which this occurs is poorly understood.
In the first part of my thesis, I have characterized the role of conserved mitotic kinase Aurora A in proper polarity establishment in the one-cell C. elegans embryo. In the second part of this work, for the first time, we link the function of phosphatase with the NEBD. We show that the PP2A-B55/SUR-6 (hereafter referred to as B55/SUR-6) is essential for proper NEBD in C. elegans embryos.
(1) Aurora A kinase/AIR-1 gradient at the centrosomes ensures singularity in the polarity axis in the one-cell C. elegans embryo
Proper cell polarization is vital for generating functional asymmetry within cells, which is crucial for development. In one-cell C. elegans embryo, centrosomes are responsible for polarity establishment, i.e., anterior-posterior body axis formation. Centrosomes are hypothesized to form a protein gradient that diffuses out to the cortex and disassembles the actomyosin network, thereby breaking symmetry and concomitantly establishing distinct domains of anterior and posterior conserved polarity proteins, PAR proteins. Primary candidate/s and the precise mechanism by which the centrosome achieves symmetry breaking remained elusive. We uncovered that RNAi-mediated depletion of conserved mitotic kinase, Aurora A kinase (AIR-1 in C. elegans) in the one-cell embryo disrupts stereotypical actomyosin-based cortical flows that occur at the time of polarity establishment. This misregulation of actomyosin dynamics leads to the formation of two posterior polarity axes. Also, we found that this function of Aurora A in polarity establi= shment is dependent on its kinase activity. Notably, this impact of Aurora A depletion is independent of its central role in microtubule nucleation. Interestingly, centrosome positioning in dictating the posterior polarity axis (or PAR-2 localization) is no longer important when Aurora A is depleted in the one-cell embryo. The mechanism by which Aurora A directs symmetry breaking is likely through direct regulation of RhoA-dependent contractility since we observed rescue in the formation of a single polarity axis in Aurora A (RNAi) embryos that are co-depleted of Rho-GEF, ECT-2. Further, a previous study showed that ECT-2 de-localizes from the posterior cortex at the time of polarity establishment, presumably under the influence of centrosome-mediated polarity cue. Our study shows that in the absence of Aurora A, ECT-2 fails to de-localize from the posterior cortex at polarity establishment, providing a possible explanation for the impaired actomyosin flow seen in Aurora A (RNAi) embryos. In summary, our work has contributed to uncovering an unconventional role of Aurora A kinase in polarity establishment in C. elegans. Thus, we propose that Aurora A gradient at the centrosome is a key for symmetry breaking and thus for ensuring proper polarity set-up.
(2) B55/SUR-6 promotes nuclear envelope breakdown in the C. elegans one-cell embryo
The nucleus constrains the cell's genetic material by forming a selective barrier to the entry of macromolecules from the cytoplasm. In animal cells, NEBD enables the spindle microtubules to access and attach to the chromosomes within the nucleus during mitosis. Proper chromosome-microtubule attachment ensures faithful segregation of genetic material into the two daughter cells. NEBD is regulated by the activity of critical kinases such as CDK-1, AIR-1 (Aurora A), and PLK-1. While these mitotic kinases are crucial for NEBD, no phosphatase has yet been linked with NEBD at the mitotic entry. Here, we identified B55/SUR-6 as an essential regulatory subunit of PP2A phosphatase critical for timely NEBD in the one-cell C. elegans embryo. We found that in embryos that are depleted for B55/SUR-6, nuclear membrane permeabilization (NEP) is significantly delayed, and nuclear lamin and nucleoporins persist throughout mitosis. As a result, chromosomes' segregation is impaired. Notably, we found that the impact of B55/SUR-6 depletion on NEBD is not because of its effect on cell cycle progression or mislocalization of essential kinases such as PlK-1 or AIR-1. We uncovered that B55/SUR-6 acts redundantly with microtubule-generated pulling forces to promote NEBD efficiently. Further, genetic epistasis experiments suggest that nuclear lamin (LMN-1), but not nucleoporin/s, is the target of B55/SUR-6. Notably, genomically-tagged GFP-B55/SUR-6 localizes to the nucleus before the onset of NEBD, suggesting that B55/SUR-6 nuclear import may directly promote NEBD. Overall, these findings link the PP2A phosphatase complex to a critical process of NEBD in animal cells.
In summary, my work has contributed to the mechanistic aspects of the two processes: polarity set-up and nuclear envelope breakdown, which are vital for the establishment and the continuity of life. | en_US |
