Understanding the mechanisms of polarity establishment and nuclear envelope breakdown in Caenorhabditis elegans embryos

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PhD; 2023; Microbiology and Cell Biology

Polarity establishment is critical for the development of multicellularity and in stem cell
division. The one-cell stage of the Caenorhabditis elegans embryo establishes
anterior-posterior polarity 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.
Concomitant to 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 cells. 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-B55SUR-6 (hereafter referred to as B55SUR-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 establishment 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 delocalizes 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) B55SUR-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 (Cyclin-dependent kinase 1), AIR-1 (Aurora
A kinase), and PLK-1 (Polo-like kinase 1). It is well established that kinase-dependent
hyperphosphorylation of nuclear envelope components results in the disassembly of
the nuclear envelope during mitosis. Thus, while these mitotic kinases are crucial for
NEBD, phosphatases have never been associated with NEBD at the mitotic entry. Here, we identified B55SUR-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 B55SUR-6, nuclear membrane permeabilization (NEP) is
significantly delayed, and nuclear lamina and several nucleoporins persist throughout
mitosis. As a result, chromosomes are missegregated and the union of maternal and
paternal genomes does not occur efficiently. Notably, we found that the impact of
B55SUR-6 depletion on NEBD is not because of its effect on general cell cycle
progression or mislocalization of essential kinases such as PlK-1 or AIR-1. We
uncovered that B55SUR-6 acts in concert with the mechanical 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 B55SUR-6. In
in-vitro pulldown experiments, we also show that B55SUR-6 interacts with the tail domain
of LMN-1, suggesting the presence of potential residues that may be
dephosphorylated at the tail region of LMN-1. Notably, genomically-tagged GFPB55SUR-6 localizes to the nucleus before the onset of NEBD, suggesting that B55SUR6 nuclear import may directly promote NEBD. Overall, these findings describe the
unexpected role of PP2A phosphatase complex for efficient breakdown of the nuclear
envelope during cell division in animal cells.
In summary, my work has contributed to the mechanistic aspects of the two
critical processes during development and division, i.e., polarity establishment and
nuclear envelope breakdown, which are vital for the continuity of life.