Zyg-11 and cul-2 regulate progression through meiosis II and polarity establishment in C. elegans

The mechanisms that ensure coupling between meiotic cell cycle progression and subsequent developmental events, including specification of embryonic axes, are poorly understood. Here, we establish that zyg-11 and the cullin cul-2 promote the metaphase-to-anaphase transition and M phase exit at meiosis II in Caenorhabditis elegans. Our results indicate that ZYG-11 acts with a CUL-2-based E3 ligase that is essential at meiosis II and that functions redundantly with the anaphase-promoting complex/cyclosome at meiosis I. Our data also indicate that delayed M phase exit in zyg-11(RNAi) embryos is due to accumulation of the B type cyclin CYB-3. We demonstrate that PAR proteins and P granules become polarized in an inverted manner during the meiosis II delay resulting from zyg-11 or cul-2 inactivation, and that zyg-11 and cul-2 can regulate polarity establishment independently of a role in cell cycle progression. Furthermore, we find that microtubules appear dispensable for ectopic polarity during the meiosis II delay in zyg-11(RNAi) embryos, as well as for AP polarity during the first mitotic cell cycle in wild-type embryos. Our findings suggest a model in which a CUL-2-based E3 ligase promotes cell cycle progression and prevents polarity establishment during meiosis II, and in which the centrosome acts as a cue to polarize the embryo along the AP axis after exit from the meiotic cell cycle.

Differential activation of the DNA replication checkpoint contributes to asynchrony of cell division in C. elegans embryos

Pierre Gönczy

BACKGROUND: Acquisition of lineage-specific cell cycle duration is a central feature of metazoan development. The mechanisms by which this is achieved during early embryogenesis are poorly understood. In the nematode Caenorhabditis elegans, differential cell cycle duration is apparent starting at the two-cell stage, when the larger anterior blastomere AB divides before the smaller posterior blastomere P(1). How anterior-posterior (A-P) polarity cues control this asynchrony remains to be elucidated.RESULTS: We establish that early C. elegans embryos possess a hitherto unrecognized DNA replication checkpoint that relies on the PI-3-like kinase atl-1 and the kinase chk-1. We demonstrate that preferential activation of this checkpoint in the P(1) blastomere contributes to asynchrony of cell division in two-cell-stage wild-type embryos. Furthermore, we show that preferential checkpoint activation is largely abrogated in embryos that undergo equal first cleavage following inactivation of Galpha signaling.CONCLUSION: Our findings establish that differential checkpoint activation contributes to acquisition of distinct cell cycle duration in two-cell-stage C. elegans embryos and suggest a novel mechanism coupling asymmetric division to acquisition of distinct cell cycle duration during development.

2003

PLK-1 asymmetry contributes to asynchronous cell division of C. elegans embryos

Yemima Suryani Budirahardja, Pierre Gönczy

Acquisition of lineage-specific cell cycle duration is an important feature of metazoan development. In Caenorhabditis elegans, differences in cell cycle duration are already apparent in two-cell stage embryos, when the larger anterior blastomere AB divides before the smaller posterior blastomere P1. This time difference is under the control of anterior-posterior (A-P) polarity cues set by the PAR proteins. The mechanisms by which these cues regulate the cell cycle machinery differentially in AB and P1 are incompletely understood. Previous work established that retardation of P1 cell division is due in part to preferential activation of an ATL-1/CHK-1 dependent checkpoint in P1, but how the remaining time difference is controlled is not known. Here, we establish that differential timing relies also on a mechanism that promotes mitosis onset preferentially in AB. The polo-like kinase PLK-1, a positive regulator of mitotic entry, is distributed in an asymmetric manner in two-cell stage embryos, with more protein present in AB than in P1. We find that PLK-1 asymmetry is regulated by A-P polarity cues through preferential protein retention in the embryo anterior. Importantly, mild inactivation of plk-1 by RNAi delays entry into mitosis in P1, but not in AB, in a manner that is independent of ATL-1/CHK-1. Together, our findings support a model in which differential timing of mitotic entry in C. elegans embryos relies on two complementary mechanisms: ATL-1/CHK-1-dependent preferential retardation in P1 and PLK-1-dependent preferential promotion in AB, which together couple polarity cues and cell cycle progression during early development

2008

Asymmetric Spindle Positioning and Intracellular Trafficking in C. Elegans Embryos

Kalyani Thyagarajan

Asymmetric cell division is critical for generating daughter cells that possess distinct fates during development and in stem cell lineages. Correct orientation of the mitotic spindle with respect to the polarity axis is crucial to this process. In the one-cell stage embryo of the nematode C. elegans, polarity cues define an anterior-posterior axis, and the mitotic spindle is positioned asymmetrically towards the posterior of the embryo in response to these cues, resulting in daughter cells that differ not only in fates, but also in sizes. Although the way by which force is generated has becoming increasingly well understood, the mechanisms by which polarity cues regulate the asymmetric distribution of active cortical force generators that pull on astral microtubules to position the spindle remained unclear at the onset of my thesis. During the course of our work, we found that intracellular trafficking plays a crucial role in the distribution and levels of proteins involved in asymmetric spindle positioning. Firstly, we found that the distribution of the Gβ protein GPB-1, a negative regulator of pulling forces, varies across the cell cycle, with levels at the cell membrane being lowest during mitosis. GPB-1 transits through the endosomal network in a dynamin-, clathrin- and RAB-5-dependent manner. We also found that GPB-1 trafficking is more pronounced on the anterior side and that this asymmetry is regulated by A-P polarity cues. In addition, we demonstrate that GPB-1 depletion results in the loss of GPR-1/2 asymmetry during mitosis, and thus symmetric pulling forces during mitosis. Secondly, we found that the clathrin heavy chain modulates spindle-positioning forces as well as the cortical distribution of positive (GPR-1/2 and DHC-1) and negative (GPB-1 and LET-99) regulators of pulling forces. Although the mechanisms by which it does so remain unclear, our data suggests that clathrin plays multiple roles in the one-cell stage C. elegans embryos, including in recycling GPB-1 to the plasma membrane. Overall, our results lead us to propose that the clathrin heavy chain and the modulation of Gβγ trafficking play a critical role during asymmetric division of one-cell stage C. elegans embryos.

EPFL2011

Asymmetric Spindle Positioning and Intracellular Trafficking in C. Elegans Embryos

Kalyani Thyagarajan

EPFL2011