The function of LEM-3 endonuclease in regulating cell fate determination

Abstract

Ontogenesis of multicellular organisms from zygotes to adults involves multiple rounds of cell fate determination. The fundamental question of how cells acquire their specific identities has intrigued researchers for centuries. In this context, 'cell fate' is defined as a unique combination of morphological, biophysical, biochemical, and functional characteristics. Once a cell commits to a specific fate, it follows a predefined developmental pathway that is often resistant to fate alteration at later stage of diTerentiation. Various factors play crucial roles in this intricate process of cell fate decision, yet the contribution of DNA endonucleases to this process has been relatively understudied. This study delves into the functional role of the LEM-3 endonuclease in regulating cell fate determination in terminally diTerentiated neurons. Using Caenorhabditis elegans as an in vivo model to investigate the fate determination process of post-mitotic cells, I found that the loss of lem-3 resulted in the emergence of additional cells that exhibited a TRN-like fate and were connected with the original TRNs via intercellular canals (Chapter III). These extra cells are the sister cells of TRNs and form binuclear syncytium with the TRNs due to failure of cytokinesis at the last round of cell division. Notably, the lem-3(-) mutants showed diTerent penetrance on cell fate determination among diTerent TRN subtypes likely because their sister cells are diTerent. Intriguingly, in some cases, the two nuclei in the syncytium can carry out distinct transcriptional programs, suggesting that connected neurons could still maintain certain levels of autonomy in transcriptional regulation. At the translational level, the formation of intercellular canals facilitates the exchange of various types of proteins that contribute to cellular diTerentiation (Chapter IV). As a result, although the transcriptional programs of the two connected cells in the syncytium can be independent, their cellular content are mostly mixed, leading to a mixed cell fate. The absence of LEM-3 disrupted proper resolution of chromatin bridges during anaphase, leading to the formation of intercellular canal. Pharmacologically inducing DNA replication errors increased the frequency of syncytium formation, and histone and nuclear envelope markers were found in the canal, supporting that the canal is a result of unresolved DNA bridges. The enzymatic activity of LEM-3 is important for its regulatory role in cell fate determination. The canal can grow and persist into late adult stages, suggesting a cytoskeleton-dependent maintenance mechanism. In addition to the TRNs, LEM-3 also regulates the fate determination of other cell types (Chapter V). By analyzing several examples of fate decision between sister cell pairs, I found that LEM-3 plays a role in the fate choice between apoptotic and non-apoptotic cells and between two distinct neuronal fates. Nevertheless, its role in the choice between neuronal and non-neuronal fates is not clear. In conclusion, this research revealed a previously underappreciated function of LEM-3/ANKLE1 endonuclease in safeguarding cellular diTerentiation by processing unresolved DNA connections during mitosis and enabling cytoplasmic separation. Our analysis of the mechanisms underlying intercellular canal formation and persistence contribute to the understanding of the repercussions of sustained connections between post-mitotic sister cells.