[
International Worm Meeting,
2015]
Cytokinesis is the last step required to separate the daughter cells after mitosis. In the germline of most animals, cytokinesis often fails, leading to the formation of a stable intercellular bridge and, eventually, a syncytium. Whereas the C. elegans adult germline is syncytial, the regulators implicated in its nucleation remain unknown. ANI-2, a non-canonical form of the scaffold protein ANI-1/Anillin, is specifically expressed at the cortex of the last germline blastomere, P4, during C. elegans embryogenesis. During the division of P4 into the two primordial germ cells (PGCs), Z2 and Z3, ANI-2 is redistributed and accumulates at the midbody between the daughter cells and is enriched throughout the subsequent stages of larval development. The presence of ANI-2 in the C. elegans germline is essential, its depletion leading to a loss of stable intercellular bridges, a severe gonad disorganization and sterility, suggesting an important role for ANI-2 in the nucleation of the syncytial germline. To address this, we first used immunofluorescence to determine which genes are implicated in ANI-2 cortical loading to P4, using conditions that perturb contractility/cytokinesis regulators (RNAi or conditional mutants). Our results show that the guanine exchange factor ECT-2, the GTPase activating protein CYK-4/MgcRacGAP and the GTPase RHO-1/RhoA are required for ANI-2 loading to the P4 cortex, whereas other contractility regulators, such as NMY-2/non-muscle myosin II and the actin filament nucleator CYK-1 are not. Further, we found that, unlike many components of the cytokinetic machinery undergoing shedding during cellular abscission, the presence of ANI-2 at the PGC midbody is stable during gastrulation and extends in size during embryonic elongation. We found that ECT-2, CYK-4 and RHO-1 are important for the proper accumulation and localization of the ANI-2 focus during gastrulation. Moreover, we found that other midbody markers, such as NMY-2, also remain at the midbody of the PGCs and undergo changes in shape similar to ANI-2 during embryogenesis, and that ANI-2 is important for NMY-2 persistence at the PGCs' midbody. Our results suggest that different sets of genes regulate ANI-2's loading to the cortex of P4 and its subsequent redistribution to the midbody of the PGCs. Furthermore, ANI-2 reorganization during elongation could be an important step to stabilize contractility regulators at the intercellular bridge and control to syncytium formation.
[
International Worm Meeting,
2019]
Cytokinesis occurs after mitosis, when the two daughter cells are physically separated. The last step of cytokinesis, termed abscission, involves numerous conserved regulators that coordinate all processes enabling resolution of the transient intercellular bridge, including midbody processing, cytoskeletal rearrangements membrane trafficking and scission. Certain cell types however undergo incomplete cytokinesis, generating cells that remain connected by a stable intercellular bridge, thus forming a syncytium. Stable intercellular bridges are a conserved feature during metazoan gametogenesis. In dividing mouse spermatocytes for instance, the protein TEX14 inhibits the recruitment of ESCRT-I regulators at the midbody ring, leading to a block of abscission and stabilization of the intercellular bridge. TEX14 is only found in vertebrates however, suggesting that this molecular mechanism of cytokinesis incompletion and syncytium formation is not conserved in most metazoans. The C. elegans syncytial gonad represents a relevant model to study how differential regulation of cytokinesis impacts syncytial organization. In C. elegans, all germ cells originate from the unique embryonic blastomere, P4, which divides incompletely to form two primordial germ cells that remain stably interconnected, Z2 and Z3. We hypothesize that an active mechanism prevents the completion of P4 cytokinesis during embryogenesis, and that this is required for proper syncytiogenesis of the larval and adult gonad. To define this mechanism and identify the step where abscission is blocked, we are using live-imaging approaches to monitor abscission regulators and compare their dynamics in P4 to those in neighboring somatic cells, which undergo complete cytokinesis. We found that the kinetics of Aurora B and midbody microtubules processing are identical in P4 and somatic cells. This indicates that the block of abscission in P4 is unlikely to be due to a defect in midbody maturation. We also found that the ESCRT-I regulator TSG-101 is loaded at the midbody ring with comparable timing in P4 and somatic cells, indicating that the mechanism of abscission block is different from that described in mouse spermatocytes. We are currently monitoring the dynamics of regulators that act at later steps in the process. Our work will help to define the mechanisms that enable differential cytokinetic regulation during gonad development.