Binucleated polyploid cells are common in many animal tissues, where they arise by endomitosis, a non-canonical cell cycle in which cells enter M phase but do not undergo cytokinesis. Different steps of cytokinesis have been shown to be inhibited during endomitosis M phase in rodents, but it is currently unknown how human cells undergo endomitosis. In this study, we use fetal-derived human hepatocyte organoids (Hep-Orgs) to investigate how human hepatocytes initiate and execute endomitosis. We find that cells in endomitosis M phase have normal mitotic timings, but lose membrane anchorage to the midbody during cytokinesis, which is associated with the loss of four cortical anchoring proteins, RacGAP1, Anillin, SEPT9, and citron kinase (CIT-K). Moreover, reduction of WNT activity increases the percentage of binucleated cells in Hep-Orgs, an effect that is dependent on the atypical E2F proteins, E2F7 and E2F8. Together, we have elucidated how hepatocytes undergo endomitosis in human Hep-Orgs, providing new insights into the mechanisms of endomitosis in mammals.
© 2024 Darmasaputra et al.

Abscission is the last step of cell division leading to the complete separation of the two sister cells and consists of the cutting of a cytoplasmic bridge. Abscission is mediated by the ESCRT membrane remodeling machinery which also triggers the severing of a thick bundle of microtubules that needs to be cleared prior to abscission. Here, we show that rather than being passive actors in abscission, microtubules control abscission speed. Using mouse embryonic stem cells, which transition from slow to fast abscission during exit from naïve pluripotency, we investigate the molecular mechanism for the regulation of abscission dynamics and identify a feedback loop between the activity of Aurora B and microtubule stability. We demonstrate that naïve stem cells maintain high Aurora B activity after cytokinesis. This high Aurora B activity leads to transient microtubule stabilization that delays abscission. In turn, stable microtubules promote the activity of Aurora B. When cells exit naïve pluripotency, a decrease in Wnt signaling leads to a decrease in the activity of Aurora B, less stable microtubules, and a faster abscission. Overall, our data demonstrate that Aurora B-dependent microtubule stability controls abscission dynamics.

Embryo development depends upon maternally derived materials. Mammalian oocytes undergo extreme asymmetric cytokinesis events, producing one large egg and two small polar bodies. During cytokinesis in somatic cells, the midbody and subsequent assembly of the midbody remnant, a signaling organelle containing RNAs, transcription factors and translation machinery, is thought to influence cellular function or fate. The role of the midbody and midbody remnant in gametes, in particular, oocytes, remains unclear. Here, we examined the formation and function of meiotic midbodies (mMB) and mMB remnants using mouse oocytes and demonstrate that mMBs have a specialized cap structure that is orientated toward polar bodies. We show that that mMBs are translationally active, and that mMB caps are required to retain nascent proteins in eggs. We propose that this specialized mMB cap maintains genetic factors in eggs allowing for full developmental competency.
© 2023. The Author(s).

Many plant and animal cells transition from canonical to non-canonical cell cycles during development, resulting in the formation of polyploid cells. Two types of non-canonical cell cycles exist: endoreplication, where cells increase their DNA content without entering M phase, and endomitosis, where cells enter M phase but exit prematurely. Although endoreplication has been extensively studied in plants and insects, much less is known on the regulation of endomitosis, which is the most common mode of polyploidization in mammals. In this study, we use fetal-derived human hepatocyte organoids (Hep-Org), to investigate how human hepatocytes initiate and execute endomitosis. We find that cells in endomitosis M phase have normal mitotic timings, but lose membrane anchorage to the midbody during cytokinesis, resulting in regression of the cytokinetic furrow and formation of binucleate cells. Using immunofluorescence, we find that three cortical anchoring proteins, RacGAP1, anillin, and citron kinase (CIT-K), lose their association with the cell cortex during cytokinetic regression. Moreover, reduction of WNT activity by withdrawal of CHIR99021, a GSK3 inhibitor, from the culturing medium increases the percentage of binucleated cells in Hep-Orgs. This effect is lost in organoids with mutations in the atypical E2F proteins, E2F7 and E2F8, which have been implicated in binucleation of rodent hepatocytes. Together, our results identify how human hepatocytes inhibit cell division in endomitosis, and highlight an evolutionary recurrent mechanism to initiate non-canonical cell cycles in mammals.

The midbody at the center of the intercellular bridge connecting dividing cells recruits the machinery essential for the final steps of cytokinesis.1-5 Successive abscission on both sides of the midbody generates a free midbody remnant (MBR) that can be inherited and accumulated in many cancer, immortalized, and stem cells, both in culture and in vivo.6-12 Strikingly, this organelle was recently shown to contain information that induces cancer cell proliferation, influences cell polarity, and promotes dorso-ventral axis specification upon interaction with recipient cells.13-16 Yet the mechanisms by which the MBR is captured by either a daughter cell or a distant cell are poorly described.10,14 Here, we report that BST2/tetherin, a well-established restriction factor that blocks the release of numerous enveloped viruses from the surface of infected cells,17-20 plays an analogous role in retaining midbody remnants. We found that BST2 is enriched at the midbody during cytokinesis and localizes at the surface of MBRs in a variety of cells. Knocking out BST2 induces the detachment of MBRs from the cell surface, their accumulation in the extracellular medium, and their transfer to distant cells. Mechanistically, the localization of BST2 at the MBR membrane is both necessary and sufficient for the interaction between MBRs and the cell surface. We thus propose that BST2 tethers post-cytokinetic midbody remnants to the cell surface. This finding reveals new parallels between cytokinesis and viral biology21-26 that unexpectedly extend beyond the ESCRT-dependent abscission step.
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