Aim 2: Differential regulation of abscission between germline stem cells and differentiating daughter cells.
We have found that AuroraB and CyclinB localize at the midbody, and that CycB/Cdk-1 promotes abscission, while AuroraB/Survivin inhibits it. Both complexes have other functions during the cell cycle, which are mainly the control of entry into M phase and the metaphase-anaphase transition. At these two stages, the regulation of AuroraB and Cdk-1 is well-described and a list of known regulators is available 12.
The core machinery that drives the eukaryotic cell cycle is very conserved across evolution, but has only been extensively studied in a few model systems (Morgan, 2007). During animal development, the canonical cell cycle is modulated and adapted in different cell types. For example, the rapid divisions of zebrafish or Drosophila embryos are devoid of Gap phases, while mouse giant trophoblast cells or many larval cells in Drosophila become polyploid by endoreplicating their DNA without any mitosis (M) phase (Budirahardja and Gonczy, 2009). How specific developmental programs alter different steps of the cell cycle remains to be understood in most cases. In this respect, the last stages of cell division, when daughter cells become separated, are probably the most diverse, but also the least explored. In sea urchin embryos, the timing of cytokinesis is shifted, and the completion of cell division only occurs during the S phase of the next cycle (Sanger et al., 1985). Cytokinesis altogether is absent during megakaryocyte differentiation, and can also be arrested at a late stage in spermatocytes of most species (Pepling et al., 1999; Vitrat et al., 1998). Cytokinesis starts by the specification of a cleavage plane and is followed by the ingression of an actomyosin contractile ring. During this transition, the mitotic spindle rearranges at the midzone to form an electron-dense structure known as the midbody, at the center of the intercellular bridge. Finally, by a process called abscission, daughter cells become physically separated. Although abscission failure can lead to tetraploidization, it remains poorly characterized due to several technical challenges (Steigemann and Gerlich, 2009). It is difficult to synchronize cells for the transient abscission process, which hampers most biochemical studies. Proteins involved at this stage may also be required earlier in the cell cycle and genetic mutations in the corresponding genes are thus likely to mask late functions. Finally, the midbody is beyond the resolution limit of most microscopes. Nevertheless, it has become clear that abscission requires remodeling of the membrane and cytoskeleton (Guizetti and Gerlich, 2010). Endosome delivery to the midbody and severing of the microtubule bundle are essential for correct abscission. The upstream regulators that initiate abscission remain unknown however. It is also unclear what regulates the timing of abscission, which can vary from minutes to hours, or can even remain incomplete, as in germ cells (Pepling et al., 1999).In our genetic screen for mutants affecting the early steps of oogenesis, we have identified the first mutations in the Drosophila homologues of Aurora-B and Survivin, two members of the Chromosome Passenger Complex (CPC). This complex regulates multiple steps of mitosis and is widely studied for its function at the centromeres, at the initiation of cytokinesis, and more recently during abscission. We found that Aurora B and Survivin inhibit abscission during normal development of Drosophila germ cells, using an allelic series of mutations. This inhibition is mediated by an Aurora B-dependent phosphorylation of Cyclin B, as a phosphomimic form of Cyclin B rescues premature abscission caused by a loss-of-function of Aurora B. In addition, we show that cell-type specific organelles, such as the fusome, and transcriptional programs, mediated by Bam in germ cells, impinge on this regulatory loop and can block abscission completely. We propose that the mutual inhibition between Aurora B and Cyclin B that we describe in germ cells, is likely to regulate abscission timing in a wide variety of cell types (Mathieu et al., Developmental Cell, 2013 Aug 12; 26(3):250-65)
Aim 2.2: Regulation of abscission downstream of AuroraB-CycB-Cdk1: the ESCRT-III complex
AuroraB and Cdk1 do not perform the membrane scission event required for the final separation of daughter cells. The secondary constriction is thought to be formed, and then abscised, by the ESCRT machinery, and the vacuolar protein sorting 4 (VPS4) 14. The subunits of the ESCRT-III complex, CHMP4B and VPS4, are relocated at the exact site of the cut just before abscission occurs. A direct phosphorylation of the ESCRT- III CHMP4C by AuroraB has been documented recently in mammalian cells, and shown to be involved in the control of abscission timing 15-16. CHMP4C has an inhibitory effect on abscission, whereas CHMP4B, a translated splice variant, has a positive effect on abscission. We have shown that AuroraB positively regulates abscission, and CyclinB-Cdk1 inhibits it. It is tempting to speculate that AuroraB phosphorylates CHMP4C, and Cdk1 phosphorylates CHMP4B. However, there is only one homolog of CHMP4 in Drosophila called Shrub (or Shrb/SNF7) 17. We have recently shown that it acts downstream of AuroraB to regulate abscission in GSCs (Matias et al., 2015). Shrb mutants give rise to egg chambers with 32 cells due to the formation of stem cysts, confirming our previous model (Mathieu et al., 2013). Shrb/SNF7 seems, however, to have both a positive and negative effect on abscission.
We showed that the ESCRT-III protein Shrb localizes to the midbody of the dividing GSC, functioning to promote abscission. Indeed, we found that reduced levels of Shrb resulted in the blockage, or strong delay, of abscission in the GSC and formation of a structure similar to a cyst. In these so called stem- cysts, the GSC keeps dividing while interconnected to its daughter cells. Consequently, we saw the appearance of egg chambers formed of 32 cells, instead of 16. Furthermore, Shrb function in abscission seems to be counteracted by AurB, as reducing AurB levels in Shrb heterozygous resulted in decreased stem-cysts and 32-cell cysts. Finally, Lethal giant discs (lgd), required for Shrb function in the endosomal pathway, was also seen localizing at the midbody and regulating abscission in GSCs. Removing one copy of Lgd from Shrb heterozygous increased the number of stem-cysts, but surprisingly the number of 32- cell cysts was reduced. This paradoxical result was explained with the observation of late abscission events in mitotic cysts, which divided the 32-cell cysts in the middle, leading to the formation of two cysts of 16 cells. (Matias et al., PLoS Genetics, 2015 Feb 3;11(2):e1004653)