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Spatial-temporal regulation of APC/C, its role on G1 arrest and impact on terminal differentiation

Periodic Reporting for period 1 - STOPAPCG1IMP (Spatial-temporal regulation of APC/C, its role on G1 arrest and impact on terminal differentiation)

Reporting period: 2015-09-01 to 2017-08-31

Cells are the basic building blocks for living organisms, including us, humans. For an organism to grow and develop into adult forms, cells need to duplicate through a coordinated process called “cell cycle”. Through this process, a single mother cell divides to generate two genetically identical daughter cells. After division, cells need to respond to external cues from the surrounding tissue and decide whether to continue dividing or to halt the cell cycle to become specialised in a task required for the proper function of the tissue/organ where they reside. The right balance between these two fates of cells is essential not only for forming proper shapes of organs and tissues but also for maintaining their functions throughout the life of the organisms. When this balance is perturbed, cells may continue dividing uncontrollably, leading to tumorigenesis. Alternatively, all the cells may become specialised and stop producing new cells, which accelerates the degeneration of tissues or the ageing process. Therefore, understanding how the division of the cells, i.e. the cell cycle, is coordinated with their responses to the external clues is key to understanding the causes of the formation and progression of tumours as well as the pathology of degenerative disorders, which will ultimately help develop new therapeutic strategies to these diseases.

To control the cell cycle, the cell has several effectors composed of proteins, collectively called “cell cycle regulators”. The levels of these players within the cell are controlled through production and destruction. One of the critical factors for the control of the protein levels during the cell cycle is the Anaphase Promoting Complex/Cyclosome (APC/C). This complex recognises proteins with specific signatures (or degrons) and targets them for degradation via the protein destruction machinery called the proteasome.
Accumulating evidence suggests that the APC/C is involved in the control of the balance between cell division and specialisation, however, the knowledge about how the APC/C accomplishes such a task remains scarce.
In this project, I aimed to understand how the APC/C coordinates the cell cycle with the cell specialisation process using the fruit fly, Drosophila melanogaster, as a model organism.
During the natural developmental process of Drosophila, the developing eye shows a remarkable, tight coordination between cell proliferation and the steps for cell specialisation. I took full advantage of this unique feature to investigate the role of the APC/C in the coordination between cell cycle and specialisation. The majority of cell cycle regulators, including APC/C, as well as the genes regulating cells’ responses to external cues, are very similar between Drosophila and mammals, although the genetic circuit in Drosophila is relatively simple and less complex compared to mammals. Therefore, using the fruit fly allows us to faster and more easily obtain results that are likely to be applicable to mammals.
In task 1, we evaluated the requirement of the APC/C for the formation of the photo detecting cells, the photoreceptors. We found that when the function of the APC/C is compromised, the cells that normally become photoreceptors fail to acquire their fate and unspecialised masses of cells are formed in the middle of the adult fly eyes. Further analysis revealed that this defect is caused by an abnormal activation of the Wingless/Wnt (Wg/Wnt) signalling pathway. An intrinsic mechanism that through the secretion of a specific molecule called Wingless by the neighbouring cells, instruct the receiving cells to keep their proliferative state.

In task 2, we aimed to identify the targets of the APC/C that when APC/C function is compromised, accumulate to cause the activation of the Wg signalling pathway. To this end, we employed a mixture of classical genetic approaches with biochemical approaches. Through these approaches, we successfully identified a cohort of proteins that present the APC/C degrons and with the potential to affect the transition from the unspecialised dividing cells to non-dividing specialised cells. Next, to identify the direct targets of the APC/C, we downregulated these candidates individually and tested whether their reduction can prevent the abnormal Wg activation caused by the inactivation of the APC/C. Through this analysis, we showed that the mitotic kinase Nek2 is directly targeted for degradation by the APC/C upon its activation with the co-activator Fzr/Cdh1.

Finally, in Task 3, we found that Nek2 is the protein that mediates the control of the Wg signalling pathway by the APC/C. By observing the behaviour of fluorescently labelled Nek2 in the eye tissue, we found that Nek2 is specifically degraded by the APC/C in the transitional zone between unspecialised cells and differentiated cells, called the morphogenetic furrow, where cells temporally stop dividing and stay in the G1 cell cycle phase. It is known that the Wg signalling pathway functions to keep the cells dividing and preventing cells to become specialised into photoreceptors. We found that when APC/C function is compromised in the transitional zone, it accumulates Nek2. This Nek2 accumulation, in turn, abnormally activates Wg signalling, which prevents cells to become photoreceptors. Altogether, we uncovered a novel function of the APC/C, by which APC/C keeps in check Wg signalling to initiate the specialisation of photoreceptors whilst stopping these cells dividing further.
In addition, we also found the evidence that this novel function of the APC/C is regulated by the Dpp signalling pathway, another signalling pathway known to promote specialisation process and antagonise Wg signalling in the transitional zone. We showed that for differentiation to proceed, Dpp signalling induces the stabilisation of a component of the APC/C. Moreover, if APC/C function is inhibited in the cells responding to Dpp, it activates Wg signalling. Thus, our results suggest that Dpp signalling uses the APC/C to inhibit Wg signalling.
To conclude, we believe that this action completely fulfilled the objectives proposed and potentially translated as the findings reported in this project are likely to be applicable to mammals and humans, as the degradation of Nek2 by the APC/C as well as the capacity of Nek2 to promote Wg signalling is conserved in humans. Since Nek2 is known to be overexpressed in various types of cancer, the pathway we identified may be involved in the formation and/or progression of tumours. Importantly, most Colorectal Cancers (CRCs) present mutations in the Wg signalling pathway, the main pathway upregulated by this APC/C-Nek2 axis. Thus, understanding the mechanisms underlying Wg/Wnt regulation during development to promote cell differentiation is crucial to develop new pharmacological approaches.
Final model of the cell cycle vs differentiation coordination by the APC/C