How Ubiquitylation Coordinates Cell Division and Differentiation - Function and Regulation of E3 Ubiquitin Ligases Beyond the Cell Cycle
Final Report Abstract
The here-described Emmy Noether project aimed to uncover “How Ubiquitylation Coordinates Cell Division and Differentiation” and to develop the required tools to investigate the “Function and Regulation of E3 Ubiquitin Ligases Beyond the Cell Cycle”. Because the transition from the proliferative/dividing state of cells into the dormant quiescent state as well as the transition from pluripotency to differentiation are highly dynamic processes, we first developed an all-in-one cell cycle reporter to follow these transitions in living cells by time-lapse microscopy. By means of genome-editing we labelled one allele of the endogenous proliferating cell nuclear antigen (PCNA) protein with a fluorescent probe allowing us to precisely classify all cell cycle stages and in addition monitor the transition into quiescence. Features, that together are unique for current cell cycle reporters for living cells. Furthermore, we developed the accompanying image analyses pipeline for PCNA-based segmentation, classification and tracking that enables visualizing the behavior of three further proteins in parallel. Notably, our approach was selected as a “Research Highlight” in Nature Methods 14, 2017 (768). We first employed the all-in-one cell cycle reporter to study the regulation of G1 phase length, in particular by cyclin D1, which has been proposed to be a key determinant of the “proliferation versus differentiation” decision. Our findings contradict the classical cell cycle model proposing that elevated cyclin D1 expression accelerates the cell cycle and drives cells toward S phase. Instead, in agreement with a revised cell cycle model postulated Dowdy and colleagues, we propose that the main function of cyclin D1 is to maintain G1 phase and prevent the transition into quiescence. This finding also might have consequence for our understanding of carcinogenesis, since almost half of transformed cells display elevated levels of cyclin D. Accordingly, rather than accelerating the speed of the cell cycle directly, elevated levels of cyclin D in cancer cells might “prime” cells for cell cycle progression and thereby tilt the balance between proliferation and quiescence. To understand how cells make the decision between proliferation, quiescence and differentiation it is not sufficient to just visualize dynamic cellular behavior during the execution of these decisions, but also to be able to control and manipulate cellular decision-making. Since decision-making is ultimately carried out by proteins that act at the right time at the right place, we developed an inducible and reversible nanobodybased degradation approach targeting any protein tagged with GFP or GFP-like fluorophores (e.g. Venus, Citrine). This allows to first visualize the behavior of a (ideally endogenously tagged) protein, then to reveal a loss-of-function phenotype at a given time and condition, followed by the recovery of the targeted protein – all within the same cell and in a single time-lapse experiment. Importantly our inducible-nanobody degradation approach can be not only be applied in tissue cell culture models but also in a vertebrate model – the zebrafish. To our knowledge, this is was the first demonstration of auxin-dependent degradation in zebrafish and likely due to its applicability to a multitude of experimental questions has not only resulted in several reagent requests, but was also picked up positively by relevant scientific social media and news outlets. Finally, to identify the substrates of ubiquitin enzymes crucial to the coordination of cell division, quiescence and differentiation, we developed E2~dID, an E2~ubiquitin thioester driven mass spectrometry identification method, which is in principle applicable to cell extracts derived from any experimental model. Focusing on the Anaphase Promoting Complex/Cyclosome (APC/C) – a crucial cell cycle enzyme that is also important for quiescence and differentiation - as a proof of principle, we showed that E2~dID performs well compared to alternative approaches and in addition reveals several unexpected APC/C substrates that shed a new light on APC/C function.
Publications
- (2015). A human interactome in three quantitative dimensions organized by stoichiometries and abundances. Cell 163: 712–723.
Hein MY, Hubner NC, Poser I, Cox J, Nagaraj N, Toyoda Y, Gak IA, Weisswange I, Mansfeld J, Buchholz F, Hyman AA & Mann M
(See online at https://doi.org/10.1016/j.cell.2015.09.053) - (2015). Realtime deformability cytometry: on-the-fly cell mechanical phenotyping. Nature Methods 12:199- 202
Otto O, Rosendahl P, Mietke A, Golfier S, Herold C, Klaue D, Girardo S, Pagliara S, Ekpenyong A, Jacobi A, Wobus M, Töpfner N, Keyser UF, Mansfeld J, Fischer-Friedrich E & Guck J
(See online at https://doi.org/10.1038/nmeth.3281) - (2015). The ABBA Motif Binds APC/C Activators and Is Shared by APC/C Substrates and Regulators. Developmental Cell 32: 358–372
Di Fiore B, Davey NE, Hagting A, Izawa D, Mansfeld J, Gibson TJ & Pines J
(See online at https://doi.org/10.1016/j.devcel.2015.01.003) - (2017). Distinct Levels of Reactive Oxygen Species Coordinate Metabolic Activity with Beta-cell Mass Plasticity. Scientific Reports 7:3994
Alfar, E.A., Kirova, D., Konantz, J., Birke S., Mansfeld, J. and Ninov N.
(See online at https://doi.org/10.1038/s41598-017-03873-9) - (2017). DNA damage during S-phase mediates the proliferation-quiescence decision in the subsequent G1 via p21 expression. Nature Communications 8:14728
Barr AR, Cooper S, Heldt FS, Butera F, Stoy H, Mansfeld J, Novák B & Bakal C
(See online at https://doi.org/10.1038/ncomms14728) - (2017). Plk1 activation in late G2 sets up commitment to mitosis. Cell Reports 19:2060-1973
Gheghiani L., Loew D., Lombard B., Mansfeld J., Gavet O.
(See online at https://doi.org/10.1016/j.celrep.2017.05.031) - (2017). Quantitative cell cycle analysis based on an endogenous all-in-one reporter for cell tracking and classification. Cell Reports 19:1953-1966
Zerjatke, T., Gak I.A., Kirova D., Fuhrmann M., Daniel K., Gonciarz M., Müller D., Glauche I. and Mansfeld J.
(See online at https://doi.org/10.1016/j.celrep.2017.05.022) - (2018). An E2-ubiquitin thioester-driven approach to identify substrates modified with ubiquitin and ubiquitin-like molecules. Nature Communications 9:4776
Bakos, G., Yu l., Gak IA., Roumeliotis, TI., Liakopoulos D., Choudhary JS. and Mansfeld J
(See online at https://doi.org/10.1038/s41467-018-07251-5) - (2018). Conditional control of fluorescent protein degradation by an auxin-dependent nanobody. Nature Communications 9:3297
Daniel K., Icha, J., Horenburg, C., Müller D., Norden C., and Mansfeld J.
(See online at https://doi.org/10.1038/s41467-018-05855-5) - (2018). Constitutive regulation of mitochondrial morphology by Aurora A kinase depends on a predicted cryptic targeting sequence at the N-terminus. Open Biology. 8:170272
Grant R., Abdelbaki A., Bertoldi A., Gavilan MP., Mansfeld J., Glover DM. and Lindon C.
(See online at https://doi.org/10.1098/rsob.170272)
