Proteolysis in Cell Cycle and Cell Fate Decisions: How Ubiquitylation Coordinates Proliferation and Differentiation
Previous and Current Research
The coordination of proliferation with quiescence and differentiation is fundamental for multicellular life and development. The decision whether cells keep dividing, or whether they temporally or permanently exit from the cell cycle is tightly controlled by ubiquitin-mediated proteolysis of key cell cycle regulators. Ubiquitin-mediated proteolysis requires the sequential interplay of three enzymes: E1, E2, and ubiquitin E3 ligases. E3 ligases recognise and covalently modify crucial substrates, such as cell cycle regulators, with chains of ubiquitin molecules and thereby target them for degradation by the 26S proteasome. Emerging evidence indicates that mutations in components of the ubiquitin proteasome system are a hallmark of multiple cancers and in addition interfere with faithful development and differentiation.
Within three lines of research our lab combines cell biological and biochemical approaches to reveal how the ubiquitin system controls cell cycle progression and the exit into quiescence and differentiation using human cells a model.
1) Ubiquitination during the transition into quiescence
Focusing on key ubiquitin E3 ligases including the APC/C (Anaphase-Promoting Complex/Cyclosome) and SCF complexes (Skp, Cullin, F-Box) we investigate how proteolysis contributes to cell cycle re-entry in quiescent or differentiated cells. Therefore, we develop tools to identify ubiquitinated proteins by mass spectrometry, reconstitute ubiquitination in vitro using purified components, genetically and chemically interfere with ubiquitination, and employ single cell live cell imaging to monitor the transition into quiescence in unperturbed and perturbed conditions. — see figure below
2) A quantitative view on the cell cycle
Taking advantage of genome engineering by homologous recombination we visualize endogenous cell cycle regulators and substrates of ubiquitin E3 ligases with fluorescent probes. Based on quantitative single cell imaging we extract total and relative molecule numbers as cells progress through the cell cycle or undergo the transition into quiescence. We collaborate with Ingmar Glauche at the Institute for Medical Informatics and Biometry (IMB) in Dresden to cast our imaging data into predictive mathematical models and advance our automatic imaging analysis pipeline. — see video below
3) Cell cycle and redox regulation
As a new research area in the lab funded by an ERC starters grant we investigate how the redox system feeds back on the cell cycle machinery. We are in particular interested in understanding how reactive oxygen species (ROS) regulate cell cycle proteins during proliferation and revealing how this contributes to cell cycle control. Focusing first on physiological ROS signaling in normal cells we subsequently aim to unravel nodes between the cell cycle and redox system that cancer cells can hijack.
For more information on our projects and to get to know us better please visit our lab page.
Future Projects and Goals
In particular, we aim to understand:
- What are the key substrates of E3 ligases to initiate and to manifest cell fate decisions?
- How is the activity of E3 ligases is regulated during the cell cycle and upon the transition into quiescence and differentiation?
- How does proteolysis contribute to cell cycle re-entry in quiescent or differentiated cells, e.g. during stem cell divisions and regeneration?
- How does the redox system feed back in the cell cycle machinery and ubiquitination?
Methodological and Technical Expertise
- gene targeting in human somatic cells
- cell cycle and redox analyses
- high-throughput imaging
- reconstitution of protein ubiquitylation in vitro
Zerjatke T*, Gak IA*, Kirova D*, Fuhrmann M, Daniel K, Gonciarz M, Müller D, Glauche I, Mansfeld J
Quantitative cell cycle analysis based on an endogenous all-in-one reporter for cell tracking and classification.
Cell Reports 19:1953-1966 (2017), Selected as a “Research Highlight” in Nature Methods 14, 768 (2017)
Alfar EA*, Kirova D*, Konantz J, Birke S, Mansfeld J#, Ninov N#
Distinct Levels of Reactive Oxygen Species Coordinate Metabolic Activity with Beta-cell Mass Plasticity.
Scientific Reports 7:3994 (2017)
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
Real-time deformability cytometry: on-the-fly cell mechanical phenotyping.
Nat. Methods 12:199–202 (2015)
Hein MY, Hubner NC, Poser I, Cox J, Nagaraj N, Toyoda Y, Gak IA, Weisswange I, Mansfeld J, Buchholz F, Hyman AA, Mann M
A human interactome in three quantitative dimensions organized by stoichiometries and abundances.
Cell 163: 712–723. (2015)
Mansfeld J, Collin P, Collins MO, Choudhary JS, Pines J
APC15 drives the turnover of MCC-CDC20 to make the spindle assembly checkpoint responsive to kinetochore attachment.
Nat Cell Biol 13: 1234–1243 (2011)
*shared first authorship