Gruppenleiter


Stephan Diekmann
Stephan Diekmann

Wissenschaftliche Mitarbeiter

Christian Hoischen

Volker Döring

Shiva Marthandan

Doktoranden

Ivana Sumanovac

Technische Assistenten

Sabine Gallert

Sabine Ohndorf

Sylke Pfeifer



 

Forschungsgruppe Diekmann

(Zur Zeit nur in Englisch verfügbar)

Stephan Diekmann, Professor fr Biophysikalische Chemie, emeritierte im September 2014. Er ist als Gastwissenschaftler noch weiter mit dem FLI verbunden.

Molekularbiologie

Human Kinetochores

We focus on the structure/function relationship of the human centromere/kinetochore complex. The centromere is a chromosomal substructure responsible for DNA segregation. The kinetochore protein complex settles at the centromere and, during mitosis, attaches the centromere to spindle microtubuli. Microtubule attachment is regulated by the mitotic checkpoint. Faithful chromosome segregation is essential for genome integrity; malfunction of this structure leads to aneuploidy. We are studying the proteins involved in kinetochore and checkpoint function by investigating their assembly and complex binding behaviour in time and space in living human cells, increasingly also in vitro and by pull-down analyses.

 

Cellular Ageing

We study cellular senescence. We carry out deep transcriptome analyses of cellular senescence within the BMBF-funded JenAge research consortium. In primary human fibroblast, we study the effects of mild stress. Based on these studies, we are establishing a quantitative model for the cellular transition from proliferation to senescence.

Projects

  • The centromere/kinetochore complex

  • We analyse, mainly in vivo, the properties of the human inner kinetochore complex. Little is known about kinetochore complex topology, protein concentrations and the presence and assembly dynamics of kinetochore proteins during the cell cycle. In collaboration with the Leonhardt lab (LMU Munich) we determined inner kinetochore protein interactions in the human cell nucleus. Furthermore, we determined the protein proximities within the inner kinetochore by in vivo FRET. Combining these interaction and proximity data, we are constructing a 3-dimensional model of the interaction network of the human inner kinetochore proteins (collaboration with P. Dittrich, FSU Jena). Parallel to these association studies, we also determine the cell cycle dependent (i) binding dynamics of kinetochore proteins to the centromere (by FRAP, FCCS) and (ii) protein concentrations. We observe a surprisingly large variability of both values.
    We pulled-down centromeric nucleosomes containing tagged CENP-A and analysed their structure by EM and AFM. In collaboration with the Dalal lab (NIH, USA) we found that human CENP-A containing nucleosomes can adopt a hemisome structure consisting of only one copy of histones CENP-A, H4, H2A and H2B each (Dimitriadis et al., 2010). In a following study we found that CENP-A containing nucleosomal structures form octamers only during S-phase, but aggregate to tetramers during the rest of the cell cycle (Bui et al., 2012).
    We detected that CENP-N assembly into the CCAN constantly changes over the cell cycle (Hellwig et al., 2011). Next we studied the properties of CENP-T and CENP-W (collaboration with K. Sullivan, Galway, Ireland). We analysed the cell cycle regulation, timing of assembly, generational persistence, and requirement for function of CENP-T and CENP-W in the cell cycle in human cells. The CENP-T/W complex assembles through a dynamic exchange mechanism in late S-phase and G2, is required for mitosis in each cell cycle and does not persist across cell generations (Prendergast et al., 2011), properties reciprocal to those of CENP-A. We thus proposed that the CENP-A and H3-CENP-T/W nucleosome components of the centromere are specialized for centromeric and kinetochore activities, respectively. We speculate that these two separate functions allow the cell to switch between chromatin configurations that reciprocally support (i) the replication of the centromere and (ii) its conversion to a mitotic state on post-replicative chromatin. We continued this work by analyzing the behavior of CENP-S and CENP-X which associate with CENP-T and CENP-W at the kinetochore (Dornblut et al., in revision). Additionally, we studied the CENP-Q/U/P/O/R sub-complex of the inner kinetochore (Eskat et al., 2012). We found that the proteins of this sub-complex bind to the kinetochore as single units mostly in S-phase. Once bound to the kinetochore, two of these proteins (CENP-Q and -U) multimerise suggesting a maturatioin step of centromeric chromatin before mitosis.
  • Mechanisms of cellular senescence

  • Cellular senescence is a permanent cell cycle arrest that can be induced by different mechanisms, including shortened telomeres, accumulation of DNA damage and oncogenic or oxidative stress. Senescent cells also arise in tissues with ageing where they may exert anti-tumor functions and/or contribute to ageing phenotypes. As an additional marker for senescent cells, we established the accumulation of annexin V at the nuclear envelope as a new tool to detect senescent cells in vitro and in human skin (Klement et al., 2012). We also demonstrated that depletion of annexin V accelerates cellular ageing of primary human fibroblasts.
    Within the BMBF-funded JenAge (Jena Centre for Systems Biology of Ageing) research project we analysed the transcriptome(s) of human primary fibroblasts during their ageing in vitro. Massive parallel mRNA sequencing was performed for two human primary fibroblast cell types at 5 different time points of their lifespan. Ageing-associated differentially expressed genes (aaDEGs) are currently being compared to aaDEGs from other model organisms of the JenAge consortium (H. sapiens, M. musculus, D. rerio, N. furzeri, C. elegans). We hope to identify commonly regulated gene expression networks which might reveal new pathways of ageing.
    In parallel we finished analyses of anti-ageing effects of anti-oxidative compounds on primary human blood cells (Marthandan et al., 2011).
    We developed a systems-biology cellular senescence model (collaboration with S. Schuster, FSU Jena) establishing a new mathematical model which quantitatively describes the ageing process of human fibroblast during replicative senescence (Schäuble et al., 2012). Applying this model, we quantitatively described the growth curve of several cell lines and their transition into senescence due to different origins of stress. The model makes specific predictions which we are testing experimentally.

 

Ausgewählte Publikationen

  • Eskat A, Deng W, Hofmeister A, Rudolphi S, Emmerth S, Hellwig D, Ulbricht T, Döring V, Bancroft JM, McAinsh AD, Cardoso MC, Meraldi P, Hoischen C, Leonhardt H, Diekmann S (2012) Step-wise assembly, maturation and dynamic behavior of the human CENP-P/O/R/Q/U kinetochore sub-complex. PLoS One, 7, e44717 [PLos One]
  • Bui M, Dimitriadis E K, Hoischen C, An E, Quénet D, Giebe S, Nita-Lazar A, Diekmann S, Dalal Y (2012) Cell-cycle-dependent structural transitions in the human CENP-A nucleosome in vivo. Cell, 150, 317-326 [PubMed]
  • Hellwig D, Emmerth S, Ulbricht T, Döring V, Hoischen C, Martin R, Samora CP, McAinsh AD, Carroll CW, Straight AF, Meraldi P, Diekmann S (2011) Dynamics of CENP-N kinetochore binding during the cell cycle. J Cell Sci, 124, 3871-3883 [PubMed]
  • Prendergast L, van Vuuren C, Kaczmarczyk A, Doering V, Hellwig D, Quinn N, Hoischen C, Diekmann S, Sullivan KF (2011) Premitotic assembly of human CENPs -T and -W switches centromeric chromatin to a mitotic state. PLoS Biol, 9, e1001082 [PubMed]
  • Dimitriadis EK, Weber C, Gill RK, Diekmann S, Dalal Y (2010) Tetrameric organization of vertebrate centromeric nucleosomes. Proc Natl Acad Sci U S A, 107, 20317-20322 [PubMed]

 


Last update: October 14, 2014

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