Advanced cellular imaging to study functional nuclear organization
|Current Lab Members:||
Mr Ezequiel Miron Sardiello (DPhil Student - Biochemistry)
Mr Justin Demmerle (DPhil Student - NDM NIH-Oxford)
Dr Cassandravictoria Innocent (Postdoctoral Research Assistant)
|Former Lab Members:||
Mr Quentin Alle (Visiting MSci Student - University of Nimes)
Chromatin of higher eukaryotes is folded in a complex manner to form higher-order domains and fibres of variable compaction, demarcated by a network of largely chromatin-void interchromatin spaces and channels leading up to nuclear pores. Compartmentalization into restrictive and permissive local chromatin environments and their confinements to specific nuclear sites are thought to play a major role in the regulation of gene activity, mRNA transport, DNA replication and repair. This large-scale organization is determined by an intricate network of epigenetic factors (histone variants and modifications, DNA methylation, PcG proteins, non-translated RNAs), but also involves numerous structural proteins (e.g., Lamins, NUPs, HP1, Cohesins).
We apply and refine advanced optical imaging methods (e.g., super-resolution structured illumination, single molecule localization, FRAP, laser-microirradiation) to address general rules and dynamic aspects of higher order chromosomal organization in mammalian cell nuclei and its contribution to control genome function.
We combine optimized immunolabelling, FISH and GFP-based protocols with customized statistical tools to quantitatively analyse spatial distribution pattern and dynamic properties of factors involved. Comparative super-resolution 3D-mapping data from single cells in defined states are complementing population-wide ChIP-sequencing and chromosome conformation capturing approaches and will contribute to a better understanding of fundamental nuclear processes.
As an integral member of the Micron Advanced Bioimaging Unit (www.micronoxford.com), we also work on new correlative super-resolution approaches for live and fixed cell imaging that are expected to be of use for many collaborative research projects within the Oxford research community.
- Smeets D, Markaki Y, Schmid VJ, Krause F, Tattermusch A, Cerase A, Sterr M, Fiedler S, Demmerle J, Popken J, Leonhardt H, Brockdorff N, Cremer T, Schermelleh L, Cremer M. 2014. Three-dimensional super-resolution microscopy of the inactive X chromosone territory reveals a collapse of its active nucelar compartment harboring sitinct Xist RNA foci. Epigenetics Chromatin. 7:8.
- Lesterlin C, Ball G, Schermelleh L, Sherratt D. 2014. RecA bundles mediate homology pairing between distant sisters during DNA break repair. Nature 506: 249-253.
- Schneider K, Fuchs C, Dobay A, Rottach A, Qin W, Álvarez-Castro JM, Nalaskowski MM, Schmid V, Leonhardt H, Schermelleh L. 2013. Dissection of cell cycle dependent dynamics of Dnmt1 by FRAP and diffusion-coupled modelling. Nucleic Acids Res. 41: 4860-76.
- Schermelleh L, Heintzmann R, Leonhardt H. 2010. A guide to super-resolution fluorescence microscopy. J Cell Biol. 190: 165-175.
- Schermelleh L, Carlton PM, Haase S, Shao L, Winoto L, Kner P, B. Burke, Cardoso MC, Agard DA, Gustafsson MG, Leonhardt H, and Sedat JW. 2008. Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320: 1332-1336.
- Schermelleh L, Haemmer A, Spada F, Rosing N, Meilinger D, Rothbauer U, Cardoso MC, and Leonhardt H. 2007. Dynamics of Dnmt1 interaction with the replication machinery and its role in postreplicative maintenance of DNA methylation. Nucleic Acids Res. 35: 4301-4312.
Figure 1: (a) Super-resolution imaging of the nuclear periphery with 3D-SIM, compared to conventional confocal laser-scanning microscopy (CLSM). Mouse C2C12 cells are immunostained with antibodies against Lamin B (green) and antibodies recognizing the cytoplasmic face of the nuclear pore complex (NPC, red). DNA is counterstained with DAPI (blue). 3D-SIM reveals a triple layered structural organization as well as chromatin channels underneath individual nuclear pores, which is not resolved with CLSM. Bars: 5 µm and 1 µm (insets). Modified from Schermelleh et al. (2008), Science 320. (b) Chromatin compartmentalization at the nuclear periphery. Schematic overlay of topologically distinct chromatin zones. The stroke width is equivalent to 100 nm, which is about the resolution limit of 3D-SIM. Bar: 1 µm.
Figure 2: Nuclear topography of RNA Pol II studied with 3D SIM, compared to deconvolution wide-field microscopy. Immunolabelling of mouse C127 cell nucleus with Ser-2P RNA Pol II and Pol 3.3 specific antibodies shows the enrichment of RNA Pol II signals at the periphery or on fibrillar protrusions of chromatin domains. Modified from Markaki et al. (2010), Cold Spring Harb Symp Quant Biol. 75.
Graduate Student and Postdoctoral Positions: Enquiries with CV welcome