Datenaufnahmesystem
Zusammenfassung der Projektergebnisse
In the electron tomography projects (Prof. Dr. Frangakis) the "Datenaufnahmesystem" has mainly been used in the fields of electron tomogram on-line alignment, high-throughput volume reconstruction, visualization, template matching, and subtomogram averaging. As a new field, also the correlation of light and electron microscopy data have been facilitated by the "Datenaufnahmesystem". The group correlated the bright, well-resolved signals of synthetic fluorophores in super-resolution light microscopy with high precision on volume data generated by both transmission and scanning electron microscopy. A new heuristics has been established and used in template matching and subtomogram averaging of whole cells. Using subtomogram averaging, the group defined the biophysical properties of the centromeric histone H3 variant centromeric protein A (CENP-A) which is responsible for specifying the location of the centromere. Two studies reconstructed individual nucleosomes both in in vitro arrays and in vivo, in cryosections. Using subtomogram averaging approaches, the studies showed nucleosome stacking as an important mechanism for generating chromatin compaction and explained how transcription and regulation factors can access nucleosomes in vivo. Collaborative projects that made use of the "Datenaufnahmesystem" included the analysis of the 3D arrangement and macromolecular structure of yeast prions, the study of the 3D ultrastructural organization of a unicellular green alga, and determined that the communication between endothelial cells and smooth muscle cells is based on an miRNA- and extracellular-vesicle-mediated mechanism. Within the group of Prof. Stelzer the "Datenaufnahmesystem" has mainly been used to align and 4- dimensionally reconstruct data from light sheet-based fluorescence microscopes (LSFM). By combining LSFM with a novel mounting method, the group achieved complete, continuous and non-invasive fluorescence live imaging of Tribolium embryogenesis at high spatiotemporal resolution. In addition, the group presented protocols for growing and embedding of cell cultures in 3D and imaging these cellular spheroides by LSFM and thus making optimal use of the optical sectioning and minimal phototoxic damage or photobleaching outside the focal plane properties of the LSFM. Also by LSFM, the group recorded the growth of primary root tips and lateral root primordia of Arabidopsis thaliana over several hours. This allowed them to quantify the contribution of cell elongation to the early morphogenesis of lateral root primordia and uncover the diurnal growth rhythm of lateral roots. Additionally, the group developed a detailed drug assay protocol performed with live cellular spheroids imaged by automated epifluorescence live microscopy and LSFM to investigate the effects of drugs on the spheroids over several days. The LSFM systems of the group have been successfully used in several collaborative projects specifically in the analysis of lateral root development, the determination of an auxin transport-dependent regulation of asymmetric growth that determines the gravitropic set-point angle of lateral roots, and in cellular junction assembly in 3D. The group of Prof. Reichert used the "Datenaufnahmesystem" for the storage and processing of 3D light microscopy, for EM tomography data and for LC-MS/MS analysis. They used LC-MS/MS based complexome profiling and identified apolipoprotein O (APOO) and apolipoprotein O-like protein (APOOL) as putative components of the Mitofilin/MINOS protein complex. By fluorescent light microscopy and electron microscopy the group analyzed the role of mitochondrial fission in the removal of mitochondria after mild and transient oxidative stress and showed that applying such conditions led to fragmentation of mitochondria and induction of DRP1-dependent mitophagy in mouse and human cells. Additionally, the group investigated the functional roles of individual domains of an inner mitochondrial membrane protein that plays a crucial role in cristae junction formation by biochemistry, as well as fluorescent light and electron microscopy. The group of Prof. Fucini, that moved to Portugal in 2012, used the "Datenaufnahmesystem" to store and process solid-state NMR data enhanced by dynamic nuclear polarization to analyze ribosome structure.
Projektbezogene Publikationen (Auswahl)
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(2013): Threedimensional visualization of the molecular architecture of cell-cell junctions in situ by cryo-electron tomography of vitreous sections. In: Methods Mol. Biol. 961, S. 97–117
Al-Amoudi, Ashraf; Frangakis, Achilleas S.
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(2014): CENP-A arrays are more condensed than canonical arrays at low ionic strength. In: Biophys. J. 106 (4), S. 875–882
Geiss, Christian P.; Keramisanou, Dimitra; Sekulic, Nikolina; Scheffer, Margot P.; Black, Ben E.; Frangakis, Achilleas S.
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(2014): Correlative lightand electron microscopy with chemical tags. In: J. Struct. Biol. 186 (2), S. 205–213
Perkovic, Mario; Kunz, Michael; Endesfelder, Ulrike; Bunse, Stefanie; Wigge, Christoph; Yu, Zhou et al.
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(2014): Imaging cellular spheroids with a single (selective) plane illumination microscope. In: Cold Spring Harb Protoc 2014 (1), S. 106–113
Swoger, Jim; Pampaloni, Francesco; Stelzer, Ernst H K
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(2014): Imaging MDCK cysts with a single (selective) plane illumination microscope. In: Cold Spring Harb Protoc 2014 (1), S. 114–118
Swoger, Jim; Pampaloni, Francesco; Stelzer, Ernst H K
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(2014): Light-sheet-based fluorescence microscopy for three-dimensional imaging of biological samples. In: Cold Spring Harb Protoc 2014 (1), S. 1–8
Swoger, Jim; Pampaloni, Francesco; Stelzer, Ernst H K
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(2014): M-free: Scoring the reference bias in sub-tomogram averaging and template matching. In: J. Struct. Biol.
Yu, Zhou; Frangakis, Achilleas S.
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(2014): Non-invasive long-term fluorescence live imaging of Tribolium castaneum embryos. In: Development 141 (11), S. 2331–2338
Strobl, Frederic; Stelzer, Ernst H K
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(2014): Novel intracellular functions of apolipoproteins: the ApoO protein family as constituents of the Mitofilin/MINOS complex determines cristae morphology in mitochondria. In: Biol. Chem. 395 (3), S. 285– 296
Koob, Sebastian; Reichert, Andreas S.
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(2014): Quantifying the autophagy-triggering effects of drugs in cell spheroids with live fluorescence microscopy. In: Methods Mol. Biol. 1165, S. 19–29
Ansari, Nariman; Hardung, Stefanie; Hötte, Katharina; Rakel, Stefanie; Antonietti, Patrick; Kögel, Donat et al.
