Weitfeld-Mikroskop zur Lebendmikroskopie
Final Report Abstract
Live cell microscopy follows dynamic processes and gives new insights to unravel biological processes. The widefield live cell microscope (AF7000/Leica) is applied in many projects in the Microscopy Core Facility at the Institute of Molecular Biology (IMB gGmbH) in Mainz. Exemplarily, two projects are described. Cellular decision-making and environmental adaptation are dependent upon a heterogeneous response of gene expression to external cues. Heterogeneity arises in transcription from random switching between transcriptionally active and inactive states, resulting in bursts of RNA synthesis. Furthermore, the cellular state influences the competency of transcription, thereby globally affecting gene expression in a cell-specific manner. We determined how external stimuli interplay with cellular state to modulate the kinetics of bursting. To this end, single-cell dynamics of nascent transcripts were monitored at the endogenous estrogen-responsive GREB1 locus in a fluorescent widefield microscope. Stochastic modeling of gene expression implicated a two-state promoter model in which the estrogen stimulus modulates the frequency of transcriptional bursting. The cellular state affects transcriptional dynamics by altering initiation and elongation kinetics and acts globally, as GREB1 alleles in the same cell correlate in their transcriptional output. Our results suggest that cellular state strongly affects the first step of the central dogma of gene expression, to promote heterogeneity in the transcriptional output of isogenic cells. The Epigenetics of Aging: Autophagy is a ubiquitous catabolic process, which causes cellular bulk degradation through vesicular engulfment of obsolete, damaged or harmful cytoplasmic components. While autophagy regulates cellular homeostasis during development and in youth, there is mounting evidence that autophagy becomes increasingly dysfunctional with age. Recent work in "Caenorhabditis elegans" even suggests that late-life dysfunctional autophagy exhibits detrimental effects that drive the ageing process. Other studies link elevated autophagy closely to increased health and longevity. In a journal these apparently opposing views were put into perspective and define the current understanding of the role of autophagy during ageing. In order to identify genes that have negative effects late in life, a high-throughput RNAi screen enclosing around 400 genes (such as chromatin factors, members of the RNAi machinery, and ubiquitin pathways) was designed. Therefore, age synchronized "C. elegans" were cultured in transwell plates. Within the transwell, the worms are sitting on a transparent polyester membrane with 8 micrometer pores in a liquid medium. The media contains RNAi feeding bacteria, which can freely diffuse to the worms. Thus, a gene-inactivation in "C. elegans" is achieved via RNAi feeding. A screening platform implemented in the microscope software, was used to set up the screen. The software allows the imaging of different plate geometries in an automated way. The plates are imaged from below with a 1.25x objective to capture the entire well with one image.
Publications
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The Replisome-Coupled E3 Ubiquitin Ligase Rtt101Mms22 Counteracts Mrc1 Function to Tolerate Genotoxic Stress. PLOS Genetics 12(2): e1005843
Buser R, Kellner V, Melnik A, Wilson- Zbinden C, Schellhaas R, Kastner L, Piwko W, Dees M, Picotti P, Maric M, Labib K, Luke B, Peter M
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Zrf1 controls mesoderm lineage genes and cardiomyocyte differentiation. Cell Cycle. 2016; Dec15(23):3306-3317
Kaymak A, Richly H.
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ZRF1 mediates remodeling of E3 ligases at DNA lesion sites during nucleotide excision repair. The Journal of Cell Biology. 2016;213(2):185-200
Gracheva E, Chitale S, Wilhelm T, et al.
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Autophagy during ageing–from Dr Jekyll to Mr Hyde. FEBS Journal, Vol. 285.2018, Issue 13, pp. 2367-2376.
Wilhelm, T., Richly, H.
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Estrogen-dependent control and cell-to-cell variability of transcriptional bursting. Molecular Systems Biology, Vol. 14.2018,
Issue 2: e7678.
Fritzsch, T. et al.
