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Live Cell Imaging on Single Cell Arrays (LISCA)

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One of the most prominent phenomena in biological systems is their capability to adopt to environmental changes. The underlying signaling networks provide strategies of optimal response. A common observation is the fact that cell decisions are heterogeneous among a isogenic population. This fact can be traced back to natural number fluctuations in signalling elements and can be treated as noise in biochemical networks.

We are interested the timing and dynamics of single cell gene expression using single cell time-lapse microscopy. We studied various bacterial model systems the (1) arabinose utilization, (2) the competence state and (3) the onset of biofilm formation.

 A high-throughput microscopy method for single-cell analysis of event-time correlations in nanoparticle-induced cell death

Murschhauser Alexandra, Röttgermann Peter J. F., Woschée Daniel, Ober Martina F., Yan Yan, Dawson Kenneth A., Rädler Joachim O.

results

The temporal context of cell death decisions remains generally hidden in ensemble mea- surements with endpoint readouts. Here, we describe a method to extract event times from fluorescence time traces of cell death-related markers in automated live-cell imaging on single-cell arrays (LISCA) using epithelial A549 lung and Huh7 liver cancer cells as a model system. In pairwise marker combinations, we assess the chronological sequence and delay times of the events lysosomal membrane permeabilization, mitochondrial outer membrane permeabilization and oxidative burst after exposure to 58 nm amino-functionalized poly- styrene nanoparticles (PS-NH2 nanoparticles). From two-dimensional event-time scatter plots we infer a lysosomal signal pathway at a low dose of nanoparticles (25 µgmL−1) for both cell lines, while at a higher dose (100 µgmL−1) a mitochondrial pathway coexists in A549 cells, but not in Huh7. In general, event-time correlations provide detailed insights into heterogeneity and interdependencies in signal transmission pathways.

  • Murschhauser, A., Röttgermann, P. J., Woschée, D., Ober, M. F., Yan, Y., Dawson, K. A., & Rädler, J. O. (2019). A high-throughput microscopy method for single-cell analysis of event-time correlations in nanoparticle-induced cell death. Communications biology, 2(1), 35.


SINGLE CELL MICROARRAYS FABRICATED BY MICROSCALE PLASMA- INITIATED PROTEIN PATTERNING (ΜPIPP)
Micropatterned arrays considerably advanced single cell fluorescence time-lapse measurements by providing standardized boundary conditions for thousands of cells in parallel. In these assays, cells are forced to adhere to defined microstructured protein islands separated by passivated, nonadhesive areas. Here we provide a detailed protocol on how to reproducibly fabricate high quality single cell arrays by microscale plasma-initiated protein patterning (μPIPP). Advantages of μPIPP arrays are the ease of preparation and the unrestricted choice of substrates as well as proteins. We demonstrate how the arrays enable the efficient measurement of single cell time trajectories using automated data acquisition and data analysis by example of single cell gene expression after mRNA transfection and time courses of single cell apoptosis. We discuss the more general use of the protocol for assessment of single cell dynamics with the help of fluorescent reporters.
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  • Anita Reiser, Matthias Lawrence Zorn, Alexandra Murschhauser, Joachim Oskar Rädler
    Part of the Methods in Molecular Biology book series (MIMB, volume 1771)

Optimization of sugar utilization strategies

Sonja Westermayer, Judith Mergerle, Georg Fritz, Uli Gerland, Joachim Rädler

Bacteria can rapidly react to environmental changes by adapting gene expression of certain genes. We investigate the time-dependent response of sugar utilization systems in Escherichia coli on the single-cell level. We study both individual systems and the situation of two competing sugar utilization systems, with the goal to characterize properties that may have been optimized by evolution. Using microfluidic set-ups we expose bacterial cultures to systematically variable environments and use time-lapse microscopy and single cell tracking to acquire single-cell expression kinetics. In mathematical models using cost-benefit analysis and game theoretical concepts, we compare different regulatory schemes as strategies to cope with variable environments.

This work is supported by the DFG through the priority program SPP

related publications:

  • J.A. Megerle, G. Fritz, U. Gerland, K. Jung, J.O. Rädler
    "Timing and dynamics of single cell gene expression in the arabinose utilization system"
    Biophysical Journal 95, p. 2103-2115 (2008)
  • M. Leisner, J.-T. Kuhr, J.O. Rädler, E. Frey, and B. Maier
    Kinetics of genetic switching into the state of bacterial competence
    Biophys. Journal 96(3) pp. 1178 - 1188 (2009)
  • A. Meyer, J. Megerle, C. Kuttler, J. Müller, C. Aguilar, L. Eberl, B. Hense, J.O. Rädler
    "Dynamics of AHL mediated quorum sensing under flow and non-flow conditions"
    Physical Biology, 9 (2012) 026007.

Collaborations:

  • Prof. Dr. Ulrich Gerland, LMU
  • Prof. Dr. Karin Schnetz, Universität zu Köln
  • Prof. Dr. Kirsten Jung, LMU

Stem cell decision making

 

FURTHER READING

  • A. Meyer, J. Megerle, C. Kuttler, J. Müller, C. Aguilar, L. Eberl, B. Hense, J.O. Rädler
    "Dynamics of AHL mediated quorum sensing under flow and non-flow conditions"
    Physical Biology, 9 (2012) 026007.
  • M. Leisner, J.-T. Kuhr, J.O. Rädler, E. Frey, and B. Maier
    Kinetics of genetic switching into the state of bacterial competence
    Biophys. Journal 96(3) pp. 1178 - 1188 (2009)
  • J.A. Megerle, G. Fritz, U. Gerland, K. Jung, J.O. Rädler
    "Timing and dynamics of single cell gene expression in the arabinose utilization system"
    Biophysical Journal_ 95, p. 2103-2115 (2008)
  • Leisner, M., K. Stingl, J.O. Rädler, and B. Maier
    "Basal expression rate of comK sets a 'switching-window' into the K-state of Bacillus subtilis"
    Molecular Microbiology, 63(6), 1806-1816 (2007).