Assay Technology
Coordination: Dr. Holger Erfle | Interdisciplinary Center for Scientific Computing, ViroQuant-CellNetworks RNAi Screening Facility | Holger.Erfle (at) bioquant.uni-heidelberg.de
Novel technologies and resources for the systematic functional analysis of miRNAs and its targets in living cells
An integrated High-Throughput and Super High Resolution Microscopy Platform for analysing miRNA Targets
The role of microRNAs (miRNAs) as potent regulators of many diverse and essential cellular functions is becoming increasingly apparent. They have the ability to affect and regulate hundreds of different mRNAs (messenger RNA) simultaneously. This induces well-balanced modulations in protein synthesis depending on ever changing cellular needs. In spite of the important role of miRNAs in these key processes, our current knowledge of the function of miRNAs in the cell, and especially of their localisation and their interactions with their target mRNAs, remains surprisingly limited. One major reason is the lack of highly sensitive and specific detection methods for miRNAs and their cellular targets on single cell levels.
In fact, existing methods can only yield static and global snapshots of miRNA expression profiles within cell populations, without taking into account the highly dynamic behaviour within the individual cell. The novel technology platform proposed here will close this critical gap and thereby substantially further deepen our understanding of the role and function of endogenous miRNAs. Most notably, the particular application of super-resolution fluorescence microscopy for quantitative and qualitative “real-time” detection of miRNA-mRNA interactions on a single cell level will allow for unprecedented sensitivities and specificities.
The experimentally confirmed interactions of miRNAs and their targets will be refined by co-localisation measurements to cellular structures. Thereafter, the dynamics of the miRNA changes upon given biological system under normal and altered conditions will be determined. Combined with the construction of regulatory networks models of the miRNA regulatory network with the biological accuracy not possible before will be created.
Scheme of the workflow:
Project partners
- Virus-Cell-Interactions (Dirk Grimm | Hygiene Institut, Universitätsklinikum Heidelberg)
- Screening of Cellular Networks (Vytaute Starkuviene | Interdisciplinary Center for Scientific Computing, University of Heidelberg)
- ViroQuant-CellNetworks RNAi Screening Facility (Holger Erfle | Interdisciplinary Center for Scientific Computing, University of Heidelberg)
The miRNA-mRNA interactions will be studied via automated correlative microscopy, linking high-throughput wide field with confocal microscopy, and further with high-resolution STORM technology (Mike Heilemann) using cellarrays. The fast wide field and the confocal microscopes hereby act as filters that serve to drastically reduce the number of “interesting” cells for downstream analysis on the STORM microscope. The newly developed cellarrays arising from a collaboration between Graffinity Pharmaceuticals GmbH will allow a higher number of clearly spatial addressable spots.
The most advanced methodologies in statistical data analysis, machine-based learning and image processing (Fred Hamprecht) will be applied to extract the maximum possible information from the experimental data as well as to efficiently streamline experimental work (confocal microscopy and STORM).
- Single Molecule Biology (Mike Heilemann | Interdisciplinary Center for Scientific Computing, University of Heidelberg)
We will provide a technological platform for super-resolution microscopy with nanometer resolution which will complement the automated correlative microscopy (Holger Erfle). It is our aim to apply super-resolution microscopy to study the spatial organisation, concentration and dynamics of specific mRNA and miRNA, as well as their interaction.
We will identify suitable fluorescent probes that can be operated as photoswitchable units and as such be used in localization-based super-resolution microscopy such as stochastic optical reconstruction microscopy (STORM) and direct STORM (dSTORM). At the same time and in cooperation with Dirk Grimm and Vytaute Starkuviene, we will develop labelling techniques for specific mRNA and miRNA, which will include direct labelling as well as sequence-specific probes.
Novel algorithmic approaches that are crucial to interpret localization microscopic data will be developed together with Fred Hamprecht.
- Multidimensional Image Processing Group (Fred Hamprecht | Interdisciplinary Center for Scientific Computing, University of Heidelberg)
High-throughput, high-resolution microscopy produces huge amounts of data that have to be processed automatically. We will provide suitable image analysis algorithms that are largely based on modern machine learning methods so that they can be easily adapted to different experimental setups. A major task of these algorithms will be to identify regions-of-interest at lower resolution which are worth exploring at the next higher resolution (i.e. from wide field to confocal, from confocal to STORM). Co-localization of flourescent molecules will be determined in the resulting multi-modal and multi-resolution representation with high accuracy and reproducability. Furthermore, we will contribute algorithmic improvements to STORM data processing in order to fully utilize the information content of this modality. Automatic quality control will ensure that defective images are excluded from further analysis.
- Modeling of Biological Processes (Ursula Kummer | Heidelberg Institute of Zoology, University of Heidelberg)