Multidimensional super-resolution imaging of membrane receptors
Membrane receptor-activated signal transduction pathways are integral to cellular functions and disease mechanisms in humans. However, until now two obstacles impede the exploitation of quantitative data about the dynamic architecture of membrane receptors: selective and efficient labeling of membrane receptors and the resolution limit of classical optical microscopy. Such quantitative data is of special importance considering the existence of confined plasma membrane compartments, i.e. nanodomains or clusters with a supposed size of 5-300 nm required for subcompartimentalization and associated function. We intend to map the distribution and quantify the spatial organization of membrane receptors in the context of other extra- and intracellular molecules at close to molecular resolution using multidimensional single-molecule based super-resolution imaging methods such as PhotoActivated Localization Microscopy (PALM) and direct Stochastic Optical Reconstruction Microscopy (dSTORM).
We will focus our attention on membrane receptors studied within the framework of the CRC/TR but will include also other membrane and membrane-associated cytoskeletal proteins and lipids to investigate the dynamic organization of extracellular membrane molecules proposed to neighboring cells. Our studies include single isolated adherent cells as well as tissue slices and tumor spheroids using wide-field, total-internal reflection (TIR), and single-plane illumination (SPIM) localization microscopy to decipher the influence of cell-cell interactions on the membrane protein network within a realistic setting. In order to achieve this ambitious goal and quantify the spatial distribution of more than 10 different target molecules in a single experiment, we aim to develop a new method for high-throughput protein localization and quantification studies that runs cycles of fluorescence tagging and super-resolution imaging using the same fluorophore. In order to remove the fluorophore from the sample before the next round of fluorescence tagging and imaging, we will develop and optimize different approaches including photobleaching, and photocleaving of fluorophore linkers. Alternatively, we intend to use the transient binding of short fluorescently labeled oligonucleotides for multiplexed super-resolution imaging. Here, the molecules of interest will be labeled with a short specific DNA sequence. Upon transient binding of the complementary fluorophore-labeled oligonucleotide the molecules will be localized. Using different DNA sequences for unequivocal labeling of the molecules of interest, cycles of washing, addition of the next unique fluorophore-labeled oligonucleotide, and localization microscopy can be run. In parallel labeling protocols have to be optimized to enable efficient multi-target labeling and imaging with maximal structure preservation. The use of the same fluorophore for all target molecules ensures comparable detection efficiencies and simplifies quantification of localization data. That is, we aim to introduce a powerful method for the quantitative visualization of the entire dynamic toponome map of membrane receptors.
Rainer Heintzmann will coordinate and supervise the project together with other members of the team.
Markus Sauer will coordinate and supervise the project together with other members of the team.
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