Growth factor receptors in cancer cells
Greater understanding of the spatial distribution of transmembrane receptor tyrosine kinases may provide new insights into signaling mechanisms controlling cell growth. Our research aims to study the role of these receptors in cancer cells using the unique characterization possibilities provided by liquid-phase electron microscopy, capable of studying tens of cells in their native liquid environment with nanometer resolution as needed to resolve the individual constituents of protein complexes. Our recent research involved members of the epidermal growth factor receptor (EGFR) family. HER2, one of the EGFR family members, is overexpressed in certain types of breast cancer. Although HER2 is considered an orphan receptor because it has no ligand, it can form homodimers, thereby contributing significantly to a dysregulation of intracellular signaling and of cell growth. In our latest research, we have studied the intra- and intercellular variation of HER2 at the single-molecule level in intact SKBR3 breast cancer cells. The unique experimental capabilities of liquid-phase scanning transmission electron microscopy (STEM) allowed quantifying the stoichiometry of HER2 complexes, distinguishing between monomers, dimers, and higher order clusters, while mapping its location in the cell. Compared to biochemical methods providing information about the average of many cells in pooled cellular material, our methodology provides unique information at the molecular level. Of major interest is to analyze differences in protein function between individual cancer cells (cancer cell heterogeneity) and between distinct functional membrane regions within the same cell. With our approach it is possible to study the effect of cancer drugs on small sub-populations of cells, aiming to increase the effectiveness of HER2 targeting drugs. Small sub-populations of breast cancer cells were found to respond differently to the prescription drug trastuzumab than bulk cancer cells, see Peckys et al., Mol. Biol. Cell 28, 3193-3202, 2017.
This research is funded by the Else Kröner-Fresenius-Stiftung in the project entitled “Investigation of the Influence of Breast Cancer Drugs on HER2 Dimerization at the Molecular Level in Individual Cells Aiming to Find Clues for Causes of Drug Resistance: HERe” with projects partners Prof. Stefan Wieman of the Deutsches Krebsforschungszentrum in Heidelberg, Dr. Diana Peckys, Biophysics Department, Saarland University, and Dr. Gilda Schmidt and Prof. Erich-Franz Solomayer of the Universitätsklinikum des Saarlandes.
Determination of ORAI channel subunit stoichiometry
The relative ratio of the different ORAI Ca2+ channels is highly relevant for cell function. However, their heterometric assembly and their stoichiometry are not known. For example, although experimental approaches pointed towards a dimeric structure at rest and a tetrameric structure upon activation, a crystal structure of truncated Drosophila ORAI1 was hexameric. Thus ORAI channel stoichiometry most likely changes during different functional states (i.e. at rest, during initial- and sustained channel activation). For ORAI2 or ORAI3, no structure is known.
Together with Prof. Barbara Niemeyer, Molecular Biophysics, Saarland University, Homburg, Germany, we explore the stoichiometry of ORAI channels. We use correlative light- and electron microscopy to visualize single ORAI ion channel subunits labeled with fluorescent nanoparticles in intact cells in their liquid state. The cellular location and the stoichiometry of these proteins will thus be studied in the intact plasma membrane, contrasting conventional approaches based on extracting the proteins from the membrane or inferring channels stoichiometry from fluorescence measurements.
This research is funded by the DFG and is part of the Collaborative Research Centre 1027 entitled “Physical modeling of non-equilibrium processes in biological systems”.
Stoichiometry of calcium channels
Liquid STEM is being used to study the stoichiometry of complexes formed by TRPV6 proteins in COS7 cells. TRPV6 proteins oligomerize into calcium-selective cation channels in the plasma membrane, which are essential for maintenance of the luminal Ca2+ concentration in the epididymal duct. Of particular interest is that the TRPV6 gene appears to be up-regulated in prostate and breast cancer. TRPV6 proteins thus could be a prognostic marker and a target for therapy under these conditions. Each TRPV6 protein consists of six predicted transmembrane helices and in analogy to the crystal structure of the six transmembrane potassium channels it is assumed that four individual TRPV6 proteins form a tetramer. However, X-ray crystallographic data is so far not available. We aim to determine the subunit stoichiometry via Liquid STEM. Such data is crucial for the understanding of ion channel function and their targeting by drugs. In addition, we have studied TMEM16A, a membrane protein forming a calcium-activated chloride channel. It was concluded that hTMEM16A resides in the plasma membrane as dimer only and is not present as monomer (J. Struct. Biol. 199, 102-113, 2017. link)This research is funded by a grant of the Leibniz competition 2014 entitled: “Electron microscopy of labeled protein complex subunits in whole cells in aqueous environment”.