Gilbert O. Fruhwirth

RhoA and RhoC have distinct roles in migration and invasion by acting through different targets

Although closely related, RhoA and RhoC have distinct molecular targets and functional roles in cell migration and invasion.

3D super-resolution imaging with blinking quantum dots.

Quantum dots are promising candidates for single molecule imaging due to their exceptional photophysical properties, including their intense brightness and resistance to photobleaching. They are also notorious for their blinking. Here we report a novel way to take advantage of quantum dot blinking to develop an imaging technique in three-dimensions with nanometric resolution. We first applied this method to simulated images of quantum dots and then to quantum dots immobilized on microspheres. We achieved imaging resolutions (fwhm) of 8-17 nm in the x-y plane and 58 nm (on coverslip) or 81 nm (deep in solution) in the z-direction, approximately 3-7 times better than what has been achieved previously with quantum dots. This approach was applied to resolve the 3D distribution of epidermal growth factor receptor (EGFR) molecules at, and inside of, the plasma membrane of resting basal breast cancer cells.

The potential of optical proteomic technologies to individualize prognosis and guide rational treatment for cancer patients

Genomics and proteomics will improve outcome prediction in cancer and have great potential to help in the discovery of unknown mechanisms of metastasis, ripe for therapeutic exploitation. Current methods of prognosis estimation rely on clinical data, anatomical staging and histopathological features. It is hoped that translational genomic and proteomic research will discriminate more accurately than is possible at present between patients with a good prognosis and those who carry a high risk of recurrence. Rational treatments, targeted to the specific molecular pathways of an individual’s high-risk tumor, are at the core of tailored therapy. The aim of targeted oncology is to select the right patient for the right drug at precisely the right point in their cancer journey. Optical proteomics uses advanced optical imaging technologies to quantify the activity states of and associations between signaling proteins by measuring energy transfer between fluorophores attached to specific proteins. Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) assays are suitable for use in cell line models of cancer, fresh human tissues and formalin-fixed paraffin-embedded tissue (FFPE). In animal models, dynamic deep tissue FLIM/FRET imaging of cancer cells in vivo is now also feasible. Analysis of protein expression and post-translational modifications such as phosphorylation and ubiquitination can be performed in cell lines and are remarkably efficiently in cancer tissue samples using tissue microarrays (TMAs). FRET assays can be performed to quantify protein-protein interactions within FFPE tissue, far beyond the spatial resolution conventionally associated with light or confocal laser microscopy. Multivariate optical parameters can be correlated with disease relapse for individual patients. FRET-FLIM assays allow rapid screening of target modifiers using high content drug screens. Specific protein-protein interactions conferring a poor prognosis identified by high content tissue screening will be perturbed with targeted therapeutics. Future targeted drugs will be identified using high content/throughput drug screens that are based on multivariate proteomic assays. Response to therapy at a molecular level can be monitored using these assays while the patient receives treatment: utilizing re-biopsy tumor tissue samples in the neoadjuvant setting or by examining surrogate tissues. These technologies will prove to be both prognostic of risk for individuals when applied to tumor tissue at first diagnosis and predictive of response to specifically selected targeted anticancer drugs. Advanced optical assays have great potential to be translated into real-life benefit for cancer patients.

Kinase-mediated quasi-dimers of EGFR

Ligand-induced dimerization of the epidermal growth factor receptor (ErbB-1/EGFR) involves conformational changes that expose an extracellular dimerization interface. Subsequent alterations within the cytoplasmic kinase domain, which culminate in tyrosine phosphorylation, are less understood. Our study addressed this question by using two strategies: a chimeric receptor approach employed ErbB-3, whose defective kinase domain was replaced by the respective part of EGFR. The implanted full-length kinase, unlike its subdomains, conferred dimerization and catalysis. The data infer that the kinase function of EGFR is restrained by the carboxyl tail; once grafted distally to the ectopic tail of ErbB-3, the kinase domain acquires quasi-dimerization and activation. In an attempt to alternatively refold the cytoplasmic tail, our other approach employed kinase inhibitors. Biophysical measurements and covalent cross-linking analyses showed that inhibitors targeting the active conformation of EGFR, in contrast to a compound recognizing the inactive conformation, induce quasi-dimers in a manner similar to the chimeric ErbB-3 molecule. Collectively, these observations unveil kinase domain-mediated quasi-dimers, which are regulated by an autoinhibitory carboxyl tail. On the basis of these observations, we propose that quasi-dimers precede formation of ligand-induced, fully active dimers, which are stabilized by both extracellular and intracellular receptor-receptor interactions.

How Forster Resonance Energy Transfer Imaging Improves the Understanding of Protein Interaction Networks in Cancer Biology

Herein we discuss how FRET imaging can contribute at various stages to delineate the function of the proteome. Therefore, we briefly describe FRET imaging techniques, the selection of suitable FRET pairs and potential caveats. Furthermore, we discuss state-of-the-art FRET-based screening approaches (underpinned by protein interaction network analysis using computational biology) and preclinical intravital FRET-imaging techniques that can be used for functional validation of candidate hits (nodes and edges) from the network screen, as well as measurement of the efficacy of perturbing these nodes/edges by short hairpin RNA (shRNA) and/or small molecule-based approaches.

Mediation of Apoptosis by Oxidized Phospholipids

Free radical-mediated oxidation of (poly)unsaturated glycerophospholipids in membranes and lipoproteins leads to the formation of a plethora of products. Some of these oxidized phospholipids, especially the truncated forms, induce apoptosis depending on their chemical structure, concentration and cell type. Depending on the phospholipid and the cell type, two pathways have so far been identified for the intracellular transmission of the apoptotic signals. One pathway involves activation of acid sphingomyelinase, which gives rise to the formation of ceramide and is followed by phosphorylation of pro-apoptotic mitogen-activated protein kinases. Alternatively, oxidized phospholipids act directly on mitochondria leading to efflux of pro-apoptotic effectors in endothelial cells. During the execution of the apoptotic program additional oxidized phospholipids are generated. The apoptotic cascade itself leads to oxidation and exposure of e.g. membrane phosphatidylserine. Oxidized phospholipids on the outer leaflet of the plasma membrane can form surface lipid patterns that specifically bind to phagocytic cells, e.g. macrophages.In this manuscript we review the recent literature reporting on apoptosis-inducing glycerophospholipids. In addition, we describe the cellular processes that lead to phospholipid oxidation as part of the apoptotic mode of cell death and are likely to enhance the recognition of apoptotic cells by phagocytic macrophages.

Expression of a chimeric antigen receptor specific for donor HLA class I enhances the potency of human regulatory T cells in preventing human skin transplant rejection

Regulatory T cell (Treg) therapy using recipient‐derived Tregs expanded ex vivo is currently being investigated clinically by us and others as a means of reducing allograft rejection following organ transplantation. Data from animal models has demonstrated that adoptive transfer of allospecific Tregs offers greater protection from graft rejection compared to polyclonal Tregs. Chimeric antigen receptors (CAR) are clinically translatable synthetic fusion proteins that can redirect the specificity of T cells toward designated antigens. We used CAR technology to redirect human polyclonal Tregs toward donor‐MHC class I molecules, which are ubiquitously expressed in allografts. Two novel HLA‐A2‐specific CARs were engineered: one comprising a CD28‐CD3ζ signaling domain (CAR) and one lacking an intracellular signaling domain (ΔCAR). CAR Tregs were specifically activated and significantly more suppressive than polyclonal or ΔCAR Tregs in the presence of HLA‐A2, without eliciting cytotoxic activity. Furthermore, CAR and ΔCAR Tregs preferentially transmigrated across HLA‐A2‐expressing endothelial cell monolayers. In a human skin xenograft transplant model, adoptive transfer of CAR Tregs alleviated the alloimmune‐mediated skin injury caused by transferring allogeneic peripheral blood mononuclear cells more effectively than polyclonal Tregs. Our results demonstrated that the use of CAR technology is a clinically applicable refinement of Treg therapy for organ transplantation.

Fluorescence lifetime endoscopy using TCSPC for the measurement of FRET in live cells

Development of remote imaging for diagnostic purposes has progressed dramatically since endoscopy began in the 1960’s. The recent advent of a clinically licensed intensity-based fluorescence micro-endoscopic instrument has offered the prospect of real-time cellular resolution imaging. However, interrogating protein-protein interactions deep inside living tissue requires precise fluorescence lifetime measurements to derive the Förster resonance energy transfer between two tagged fluorescent markers. We developed a new instrument combining remote fiber endoscopic cellular-resolution imaging with TCSPC-FLIM technology to interrogate and discriminate mixed fluorochrome labeled beads and expressible GFP/TagRFP tags within live cells. Endoscopic-FLIM (e-FLIM) data was validated by comparison with data acquired via conventional FLIM and e-FLIM was found to be accurate for both bright bead and dim live cell samples. The fiber based micro-endoscope allowed remote imaging of 4 µm and 10 µm beads within a thick Matrigel matrix with confident fluorophore discrimination using lifetime information. More importantly, this new technique enabled us to reliably measure protein-protein interactions in live cells embedded in a 3D matrix, as demonstrated by the dimerization of the fluorescent protein-tagged membrane receptor CXCR4. This cell-based application successfully demonstrated the suitability and great potential of this new technique for in vivo pre-clinical biomedical and possibly human clinical applications.

Two-photon 3D FIONA of individual quantum dots in an aqueous environment.

We report the first two-photon (2P) microscopy of individual quantum dots (QDs) in an aqueous environment with both widefield and point-scan excitations at nanometer accuracy. Thiol-containing reductants suppress QD blinking and enable measurement of the 36 nm step size of individual Myosin V motors in vitro. We localize QDs with an accuracy of 2-3 nm in all three dimensions by using a 9 × 9 matrix excitation hologram and an array detector, which also increases the 3D scan imaging rate by 80-fold. With this 3D microscopy we validate the LamB receptor distribution on E. coli and the endocytosis of EGF-receptors in breast cancer cells.

The ErbB4 CYT2 variant protects EGFR from ligand-induced degradation to enhance cancer cell motility

Dimerization of EGFR with an ErbB4 receptor variant increases growth factor–induced migration of breast cancer cells. Drug Resistance Through Dimerization The epidermal growth factor receptor (EGFR) is often targeted in various cancers, including breast cancer. The EGFR can dimerize with related receptors in the ErbB family, and formation of these heterodimers is associated with the development of resistance to EGFR inhibitors. Kiuchi et al. found that binding of EGFR to a naturally occurring variant of the receptor ErbB4 prevented a ubiquitin E3 ligase from associating with EGFR and triggering its breakdown. The migration of breast cancer cells to EGFR ligands was increased when EGFR was overexpressed with the ErbB4 variant, but not with a mutant that could not dimerize with EGFR. Furthermore, the transcript for this ErbB4 variant was increased in a subset of breast cancer patients. The epidermal growth factor receptor (EGFR) is a member of the ErbB family that can promote the migration and proliferation of breast cancer cells. Therapies that target EGFR can promote the dimerization of EGFR with other ErbB receptors, which is associated with the development of drug resistance. Understanding how interactions among ErbB receptors alter EGFR biology could provide avenues for improving cancer therapy. We found that EGFR interacted directly with the CYT1 and CYT2 variants of ErbB4 and the membrane-anchored intracellular domain (mICD). The CYT2 variant, but not the CYT1 variant, protected EGFR from ligand-induced degradation by competing with EGFR for binding to a complex containing the E3 ubiquitin ligase c-Cbl and the adaptor Grb2. Cultured breast cancer cells overexpressing both EGFR and ErbB4 CYT2 mICD exhibited increased migration. With molecular modeling, we identified residues involved in stabilizing the EGFR dimer. Mutation of these residues in the dimer interface destabilized the complex in cells and abrogated growth factor–stimulated cell migration. An exon array analysis of 155 breast tumors revealed that the relative mRNA abundance of the ErbB4 CYT2 variant was increased in ER+ HER2– breast cancer patients, suggesting that our findings could be clinically relevant. We propose a mechanism whereby competition for binding to c-Cbl in an ErbB signaling heterodimer promotes migration in response to a growth factor gradient.