Activation of the p75 neurotrophin receptor through conformational rearrangement of disulphide-linked receptor dimers

Ligand-mediated dimerization has emerged as a universal mechanism of growth factor receptor activation. Neurotrophins interact with dimers of the p75 neurotrophin receptor (p75(NTR)), but the mechanism of receptor activation has remained elusive. Here, we show that p75(NTR) forms disulphide-linked dimers independently of neurotrophin binding through the highly conserved Cys(257) in its transmembrane domain. Mutation of Cys(257) abolished neurotrophin-dependent receptor activity but did not affect downstream signaling by the p75(NTR)/NgR/Lingo-1 complex in response to MAG, indicating the existence of distinct, ligand-specific activation mechanisms for p75(NTR). FRET experiments revealed a close association of p75(NTR) intracellular domains that was transiently disrupted by conformational changes induced upon NGF binding. Although mutation of Cys(257) did not alter the oligomeric state of p75(NTR), the mutant receptor was no longer able to propagate conformational changes to the cytoplasmic domain upon ligand binding. We propose that neurotrophins activate p75(NTR) by a mechanism involving rearrangement of disulphide-linked receptor subunits

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

Coordinate-based colocalization analysis of single-molecule localization microscopy data

Malkusch S, Endesfelder U, Mondry J, Gelléri M, Verveer PJ, Heilemann M. Coordinate-based colocalization analysis of single-molecule localization microscopy data. Histochemistry and Cell Biology. 2012;137(1):1-10

Single Particle Tracking Reveals that EGFR Signaling Activity Is Amplified in Clathrin-Coated Pits.

Signaling from the epidermal growth factor receptor (EGFR) via phosphorylation on its C-terminal tyrosine residues requires self-association, which depends on the diffusional properties of the receptor and its density in the plasma membrane. Dimerization is a key event for EGFR activation, but the role of higher order clustering is unknown. We employed single particle tracking to relate the mobility and aggregation of EGFR to its signaling activity. EGFR mobility alternates between short-lived free, confined and immobile states. In the immobile state, EGFR tends to aggregate in clathrin-coated pits, which is further enhanced in a phosphorylation-dependent manner and does not require ligand binding. EGFR phosphorylation is further amplified by cross-phosphorylation in clathrin-coated pits. Because phosphorylated receptors can escape from the pits, local gradients of signaling active EGFR are formed. These results show that amplification of EGFR phosphorylation by receptor clustering in clathrin-coated pits supports signal activation at the plasma membrane

Co-imaging extrinsic, intrinsic and effector caspase activity by fluorescence anisotropy microscopy

In order to overcome intercellular variability and thereby effectively assess signal propagation in biological networks it is imperative to simultaneously quantify multiple biological observables in single living cells. While fluorescent biosensors have been the tool of choice to monitor the dynamics of protein interaction and enzymatic activity, co-measuring more than two of them has proven challenging. In this work, we designed three spectrally separated anisotropy-based Förster Resonant Energy Transfer (FRET) biosensors to overcome this difficulty. We demonstrate this principle by monitoring the activation of extrinsic, intrinsic and effector caspases upon apoptotic stimulus. Together with modelling and simulations we show that time of maximum activity for each caspase can be derived from the anisotropy of the corresponding biosensor. Such measurements correlate relative activation times and refine existing models of biological signalling networks, providing valuable insight into signal propagation. Keywords: Caspase activity, Apoptotic network, Anisotropy FRET biosensor, Co-monitoring, Imaging, Polarization microscop

Ezrin is a downstream effector of trafficking PKC–integrin complexes involved in the control of cell motility

Protein kinase C (PKC) α has been implicated in β1 integrin-mediated cell migration. Stable expression of PKCα is shown here to enhance wound closure. This PKC-driven migratory response directly correlates with increased C-terminal threonine phosphorylation of ezrin/moesin/radixin (ERM) at the wound edge. Both the wound migratory response and ERM phosphorylation are dependent upon the catalytic function of PKC and are susceptible to inhibition by phosphatidylinositol 3-kinase blockade. Upon phorbol 12,13-dibutyrate stimulation, green fluorescent protein–PKCα and β1 integrins co-sediment with ERM proteins in low-density sucrose gradient fractions that are enriched in transferrin receptors. Using fluorescence lifetime imaging microscopy, PKCα is shown to form a molecular complex with ezrin, and the PKC-co-precipitated endogenous ERM is hyperphosphorylated at the C-terminal threonine residue, i.e. activated. Electron microscopy showed an enrichment of both proteins in plasma membrane protrusions. Finally, overexpression of the C-terminal threonine phosphorylation site mutant of ezrin has a dominant inhibitory effect on PKCα-induced cell migration. We provide the first evidence that PKCα or a PKCα-associated serine/threonine kinase can phosphorylate the ERM C-terminal threonine residue within a kinase–ezrin molecular complex in vivo