Monitoring the Size and Lateral Dynamics of ErbB1 Enriched Membrane Domains through Live Cell Plasmon Coupling Microscopy

To illuminate the role of the spatial organization of the epidermal growth factor receptor (ErbB1) in signal transduction quantitative information about the receptor topography on the cell surface, ideally on living cells and in real time, are required. We demonstrate that plasmon coupling microscopy (PCM) enables to detect, size, and track individual membrane domains enriched in ErbB1 with high temporal resolution. We used a dendrimer enhanced labeling strategy to label ErbB1 receptors on epidermoid carcinoma cells (A431) with 60 nm Au nanoparticle (NP) immunolabels under physiological conditions at 37°C. The statistical analysis of the spatial NP distribution on the cell surface in the scanning electron microscope (SEM) confirmed a clustering of the NP labels consistent with a heterogeneous distribution of ErbB1 in the plasma membrane. Spectral shifts in the scattering response of clustered NPs facilitated the detection and sizing of individual NP clusters on living cells in solution in an optical microscope. We tracked the lateral diffusion of individual clusters at a frame rate of 200 frames/s while simultaneously monitoring the configurational dynamics of the clusters. Structural information about the NP clusters in their membrane confinements were obtained through analysis of the electromagnetic coupling of the co-confined NP labels through polarization resolved PCM. Our studies show that the ErbB1 receptor is enriched in membrane domains with typical diameters in the range between 60–250 nm. These membrane domains exhibit a slow lateral diffusion with a diffusion coefficient of  = |0.0054±0.0064| µm2/s, which is almost an order of magnitude slower than the mean diffusion coefficient of individual NP tagged ErbB1 receptors under identical conditions

The architecture of EGFR's basal complexes reveals autoinhibition mechanisms in dimers and oligomers

Our current understanding of epidermal growth factor receptor (EGFR) autoinhibition is based on X-ray structural data of monomer and dimer receptor fragments and does not explain how mutations achieve ligand-independent phosphorylation. Using a repertoire of imaging technologies and simulations we reveal an extracellular head-to-head interaction through which ligand-free receptor polymer chains of various lengths assemble. The architecture of the head-to-head interaction prevents kinase-mediated dimerisation. The latter, afforded by mutation or intracellular treatments, splits the autoinhibited head-to-head polymers to form stalk-to-stalk flexible non-extended dimers structurally coupled across the plasma membrane to active asymmetric tyrosine kinase dimers, and extended dimers coupled to inactive symmetric kinase dimers. Contrary to the previously proposed main autoinhibitory function of the inactive symmetric kinase dimer, our data suggest that only dysregulated species bear populations of symmetric and asymmetric kinase dimers that coexist in equilibrium at the plasma membrane under the modulation of the C-terminal domain

AB-plot assisted determination of fluorophore mixtures in a fluorescence lifetime microscope using spectra or quenchers

Most treatments of frequency domain lifetime measurements indicate that a set of measurements must be made at multiple frequencies in order to determine the lifetimes of the components in a mixture. Although this is the case in general, under special conditions, single-frequency data can resolve multiple lifetimes. Here, data are presented showing several approaches to determining fluorescence lifetimes in two-component mixtures using single-frequency data. Common to all of the procedures presented is exploitation of variations in the relative contributions of a particular fluorophore to the total fluorescence from a mixture of fluorophores. This variation can be produced intentionally by observing a number of samples which contain different relative amounts of the fluorophores. It can be produced fortuitously by observing spatial variations in a mixture of fluorophores in a specimen or set of specimens observed with a lifetime imaging system. It can also be produced by examination of the lifetime spectrum obtained from a fluorophore mixture or by varying the concentration of a quencher in a fluorophore mixture, in which the two fluorophores have different rate constants for quenching. In many instances, the set of approaches presented here will be unsuitable for examination of arbitrary samples of unknown composition for which the multifrequency approach should be used. However, measurements produced using single-frequency methods may be applied to good effect for controlled experiments having defined fluorophores or sets of fluorophores, particularly in the case of biological lifetime imaging studies

Photophysics of green and red fluorescent proteins: Implications for quantitative microscopy

This chapter describes the implications for quantitative microscopy. The green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its mutants constitute a class of fluorophores that has revolutionized the evaluation of molecular interactions and visualization of biological systems. When fused to proteins of interest and expressed in vivo, fluorescent proteins (FPs) act as versatile indicators of structure and function within cells and can be imaged with the full repertoire of fluorescence microscopy techniques. FPs and their constructs are finding increasing use in fluorescence lifetime imaging microscopy (FLIM) and fluorescence resonance energy transfer (FRET) modes of microspectroscopy for the elucidation of protein-protein interactions, signaling, and trafficking in cellular systems. The simple and the most common, application of FPs is as a passive marker fused to a target protein of interest for visualizing its spatiotemporal distribution. Mutations in and around the chromophore have systematic effects on the spectra. Green and red fluorescent proteins exhibit photophysical properties that are generally more complex than those of traditional fluorophores, for example, fluorescein and rhodamine

Acta mathematica Universitatis Comenianae

Adaptor protein Grb2 binds phosphotyrosines in the epidermal growth factor (EGF) receptor (EGFR) and thereby links receptor activation to intracellular signaling cascades. Here, we investigated how recruitment of Grb2 to EGFR is affected by the spatial organization and quaternary state of activated EGFR. We used the techniques of image correlation spectroscopy (ICS) and lifetime-detected Förster resonance energy transfer (also known as FLIM-based FRET or FLIM-FRET) to measure ligand-induced receptor clustering and Grb2 binding to activated EGFR in BaF/3 cells. BaF/3 cells were stably transfected with fluorescently labeled forms of Grb2 (Grb2-mRFP) and EGFR (EGFR-eGFP). Following stimulation of the cells with EGF, we detected nanometer-scale association of Grb2-mRFP with EGFR-eGFP clusters, which contained, on average, 4 ± 1 copies of EGFR-eGFP per cluster. In contrast, the pool of EGFR-eGFP without Grb2-mRFP had an average cluster size of 1 ± 0.3 EGFR molecules per punctum. In the absence of EGF, there was no association between EGFR-eGFP and Grb2-mRFP. To interpret these data, we extended our recently developed model for EGFR activation, which considers EGFR oligomerization up to tetramers, to include recruitment of Grb2 to phosphorylated EGFR. The extended model, with adjustment of one new parameter (the ratio of the Grb2 and EGFR copy numbers), is consistent with a cluster size distribution where 2% of EGFR monomers, 5% of EGFR dimers, <1% of EGFR trimers, and 94% of EGFR tetramers are associated with Grb2. Together, our experimental and modeling results further implicate tetrameric EGFR as the key signaling unit and call into question the widely held view that dimeric EGFR is the predominant signaling unit

Quality and stability of extemporaneous pyridoxal phosphate preparations used in the treatment of paediatric epilepsy

Objectives To assess pyridoxal 5’-phosphate (PLP) content and stability of extemporaneous PLP liquids prepared from dietary supplements used for the treatment of vitamin B6-dependent epilepsy. Methods PLP liquids were prepared in accordance with guidelines given to patients from marketed 50 mg PLP dietary capsules and tablets. The PLP content and stability was evaluated under conditions resembling the clinic setting using reverse phase HPLC and mass spectrometry. Key findings PLP content in most of the extemporaneously prepared liquids from dietary supplements was found to be different to the expected amount (~16-60 mg). Most of these PLP extemporaneous liquids were stable at room temperature (protected from light) after 24 h but unstable after 4 h when exposed to light. A key photo-degradation product of PLP in water was confirmed as 4-pyridoxic acid 5'-phosphate (PAP). Conclusion PLP tablets from Solgar® were found to be the most reliable product for the preparation of extemporaneous PLP liquids. This work highlighted the difference between the marketed PLP dietary supplements quality and the importance of proper storage of aqueous PLP. There is a need to develop pharmaceutical forms of PLP that ensure dose accuracy and avoid potentially unsafe impurities with the aim of enhancing safety and compliance

AIS transactions on human-computer interaction : THCI

Antimicrobial peptides hold promise as broad-spectrum alternatives to conventional antibiotics. The mechanism of action of this class of peptide is a topical area of research focused predominantly on their interaction with artificial membranes. Here we compare the interaction mechanism of a model antimicrobial peptide with single artificial membranes and live bacterial cells. The interaction kinetics was imaged using time-lapse fluorescence lifetime imaging of a fluorescently-tagged melittin derivative. Interaction with the synthetic membranes resulted in membrane pore formation. In contrast, the interaction with bacteria led to transient membrane disruption and corresponding leakage of the cytoplasm, but surprisingly with a much reduced level of pore formation. The discovery that pore formation is a less significant part of lipid-peptide interaction in live bacteria highlights the mechanistic complexity of these interactions in living cells compared to simple artificial systems

Stoichiometry of at the During Their Using Total Internal Reflection Fluorescent (TIRF) Live Imaging and Single-Molecule Tracking

International audienceDetermination of protein stoichiometry in living cells is key to understanding basic biological processes. This is particularly important for receptor-mediated endocytosis, a highly regulated mechanism that requires the sequential assembly of numerous factors. Here, we describe a quantitative approach to analyze receptor clustering dynamics at the plasma membrane. Our workflow combines TIRF live imaging of a CRISPR-Cas9-edited cell line expressing a GFP-tagged receptor in a physiological relevant environment, a calibration technique for single-molecule analysis of GFP, and detection and tracking with an open-source software. This method allows to determine the number of receptor molecules at the plasma membrane in real time