Elizabeth A. Jares-Erijman

Quantum dot ligands provide new insights into erbB/HER receptor–mediated signal transduction

The erbB/HER family of transmembrane receptor tyrosine kinases (RTKs) mediate cellular responses to epidermal growth factor (EGF) and related ligands. We have imaged the early stages of RTK-dependent signaling in living cells using: (i) stable expression of erbB1/2/3 fused with visible fluorescent proteins (VFPs), (ii) fluorescent quantum dots (QDs) bearing epidermal growth factor (EGF-QD) and (iii) continuous confocal laser scanning microscopy and flow cytometry. Here we demonstrate that EGF-QDs are highly specific and potent in the binding and activation of the EGF receptor (erbB1), being rapidly internalized into endosomes that exhibit active trafficking and extensive fusion. EGF-QDs bound to erbB1 expressed on filopodia revealed a previously unreported mechanism of retrograde transport to the cell body. When erbB2-monomeric yellow fluorescent protein (mYFP) or erbB3-monomeric Citrine (mCitrine) were coexpressed with erbB1, the rates and extent of endocytosis of EGF-QD and the RTK-VFP demonstrated that erbB2 but not erbB3 heterodimerizes with erbB1 after EGF stimulation, thereby modulating EGF-induced signaling. QD-ligands will find widespread use in basic research and biotechnological developments.

NMR of α-synuclein-polyamine complexes elucidates the mechanism and kinetics of induced aggregation

The aggregation of α‐synuclein is characteristic of Parkinsons disease (PD) and other neurodegenerative synucleinopathies. The 140‐aa protein is natively unstructured; thus, ligands binding to the monomeric form are of therapeutic interest. Biogenic polyamines promote the aggregation of α‐synuclein and may constitute endogenous agents modulating the pathogenesis of PD. We characterized the complexes of natural and synthetic polyamines with α‐synuclein by NMR and assigned the binding site to C‐terminal residues 109–140. Dissociation constants were derived from chemical shift perturbations. Greater polyamine charge (+2 → +5) correlated with increased affinity and enhancement of fibrillation, for which we propose a simple kinetic mechanism involving a dimeric nucleation center. According to the analysis, polyamines increase the extent of nucleation by ∼104 and the rate of monomer addition ∼40‐fold. Significant secondary structure is not induced in monomeric α‐synuclein by polyamines at 15°C. Instead, NMR reveals changes in a region (aa 22–93) far removed from the polyamine binding site and presumed to adopt the β‐sheet conformation characteristic of fibrillar α‐synuclein. We conclude that the C‐terminal domain acts as a regulator of α‐synuclein aggregation.

Fluorescence imaging of amyloid formation in living cells by a functional, tetracysteine-tagged |[alpha]|-synuclein

α-synuclein is a major component of intraneuronal protein aggregates constituting a distinctive feature of Parkinson disease. To date, fluorescence imaging of dynamic processes leading to such amyloid deposits in living cells has not been feasible. To address this need, we generated a recombinant α-synuclein (α-synuclein-C4) bearing a tetracysteine target for fluorogenic biarsenical compounds. The biophysical, biochemical and aggregation properties of α-synuclein-C4 matched those of the wild-type protein in vitro and in living cells. We observed aggregation of α-synuclein-C4 transfected or microinjected into cells, particularly under oxidative stress conditions. Fluorescence resonance energy transfer (FRET) between FlAsH and ReAsH confirmed the close association of fibrillized α-synuclein-C4 molecules. α-synuclein-C4 offers the means for directly probing amyloid formation and interactions of α-synuclein with other proteins in living cells, the response to cellular stress and screening drugs for Parkinson disease.

Photoswitchable water-soluble quantum dots: pcFRET based on amphiphilic photochromic polymer coating.

A novel surface architecture was developed to generate biocompatible and stable photoswitchable quantum dots (psQDs). Photochromic diheteroarylethenes, which undergo thermally stable photoconversions between two forms with different spectral properties in organic solvents, were covalently linked to an amphiphilic polymer that self-assembles with the lipophilic chains surrounding commercial hydrophobic core-shell CdSe/ZnS QDs. This strategy creates a small (∼7 nm diameter) nanoparticle (NP) that is soluble in aqueous medium. The NP retains and even enhances the desirable properties of the original QD (broad excitation, narrow emission, photostability), but the brightness of its emission can be tailored by light. The modulation of emission monitored by steady-state and time-resolved fluorescence was 35-40%. The psQDs exhibit unprecedented photostability and fatigue resistance over at least 16 cycles of photoconversion.

Fluorescent Ratiometric MFC Probe Sensitive to Early Stages of α-Synuclein Aggregation

We introduce a sensor molecule, AS140-MFC, consisting of a covalent adduct of an Ala-to-Cys mutant of alpha-synuclein with the 3-hydroxychromone dual emission dye MFC. We show that the AS140-MFC construct is a multiparametric fluorescent probe suitable for the continuous monitoring of protein aggregation and is sensitive to the early and intermediate stages of alpha-synuclein aggregation, a process associated with Parkinsons disease.

Quantum Dots As Ultrasensitive Nanoactuators and Sensors of Amyloid Aggregation in Live Cells

Quantum dots multifunctionalized with the amyloid protein alpha-synuclein act at nanomolar concentrations as very potent inducers of the aggregation of micromolar-millimolar bulk concentrations of the protein in vitro and in cells. Fibrillation in live cells, a process diagnostic of Parkinsons disease, is accelerated up to 15-fold with only approximately 100 nanoparticles. The combination with a tetracysteine-tagged form of alpha-synuclein specific for fluorogenic biarsenicals constitutes a very sensitive system for studying pathological amyloid formation in cells.

Modulation of a photoswitchable dual-color quantum dot containing a photochromic FRET acceptor and an internal standard.

Photoswitchable semiconductor nanoparticles, quantum dots (QDs), couple the advantages of conventional QDs with the ability to reversibly modulate the QD emission, thereby improving signal detection by rejection of background signals. Using a simple coating methodology with polymers incorporating a diheteroarylethene photochromic FRET acceptor as well as a spectrally distinct organic fluorophore, photoswitchable QDs were prepared that are small, biocompatible, and feature ratiometric dual emission. With programmed irradiation, the fluorescence intensity ratio can be modified by up to ∼100%.

Highly Solvatochromic 7-Aryl-3-hydroxychromones.

Introduction of the dialkylaminophenyl group in position 7 of 3-hydroxychromone changes the orientation of the excited-state dipole moment and leads to superior solvatochromic properties (>170 nm emission shift in aprotic media). The excited-state intramolecular proton-transfer (ESIPT) reaction of 7-aryl-3-hydroxychromones is almost completely inhibited in most solvents. Methylation of the 3-OH abolishes ESIPT completely and also leads to improved photostability. The probes exhibit a ∼100-fold increase in fluorescence intensity and large Stokes shifts upon binding to membranes, reflecting differences in membrane phase and charge by a >40 nm spread in the emission band position.

Multiparametric Fluorescence Detection of Early Stages in the Amyloid Protein Aggregation of Pyrene-labeled α-Synuclein

The aggregation of alpha-synuclein, a presynaptic protein, has an important role in the etiology of Parkinsons disease. Oligomers or protofibrils adopting the cross-beta-sheet structure characteristic of fibrillating amyloid proteins are presumed to be the primary cytotoxic species. Current techniques for monitoring the kinetics of alpha-synuclein aggregation based on fluorescent dyes such as Thioflavin-T and Congo red detect only the terminal fibrillar species, are discontinuous and notoriously irreproducible. We have devised a new fluorescence aggregation assay that is continuous and provides a large set of fluorescence parameters sensitive to the presence of oligomeric intermediates as well as fibrils. The approach involves tagging functionally neutral Ala-to-Cys variants of alpha-synuclein with the long-lifetime fluorophore pyrene. Upon induction of aggregation at 37 degrees C, the entire family of steady-state descriptors of pyrene emission (monomer intensity, solvent polarity ratio (I(I)/I(III)), and anisotropy; and excimer intensity) change dramatically, particularly during the early stages in which oligomeric intermediates form and evolve. The pyrene probe senses a progressive decrease in polarity, an increase in molecular mass and close intermolecular association in a manner dependent on position in the sequence and the presence of point mutations. The time-resolved decays (0-160 ns) of intensity and anisotropy exhibited complex, characteristic features. The new assay constitutes a convenient platform for the high-throughput screening of agents useful in the diagnosis and therapy of Parkinsons disease as well as in basic investigations.

Imaging Quantum Dots Switched On and Off by Photochromic Fluorescence Resonance Energy Transfer (pcFRET)

ABSTRACT The reversible modulation of the emission of CdSe/ZnS semiconductor nanocrystals (quantum dots) was achieved by binding photochromic diheteroarylethenes and switchable acceptors for fluorescence resonance energy transfer. A biotinylated diheteroarylethene derivative was bound to quantum dots bearing conjugated streptavidin, leading to an intensity decrease as a consequence of energy transfer to the closed form of the acceptor. Interconversion between the open and closed forms by irradiation with 365 and 546 nm light enabled deactivation and activation, respectively, of the FRET process with a corresponding modulation of quantum dot emission, observed both in solution and by sequential wide-field imaging.