Förster (or Fluorescence) Resonance Energy Transfer (FRET) is unique in generating fluorescence signals sensitive to molecular conformation, association, and separation in the 1-10 nm range. We introduce a revised photophysical framework for the phenomenon and provide a systematic catalog of FRET techniques adapted to imaging systems, including new approaches proposed as suitable prospects for implementation. Applications extending from a single molecule to live cells will benefit from multidimensional microscopy techniques, particularly those adapted for optical sectioning and incorporating new algorithms for resolving the component contributions to images of complex molecular systems.

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.

The aggregation of a-synuclein is characteristic of Parkinson's 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 a-synuclein and may constitute endogenous agents modulating the pathogenesis of PD. We characterized the complexes of natural and synthetic polyamines with a-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 ( þ 2-þ 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 B10 4 and the rate of monomer addition B40-fold. Significant secondary structure is not induced in monomeric a-synuclein by polyamines at 151C. Instead, NMR reveals changes in a region (aa 22-93) far removed from the polyamine binding site and presumed to adopt the b-sheet conformation characteristic of fibrillar a-synuclein. We conclude that the C-terminal domain acts as a regulator of a-synuclein aggregation.