Visualization of vesicle transport along and between distinct pathways in neurites of living cells.

Trafficking of secretory vesicles along neurites of PC12 cells was visualized by 2D and 3D real-time imaging using fluorescence microscopy. Vesicle motion along distinct pathways was directly seen. From an overlay of individual pathways, the underlying cytoskeletal filament could be imaged at a subwavelength resolution. Continuous vesicle transport was interrupted by periods of diffusive motion with concomitant pathway changes. Statistical analysis shows that such interruptions were distributed stochastically along the filament, indicating a limited processivity of motor proteins also in a cellular context. Periods of diffusive motion facilitated the interaction with actively transported vesicles. Frequent associations and dissociations of vesicles have been observed consistently, pointing to a functional relevance of vesicle cotransport.

Single-molecule reader for proteomics and genomics.

Recent developments in ultrasensitive fluorescence microscopy enabled the detection and detailed characterization of individual biomolecules in their native environment. New types of information can be obtained from studying individual molecules, which is not accessible from ensemble measurements. Moreover, this methodological advance matches the need of bioscience to downscale the sample amount required for screening devices. It is envisioned that concentrations as low as approximately 1000 molecules contained in a sample of 1 nl can be detected in a chip-based assay. In this review, we overview state-of-the-art single molecule microscopy with respect to its applicability to ultrasensitive screening. Quantitative estimations will be given, based on a novel apparatus designed for large area screening at single molecule sensitivity.

Ultrasensitive pharmacological characterisation of the voltage-gated potassium channel K(V)1.3 studied by single-molecule fluorescence microscopy.

The determination of pharmacologically relevant constants is crucial in order to understand the effects of compounds interacting with various membrane receptors. In this report we study a venom component of the Central American scorpion Centruroides limbatus, a short peptide termed hongotoxin(1) (HgTX(1)), which specifically binds to the voltage-gated potassium channel K(V)1.3 at a molecular stoichiometry of 1:1. A toxin analogue (HgTX(1)-A19C) was subjected to fluorescence labelling studies with Cy5. Utilising an ultrasensitive microscopic method (single-dye tracing; SDT) we were able to directly visualise HgTX(1)-A19C-Cy5 binding to the voltage-gated potassium channel K(V)1.3 on Jurkat cells at the single molecule level. For the first time, this approach allowed the determination of both the dissociation constant (K(D)) and the off-rate (k(off)) of HgTX(1)-A19C-Cy5 on living cells. In order to validate this novel approach, the data obtained with SDT were correlated to radioligand binding studies performed under identical conditions using a radioiodinated HgTX(1) analogue.

Single-molecule reader for high-throughput bioanalysis.

We report here the development of a device for single-molecule imaging on large surface areas. A CCD camera operated in time delay and integration mode is synchronized with the movement of a sample scanning stage, enabling continuous data acquisition. Experiments on single fluorescent lipid molecules in supported lipid bilayers and on stained living cells demonstrate the capabilities of the method. Areas of up to 5 x 5 mm(2) were recorded within 11 min at a pixel size of 129 nm.

Apo AI/ABCA1-dependent and HDL3-mediated lipid efflux from compositionally distinct cholesterol-based microdomains.

We have investigated whether a raft heterogeneity exists in human monocyte-derived macrophages and fibroblasts and whether these microdomains are modulated by lipid efflux. Triton X-100 (Triton) or Lubrol WX (Lubrol) detergent-resistant membranes from cholesterol-loaded monocytes were associated with the following findings: (i) Lubrol-DRM contained most of the cellular cholesterol and at least 75% of Triton-detergent-resistant membranes. (ii) 'Lubrol rafts', defined by their solubility in Triton but insolubility in Lubrol, were enriched in unsaturated phosphatidylcholine and showed a lower cholesterol to choline-phospholipid ratio compared to Triton rafts. (iii) CD14 and CD55 were recovered in Triton- and Lubrol-detergent-resistant membranes, whereas CD11b was found exclusively in Triton DRM. ABCA1 implicated in apo AI-mediated lipid efflux and CDC42 were partially localized in Lubrol- but not in Triton-detergent-resistant membranes. (iv) Apo AI preferentially depleted cholesterol and choline-phospholipids from Lubrol rafts, whereas HDL3 additionally decreased the cholesterol content of Triton rafts. In fibroblasts, neither ABCA1 nor CDC42 was found in Lubrol rafts, and both apo AI and HDL3 reduced the lipid content in Lubrol- as well as in Triton-detergent-resistant membranes. In summary, we provide evidence for the existence of compositionally distinct membrane microdomains in human cells and their modulation by apo AI/ABCA1-dependent and HDL3-mediated lipid efflux.

Single molecule recognition of protein binding epitopes in brush border membranes by force microscopy.

Sidedness and accessibility of protein epitopes in intact brush border membrane vesicles were analyzed by detecting single molecule interaction forces using molecular recognition force microscopy in aqueous physiological solutions. Frequent antibody-antigen recognition events were observed with a force microscopy tip carrying an antibody directed against the periplasmically located gamma-glutamyltrans- peptidase, suggesting a right side out orientation of the vesicles. Phlorizin attached to the tips bound to NA+/D-glucose cotransporter molecules present in the vesicles. The recognition was sodium dependent and inhibited by free phlorizin and D-glucose, and revealed an apparent K(D) of 0.2 microM. Binding events were also observed with an antibody directed against the epitope aa603-aa630 close to the C terminus of the transporter. In the presence of phlorizin the probability of antibody binding was reduced but the most probable unbinding force f(u) = 100 pN remained unchanged. In the presence of D-glucose and sodium, however, both the binding probability and the most probable binding force (f(u) = 50 pN) were lower than in its absence. These studies demonstrate that molecular recognition force microscopy is a versatile tool to probe orientation and conformational changes of epitopes of membrane components during binding and trans-membrane transport.