Delayed emergence of subdiffraction-sized mutant huntingtin fibrils following inclusion body formation.

Aberrant aggregation of improperly folded proteins is the hallmark of several human neurodegenerative disorders, including Huntington's Disease (HD) with autosomal-dominant inheritance. In HD, expansion of the CAG-repeat-encoded polyglutamine (polyQ) stretch beyond ~40 glutamines in huntingtin (Htt) and its N-terminal fragments leads to the formation of large (up to several µm) globular neuronal inclusion bodies (IBs) over time. We report direct observations of aggregating Htt exon 1 in living and fixed cells at enhanced spatial resolution by stimulated emission depletion (STED) microscopy and single-molecule super-resolution optical imaging. Fibrils of Htt exon 1 arise abundantly across the cytosolic compartment and also in neuritic processes only after nucleation and aggregation into a fairly advanced stage of growth of the prominent IB have taken place. Structural characterizations of fibrils by STED show a distinct length cutoff at ~1·5 µm and reveal subsequent coalescence (bundling/piling). Cytosolic fibrils are observed even at late stages in the process, side-by-side with the mature IB. Htt sequestration into the IB, which in neurons has been argued to be a cell-protective phenomenon, thus appears to saturate and over-power the cellular degradation systems and leaves cells vulnerable to further aggregation producing much smaller, potentially toxic, conformational protein species of which the fibrils may be comprised. We further found that exogenous delivery of the apical domain of the chaperonin subunit CCT1 to the cells via the cell medium reduced the aggregation propensity of mutant Htt exon 1 in general, and strongly reduced the occurrence of such late-stage fibrils in particular.

STED microscopy with optimized labeling density reveals 9-fold arrangement of a centriole protein.

Super-resolution fluorescence microscopy can achieve resolution beyond the optical diffraction limit, partially closing the gap between conventional optical imaging and electron microscopy for elucidation of subcellular architecture. The centriole, a key component of the cellular control and division machinery, is 250 nm in diameter, a spatial scale where super-resolution methods such as stimulated emission depletion (STED) microscopy can provide previously unobtainable detail. We use STED with a resolution of 60 nm to demonstrate that the centriole distal appendage protein Cep164 localizes in nine clusters spaced around a ring of ∼300 nm in diameter, and quantify the influence of the labeling density in STED immunofluorescence microscopy. We find that the labeling density dramatically influences the observed number, size, and brightness of labeled Cep164 clusters, and estimate the average number of secondary antibody labels per cluster. The arrangements are morphologically similar in centrioles of both proliferating cells and differentiated multiciliated cells, suggesting a relationship of this structure to function. Our STED measurements in single centrioles are consistent with results obtained by electron microscopy, which involve ensemble averaging or very different sample preparation conditions, suggesting that we have arrived at a direct measurement of a centriole protein by careful optimization of the labeling density.

Plasmonic substrates for multiplexed protein microarrays with femtomolar sensitivity and broad dynamic range.

Protein chips are widely used for high-throughput proteomic analysis, but to date, the low sensitivity and narrow dynamic range have limited their capabilities in diagnostics and proteomics. Here we present protein microarrays on a novel nanostructured, plasmonic gold film with near-infrared fluorescence enhancement of up to 100-fold, extending the dynamic range of protein detection by three orders of magnitude towards the fM regime. We employ plasmonic protein microarrays for the early detection of a cancer biomarker, carcinoembryonic antigen, in the sera of mice bearing a xenograft tumour model. Further, we demonstrate a multiplexed autoantigen array for human autoantibodies implicated in a range of autoimmune diseases with superior signal-to-noise ratios and broader dynamic range compared with commercial nitrocellulose and glass substrates. The high sensitivity, broad dynamic range and easy adaptability of plasmonic protein chips presents new opportunities in proteomic research and diagnostics applications.

STED Super-resolution Microscopy in Drosophila Tissue and in Mammalian Cells.

Far-field super-resolution microscopy is a rapidly emerging method that is opening up opportunities for biological imaging beyond the optical diffraction limit. We have implemented a Stimulated Emission Depletion (STED) microscope to image single dye, cell, and tissue samples with 50-80 nm resolution. First, we compare the STED performance imaging single molecules of several common dyes and report a novel STED dye. Then we apply STED to image planar cell polarity protein complexes in intact fixed Drosophila tissue for the first time. Finally, we present a preliminary study of the centrosomal protein Cep164 in mammalian cells. Our images suggest that Cep164 is arranged in a nine-fold symmetric pattern around the centriole, consistent with findings suggested by cryoelectron tomography. Our work demonstrates that STED microscopy can be used for superresolution imaging in intact tissue and provides ultrastructural information in biological samples as an alternative to immuno-electron microscopy.

Nanoliter dispensing method by degassed poly(dimethylsiloxane) microchannels and its application in protein crystallization.

This paper describes a method of dispensing a nanoliter volume of liquid into arrays of microwells through degassed poly(dimethylsiloxane) (PDMS) microchannels. In this method, the PDMS microchannels were reversibly bound to arrays of microwells. The PDMS elastomer was predegassed and served as an internal vacuum pumping source. Various aqueous solutions were infused into arrays of microwells through the reversibly sealed PDMS microchannels. Microwells fabricated in PDMS, poly(methyl methacrylate) (PMMA), and glass were all compatible with this dispensing method. By removing the PDMS microchannels, arrays of droplets confined in the microwells were obtained. Multiplex reaction and screening at the nanoliter scale were carried out by binding two such arrays of microwells to form microchambers. We applied this method to screening the crystallization conditions of four known proteins. Long-term incubation of over 2 months was achieved by employing glass microwells. An unknown protein was then crystallized using the screening method in microwells. The crystals with sufficient size were harvested from the reversibly bound microwells. X-ray diffraction with a resolution of 3.1 Angstrom was obtained.

Coherent ultrafast photoelectron spectroscopy in Br2: an alternative method for measuring Delta v not equal 0 transitions in Rydberg-to-Rydberg excitations.

A two-state vibrational wave packet is prepared in a low-lying 4d[12](1 or 2) Rydberg state of jet cooled Br(2) (4d, v(')=3 and v(')=4) by two-photon excitation with 266.5 nm pulses from an ultrafast laser. The wave packet is detected by autoionization following excitation with time-delayed 800 nm pulses to the n=8 (v(+)=4) and n=9 (v(+)=3) Rydberg states in the (2)Pi(12) angular momentum core state. Autoionization of each state occurs to the (2)Pi(32) state of the ion through spin-orbit ionization. Photoelectron spectroscopy is used to differentiate between the n=8 and n=9 ejected photoelectrons. Detection of the wave packet recurrences via the n=8 and n=9 Rydberg states reveals a pi phase-shift difference of the recurrences between the two final states. In each case, Delta v not equal 0 transitions are observed since wave packet recurrences are detected. By fitting the observed phase change of the recurrences with a simple model for the overlap amplitudes and assumptions about the potentials, we estimate, within the context of the model, that approximately 0.6% of the transitions may be attributed to Delta v= +/- 1 transitions between the initial Rydberg superposition state and the final Rydberg detection state.

Acid-degradable particles for protein-based vaccines: enhanced survival rate for tumor-challenged mice using ovalbumin model.

Acid-degradable protein-loaded polymer particles show promise for antigen-based vaccines due to their ability to activate cytotoxic T lymphocytes (CTLs) in vitro. Protein loadings and cytotoxic T lymphocyte activation efficiencies have now been enhanced through novel delivery vehicle designs. In particular, the use of a more hydrophilic acid-degradable cross-linker leads to increased water dispersibility and increased protein loading efficiency for the particles. A 2.5-fold increase in protein encapsulation allows the delivery of more protein antigen to antigen presenting cells (APCs) leading to a 20-fold rise in antigen presentation levels. The mechanism by which APCs internalize these particles was explored using the phagocytosis inhibitor, cytochalasin B. In addition, preliminary in vivo experiments were conducted to investigate the ability of the protein-loaded particles to provide immunity against tumors in mice, and an enhanced survival rate over the use of protein alone was observed, indicating that this vaccine delivery strategy has great practical potential.