Clemens F. Kaminski

ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function

Summary The mechanisms by which mutations in FUS and other RNA binding proteins cause ALS and FTD remain controversial. We propose a model in which low-complexity (LC) domains of FUS drive its physiologically reversible assembly into membrane-free, liquid droplet and hydrogel-like structures. ALS/FTD mutations in LC or non-LC domains induce further phase transition into poorly soluble fibrillar hydrogels distinct from conventional amyloids. These assemblies are necessary and sufficient for neurotoxicity in a C. elegans model of FUS-dependent neurodegeneration. They trap other ribonucleoprotein (RNP) granule components and disrupt RNP granule function. One consequence is impairment of new protein synthesis by cytoplasmic RNP granules in axon terminals, where RNP granules regulate local RNA metabolism and translation. Nuclear FUS granules may be similarly affected. Inhibiting formation of these fibrillar hydrogel assemblies mitigates neurotoxicity and suggests a potential therapeutic strategy that may also be applicable to ALS/FTD associated with mutations in other RNA binding proteins.

Cavity enhanced absorption spectroscopy of multiple trace gas species using a supercontinuum radiation source

Supercontinuum radiation sources are attractive for spectroscopic applications owing to their broad wavelength coverage, which enables spectral signatures of multiple species to be detected simultaneously. Here we report the first use of a supercontinuum radiation source for broadband trace gas detection using a cavity enhanced absorption technique. Spectra were recorded at bandwidths of up to 100 nm, encompassing multiple absorption bands of H(2)O, O(2) and O(2)-O(2). The same instrument was also used to make quantitative measurements of NO(2) and NO(3). For NO(3) a detection limit of 3 parts-per-trillion in 2 s was achieved, which corresponds to an effective 3sigma sensitivity of 2.4 x 10(-9) cm(-1)Hz(-1/2). Our results demonstrate that a conceptually simple and robust instrument is capable of highly sensitive broadband absorption measurements.

From microdroplets to microfluidics: selective emulsion separation in microfluidic devices.

Microdroplets show great promise as a new high-throughput technology in chemistry, biochemistry, and molecular biology. Microdroplets can be generated at rates in excess of several thousands per second and accurately formulated using minute amounts of small molecules, DNA, proteins, or cells. Furthermore, integrated active elements can be used to control individual droplets. With the technology for creating, dividing, fusing, interrogating, and even sorting microdroplets already developed, one of the main problems to be resolved is how to access their contents. Droplets are naturally self-contained microreactors that prevent sample loss, diffusion, and cross-contamination, general issues that afflict traditional microfluidics. However, the isolated nature of droplets prevents physical access of their contents on-chip. Even though this does not represent a problem for many of the applications that have already been demonstrated, it limits the integration of microdroplets with other platforms. Analytical techniques such as mass spectrometry, capillary electrophoresis, and liquid chromatography have been successfully integrated with continuous-flow microfluidic devices, but their integration with microdroplets remains challenging. If the contents of microdroplets could be readily extracted on demand, the carrier fluid discarded, and the microdroplets converted into a continuous stream, microfluidic functionality could be combined with the advantages of microdroplets. In this paper we present a technology that bridges the fields of microdroplets and continuous-flow microfluidics by extracting on-chip the contents of microdroplets and incorporating them into a continuous stream. The extraction is achieved through electrocoalescence: droplets are forced to coalesce with an aqueous stream by application of an electric field across the channel. The extraction is controlled through the voltage applied at microfabricated electrodes on each side of the channel and can be performed in a continuous or discrete fashion. The discrete collection of droplets can be controlled by an external electrical signal related to the contents of the droplets. As a proof of principle, we have implemented a fluorescence intensity based detection system to control the collection of the droplets, resulting in a device capable of selectively incorporating the contents of droplets of interest to a continuous microfluidic stream. We used flow-focusing to generate microdroplets. An aqueous stream was focused between two oil streams as they pass through a junction. Shear forces make the aqueous thread break up into monodisperse droplets. Droplet size and frequency were controlled by a combination of channel dimensions and flow rates. We used a mixture of fluorous oil (FC-77) and 1H,1H,2H,2H-perfluorooctanol (70:30 by weight) as the carrier phase. The oil and aqueous flows at the flow-focusing device were adjusted to generate the desired droplet frequency, typically ranging from 10–250 Hz. The flow of the lateral aqueous phase was adjusted so an interface was held in the region between the electrodes without overflow in either direction. Figure 1a shows a scheme of a typical device where droplets flow parallel to a stream of water between two electrodes. In the absence of an electric field, the droplets are not perturbed by the presence of the aqueous stream and follow the geometrically determined flow lines. Figure 1b and c show micrographs of such a device in operation. Droplets of a dye generated at the flow-focusing device flow past the

In situ measurements of the formation and morphology of intracellular β-amyloid fibrils by super-resolution fluorescence imaging.

Misfolding and aggregation of peptides and proteins is a characteristic of many neurodegenerative disorders, including Alzheimers disease (AD). In AD the β-amyloid peptide (Aβ) aggregates to form characteristic fibrillar structures, which are the deposits found as plaques in the brains of patients. We have used direct stochastic optical reconstruction microscopy, dSTORM, to probe the process of in situ Aβ aggregation and the morphology of the ensuing aggregates with a resolution better than 20 nm. We are able to distinguish different types of structures, including oligomeric assemblies and mature fibrils, and observe a number of morphological differences between the species formed in vitro and in vivo, which may be significant in the context of disease. Our data support the recent view that intracellular Aβ could be associated with Aβ pathogenicity in AD, although the major deposits are extracellular, and suggest that this approach will be widely applicable to studies of the molecular mechanisms of protein deposition diseases.

Fluorescence intensity and lifetime imaging of free and micellar-encapsulated doxorubicin in living cells

Frequency domain fluorescence lifetime imaging microscopy (FLIM) has been used in combination with laser scanning confocal microscopy to study the cellular uptake behavior of the antitumor drug doxorubicin (DOX) and micellar-encapsulated DOX (PLyAd-DOX). The endocytosis uptake process of PLyAd-DOX was monitored over 72 hours using confocal microscopy, with a maximum fluorescence recorded at incubation periods around 24 hours. The micellar structure was not found to release the encapsulated DOX during the time course of imaging. FLIM revealed single lifetime distributions of PLyAd-DOX during accumulation in the cytoplasm. The free DOX in contrast was observed both in the cytoplasm and the nuclear domain of the cell, showing bimodal lifetime distributions. There was a marked dependence of the measured free-DOX lifetime on concentration within the cell, in contrast to reference experiments in aqueous solution, where no such dependence was found. The results suggest the formation of macromolecular structures inside the living cells.

High bandwidth absorption spectroscopy with a dispersed supercontinuum source.

An optical gas sensor is presented, making use of a dispersed supercontinuum source, capable of acquiring broad bandwidth spectra at ultrahigh wavelength sweep and repetition rates. Wavelength sweeps from 1100 nm to 1700 nm can be performed in 800 ns at a spectral resolution of 40 pm. This is comparable to line-widths of molecular spectra at atmospheric pressure. Quantitative measurements are presented of CH(4) employing 80 nm wide sweeps over the P- Q- and R-branches of the 2nu(3) transition near 1665 nm, at rates exceeding 100 kHz. The effective acquisition rate is determined by the amount of averaging required, and the effect of this averaging on observed precision is investigated.

Rotaviruses Associate with Cellular Lipid Droplet Components To Replicate in Viroplasms, and Compounds Disrupting or Blocking Lipid Droplets Inhibit Viroplasm Formation and Viral Replication

ABSTRACT Rotaviruses are a major cause of acute gastroenteritis in children worldwide. Early stages of rotavirus assembly in infected cells occur in viroplasms. Confocal microscopy demonstrated that viroplasms associate with lipids and proteins (perilipin A, ADRP) characteristic of lipid droplets (LDs). LD-associated proteins were also found to colocalize with viroplasms containing a rotaviral NSP5-enhanced green fluorescent protein (EGFP) fusion protein and with viroplasm-like structures in uninfected cells coexpressing viral NSP2 and NSP5. Close spatial proximity of NSP5-EGFP and cellular perilipin A was confirmed by fluorescence resonance energy transfer. Viroplasms appear to recruit LD components during the time course of rotavirus infection. NSP5-specific siRNA blocked association of perilipin A with NSP5 in viroplasms. Viral double-stranded RNA (dsRNA), NSP5, and perilipin A cosedimented in low-density gradient fractions of rotavirus-infected cell extracts. Chemical compounds interfering with LD formation (isoproterenol plus isobutylmethylxanthine; triacsin C) decreased the number of viroplasms and inhibited dsRNA replication and the production of infectious progeny virus; this effect correlated with significant protection of cells from virus-associated cytopathicity. Rotaviruses represent a genus of another virus family utilizing LD components for replication, pointing at novel therapeutic targets for these pathogens.

Surface enhanced coherent anti-Stokes Raman scattering on nanostructured gold surfaces

Coherent anti-Stokes Raman spectroscopy (CARS) is a well-known tool in multiphoton imaging and nonlinear spectroscopy. In this work we combine CARS with plasmonic surface enhancement on reproducible nanostructured surfaces. We demonstrate strong correlation between plasmon resonances and surface-enhanced CARS (SECARS) intensities on our nanostructured surfaces and show that an enhancement of ∼10(5) can be obtained over standard CARS. Furthermore, we find SECARS to be >10(3) times more sensitive than surface-enhanced Raman Spectroscopy (SERS). We also demonstrate SECARS imaging of molecular monolayers. Our work paves the way for reliable single molecule Raman spectroscopy and fast molecular imaging on plasmonic surfaces.

Effect of heat release on turbulence and scalar-turbulence interaction in premixed combustion

Stereoscopic particle image velocimetry and planar laser induced fluorescence measurements of hydroxyl radical are simultaneously applied to measure, respectively, local turbulence intensities and flame front position in premixed ethylene-air flames stabilized on a bluff body. Three different equivalence ratios, 0.55, 0.63, and 0.7, and three different Reynolds numbers, 14 000, 17 000, and 21 000, are considered. Laser measurements were made for five different flame configurations within the ranges above and in the corresponding cold flows. By comparing the measurements of the cold and the corresponding hot flows, the effect of heat release on the turbulence and its interaction with the flame front is studied. All the flames are in the thin reaction zone regime. Typical flow features forming behind the bluff body are observed in the cold flows, whereas in the reacting flows the mean velocities and thus the shape, size, and characteristics of the recirculating eddy behind the bluff body are strongly influenced by the heat release. The strong acceleration across the mean flame and the radial outward shift of the stagnation plane of the recirculating eddy yield negative radial velocities which are absent in the corresponding cold flow cases. The spatial intermittency of the flame front leads to an increase in the turbulent kinetic energy. Although a decrease in the mean and rms values of the strain rate tensor eij components is observed for the reacting case as one would expect, the local flow acceleration across the flame front leads to a substantial increase in the skewness and the kurtosis of the probability density functions (PDFs) of eij components. The turbulence-scalar interaction is studied by analyzing the orientation of the flame front normal with the eigenvectors of eij. The PDFs of this orientation clearly show that the normals have an increased tendency to align with the extensive strain rate, which implies that the scalar gradients are destroyed by the turbulence as the scalar isosurfaces are pulled apart. This result questions the validity of passive scalar turbulence physics commonly used for premixed flame modeling. However, the influence of Lewis number on this alignment behavior is not clear at this time.Stereoscopic particle image velocimetry and planar laser induced fluorescence measurements of hydroxyl radical are simultaneously applied to measure, respectively, local turbulence intensities and flame front position in premixed ethylene-air flames stabilized on a bluff body. Three different equivalence ratios, 0.55, 0.63, and 0.7, and three different Reynolds numbers, 14 000, 17 000, and 21 000, are considered. Laser measurements were made for five different flame configurations within the ranges above and in the corresponding cold flows. By comparing the measurements of the cold and the corresponding hot flows, the effect of heat release on the turbulence and its interaction with the flame front is studied. All the flames are in the thin reaction zone regime. Typical flow features forming behind the bluff body are observed in the cold flows, whereas in the reacting flows the mean velocities and thus the shape, size, and characteristics of the recirculating eddy behind the bluff body are strongly influe...

Intrinsically disordered proteins as molecular shields

The broad family of LEA proteins are intrinsically disordered proteins (IDPs) with several potential roles in desiccation tolerance, or anhydrobiosis, one of which is to limit desiccation-induced aggregation of cellular proteins. We show here that this activity, termed molecular shield function, is distinct from that of a classical molecular chaperone, such as HSP70 - while HSP70 reduces aggregation of citrate synthase (CS) on heating, two LEA proteins, a nematode group 3 protein, AavLEA1, and a plant group 1 protein, Em, do not; conversely, the LEA proteins reduce CS aggregation on desiccation, while HSP70 lacks this ability. There are also differences in interaction with client proteins - HSP70 can be co-immunoprecipitated with a polyglutamine-containing client, consistent with tight complex formation, whereas the LEA proteins can not, although a loose interaction is observed by Förster resonance energy transfer. In a further exploration of molecular shield function, we demonstrate that synthetic polysaccharides, like LEA proteins, are able to reduce desiccation-induced aggregation of a water-soluble proteome, consistent with a steric interference model of anti-aggregation activity. If molecular shields operate by reducing intermolecular cohesion rates, they should not protect against intramolecular protein damage. This was tested using the monomeric red fluorescent protein, mCherry, which does not undergo aggregation on drying, but the absorbance and emission spectra of its intrinsic fluorophore are dramatically reduced, indicative of intramolecular conformational changes. As expected, these changes are not prevented by AavLEA1, except for a slight protection at high molar ratios, and an AavLEA1-mCherry fusion protein is damaged to the same extent as mCherry alone. A recent hypothesis proposed that proteomes from desiccation-tolerant species contain a higher degree of disorder than intolerant examples, and that this might provide greater intrinsic stability, but a bioinformatics survey does not support this, since there are no significant differences in the degree of disorder between desiccation tolerant and intolerant species. It seems clear therefore that molecular shield function is largely an intermolecular activity implemented by specialist IDPs, distinct from molecular chaperones, but with a role in proteostasis.