Cross-linking of high-affinity immunoglobulin E (IgE) results in the life-threatening allergic reaction anaphylaxis. Yet the cellular mechanisms that induce B cells to produce IgE in response to ...allergens remain poorly understood. T follicular helper (T
) cells direct the affinity and isotype of antibodies produced by B cells. Although T
cell-derived interleukin-4 (IL-4) is necessary for IgE production, it is not sufficient. We report a rare population of IL-13-producing T
cells present in mice and humans with IgE to allergens, but not when allergen-specific IgE was absent or only low-affinity. These "T
13" cells have an unusual cytokine profile (IL-13
IL-4
IL-5
IL-21
) and coexpress the transcription factors BCL6 and GATA3. T
13 cells are required for production of high- but not low-affinity IgE and subsequent allergen-induced anaphylaxis. Blocking T
13 cells may represent an alternative therapeutic target to ameliorate anaphylaxis.
Raman fingerprints of amyloid structures Flynn, Jessica D; Lee, Jennifer C
Chemical communications (Cambridge, England),
06/2018, Letnik:
54, Številka:
51
Journal Article
Recenzirano
Odprti dostop
Structural differences in pathological and functional amyloid fibrils have been investigated by Raman microspectroscopy. Second-derivative analyses of amide-I and amide-III bands distinguish parallel ...in-register β-sheets from a β-solenoid. Further, spatially resolved Raman spectra reveal molecular heterogeneity in amyloid structures.
The greatly enhanced fields near metal nanoparticles have demonstrated remarkable optical properties and are promising for applications from solar energy to biosensing. However, direct experimental ...study of these light-matter interactions at the nanoscale has remained difficult due to the limitations of optical microscopy. Here, we use single-molecule fluorescence imaging to probe how a plasmonic nanoantenna modifies the fluorescence emission from a dipole emitter. We show that the apparent fluorophore emission position is strongly shifted upon coupling to an antenna and that the emission of dyes located up to 90 nm away is affected by this coupling. To predict this long-ranged effect, we present a framework based on a distance-dependent partial coupling of the dye emission to the antenna. Our direct interpretation of these light-matter interactions will enable more predictably optimized, designed, and controlled plasmonic devices and will permit reliable plasmon-enhanced single-molecule nanoscopy.
Parkinson’s disease (PD) is associated with the formation of α-synuclein amyloid fibrils. Elucidating the role of these β-sheet-rich fibrils in disease progression is crucial; however, collecting ...detailed structural information on amyloids is inherently difficult because of their insoluble, non-crystalline, and polymorphic nature. Here, we show that Raman spectroscopy is a facile technique for characterizing structural features of α-synuclein fibrils. Combining Raman spectroscopy with aggregation kinetics and transmission electron microscopy, we examined the effects of pH and ionic strength as well as four PD–related mutations (A30P, E46K, G51D, and A53T) on α-synuclein fibrils. Raman spectral differences were observed in the amide-I, amide-III, and fingerprint regions, indicating that secondary structure and tertiary contacts are influenced by pH and to a lesser extent by NaCl. Faster aggregation times appear to facilitate unique fibril structure as determined by the highly reproducible amide-I band widths, linking aggregation propensity and fibril polymorphism. Importantly, Raman spectroscopy revealed molecular-level perturbations of fibril conformation by the PD–related mutations that are not apparent through transmission electron microscopy or limited proteolysis. The amide-III band was found to be particularly sensitive, with G51D exhibiting the most distinctive features, followed by A53T and E46K. Relating to a cellular environment, our data would suggest that fibril polymorphs can be formed in different cellular compartments and potentially result in distinct phenotypes. Our work sets a foundation toward future cellular Raman studies of amyloids.
Skin color patterns are ubiquitous in nature, impact social behavior, predator avoidance, and protection from ultraviolet irradiation. A leading model system for vertebrate skin patterning is the ...zebrafish; its alternating blue stripes and yellow interstripes depend on light-reflecting cells called iridophores. It was suggested that the zebrafish's color pattern arises from a single type of iridophore migrating differentially to stripes and interstripes. However, here we find that iridophores do not migrate between stripes and interstripes but instead differentiate and proliferate in-place, based on their micro-environment. RNA-sequencing analysis further reveals that stripe and interstripe iridophores have different transcriptomic states, while cryogenic-scanning-electron-microscopy and micro-X-ray diffraction identify different crystal-arrays architectures, indicating that stripe and interstripe iridophores are different cell types. Based on these results, we present an alternative model of skin patterning in zebrafish in which distinct iridophore crystallotypes containing specialized, physiologically responsive, organelles arise in stripe and interstripe by in-situ differentiation.
Coupling to metal nanoparticles can increase the fluorescence intensity and photostability of fluorescent probes, and this plasmon-enhanced fluorescence is particularly promising for the dimmer ...fluorescent proteins common in biological imaging. Here, we measure the intensity distribution of single Cy3.5 dye molecules and mCherry fluorescent proteins one at a time as they adsorb on a conformal surface 4.8–61.0 nm thick over a gold nanorod (NR). The emission intensities for both types of fluorophores depend nonmonotonically on the spacer thickness, and an optimal spacer thickness of ∼10 nm is observed for both fluorophores using two different spacer layer materials. Emission from fluorophores coupled to metal nanoparticles is affected by two competing processes: an enhanced spontaneous decay rate and quenching via nonradiative antenna modes. After averaging over a conformal surface, the product of the simulated enhanced local electric field intensity and the quantum efficiency modification reproduces the experimental 10 nm ideal spacer thickness. Overall, up to a 3.4-fold average enhancement in fluorescence intensity was achieved despite the simple geometry, based on biocompatible, tunable, and economic colloidal gold NRs. This study of the distance dependence of single-molecule plasmon-enhanced fluorescence shows promise for super-resolving cellular membrane proteins naturally positioned above an extracellular substrate.
α-Synuclein (α-Syn) is a presynaptic protein that is accumulated in its amyloid form in the brains of Parkinson's patients. Although its biological function remains unclear, α-syn has been suggested ...to bind to synaptic vesicles and facilitate neurotransmitter release. Recently, studies have found that α-syn induces membrane tubulation, highlighting a potential mechanism for α-syn to stabilize highly curved membrane structures which could have both functional and dysfunctional consequences. To understand how membrane remodeling by α-syn affects amyloid formation, we have studied the α-syn aggregation process in the presence of phosphatidylglycerol (PG) micellar tubules, which were the first reported example of membrane tubulation by α-syn. Aggregation kinetics, β-sheet content, and macroscopic protein-lipid structures were observed by Thioflavin T fluorescence, circular dichroism spectroscopy and transmission electron microscopy, respectively. Collectively, the presence of PG micellar tubules formed at a stochiometric (L/P = 1) ratio was found to stimulate α-syn fibril formation. Moreover, transmission electron microscopy and solid-state nuclear magnetic resonance spectroscopy revealed the co-assembly of PG and α-syn into fibril structures. However, isolated micellar tubules do not form fibrils by themselves, suggesting an important role of free α-syn monomers during amyloid formation. In contrast, fibrils did not form in the presence of excess PG lipids (≥L/P = 50), where most of the α-syn molecules are in a membrane-bound α-helical form. Our results provide new mechanistic insights into how membrane tubules modulate α-syn amyloid formation and support a pivotal role of protein–lipid interaction in the dysfunction of α-syn.
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•α-Syn remodels anionic PG, PA, and PS membranes into micellar tubules.•Effect of POPG on α-syn amyloid formation was probed by CD, ThT fluorescence, and TEM.•POPG micellar tubules shorten the lag phase and enhanced β-sheet formation.•POPG micellar tubules are co-assembled into α-syn fibrils.•POPG micellar tubes do not to convert into amyloids in the absence of free α-syn.
The sensitivity and resolution of single-molecule fluorescence imaging in biology are mainly limited by two known weaknesses of fluorescent proteins: label brightness and photostability. In this ...work, we use patterned gold substrates to achieve plasmon-enhanced emission from intrinsically fluorescent proteins in living pathogenic bacteria cells. By coupling membrane-bound single fluorescent protein fusions to the virulence regulator TcpP in living Vibrio cholerae bacteria to extracellular gold nanotriangle arrays, we use plasmonics to improve our measurements of this important question in pathogenesis: how does V. cholerae produce its deadly toxin? Based on a simple experimental geometry, we observe a 1.3× enhancement in the rate of emission and a 1.4× enhancement in the number of photons detected prior to photobleaching. Furthermore, by enhancing both the rate of emission and the total number of photons detected from single-molecule fluorescent probes in live cells, we show that plasmon-enhanced fluorescence is a biocompatible, generalizable path to directly improve the resolution and trajectory lengths of single molecules in live cells.
Mapping conformational changes of α-synuclein (α-syn) from soluble, unstructured monomers to β-sheet- rich aggregates is crucial towards understanding amyloid formation. Raman microspectroscopy is ...now used to spatially resolve conformational heterogeneity of amyloid aggregates and monitor amyloid formation of segmentally
C-labeled α-syn in real-time. As the
C-isotope shifts the amide-I stretching frequency to lower energy, the ligated construct,
C
C
-α-syn, exhibits two distinct bands allowing for simultaneous detection of secondary structural changes in N-terminal 1-86 and C-terminal 87-140 residues. The disordered-to-β-sheet conformational change is first observed for the N-terminal followed by the C-terminal region. Finally, Raman spectroscopic changes occurred prior to Thioflavin T fluorescence enhancement, indicating that the amide-I band is a superior probe of amyloid formation.
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