Self-Assembling Protein Surfaces for In Situ Capture of Cell-Free-Synthesized Proteins

We present a new method for the surface capture of proteins in cell-free protein synthesis (CFPS). We demonstrate the spontaneous self-assembly of the protein BslA into functionalizable surfaces on the surface of a CFPS reaction chamber. We show that proteins can be covalently captured by such surfaces, using “Catcher/Tag” technology. Importantly, proteins of interest can be captured either when synthesised in situ by CFPS above the BslA surfaces, or when added as pure protein. The simplicity and cost efficiency of this method suggest that it will find many applications in cell-free-based methods

Live‐cell super‐resolution imaging of actin using LifeAct‐14 with a PAINT‐based approach

We present direct‐LIVE‐PAINT, an easy‐to‐implement approach for the nanoscopic imaging of protein structures in live cells using labeled binding peptides. We demonstrate the feasibility of direct‐LIVE‐PAINT with an actin‐binding peptide fused to EGFP, the location of which can be accurately determined as it transiently binds to actin filaments. We show that direct‐LIVE‐PAINT can be used to image actin structures below the diffraction‐limit of light and have used it to observe the dynamic nature of actin in live cells. We envisage a similar approach could be applied to imaging other proteins within live mammalian cells

Single-molecule FRET studies on alpha-synuclein oligomerization of Parkinson's disease genetically related mutants.

Oligomers of alpha-synuclein are toxic to cells and have been proposed to play a key role in the etiopathogenesis of Parkinson's disease. As certain missense mutations in the gene encoding for alpha-synuclein induce early-onset forms of the disease, it has been suggested that these variants might have an inherent tendency to produce high concentrations of oligomers during aggregation, although a direct experimental evidence for this is still missing. We used single-molecule Förster Resonance Energy Transfer to visualize directly the protein self-assembly process by wild-type alpha-synuclein and A53T, A30P and E46K mutants and to compare the structural properties of the ensemble of oligomers generated. We found that the kinetics of oligomer formation correlates with the natural tendency of each variant to acquire beta-sheet structure. Moreover, A53T and A30P showed significant differences in the averaged FRET efficiency of one of the two types of oligomers formed compared to the wild-type oligomers, indicating possible structural variety among the ensemble of species generated. Importantly, we found similar concentrations of oligomers during the lag-phase of the aggregation of wild-type and mutated alpha-synuclein, suggesting that the properties of the ensemble of oligomers generated during self-assembly might be more relevant than their absolute concentration for triggering neurodegeneration.LT has been recipient of a grant PAT Post Doc Outgoing 2009 – 7th Framework Program Marie Curie COFUND actions. NC was funded by a Royal Society Dorothy Hodgkin Research Fellowship and is currently a Ramón y Cajal Research Fellow (Spanish Ministry of Economy and Competitiveness). MHH thanks the Royal Society of Chemistry (Analytical Chemistry Trust Fund) for his studentship. AJD is funded by the Schiff Foundation.This is the final version of the article. It first appeared from NPG via http://dx.doi.org/10.1038/srep1669

Synaptic expression of TAR-DNA-binding protein 43 in the mouse spinal cord determined using super-resolution microscopy

Funding: This work was supported by Motor Neurone Disease (MND) Association UK (Miles/Apr18/863-791), Chief Scientist Office, RS Macdonald Charitable Trust, ALS CURE Project, the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Programme (695568 SYNNOVATE), Simons Foundation Autism Research Initiative (529085), and the Wellcome Trust (Technology Development grant 202932).Amyotrophic Lateral Sclerosis (ALS) is characterised by a loss of motor neurons in the brain and spinal cord that is preceded by early-stage changes in synapses that may be associated with TAR-DNA-Binding Protein 43 (TDP-43) pathology. Cellular inclusions of hyperphosphorylated TDP-43 (pTDP-43) are a key hallmark of neurodegenerative diseases such ALS. However, there has been little characterisation of the synaptic expression of TDP-43 inside subpopulations of spinal cord synapses. This study utilises a range of high-resolution and super-resolution microscopy techniques with immunolabelling, as well as an aptamer-based TDP-43 labelling strategy visualised with single-molecule localisation microscopy, to characterise and quantify the presence of pTDP-43 in populations of excitatory synapses near where motor neurons reside in the lateral ventral horn of the mouse lumbar spinal cord. We observe that TDP-43 is expressed in approximately half of spinal cord synapses as nanoscale clusters. Synaptic TDP-43 clusters are found most abundantly at synapses associated with VGLUT1-positive presynaptic terminals, compared to VGLUT2-associated synapses. Our nanoscopy techniques showed no difference in the subsynaptic expression of pTDP-43 in the ALS mouse model, SOD1G93a, compared to healthy controls, despite prominent structural deficits in VGLUT1-associated synapses in SOD1G93a mice. This research characterizes the basic synaptic expression of TDP-43 with nanoscale precision and provides a framework with which to investigate the potential relationship between TDP-43 pathology and synaptic pathology in neurodegenerative diseases.Publisher PDFPeer reviewe

Determining the location of the α-synuclein dimer Interface using native top-down fragmentation and isotope depletion-mass spectrometry

α-Synuclein (αSyn), a 140-residue intrinsically disordered protein, comprises the primary proteinaceous component of pathology-associated Lewy body inclusions in Parkinson’s disease (PD). Due to its association with PD, αSyn is studied extensively; however, the endogenous structure and physiological roles of this protein are yet to be fully understood. Here, ion mobility-mass spectrometry and native top-down electron capture dissociation fragmentation have been used to elucidate the structural properties associated with a stable, naturally occurring dimeric species of αSyn. This stable dimer appears in both wild-type (WT) αSyn and the PD-associated variant A53E. Furthermore, we integrated a novel method for generating isotopically depleted protein into our native top-down workflow. Isotope depletion increases signal-to-noise ratio and reduces the spectral complexity of fragmentation data, enabling the monoisotopic peak of low abundant fragment ions to be observed. This enables the accurate and confident assignment of fragments unique to the αSyn dimer to be assigned and structural information about this species to be inferred. Using this approach, we were able to identify fragments unique to the dimer, which demonstrates a C-terminal to C-terminal interaction between the monomer subunits. The approach in this study holds promise for further investigation into the structural properties of endogenous multimeric species of αSyn