Kristina A. Ganzinger

The extracellular chaperone clusterin sequesters oligomeric forms of the amyloid-β 1−40 peptide

In recent genome-wide association studies, the extracellular chaperone protein, clusterin, has been identified as a newly-discovered risk factor in Alzheimers disease. We have examined the interactions between human clusterin and the Alzheimers disease–associated amyloid-β1−40 peptide (Aβ1−40), which is prone to aggregate into an ensemble of oligomeric intermediates implicated in both the proliferation of amyloid fibrils and in neuronal toxicity. Using highly sensitive single-molecule fluorescence methods, we have found that Aβ1−40 forms a heterogeneous distribution of small oligomers (from dimers to 50-mers), all of which interact with clusterin to form long-lived, stable complexes. Consequently, clusterin is able to influence both the aggregation and disaggregation of Aβ1−40 by sequestration of the Aβ oligomers. These results not only elucidate the protective role of clusterin but also provide a molecular basis for the genetic link between clusterin and Alzheimers disease.

Single molecule characterization of the interactions between amyloid-β peptides and the membranes of hippocampal cells

Oligomers of the 40 and 42 residue amyloid-β peptides (Aβ40 and Aβ42) have been implicated in the neuronal damage and impaired cognitive function associated with Alzheimer’s disease. However, little is known about the specific mechanisms by which these misfolded species induce such detrimental effects on cells. In this work, we use single-molecule imaging techniques to examine the initial interactions between Aβ monomers and oligomers and the membranes of live cells. This highly sensitive method enables the visualization of individual Aβ species on the cell surface and characterization of their oligomerization state, all at biologically relevant, nanomolar concentrations. The results indicate that oligomers preferentially interact with cell membranes, relative to monomers and that the oligomers become immobilized on the cell surface. Additionally, we observe that the interaction of Aβ species with the cell membrane is inhibited by the presence of ATP-independent molecular chaperones. This study demonstrates the power of this methodology for characterizing the interactions between protein aggregates and the membranes of live neuronal cells at physiologically relevant concentrations and opens the door to quantitative studies of the cellular responses to potentially pathogenic oligomers.

Kinetic model of the aggregation of alpha-synuclein provides insights into prion-like spreading

Significance Growing experimental evidence suggests that the pathological spreading of alpha-synuclein aggregates in Parkinson’s disease is mediated through a process of templated seeding whereby aggregates catalyze the conversion of soluble protein molecules into their aggregated forms. A molecular-level understanding of this process is still lacking. Here, we determine the concentrations and numbers of aggregates necessary for the effective seeding of alpha-synuclein, thus providing a quantitative framework to understand the conditions when its seeded propagation is favorable. We find that high concentrations of aggregates are needed for seeding yet that aggregates cause cytotoxicity at significantly lower concentrations. This suggests that templated seeding is unlikely to be the main mechanism of spreading in Parkinson’s disease but occurs together with oligomer-induced cellular stress. The protein alpha-synuclein (αS) self-assembles into small oligomeric species and subsequently into amyloid fibrils that accumulate and proliferate during the development of Parkinson’s disease. However, the quantitative characterization of the aggregation and spreading of αS remains challenging to achieve. Previously, we identified a conformational conversion step leading from the initially formed oligomers to more compact oligomers preceding fibril formation. Here, by a combination of single-molecule fluorescence measurements and kinetic analysis, we find that the reaction in solution involves two unimolecular structural conversion steps, from the disordered to more compact oligomers and then to fibrils, which can elongate by further monomer addition. We have obtained individual rate constants for these key microscopic steps by applying a global kinetic analysis to both the decrease in the concentration of monomeric protein molecules and the increase in oligomer concentrations over a 0.5–140-µM range of αS. The resulting explicit kinetic model of αS aggregation has been used to quantitatively explore seeding the reaction by either the compact oligomers or fibrils. Our predictions reveal that, although fibrils are more effective at seeding than oligomers, very high numbers of seeds of either type, of the order of 104, are required to achieve efficient seeding and bypass the slow generation of aggregates through primary nucleation. Complementary cellular experiments demonstrated that two orders of magnitude lower numbers of oligomers were sufficient to generate high levels of reactive oxygen species, suggesting that effective templated seeding is likely to require both the presence of template aggregates and conditions of cellular stress.

A Quantitative Comparison of Single-Dye Tracking Analysis Tools Using Monte Carlo Simulations

Single-particle tracking (SPT) is widely used to study processes from membrane receptor organization to the dynamics of RNAs in living cells. While single-dye labeling strategies have the benefit of being minimally invasive, this comes at the expense of data quality; typically a data set of short trajectories is obtained and analyzed by means of the mean square displacements (MSD) or the distribution of the particles’ displacements in a set time interval (jump distance, JD). To evaluate the applicability of both approaches, a quantitative comparison of both methods under typically encountered experimental conditions is necessary. Here we use Monte Carlo simulations to systematically compare the accuracy of diffusion coefficients (D-values) obtained for three cases: one population of diffusing species, two populations with different D-values, and a population switching between two D-values. For the first case we find that the MSD gives more or equally accurate results than the JD analysis (relative errors of D-values <6%). If two diffusing species are present or a particle undergoes a motion change, the JD analysis successfully distinguishes both species (relative error <5%). Finally we apply the JD analysis to investigate the motion of endogenous LPS receptors in live macrophages before and after treatment with methyl-β-cyclodextrin and latrunculin B.

Single-Molecule Imaging Reveals that Small Amyloid-β1–42 Oligomers Interact with the Cellular Prion Protein (PrPC)

Oligomers of the amyloid‐β peptide (Aβ) play a central role in the pathogenesis of Alzheimer’s disease and have been suggested to induce neurotoxicity by binding to a plethora of cell‐surface receptors. However, the heterogeneous mixtures of oligomers of varying sizes and conformations formed by Aβ42 have obscured the nature of the oligomeric species that bind to a given receptor. Here, we have used single‐molecule imaging to characterize Aβ42 oligomers (oAβ42) and to confirm the controversial interaction of oAβ42 with the cellular prion protein (PrPC) on live neuronal cells. Our results show that, at nanomolar concentrations, oAβ42 interacts with PrPC and that the species bound to PrPC are predominantly small oligomers (dimers and trimers). Single‐molecule biophysical studies can thus aid in deciphering the mechanisms that underlie receptor‐mediated oAβ‐induced neurotoxicity, and ultimately facilitate the discovery of novel inhibitors of these pathways.

Capturing resting T cells: the perils of PLL

Supported by a Royal Society University Research Fellowship (UF120277 to S.F.L.) and Research Professorship (RP150066 to D.K.); the EPSRC (EP/L027631/1 to A.P.,); the Wellcome Trust (098274/Z/12/Z to S.J.D., and WT101609MA to R.A.F.); PA Cephalosporin Fund (C.E.); the Wolfson Imaging Centre Oxford (funded by the Wolfson Foundation and Wellcome Trust; 104924/14/Z/14); the Micron Advanced BioImaging Unit (Wellcome Trust Strategic Award 091911); the Medical Research Council (MC_UU_12010/Unit Programmes G0902418 and MC_UU_12025); an MRC/BBSRC/EPSRC award (MR/K01577X/1); and a Marie Sklodowska-Curie Intra-European grant (707348 to I.U.).

Detecting RNA base methylations in single cells by in situ hybridization

Methylated bases in tRNA, rRNA and mRNA control a variety of cellular processes, including protein synthesis, antimicrobial resistance and gene expression. Currently, bulk methods that report the average methylation state of ~104–107 cells are used to detect these modifications, obscuring potentially important biological information. Here, we use in situ hybridization of Molecular Beacons for single-cell detection of three methylations (m62A, m1G and m3U) that destabilize Watson–Crick base pairs. Our method—methylation-sensitive RNA fluorescence in situ hybridization—detects single methylations of rRNA, quantifies antibiotic-resistant bacteria in mixtures of cells and simultaneously detects multiple methylations using multicolor fluorescence imaging.Methylated RNA bases influence many life processes, but current detection methods lack the ability to detect individual methylations in single cells. Here, the authors use fluorescence hybridization probes sensitive to methylation to detect specific epitranscriptomic modifications at the single-cell level.