Super-resolution microscopy allows biological systems to be studied at the nanoscale, but has been restricted to providing only positional information. Here, we show that it is possible to perform multi-dimensional super-resolution imaging to determine both the position and the environmental properties of single-molecule fluorescent emitters. The method presented here exploits the solvatochromic and fluorogenic properties of nile red to extract both the emission spectrum and the position of each dye molecule simultaneously enabling mapping of the hydrophobicity of biological structures. We validated this by studying synthetic lipid vesicles of known composition. We then applied both to super-resolve the hydrophobicity of amyloid aggregates implicated in neurodegenerative diseases, and the hydrophobic changes in mammalian cell membranes. Our technique is easily implemented by inserting a transmission diffraction grating into the optical path of a localization-based super-resolution microscope, enabling all the information to be extracted simultaneously from a single image plane.

Protein aggregation is a complex process resulting in the formation of heterogeneous mixtures of aggregate populations that are closely linked to neurodegenerative conditions, such as Alzheimer’s disease. Here, we find that soluble aggregates formed at different stages of the aggregation process of amyloid beta (Aβ42) induce the disruption of lipid bilayers and an inflammatory response to different extents. Further, by using gradient ultracentrifugation assay, we show that the smaller aggregates are those most potent at inducing membrane permeability and most effectively inhibited by antibodies binding to the C-terminal region of Aβ42. By contrast, we find that the larger soluble aggregates are those most effective at causing an inflammatory response in microglia cells and more effectively inhibited by antibodies targeting the N-terminal region of Aβ42. These findings suggest that different toxic mechanisms driven by different soluble aggregated species of Aβ42 may contribute to the onset and progression of Alzheimer’s disease.

SummaryReplisome assembly at eukaryotic replication forks connects the DNA helicase to DNA polymerases and many other factors. The helicase binds the leading-strand polymerase directly, but is connected to the Pol α lagging-strand polymerase by the trimeric adaptor Ctf4. Here, we identify new Ctf4 partners in addition to Pol α and helicase, all of which contain a “Ctf4-interacting-peptide” or CIP-box. Crystallographic analysis classifies CIP-boxes into two related groups that target different sites on Ctf4. Mutations in the CIP-box motifs of the Dna2 nuclease or the rDNA-associated protein Tof2 do not perturb DNA synthesis genome-wide, but instead lead to a dramatic shortening of chromosome 12 that contains the large array of rDNA repeats. Our data reveal unexpected complexity of Ctf4 function, as a hub that connects multiple accessory factors to the replisome. Most strikingly, Ctf4-dependent recruitment of CIP-box proteins couples other processes to DNA synthesis, including rDNA copy-number regulation.

The aberrant misfolding and subsequent conversion of monomeric protein into amyloid aggregates characterises many neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. These aggregates are highly heterogeneous in structure, generally of low abundance and typically smaller than the diffraction limit of light (≈250 nm). To overcome the challenges these characteristics pose to the study of endogenous aggregates formed in cells, we have developed a method to characterise them at the nanometre scale without the need for a conjugated fluorophore. Using a combination of DNA PAINT and an amyloid‐specific aptamer, we demonstrate that this technique is able to detect and super‐resolve a range of aggregated species, including those formed by α‐synuclein and amyloid‐β. Additionally, this method enables endogenous protein aggregates within cells to be characterised. We found that neuronal cells derived from patients with Parkinson's disease contain a larger number of protein aggregates than those from healthy controls.

To quantify and characterize the potentially toxic protein aggregates associated with neurodegenerative diseases, a high‐throughput assay based on measuring the extent of aggregate‐induced Ca2+ entry into individual lipid vesicles has been developed. This approach was implemented by tethering vesicles containing a Ca2+ sensitive fluorescent dye to a passivated surface and measuring changes in the fluorescence as a result of membrane disruption using total internal reflection microscopy. Picomolar concentrations of Aβ42 oligomers could be observed to induce Ca2+ influx, which could be inhibited by the addition of a naturally occurring chaperone and a nanobody designed to bind to the Aβ peptide. We show that the assay can be used to study aggregates from other proteins, such as α‐synuclein, and to probe the effects of complex biofluids, such as cerebrospinal fluid, and thus has wide applicability.