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Spectrally separated fluorophores allow the observation of multiple targets simultaneously inside living cells, leading to a deeper understanding of the molecular interplay that regulates cell function and fate. Chemogenetic systems combining a tag and a synthetic fluorophore provide certain advantages over fluorescent proteins since there is no requirement for chromophore maturation. Here, we present the engineering of a set of spectrally orthogonal fluorogen-activating tags based on the fluorescence-activating and absorption shifting tag (FAST) that are compatible with two-color, live-cell imaging. The resulting tags, greenFAST and redFAST, demonstrate orthogonality not only in their fluorogen recognition capabilities, but also in their one- and two-photon absorption profiles. This pair of orthogonal tags allowed the creation of a two-color cell cycle sensor capable of detecting very short, early cell cycles in zebrafish development and the development of split complementation systems capable of detecting multiple protein–protein interactions by live-cell fluorescence microscopy. The fluorescent chemogenetic reporters greenFAST and redFAST were engineered by protein engineering. They display orthogonal fluorogen recognition and spectral properties allowing efficient multicolor imaging of proteins in live cells and organisms.

Abnormal aggregation of TAR DNA‐binding protein 43 (TDP‐43) is a pathological hallmark of motor neuron disease (MND), yet current methods for quantifying these aggregates in biological samples remain limited in sensitivity and resolution. Here, single‐molecule fluorescence microscopy is applied to post‐mortem brain extracts to quantitatively characterize aggregates containing TDP‐43 at the individual particle level. The resulting aggregate fingerprints, consisting of morphological and compositional profiles, are sufficient to distinguish MND donors from neurologically normal controls and further discriminate between clinically distinct MND subgroups. Comparative proteomic analysis confirms and extends these findings, revealing convergent and complementary molecular signatures. These results demonstrate, for the first time, that single‐molecule aggregate profiling can stratify MND cases using patient‐derived tissues, paving the way for the development of sensitive minimally invasive diagnostics and mechanistically informed disease monitoring tools. Single‐molecule fluorescence microscopy is combined with proteomic profiling to fingerprint the morphology and composition of TDP‐43 protein aggregates found in post‐mortem motor neuron disease brain tissues. Aggregate fingerprints can distinguish disease from control donors and detect unexpected TDP‐43 pathology in SOD1‐MND. These findings challenge existing models and offer a new route toward sensitive diagnostics and patient stratification tools.

Background The tumour-suppressor protein p53 can form amyloid aggregates resulting in loss of tumour-suppressing functions and leading to tumour formation. The detection of p53 aggregates in cancer cells has been demonstrated but these aggregates have not been detected in liquid biopsies to date, due to the lack of sufficiently sensitive methods. Methods We developed an ultrasensitive immunoassay based on the single-molecule array (SiMoA) technology to detect p53 aggregates in plasma, based on antibody capture of the aggregates. We confirmed that the assay detects p53 aggregates using super-resolution imaging. We then investigated the p53 aggregate concentrations in the plasma of 190 pre-surgery glioblastoma (GB) patients and 22 controls using this assay. Results We found that the plasma p53 aggregate levels are significantly elevated in pre-surgery GB patients’ plasma compared to controls. Longitudinal study further reveals that p53 aggregate levels may increase before GB recurrence and decrease following treatment. We also observed raised p53 aggregate concentrations in the plasma of cancer patients with brain metastases. Conclusions This study demonstrates the detection of p53 aggregates in liquid biopsies. Our findings highlight the potential of p53 aggregates as a novel biomarker for glioblastoma. Wu et al. develop an ultrasensitive immunoassay based on the single-molecule array (SiMoA) technology to detect p53 aggregates in plasma. They show that the p53 aggregate concentrations in the plasma of pre-surgery glioblastoma patients are significantly higher compared to controls. Plain language summary p53 is an important protein molecule that prevents cancer from developing. However, under some conditions, p53 can clump together to form aggregates. These aggregates cannot stop cancer and may even help cancer grow. This makes p53 aggregates a potential marker of cancer. We have developed a very sensitive test to catch and detect the rare p53 aggregates in the blood. With this test, we find that there are more p53 aggregates in the blood of glioblastoma (a highly aggressive brain cancer) patients than people without cancer. Our test is 90.57% accurate in diagnosing glioblastoma and may also help predict cancer recurrence and monitor treatment response.

INTRODUCTION The monoclonal antibodies Aducanumab, Lecanemab, Gantenerumab, and Donanemab were developed for the treatment of Alzheimer's disease (AD). METHODS We used single‐molecule detection and super‐resolution imaging to characterize the binding of these antibodies to diffusible amyloid beta (Aβ) aggregates generated in‐vitro and harvested from human brains. RESULTS Lecanemab showed the best performance in terms of binding to the small‐diffusible Aβ aggregates, affinity, aggregate coating, and the ability to bind to post‐translationally modified species, providing an explanation for its therapeutic success. We observed a Braak stage–dependent increase in small‐diffusible aggregate quantity and size, which was detectable with Aducanumab and Gantenerumab, but not Lecanemab, showing that the diffusible Aβ aggregates change with disease progression and the smaller aggregates to which Lecanemab preferably binds exist at higher quantities during earlier stages. DISCUSSION These findings provide an explanation for the success of Lecanemab in clinical trials and suggests that Lecanemab will be more effective when used in early‐stage AD. Highlights Anti amyloid beta therapeutics are compared by their diffusible aggregate binding characteristics. In‐vitro and brain‐derived aggregates are tested using single‐molecule detection. Lecanemab shows therapeutic success by binding to aggregates formed in early disease. Lecanemab binds to these aggregates with high affinity and coats them better.

Understanding the role of small, soluble aggregates of beta-amyloid (Aβ) and tau in Alzheimer’s disease (AD) is of great importance for the rational design of preventative therapies. Here we report a set of methods for the detection, quantification, and characterisation of soluble aggregates in conditioned media of cerebral organoids derived from human iPSCs with trisomy 21, thus containing an extra copy of the amyloid precursor protein (APP) gene. We detected soluble beta-amyloid (Aβ) and tau aggregates secreted by cerebral organoids from both control and the isogenic trisomy 21 (T21) genotype. We developed a novel method to normalise measurements to the number of live neurons within organoid-conditioned media based on glucose consumption. Thus normalised, T21 organoids produced 2.5-fold more Aβ aggregates with a higher proportion of larger (300–2000 nm 2 ) and more fibrillary-shaped aggregates than controls, along with 1.3-fold more soluble phosphorylated tau (pTau) aggregates, increased inflammasome ASC-specks, and a higher level of oxidative stress inducing thioredoxin-interacting protein (TXNIP). Importantly, all this was detectable prior to the appearance of histological amyloid plaques or intraneuronal tau-pathology in organoid slices, demonstrating the feasibility to model the initial pathogenic mechanisms for AD in-vitro using cells from live genetically pre-disposed donors before the onset of clinical disease. Then, using different iPSC clones generated from the same donor at different times in two independent experiments, we tested the reproducibility of findings in organoids. While there were differences in rates of disease progression between the experiments, the disease mechanisms were conserved. Overall, our results show that it is possible to non-invasively follow the development of pathology in organoid models of AD over time, by monitoring changes in the aggregates and proteins in the conditioned media, and open possibilities to study the time-course of the key pathogenic processes taking place.

Protein aggregates are a pathological hallmark across neurodegenerative diseases. Yet, the disconnect between molecular-level aggregation and the emergence of disease severely limits mechanistic understanding of neurodegeneration. Here, we bridge this disconnect by showing that a cellular tipping point emerges as a universal feature across diseases from the competition between aggregate accumulation and removal. We map the resulting cellular phase transition with our high-throughput live-cell assay, measuring the tipping point that separates healthy cells from those with large aggregate loads. Using super-resolution imaging of brain tissue from Alzheimer's and Parkinson's disease, we quantify how the balance of accumulation and removal is shifted in disease. We validate our framework by predicting how designed aggregation inhibitors shift the tipping point to restore cellular homeostasis. Our results provide a mechanistic framework connecting molecular-level aggregation to disease, paving the way for a quantitative, unified understanding of neurodegeneration and enabling predictions of therapeutic efficacy.

Protein-protein interactions (PPI) can be detected through selective complementation of split fluorescent reporters made of two complementary fragments that reassemble into a functional fluorescent reporter when in close proximity. We previously introduced splitFAST, a chemogenetic PPI reporter with rapid and reversible complementation. Here, we present the engineering of RspA-splitFAST, an improved reporter displaying higher brightness, lower self-complementation and higher dynamic range for optimal monitoring of PPI using an original protein engineering strategy that exploits proteins with orthology relationships. Our study allowed the identification of a system with improved properties and enabled a better understanding of the molecular features controlling the complementation properties. Because of the rapidity and reversibility of its complementation, its low self-complementation, high dynamic range, and improved brightness, RspA-splitFAST is well suited to study PPI with high spatial and temporal resolution, opening great prospects to decipher the role of PPI in various biological contexts.

Inappropriate aggregation of TAR DNA-binding protein 43 (TDP-43) is a hallmark of motor neuron disease (MND). Current methods for quantifying heterogeneous aggregate populations in biofluids are limited, precluding their routine use in diagnosis and disease monitoring. Single molecule microscopy methods overcome these limitations to deliver quantitative morphological and compositional fingerprinting of molecular assemblies containing TDP-43. Here, we demonstrate the application of such methods to extracts from donor brain tissues. We show the number and morphology of aggregates derived from frontal cortex and cerebellar samples is sufficient to distinguish MND donors from neurologically normal controls, as well as between different MND cohorts. In addition, we compare proteomic and microscopic compositional profiles, demonstrating how complementary insights delivered by each technique can enhance our understanding of disease mechanisms. These single molecule assays will inform future diagnostic and stratification technologies, improving our ability to deliver patient care in a timely and targeted manner.Competing Interest StatementThe authors have declared no competing interest.Footnotes* https://doi.org/10.5281/zenodo.14960397* https://doi.org/10.5281/zenodo.14965410

Monoclonal antibodies Aducanumab, Lecanemab, Gantenerumab, and Donanemab have been developed for treatment of Alzheimer’s disease. Here, we have used single-molecule detection techniques and super-resolution imaging to characterise the binding of these antibodies to beta-amyloid aggregates including human post-mortem brain samples. Lecanemab is the best antibody in terms of binding to the small-soluble beta-amyloid aggregates, affinity, aggregate coating, and the ability to bind to post-translationally modified species, explaining its therapeutic success.