Cathodoluminescent and Characteristic X-Ray-Emissive Rare-Earth-Doped Core/Shell Protein Labels for Spectromicroscopic Analysis of Cell Surface Receptors.

Understanding the localization and the interactions of biomolecules at the nanoscale and in the cellular context remains challenging. Electron microscopy (EM), unlike light-based microscopy, gives access to the cellular ultrastructure yet results in grey-scale images and averts unambiguous (co-)localization of biomolecules. Multimodal nanoparticle-based protein labels for correlative cathodoluminescence electron microscopy (CCLEM) and energy-dispersive X-ray spectromicroscopy (EDX-SM) are presented. The single-particle STEM-cathodoluminescence (CL) and characteristic X-ray emissivity of sub-20 nm lanthanide-doped nanoparticles are exploited as unique spectral fingerprints for precise label localization and identification. To maximize the nanoparticle brightness, lanthanides are incorporated in a low-phonon host lattice and separated from the environment using a passivating shell. The core/shell nanoparticles are then functionalized with either folic (terbium-doped) or caffeic acid (europium-doped). Their potential for (protein-)labeling is successfully demonstrated using HeLa cells expressing different surface receptors that bind to folic or caffeic acid, respectively. Both particle populations show single-particle CL emission along with a distinctive energy-dispersive X-ray signal, with the latter enabling color-based localization of receptors within swift imaging times well below 2 min per µ m $\umu\text{m}$ 2 while offering high resolution with a pixel size of 2.78 nm. Taken together, these results open a route to multi-color labeling based on electron spectromicroscopy.

An evolutionarily conserved mechanism controls reversible amyloids of pyruvate kinase via pH-sensing regions.

Amyloids are known as irreversible aggregates associated with neurodegenerative diseases. However, recent evidence shows that a subset of amyloids can form reversibly and fulfill essential cellular functions. Yet, the molecular mechanisms regulating functional amyloids and distinguishing them from pathological aggregates remain unclear. Here, we investigate the conserved principles of amyloid reversibility by studying the essential metabolic enzyme pyruvate kinase (PK) in yeast and human cells. We demonstrate that yeast PK (Cdc19) and human PK (PKM2) form reversible amyloids through a pH-sensitive amyloid core. Stress-induced cytosolic acidification promotes aggregation via protonation of specific glutamate (yeast) or histidine (human) residues within the amyloid core. Mutations mimicking protonation cause constitutive PK aggregation, while non-protonatable PK mutants remain soluble even upon stress. Physiological PK aggregation is coupled to metabolic rewiring and glycolysis arrest, causing severe growth defects when misregulated. Our work thus identifies an evolutionarily conserved, potentially widespread mechanism regulating functional amyloids during stress.

Nanoparticles Dysregulate the Human Placental Secretome with Consequences on Angiogenesis and Vascularization.

Exposure to nanoparticles (NPs) in pregnancy is increasingly linked to adverse effects on embryo-fetal development and health later in life. However, the developmental toxicity mechanisms of NPs are largely unknown, in particular potential effects on the placental secretome, which orchestrates many developmental processes pivotal for pregnancy success. This study demonstrates extensive material- and pregnancy stage-specific deregulation of placental signaling from a single exposure of human placental explants to physiologically relevant concentrations of engineered (silica (SiO2) and titanium dioxide (TiO2) NPs) and environmental NPs (diesel exhaust particles, DEPs). This includes a multitude of secreted inflammatory, vascular, and endocrine placental factors as well as extracellular vesicle (EV)-associated proteins. Moreover, conditioned media (CM) from NP-exposed explants induce pronounced anti-angiogenic and anti-vasculogenic effects, while early neurodevelopmental processes are only marginally affected. These findings underscore the potential of metal oxide NPs and DEPs for widespread interference with the placental secretome and identify vascular morphogenesis as a sensitive outcome for the indirect developmental toxicity of different NPs. Overall, this work has profound implications for the future safety assessment of NPs for industrial, commercial, or medical applications in pregnancy, which should consider placenta-mediated toxicity by holistic secretomics approaches to ensure the development of safe nanotechnologies.

Protein Aggregation on Metal Oxides Governs Catalytic Activity and Cellular Uptake.

Engineering of catalytically active inorganic nanomaterials holds promising prospects for biomedicine. Catalytically active metal oxides show applications in enhancing wound healing but have also been employed to induce cell death in photodynamic or radiation therapy. Upon introduction into a biological system, nanomaterials are exposed to complex fluids, causing interaction and adsorption of ions and proteins. While protein corona formation on nanomaterials is acknowledged, its modulation of nanomaterial catalytic efficacy is less understood. In this study, proteomic analyses and nano-analytic methodologies quantify and characterize adsorbed proteins, correlating this protein layer with metal oxide catalytic activity in vitro and in vivo. The protein corona comprises up to 280 different proteins, constituting up to 38% by weight. Enhanced complement factors and other opsonins on nanocatalyst surfaces lead to their uptake into macrophages when applied topically, localizing >99% of the nanomaterials in tissue-resident macrophages. Initially, the formation of the protein corona significantly reduces the nanocatalysts' activity, but this activity can be partially recovered in endosomal conditions due to the proteolytic degradation of the corona. Overall, the research reveals the complex relationship between physisorbed proteins and the catalytic characteristics of specific metal oxide nanoparticles, providing design parameters for optimizing nanocatalysts in complex biological environments.

Boron Nitride Nanosheets Induce Lipid Accumulation and Autophagy in Human Alveolar Lung Epithelial Cells Cultivated at Air-Liquid Interface.

Hexagonal boron nitride (hBN) is an emerging 2D material attracting significant attention due to its superior electrical, chemical, and therapeutic properties. However, inhalation toxicity mechanisms of hBN in human lung cells are poorly understood. Here, cellular interaction and effects of hBN nanosheets is investigated in alveolar epithelial cells cultured on porous inserts and exposed under air-liquid interface conditions for 24 h. hBN is taken up by the cells as determined in a label-free manner via RAMAN-confocal microscopy, ICP-MS, TEM, and SEM-EDX. No significant (p > 0.05) effects are observed on cell membrane integrity (LDH release), epithelial barrier integrity (TEER), interleukin-8 cytokine production or reactive oxygen production at tested dose ranges (1, 5, and 10 µg cm-2). However, it is observed that an enhanced accumulation of lipid granules in cells indicating the effect of hBN on lipid metabolism. In addition, it is observed that a significant (p < 0.05) and dose-dependent (5 and 10 µg cm-2) induction of autophagy in cells after exposure to hBN, potentially associated with the downstream processing and breakdown of excess lipid granules to maintain lipid homeostasis. Indeed, lysosomal co-localization of lipid granules supporting this argument is observed. Overall, the results suggest that the continuous presence of excess intracellular lipids may provoke adverse outcomes in the lungs.

Development of single-cell ICP-TOFMS to measure nanoplastics association with human cells.

Nanoplastics, solid polymer particles smaller than 1 µm, are suspected to be widely present in the environment, food and air, and may pose a potential threat to human health. Detecting nanoplastics in and associated with individual cells is crucial to understand their mechanisms of toxicity and potential harm. In this context, we developed a single-cell inductively coupled plasma time-of-flight mass spectrometry (sc-ICP-TOFMS) method for the sensitive and rapid quantification of metal-doped model nanoplastics in human cells. By providing multi-elemental fingerprints of both the nanoplastics and the cells, this approach can be advantageous in laboratory toxicological studies as it allows for the simultaneous acquisition of a full mass spectrum with high time resolution. As a proof-of-concept study, we exposed two different human cell lines relevant to inhalation exposures (A549 alveolar epithelial cells and THP-1 monocytes) to Pd-doped nanoplastics. The sc-ICP-TOFMS analysis revealed a similar dose-dependent endocytotic capacity of THP-1 and A549 cells for nanoplastics uptake, and particle internalization was confirmed by transmission electron microscopy. Moreover, single-cell quantification showed that a considerable proportion of the exposed cells (72% of THP-1; 67% of A549) did not associate with any nanoplastics after exposure to 50 µg L-1 for 24 h. This highlights the importance to include single-cell analysis in the future safety assessment of nanoplastics in order to account for heterogeneous uptake within cell populations and to identify the origins and response trajectories of nanoplastics in biological tissues. In this regard, sc-ICP-TOFMS can be a powerful approach to provide quantitative data on nanoplastics-cell associations at single cell level for a large number of individual cells.

X-ray radio-enhancement by Ti3C2Tx MXenes in soft tissue sarcoma.

Radiotherapy is a cornerstone of cancer treatment. However, due to the low tissue specificity of ionizing radiation, damage to the surrounding healthy tissue of the tumor remains a significant challenge. In recent years, radio-enhancers based on inorganic nanomaterials have gained considerable interest. Beyond the widely explored metal and metal oxide nanoparticles, 2D materials, such as MXenes, could present potential benefits because of their inherently large specific surface area. In this study, we highlight the promising radio-enhancement properties of Ti3C2Tx MXenes. We demonstrate that atomically thin layers of titanium carbides (Ti3C2Tx MXenes) are efficiently internalized and well-tolerated by mammalian cells. Contrary to MXenes suspended in aqueous buffers, which fully oxidize within days, yielding rice-grain shaped rutile nanoparticles, the MXenes internalized by cells oxidize at a slower rate. This is consistent with cell-free experiments that have shown slower oxidation rates in cell media and lysosomal buffers compared to dispersants without antioxidants. Importantly, the MXenes exhibit robust radio-enhancement properties, with dose enhancement factors reaching up to 2.5 in human soft tissue sarcoma cells, while showing no toxicity to healthy human fibroblasts. When compared to oxidized MXenes and commercial titanium dioxide nanoparticles, the intact 2D titanium carbide flakes display superior radio-enhancement properties. In summary, our findings offer evidence for the potent radio-enhancement capabilities of Ti3C2Tx MXenes, marking them as a promising candidate for enhancing radiotherapy.

Nanoanalytical Insights into the Stability, Intracellular Fate, and Biotransformation of Metal-Organic Frameworks.

Metal-organic frameworks (MOFs) have found increasing applications in the biomedical field due to their unique properties and high modularity. Although the limited stability of MOFs in biological environments is increasingly recognized, analytical techniques have not yet been harnessed to their full potential to assess the biological fate of MOFs. Here, we investigate the environment-dependent biochemical transformations of widely researched nanosized MOFs (nMOFs) under conditions relevant to their medical application. We assess the chemical stability of antimicrobial zinc-based drug delivery nMOFs (Zn-ZIF-8 and Zn-ZIF-8:Ce) and radio-enhancer candidate nMOFs (Hf-DBA, Ti-MIL-125, and TiZr-PCN-415) containing biologically nonessential group IV metal ions. We reveal that even a moderate decrease in pH to values encountered in lysosomes (pH 4.5-5) leads to significant dissolution of ZIF-8 and partial dissolution of Ti-MIL-125, whereas no substantial dissolution was observed for TiZr-PCN-415 and Hf-DBA nMOFs. Exposure to phosphate-rich buffers led to phosphate incorporation in all nMOFs, resulting in amorphization and morphological changes. Interestingly, long-term cell culture studies revealed that nMOF (bio)transformations of, e.g., Ti-MIL-125 were cellular compartment-dependent and that the phosphate content in the nMOF varied significantly between nMOFs localized in lysosomes and those in the cytoplasm. These results illustrate the delicate nature and environment-dependent properties of nMOFs across all stages of their life cycle, including storage, formulation, and application, and the need for in-depth analyses of biotransformations for an improved understanding of structure-function relationships. The findings encourage the considerate choice of suspension buffers for MOFs because these media may lead to significant material alterations prior to application.

Metal-Organic Framework Mediated Radio-Enhancement Assessed in High-Throughput-Compatible 3D Tumor Spheroid Co-Cultures.

Inorganic nanomaterials have gained increasing attention in radiation oncology, owing to their radiation therapy enhancing properties. To accelerate candidate material selection and overcome the disconnect between conventional 2D cell culture and in vivo findings, screening platforms unifying high-throughput with physiologically relevant endpoint analysis based on 3D in vitro models are promising. Here, a 3D tumor spheroid co-culture model based on cancerous and healthy human cells is presented for the concurrent assessment of radio-enhancement efficacy, toxicity, and intratissural biodistribution with full ultrastructural context of radioenhancer candidate materials. Its potential for rapid candidate materials screening is showcased based on the example of nano-sized metal-organic frameworks (nMOFs) and direct benchmarking against gold nanoparticles (the current "gold standard"). Dose enhancement factors (DEFs) ranging between 1.4 and 1.8 are measured for Hf-, Ti-, TiZr-, and Au-based materials in 3D tissues and are overall lower than in 2D cell cultures, where DEF values exceeding 2 are found. In summary, the presented co-cultured tumor spheroid-healthy fibroblast model with tissue-like characteristics may serve as high-throughput platform enabling rapid, cell line-specific endpoint analysis for therapeutic efficacy and toxicity assessment, as well as accelerated radio-enhancer candidate screening.

Mre11-Rad50 oligomerization promotes DNA double-strand break repair.

The conserved Mre11-Rad50 complex is crucial for the detection, signaling, end tethering and processing of DNA double-strand breaks. While it is known that Mre11-Rad50 foci formation at DNA lesions accompanies repair, the underlying molecular assembly mechanisms and functional implications remained unclear. Combining pathway reconstitution in electron microscopy, biochemical assays and genetic studies, we show that S. cerevisiae Mre11-Rad50 with or without Xrs2 forms higher-order assemblies in solution and on DNA. Rad50 mediates such oligomerization, and mutations in a conserved Rad50 beta-sheet enhance or disrupt oligomerization. We demonstrate that Mre11-Rad50-Xrs2 oligomerization facilitates foci formation, DNA damage signaling, repair, and telomere maintenance in vivo. Mre11-Rad50 oligomerization does not affect its exonuclease activity but drives endonucleolytic cleavage at multiple sites on the 5'-DNA strand near double-strand breaks. Interestingly, mutations in the human RAD50 beta-sheet are linked to hereditary cancer predisposition and our findings might provide insights into their potential role in chemoresistance.

Reversible amyloids of pyruvate kinase couple cell metabolism and stress granule disassembly.

Cells respond to stress by blocking translation, rewiring metabolism and forming transient messenger ribonucleoprotein assemblies called stress granules (SGs). After stress release, re-establishing homeostasis and disassembling SGs requires ATP-consuming processes. However, the molecular mechanisms whereby cells restore ATP production and disassemble SGs after stress remain poorly understood. Here we show that upon stress, the ATP-producing enzyme Cdc19 forms inactive amyloids, and that their rapid re-solubilization is essential to restore ATP production and disassemble SGs in glucose-containing media. Cdc19 re-solubilization is initiated by the glycolytic metabolite fructose-1,6-bisphosphate, which directly binds Cdc19 amyloids, allowing Hsp104 and Ssa2 chaperone recruitment and aggregate re-solubilization. Fructose-1,6-bisphosphate then promotes Cdc19 tetramerization, which boosts its activity to further enhance ATP production and SG disassembly. Together, these results describe a molecular mechanism that is critical for stress recovery and directly couples cellular metabolism with SG dynamics via the regulation of reversible Cdc19 amyloids.

Regulatory control of DNA end resection by Sae2 phosphorylation.

DNA end resection plays a critical function in DNA double-strand break repair pathway choice. Resected DNA ends are refractory to end-joining mechanisms and are instead channeled to homology-directed repair. Using biochemical, genetic, and imaging methods, we show that phosphorylation of Saccharomyces cerevisiae Sae2 controls its capacity to promote the Mre11-Rad50-Xrs2 (MRX) nuclease to initiate resection of blocked DNA ends by at least two distinct mechanisms. First, DNA damage and cell cycle-dependent phosphorylation leads to Sae2 tetramerization. Second, and independently, phosphorylation of the conserved C-terminal domain of Sae2 is a prerequisite for its physical interaction with Rad50, which is also crucial to promote the MRX endonuclease. The lack of this interaction explains the phenotype of rad50S mutants defective in the processing of Spo11-bound DNA ends during meiotic recombination. Our results define how phosphorylation controls the initiation of DNA end resection and therefore the choice between the key DNA double-strand break repair mechanisms.

Extreme disorder in an ultrahigh-affinity protein complex.

Molecular communication in biology is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiological conditions or that contain large unstructured regions commonly interact with well-structured binding sites on other biomolecules. Here we demonstrate the existence of an unexpected interaction mechanism: the two intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α associate in a complex with picomolar affinity, but fully retain their structural disorder, long-range flexibility and highly dynamic character. On the basis of closely integrated experiments and molecular simulations, we show that the interaction can be explained by the large opposite net charge of the two proteins, without requiring defined binding sites or interactions between specific individual residues. Proteome-wide sequence analysis suggests that this interaction mechanism may be abundant in eukaryotes.