Photothermally Heated Asymmetric Thin Nanopores Suggest the Influence of Temperature on the Intermediate Conformational State of Cytochrome c in an Electric Field.

Nanopore sensing is a label-free single-molecule technique that enables the study of the dynamical structural properties of proteins. Here, we detect the translocation of cytochrome c (Cyt c) through an asymmetric thin nanopore with photothermal heating to evaluate the influence of temperature on Cyt c conformation during its translocation in an electric field. Before Cyt c translocates through an asymmetric thin SiNx nanopore, ∼1 ms trapping events occur due to electric field-induced denaturation. These trapping events were corroborated by a control analysis with a transmission electron microscopy-drilled pore and denaturant buffer. Cyt c translocation events exhibited markedly greater broad current blockade when the pores were photothermally heated. Collectively, our molecular dynamics simulation predicted that an increased temperature facilitates denaturation of the α-helical structure of Cyt c, resulting in greater blockade current during Cyt c trapping. Our photothermal heating method can be used to study the influence of temperature on protein conformation at the single-molecule level in a label-free manner.

Spin-State Switching of Spin-Crossover Complexes on Cu(111) Evidenced by Spin-Flip Spectroscopy.

Spin-crossover compounds can be switched between two stable states with different magnetic moments, conformations, electronic, and optical properties, which opens appealing perspectives for technological applications including miniaturization down to the scale of single molecules. Although control of the spin states is crucial their direct identification is challenging in single-molecule experiments. Here we investigate the spin-crossover complex [Fe(HB(1,2,4-triazol-1-yl)3)2] on a Cu(111) surface with scanning tunneling microscopy and density functional theory calculations. Spin crossover of single molecules in dense islands is achieved via electron injection. Spin-flip excitations are resolved in scanning tunneling spectra in a magnetic field enabling the direct identification of the molecular spin state, and revealing the existence of magnetic anisotropy in the HS molecules.

Low-affinity LFA1-dependent outside-in signaling mediates avidity modulation via the Rabin8-Rab8 axis.

Lymphocyte interactions mediated by leukocyte integrin lymphocyte function-associated antigen 1 (LFA1) and intercellular adhesion molecules (ICAMs) are important for lymphocyte trafficking and antigen recognition. Integrins are regulated by the modulation of ligand-binding affinity and avidity (valency). Although the mechanism underlying high-affinity LFA1 binding has been investigated extensively, the molecular mechanisms by which low-affinity multivalent binding initiates adhesion remain unclear. We previously showed that ICAM1 and monoclonal antibodies that recognize specific LFA1 conformations induce the accumulation of LFA1 at the contact surface. In this study, we found that the small GTPase Rab8 is critical for intracellular transport and accumulation of LFA1 at cell contact areas mediated by low-affinity LFA1-dependent outside-in signaling. Super-resolution microscopy revealed that Rab8 co-localized with LFA1 in small vesicles near the contact membrane. Inactivation of Rab8 decreased ICAM1-dependent adhesion and substantially reduced LFA1 density on the contact membrane. The GTP-bound active form of Rab8 increased cell adhesiveness and promoted LFA1 accumulation at the contact area through co-trafficking with LFA1. Rab8 activation was induced by low-affinity conformation-dependent outside-in signaling via the guanine exchange factor Rabin8, which induced Rab8 activation at the cell contact area independent of Rap1. Single-molecule imaging of ICAM1 on a supported planner lipid bilayer demonstrated that Rab8 increased the frequency of LFA1-ICAM1 interactions without affecting their binding lifetime, indicating that Rab8 is mainly involved in the modulation of LFA1 avidity rather than LFA1 affinity. The present findings underscore the importance of low-affinity conformation-dependent outside-in signaling via the Rabin8-Rab8 axis leading to the initiation of LFA1 transport to the contact area.

Secondary Nucleation of Aß Revealed by Single-Molecule and Computational Approaches.

Understanding the mechanisms underlying amyloid-ß (Aß) aggregation is pivotal in the context of Alzheimer's disease. This study aims to elucidate the secondary nucleation process of Aß42 peptides by combining experimental and computational methods. Using a newly developed nanopipette-based amyloid seeding and translocation assay, confocal fluorescence spectroscopy, and molecular dynamics simulations, the influence of the seed properties on Aß aggregation is investigated. Both fragmented and unfragmented seeds played distinct roles in the formation of oligomers, with fragmented seeds facilitating the formation of larger aggregates early in the incubation phase. The results show that secondary nucleation leads to the formation of oligomers of various sizes and structures as well as larger fibrils structured in ß-sheets. From these findings a mechanism of secondary nucleation involving two types of aggregate populations, one released and one growing on the mother fiber is proposed.

Subtype-specific conformational landscape of NMDA receptor gating.

N-methyl-D-aspartate receptors are ionotropic glutamate receptors that mediate synaptic transmission and plasticity. Variable GluN2 subunits in diheterotetrameric receptors with identical GluN1 subunits set very different functional properties. To understand this diversity, we use single-molecule fluorescence resonance energy transfer (smFRET) to measure the conformations of the ligand binding domain and modulatory amino-terminal domain of the common GluN1 subunit in receptors with different GluN2 subunits. Our results demonstrate a strong influence of the GluN2 subunits on GluN1 rearrangements, both in non-agonized and partially agonized activation intermediates, which have been elusive to structural analysis, and in the fully liganded state. Chimeric analysis reveals structural determinants that contribute to these subtype differences. Our study provides a framework for understanding the conformational landscape that supports highly divergent levels of activity, desensitization, and agonist potency in receptors with different GluN2s and could open avenues for the development of subtype-specific modulators.

Neurofilament light (NfL) concentrations in patients with epilepsy with recurrent isolated seizures: Insights from a clinical cohort study.

PURPOSE: To detect possible neuronal damage due to recurrent isolated seizures in patients with epilepsy in a clinical routine setting. METHODS: We measured the serum concentrations of neurofilament light chain (sNfL) in 46 outpatients with an at least monthly occurrence (self-reported) of generalized tonic-clonic seizures in the six months prior to the study and in 49 patients who had been seizure free (self-reported) for at least one year. We assigned the patients with seizure activity into groups with moderate and high seizure frequency. We measured sNfL with a highly sensitive single molecule array (Simoa). RESULTS: The majority (94 %) of all patients with epilepsy had sNfL values within the age adjusted reference ranges of our laboratory. Three patients with and three patients without seizure activity (each 3 %) showed elevated sNfL concentrations. Age adjusted sNfL concentrations did not differ significantly between patients with and without seizure activity in the total sample or in the female subgroup. In contrast, NfL concentrations were significantly higher in male patients with seizure activity and highest in the subgroup of those with high seizure activity, but were only above the reference range in two patients. sNfL concentrations did not differ between focal and generalized epilepsies and between genetic and structural etiologies. CONCLUSIONS: The sNfL concentrations in patients with epilepsy and healthy patients did not differ significantly. The finding of higher sNfL concentrations in males with self-reported seizure activity should be viewed with utmost caution because the difference was small and only two male patients showed sNfL concentrations above the reference range.

Detecting serologically difficult ABO blood groups using single-molecule real-time sequencing technology.

BACKGROUND AND OBJECTIVES: Recently, third-generation long-read sequencing technology has been increasingly applied to the detection of various blood group systems. Because of its long read length and use of single-molecule sequencing, it is capable of obtaining the sequences of blood group genes in their entirety as well as of distinguishing haplotypes. Therefore, here, we collected ABO blood group samples that were difficult to classify serologically and analysed the sequences of the coding regions of the ABO genes as well as the sequences upstream and downstream of the coding regions. MATERIALS AND METHODS: Samples with ABO antigen typing and reverse serum typing discrepancies were screened in a total of 21 patients. All samples were subjected to serological testing and preliminary ABO genotyping (polymerase chain reaction with sequence-specific primers [PCR-SSP]), followed by single-molecule real-time (SMRT) sequencing to obtain complete ABO gene sequences. PCR sequence-based typing (PCR-SBT) was performed to validate the results. RESULTS: Of the 21 samples, 15 had common ABO types, and 6 had rare ABO subtypes. One new allele, ABO*B.NEW (c.861C>T), and one allelic base recombination event was identified. Forty-two haplotype sequences were obtained via SMRT sequencing with intronic single-nucleotide variants (SNVs) specific to the ABO allele, and all of the exon region sequences were consistent with the PCR-SBT results. CONCLUSION: SMRT sequencing is capable of accurately obtaining complete ABO gene sequences, distinguishing haplotypes and identifying allelic recombination.

Low Vapor Pressure Solvents for Single-Molecule Junction Measurements.

Nonpolar solvents commonly used in scanning tunneling microscope-based break junction measurements exhibit hazards and relatively low boiling points (bp) that limit the scope of solution experiments at elevated temperatures. Here we show that low toxicity, ultrahigh bp solvents such as bis(2-ethylhexyl) adipate (bp = 417 °C) and squalane (457 °C) can be used to probe molecular junctions at ≥100 °C. With these, we extend solvent- and temperature-dependent conductance trends for junction components such as 4,4'-bipyridine and thiomethyl-terminated oligophenylenes and reveal the gold snapback distance is larger at 100 °C due to increased surface atom mobility. We further show the rate of surface transmetalation and homocoupling reactions using phenylboronic acids increases at 100 °C, while junctions comprising anticipated boroxine condensation products form only at room temperature in an anhydrous glovebox atmosphere. Overall, this work demonstrates the utility of low vapor pressure solvents for the comprehensive characterization of junction properties and chemical reactivity at the single-molecule limit.

Studies of DNA breathing in exciton-coupled (iCy3)2 dimer-labeled DNA constructs by polarization-sweep single-molecule fluorescence (PS-SMF) microscopy.

Local fluctuations of the sugar-phosphate backbones and bases of DNA (a form of DNA 'breathing') play a central role in the assembly of protein-DNA complexes. We present a single-molecule fluorescence method to sensitively measure the local conformational fluctuations of exciton-coupled cyanine [(iCy3)2] dimer-labeled DNA fork constructs in which the dimer probes are placed at varying positions relative to the DNA fork junction. These systems exhibit spectroscopic signals that are sensitive to the local conformations adopted by the sugar-phosphate backbones and bases immediately surrounding the dimer probe label positions. The (iCy3)2 dimer has one symmetric (+) and one anti-symmetric (-) exciton with respective transition dipole moments oriented perpendicular to one another. We excite single molecule samples using a continuous-wave, linearly polarized laser with its polarization direction rotated at a frequency of 1 MHz. The ensuing fluorescence signal is modulated as the laser polarization alternately excites the symmetric and anti-symmetric excitons of the (iCy3)2 dimer probe. Phase-sensitive detection of the signal at the photon-counting level provides information about the distribution of local conformations and conformational dynamics. We analyze our data using a kinetic network model, which we use to parametrize the free energy surface of the system. In addition to observing DNA breathing at and near ss-dsDNA junctions, the approach can be used to study the effects of proteins that bind and function at these sites.

Probing recombinant AAV capsid integrity and genome release after thermal stress by mass photometry.

Adeno-associated viruses (AAVs) are gaining traction as delivery vehicles for gene therapy although the molecular understanding of AAV-transgene release is still limited. Typically, the process of viral uncoating is investigated (in vitro) through thermal stress, revealing capsid disintegration at elevated temperatures. To assess the (in)stability of different empty and filled AAV preparations, we used the light-scattering-based interferometric microscopy technique of mass photometry that, on a single-particle basis, determines the molecular weight of AAVs. By introducing a heat-stable DNA plasmid as an internal standard, we quantitatively probed the impact of heat on AAVs. Generally, empty AAVs exhibited greater heat resistance than genome-filled particles. Our data also indicate that upon DNA release, the capsids do not transform into empty AAVs, but seem to aggregate or disintegrate. Strikingly, some AAVs exhibited an intermediate state with disrupted capsids but preserved bound genome, a feature that experimentally only emerged following incubation with a nuclease. Our data demonstrate that the thermal uncoating process is highly AAV specific (i.e., can be influenced by serotype, genome, host system). We argue that nuclease treatment in combination with MP can be used as an additional analytical tool for assessing structural integrity of recombinant and/or clinical AAV vectors.

Analysis of genomic copy number variations through whole-genome scan in Yunling cattle.

Yunling cattle is a new breed of beef cattle bred in Yunnan Province, China, which has the advantages of fast growth, excellent meat quality, improved tolerance ability, and important landscape value. Copy number variation (CNV) is a significant source of gene structural variation and plays a crucial role in evolution and phenotypic diversity. Based on the latest reference genome ARS-UCD2.0, this study analyzed the genome-wide distribution of CNVs in Yunling cattle using short-read whole-genome sequencing data (n = 129) and single-molecule long-read sequencing data (n = 1), and a total of 16,507 CNVs were detected. After merging CNVs with overlapping genomic positions, 3,728 CNV regions (CNVRs) were obtained, accounting for 0.61% of the reference genome. The functional analysis indicated significant enrichment of CNVRs in 96 GO terms and 57 KEGG pathways, primarily related to cell adhesion, signal transduction, neuromodulation, and nutritional metabolism. Additionally, 111 CNVRs overlapped with 76 quantitative trait loci (QTLs), including Subcutaneous fat thickness QTL, Longissimus muscle area QTL, and Marbling score QTL. Several CNVR-overlapping genes, including BZW1, AOX1, and LOC100138449, overlap with regions associated with meat color and quality QTLs. Furthermore, Vst analysis showed that PSMB4, ERICH1, SMC2, and PPP4R3A were highly divergent between Yunling and Brahman cattle. In summary, we have constructed the genomic CNV map of Yunling cattle for the first time using whole-genome resequencing. This provides valuable genetic variation resources for the study of the Yunling cattle genome and contributes to the study of economic traits in Yunling cattle.

Functional role of carbohydrate-binding modules in multi-modular chitinase OfChtII.

The primary distinction between insect and bacterial chitin degradation systems lies in the presence of a multi-modular endo-acting chitinase ChtII, in contrast to a processive exo-acting chitinase. Although the essential role of ChtII during insect development and its synergistic action with processive chitinase during chitin degradation have been established, the mechanistic understanding of how it deconstructs chitin remains largely elusive. Here OfChtII from the insect Ostrinia furnacalis was investigated employing comprehensive approaches encompassing biochemical and microscopic analyses. The results demonstrated that OfChtII truncations with more carbohydrate-binding modules (CBMs) exhibited enhanced hydrolysis activity, effectively yielding a greater proportion of fibrillary fractions from the compacted chitin substrate. At the single-molecule level, the CBMs in these OfChtII truncations have been shown to primarily facilitate chitin substrate association rather than dissociation. Furthermore, a greater number of CBMs was demonstrated to be essential for the enzyme to effectively bind to chitin substrates with high crystallinity. Through real-time imaging by high-speed atomic force microscopy, the OfChtII-B4C1 truncation with three CBMs was observed to shear chitin fibers, thereby generating fibrillary fragments and deconstructing the compacted chitin structure. This work pioneers in revealing the nanoscale mechanism of endo-acting multi-modular chitinase involved in chitin degradation, which provides an important reference for the rational design of chitinases or other glycoside hydrolases.

A Statistical Route to Robust SERS Quantitation Beyond the Single-Molecule Level.

Single-molecule surface-enhanced Raman spectroscopy (SM-SERS) holds great potential to revolutionize ultratrace quantitative analysis. However, achieving quantitative SM-SERS is challenging because of strong intensity fluctuation and blinking characteristics. In this study, we reveal the relation P = 1 - e-α between the statistical SERS probability P and the microscopic average molecule number α in SERS spectra, which lays the physical foundation for a statistical route to implement SM-SERS quantitation. Utilizing SERS probability calibration, we achieve quantitative SERS analysis with batch-to-batch robustness, extremely wide detection range of concentration covering 9 orders of magnitude, and ultralow detection limit far below the single-molecule level. These results indicate the physical feasibility of robust SERS quantitation through statistical route and certainly open a new avenue for implementing SERS as a practical analysis tool in various application scenarios.

Protocol for the production and reconstitution of VDAC1 for functional assays.

The voltage-dependent anion channel (VDAC) is an abundant and multifunctional outer mitochondrial membrane protein, playing key roles in neurodegeneration, apoptosis, and mitochondrial membrane biogenesis. Here, we present a protocol to produce and reconstitute high yields of detergent-solubilized VDAC, expressed as inclusion bodies in E. coli. We describe steps for purification by affinity chromatography and refolding in lauryldimethylamine-N-oxide (LDAO). We then detail procedures for reconstituting VDAC into membrane vesicles to assay its channel and phospholipid scramblase activity via fluorescence-based assays. For complete details on the use and execution of this protocol, please refer to Bergdoll et al.,1 Queralt-Martín et al., 2 and Jahn et al.3.

Persistent autonomous molecular motion of DNA walker along a single-molecule nano-track for intracellular MicroRNA imaging.

While the intracellular imaging of miRNA biomarkers is of significant importance for the diagnosis and treatment of human cancers, DNA assembled nanoprobe has recently attracted considerable attention for imaging intracellular biomolecules. However, the complex construction process, intrinsic vulnerability to nuclease degradation and the limited signal transduction efficiency hamper its widespread application. In this contribution, based on persistent autonomous molecular motion of DNAzyme walker along a nano-substrate track, a DNA nanosphere probe (PNLD) is developed for the sensitive intracellular miR-21 imaging. Specifically, DNA nanosphere (called PN, single-molecule nano-track) is assembled from only one palindromic substrate, into which the locking strand-silenced DNAzymes (LD) are installed in a controlled manner. PNLD (made of PN and LD) can protect all DNA components against nuclease attack and maintain its structural integrity in serum solution over 24 h. Upon the activation by target miRNA, DNAzyme walker can move on the substrate scattered within PNLD (or on the surface) and between different PNLD objects and cleave many DNA substrates, generating an amplified signal. As a result, miR-21 can be detected down to 6.83 pM without the detectable interference from co-existing nontarget miRNAs. Moreover, PNLD system can accurately screen the different expression levels of miR-21 within the same type of cells and different types of cells, which is consistent with gold standard polymerase chain reaction (PCR) assay. Via changing the target recognition sequence, the PNLD system can be suitable for the intracellular imaging of miR-155, exhibiting the desirable universality. In addition, the DNAzyme walker-based PNLD system can be used to distinguish cancer cells from healthy cells, implying the potential application in cancer diagnosis and prognosis.

An old dog with new tricks: TFEB promotes syncytin expression and cell fusion in the human placenta.

In the human placenta, cell fusion is crucial for forming the syncytiotrophoblast, a multinucleated giant cell essential for maintaining pregnancy and ensuring fetal health. The formation of the syncytiotrophoblast is catalyzed by the evolutionarily modern fusogens syncytin-1 and syncytin-2. In this issue of Genes & Development, Esbin and colleagues (doi:10.1101/gad.351633.124) reveal a critical role for the transcription factor TFEB in the regulation of syncytin expression and the promotion of trophoblast fusion. Notably, TFEB's pro-fusion role operates independently of its well-known functions in lysosome biogenesis and autophagy, suggesting that TFEB has acquired additional functions to promote cell fusion in the human placenta.

Application of single-molecule analysis to singularity phenomenon of cells.

Single-molecule imaging in living cells is an effective tool for elucidating the mechanisms of cellular phenomena at the molecular level. However, the analysis was not designed for throughput and requires high expertise, preventing it from reaching large scale, which is necessary when searching for rare cells that induce singularity phenomena. To overcome this limitation, we have automated the imaging procedures by combining our own focusing device, artificial intelligence, and robotics. The apparatus, called automated in-cell single-molecule imaging system (AiSIS), achieves a throughput that is a hundred-fold higher than conventional manual imaging operations, enabling the analysis of molecular events by individual cells across a large population. Here, using AiSIS, we demonstrate the single-molecule imaging of molecular behaviors and reactions related to tau protein aggregation, which is considered a singularity phenomenon in neurological disorders. Changes in the dynamics and kinetics of molecular events were observed inside and on the basal membrane of cells after the induction of aggregation. Additionally, to detect rare cells based on the molecular behavior, we developed a method to identify the state of individual cells defined by the quantitative distribution of molecular mobility and clustering. Using this method, cellular variations in receptor behavior were shown to decrease following ligand stimulation. This cell state analysis based on large-scale single-molecule imaging by AiSIS will advance the study of molecular mechanisms causing singularity phenomena.

High-Speed Atomic Force Microscopy Reveals Fluctuations and Dimer Splitting of the N-Terminal Domain of GluA2 Ionotropic Glutamate Receptor-Auxiliary Subunit Complex.

α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid glutamate receptors (AMPARs) enable rapid excitatory synaptic transmission by localizing to the postsynaptic density of glutamatergic spines. AMPARs possess large extracellular N-terminal domains (NTDs), which are crucial for AMPAR clustering at synaptic sites. However, the dynamics of NTDs and the molecular mechanism governing their synaptic clustering remain elusive. Here, we employed high-speed atomic force microscopy (HS-AFM) to directly visualize the conformational dynamics of NTDs in the GluA2 subunit complexed with TARP γ2 in lipid environments. HS-AFM videos of GluA2-γ2 in the resting and activated/open states revealed fluctuations in NTD dimers. Conversely, in the desensitized/closed state, the two NTD dimers adopted a separated conformation with less fluctuation. Notably, we observed individual NTD dimers transitioning into monomers, with extended monomeric states in the activated/open state. Molecular dynamics simulations provided further support, confirming the energetic stability of the monomeric NTD states within lipids. This NTD-dimer splitting resulted in subunit exchange between the receptors and increased the number of interaction sites with synaptic protein neuronal pentraxin 1 (NP1). Moreover, our HS-AFM studies revealed that NP1 forms a ring-shaped octamer through N-terminal disulfide bonds and binds to the tip of the NTD. These findings suggest a molecular mechanism in which NP1, upon forming an octamer, is secreted into the synaptic region and binds to the tip of the GluA2 NTD, thereby bridging and clustering multiple AMPARs. Thus, our findings illuminate the critical role of NTD dynamics in the synaptic clustering of AMPARs and contribute valuable insights into the fundamental processes of synaptic transmission.

Dynamics and Evolutionary Conservation of B Complex Protein Recruitment During Spliceosome Activation.

Spliceosome assembly and catalytic site formation (called activation) involve dozens of protein and snRNA binding and unbinding events. The B-complex specific proteins Prp38, Snu23, and Spp381 have critical roles in stabilizing the spliceosome during conformational changes essential for activation. While these proteins are conserved, different mechanisms have been proposed for their recruitment to spliceosomes. To visualize recruitment directly, we used Colocalization Single Molecule Spectroscopy (CoSMoS) to study the dynamics of Prp38, Snu23, and Spp381 during splicing in real time. These proteins bind to and release from spliceosomes simultaneously and are likely associated with one another. We designate the complex of Prp38, Snu23, and Spp381 as the B Complex Protein (BCP) subcomplex. Under splicing conditions, the BCP associates with pre-mRNA after tri-snRNP binding. BCP release predominantly occurs after U4 snRNP dissociation and after NineTeen Complex (NTC) association. Under low concentrations of ATP, the BCP pre-associates with the tri-snRNP resulting in their simultaneous binding to pre-mRNA. Together, our results reveal that the BCP recruitment pathway to the spliceosome is conserved between S. cerevisiae and humans. Binding of the BCP to the tri-snRNP when ATP is limiting may result in formation of unproductive complexes that could be used to regulate splicing.

Tailored Fabrication of 3D Nanopores Made of Dielectric Oxides for Multiple Nanoscale Applications.

Solid-state nanopores are a key platform for single-molecule detection and analysis that allow engineering of their properties by controlling size, shape, and chemical functionalization. However, approaches relying on polymers have limits for what concerns hardness, robustness, durability, and refractive index. Nanopores made of oxides with high dielectric constant would overcome such limits and have the potential to extend the suitability of solid-state nanopores toward optoelectronic technologies. Here, we present a versatile method to fabricate three-dimensional nanopores made of different dielectric oxides with convex, straight, and concave shapes and demonstrate their functionality in a series of technologies and applications such as ionic nanochannels, ionic current rectification, memristors, and DNA sensing. Our experimental data are supported by numerical simulations that showcase the effect of different shapes and oxide materials. This approach toward robust and tunable solid-state nanopores can be extended to other 3D shapes and a variety of dielectrics.