Structural insights into the multifunctionality of rabies virus P3 protein.

Viruses form extensive interfaces with host proteins to modulate the biology of the infected cell, frequently via multifunctional viral proteins. These proteins are conventionally considered as assemblies of independent functional modules, where the presence or absence of modules determines the overall composite phenotype. However, this model cannot account for functions observed in specific viral proteins. For example, rabies virus (RABV) P3 protein is a truncated form of the pathogenicity factor P protein, but displays a unique phenotype with functions not seen in longer isoforms, indicating that changes beyond the simple complement of functional modules define the functions of P3. Here, we report structural and cellular analyses of P3 derived from the pathogenic RABV strain Nishigahara (Nish) and an attenuated derivative strain (Ni-CE). We identify a network of intraprotomer interactions involving the globular C-terminal domain and intrinsically disordered regions (IDRs) of the N-terminal region that characterize the fully functional Nish P3 to fluctuate between open and closed states, whereas the defective Ni-CE P3 is predominantly open. This conformational difference appears to be due to the single mutation N226H in Ni-CE P3. We find that Nish P3, but not Ni-CE or N226H P3, undergoes liquid-liquid phase separation and this property correlates with the capacity of P3 to interact with different cellular membrane-less organelles, including those associated with immune evasion and pathogenesis. Our analyses propose that discrete functions of a critical multifunctional viral protein depend on the conformational arrangements of distant individual domains and IDRs, in addition to their independent functions.

Molecular Basis of Functional Effects of Phosphorylation of the C-Terminal Domain of the Rabies Virus P Protein.

The rabies virus (RABV) phosphoprotein (P protein) is expressed as several isoforms, which differ in nucleocytoplasmic localization and microtubule (MT) association, mediated by several sequences, including nuclear localization (NLS) and export (NES) sequences. This appears to underpin a functional diversity enabling multiple functions in viral replication and modulation of host biology. Mechanisms regulating trafficking are poorly defined, but phosphorylation by protein kinase C (PKC) in the P protein C-terminal domain (PCTD) regulates nuclear trafficking, mediated by PCTD-localized NLS/NES sequences, indicating that phosphorylation contributes to functional diversity. The molecular mechanism underlying the effects of PKC, and potential roles in regulating other host-cell interactions are unresolved. Here, we assess effects of phosphorylation on the P3 isoform, which differs from longer isoforms through an ability to localize to the nucleus and associate with MTs, which are associated with antagonism of interferon (IFN) signaling. We find that phosphomimetic mutation of the PKC site S210 inhibits nuclear accumulation and MT association/bundling. Structural analysis indicated that phosphomimetic mutation induces no significant structural change to the NLS/NES but results in the side chain of N226 switching its interactions from E228, within the NES, to E210. Intriguingly, N226 is the sole substituted residue between the PCTD of the pathogenic IFN-resistant RABV strain Nishigahara and a derivative attenuated IFN-sensitive strain Ni-CE, inhibiting P3 nuclear localization and MT association. Thus, S210 phosphorylation appears to impact on N226/E228 to regulate P protein localization, with N226 mutation in Ni-CE mimicking a constitutively phosphorylated state resulting in IFN sensitivity and attenuation. IMPORTANCE Rabies virus P protein is a multifunctional protein with critical roles in replication and manipulation of host-cell processes, including subversion of immunity. This functional diversity involves interactions of several P protein isoforms with the cell nucleus and microtubules. Previous studies showed that phosphorylation of the P protein C-terminal domain (PCTD) at S210, near nuclear trafficking sequences, regulates nucleocytoplasmic localization, indicating key roles in functional diversity. The molecular mechanisms of this regulation have remained unknown. Here, we show that phosphomimetic mutation of S210 regulates nuclear localization and MT association. This regulation does not appear to result from disrupted PCTD structure, but rather from a switch of specific side chain interactions of N226. Intriguingly, N226 was previously implicated in P protein nuclear localization/MT association, immune evasion, and RABV pathogenesis, through undefined mechanisms. Our data indicate that the S210-N226 interface is a key regulator of virus-host interactions, which is significant for pathogenesis.

Lyssavirus P Protein Isoforms Diverge Significantly in Subcellular Interactions Underlying Mechanisms of Interferon Antagonism.

Viral hijacking of microtubule (MT)-dependent transport is well understood, but several viruses also express discrete MT-associated proteins (vMAPs), potentially to modulate MT-dependent processes in the host cell. Specific roles for vMAP-MT interactions include subversion of antiviral responses by P3, an isoform of the P protein of rabies virus (RABV; genus Lyssavirus), which mediates MT-dependent antagonism of interferon (IFN)-dependent signal transducers and activators of transcription 1 (STAT1) signaling. P3 also undergoes nucleocytoplasmic trafficking and inhibits STAT1-DNA binding, indicative of intranuclear roles in a multipronged antagonistic strategy. MT association/STAT1 antagonist functions of P3 correlate with pathogenesis, indicating potential as therapeutic targets. However, key questions remain, including whether other P protein isoforms interact with MTs, the relationship of these interactions with pathogenesis, and the extent of conservation of P3-MT interactions between diverse pathogenic lyssaviruses. Using super-resolution microscopy, live-cell imaging, and immune signaling analyses, we find that multiple P protein isoforms associate with MTs and that association correlates with pathogenesis. Furthermore, P3 proteins from different lyssaviruses exhibit variation in intracellular localization phenotypes that are associated with STAT1 antagonist function, whereby P3-MT association is conserved among lyssaviruses of phylogroup I but not phylogroup II, while nucleocytoplasmic localization varies between P3 proteins of the same phylogroup within both phylogroup I and II. Nevertheless, the divergent P3 proteins retain significant IFN antagonist function, indicative of adaptation to favor different inhibitory mechanisms, with MT interaction important to phylogroup I viruses. IMPORTANCE Lyssaviruses, including rabies virus, cause rabies, a progressive encephalomyelitis that is almost invariably fatal. There are no effective antivirals for symptomatic infection, and effective application of current vaccines is limited in areas of endemicity, such that rabies causes ~59,000 deaths per year. Viral subversion of host cell functions, including antiviral immunity, is critical to disease, and isoforms of the lyssavirus P protein are central to the virus-host interface underpinning immune evasion. Here, we show that specific cellular interactions of P protein isoforms involved in immune evasion vary significantly between different lyssaviruses, indicative of distinct strategies to evade immune responses. These findings highlight the diversity of the virus-host interface, an important consideration in the development of pan-lyssavirus therapeutic approaches.

Nanoscale characterization of drug-induced microtubule filament dysfunction using super-resolution microscopy.

BACKGROUND: The integrity of microtubule filament networks is essential for the roles in diverse cellular functions, and disruption of its structure or dynamics has been explored as a therapeutic approach to tackle diseases such as cancer. Microtubule-interacting drugs, sometimes referred to as antimitotics, are used in cancer therapy to target and disrupt microtubules. However, due to associated side effects on healthy cells, there is a need to develop safer drug regimens that still retain clinical efficacy. Currently, many questions remain open regarding the extent of effects on cellular physiology of microtubule-interacting drugs at clinically relevant and low doses. Here, we use super-resolution microscopies (single-molecule localization and optical fluctuation based) to reveal the initial microtubule dysfunctions caused by nanomolar concentrations of colcemid. RESULTS: We identify previously undetected microtubule (MT) damage caused by clinically relevant doses of colcemid. Short exposure to 30-80 nM colcemid results in aberrant microtubule curvature, with a trend of increased curvature associated to increased doses, and curvatures greater than 2 rad/µm, a value associated with MT breakage. Microtubule fragmentation was detected upon treatment with ≥ 100 nM colcemid. Remarkably, lower doses (< 20 nM after 5 h) led to subtle but significant microtubule architecture remodelling characterized by increased curvature and suppression of microtubule dynamics. CONCLUSIONS: Our results support the emerging hypothesis that microtubule-interacting drugs induce non-mitotic effects in cells, and establish a multi-modal imaging assay for detecting and measuring nanoscale microtubule dysfunction. The sub-diffraction visualization of these less severe precursor perturbations compared to the established antimitotic effects of microtubule-interacting drugs offers potential for improved understanding and design of anticancer agents.

Self-assembled, optically-active {naphthalene diimide}U{cucurbit[8]uril} ensembles in an aqueous environment.

Naphthalene diimides (NDIs) are shown to arrange spontaneously co-facially with cucurbit[8]uril (CB[8]) in an aqueous environment through purely non-covalent interactions. The resultant 2 : 2 supramolecular complex of NDI and CB[8] is highly fluorescent (>30 times more than the constituent NDIs) due to the formation of NDI-NDI excimers within the supramolecular complex.

Wavelength-Dependent Fluorescent Immunosensors via Incorporation of Polarity Indicators near the Binding Interface of Antibody Fragments.

Herein, we describe a fluorescent immunosensor designed by incorporating an unnatural amino acid fluorophore into the binding site of an EGFR-specific antibody fragment, resulting in quantifiable EGFR-dependent changes in peak fluorescence emission wavelength. To date, immunosensor design strategies have relied on binding-induced changes in fluorescence intensity that are prone to excitation source fluctuations and sample-dependent noise. In this study, we used a rational design approach to incorporate a polarity indicator (Anap) into specific positions of an anti-EGFR single chain antibody to generate an emission wavelength-dependent immunosensor. We found that when incorporated within the topological neighborhood of the antigen binding interface, the Anap emission wavelength is blue-shifted by EGFR-binding in a titratable manner, up to 20 nm, with nanomolar detection limits. This approach could be applicable to other antibody/antigen combinations for integration into a wide range of assay platforms (including homogeneous, solid-phase assay, or microfluidic assays) for one-step protein quantification.

Harnessing Brightness in Naphthalene Diimides.

The development of brightly emissive compounds is of great research and commercial interest, with established and emerging applications across chemistry, biology, physics, medicine and engineering. Among the many types of molecules available, naphthalene diimides have been widely used for both fundamental photophysical studies and in practical applications that utilise fluorescence as an information readout. The monomeric naphthalene diimide is weakly fluorescent, however through various methods of core-derivatisation, it can be developed to be highly fluorescent and further functionalised to add utility. In this review, we highlight recent advances made in naphthalene diimide chemistry that have led to development of molecules with improved optical properties, and the design strategies utilised to produce bright fluorescence emission as small molecules or in supramolecular architectures.

A Multifunctional, Charge-Neutral, Chiral Octahedral M12 L12 Cage.

A chiral, octahedral M12 L12 cage, which is charge neutral and contains an internal void of about 2000 Å3 , is reported. The cage was synthesised as an enantiopure complex by virtue of amino-acid-based dicarboxylate ligands, which assemble around copper paddlewheels at the vertices of the octahedron. The cage persists in solution with retention of the fluorescence properties of the parent acid. The solid-state structure contains large pores both within and between the cages, and displays permanent porosity for the sorption of gases with retention of crystallinity. Initial tests show some enantioselectivity of the cage towards guests in solution.

Topologically Controlled Synthesis of Reversible Macrocyclic Compounds in Microemulsions.

A novel and simple approach was used to synthesize a reversible macrocyclic compound. This approach is based on capturing molecules with a photodynamic covalent bond inside microemulsions as nanoreactors and exposing them to UV radiation to facilitate a stereoselective reaction to form cyclic compounds quantitatively and selectively. The size of the microemulsion droplets used played a crucial role in synthesizing the reversible macrocyclic compounds. The formed macrocyclic compound was able to revert to monomers under UV irradiation of a shorter wavelength.

Tuning the structure, thermal stability and rheological properties of liquid crystal phases via the addition of silica nanoparticles.

The effects of adding silica nanoparticles of varying size and surface chemistry to a liquid crystal system were analysed using small-angle scattering and polarising light microscopy, with varying temperature and applied shear. It was found that nanoparticles aggregate at domain boundaries, causing a reduction in average liquid crystal domain size. These particles can inhibit phase transitions that occur at specific temperatures, ascribed to aggregates posing a kinetic barrier to rearrangement required for phase transitions. Nanoparticles can also promote the existence of specific phases, such as a deswollen hexagonal mesophase for the system studied here, suggested to be caused by silica aggregates 'templating' new phases. These findings have important implications for the application of such systems in biotechnology, and particularly the ability to completely inhibit a phase change at low temperature suggests the potential for mechanistic insight into new methods of cryopreservation.

1D Self-Assembly and Ice Recrystallization Inhibition Activity of Antifreeze Glycopeptide-Functionalized Perylene Bisimides.

Antifreeze glycoproteins (AFGPs) are polymeric natural products that have drawn considerable interest in diverse research fields owing to their potent ice recrystallization inhibition (IRI) activity. Self-assembled materials have emerged as a promising class of biomimetic ice growth inhibitor, yet the development of AFGP-based supramolecular materials that emulate the aggregative behavior of AFGPs have not yet been reported. This work reports the first example of the 1D self-assembly and IRI activity of AFGP-functionalized perylene bisimides (AFGP-PBIs). Glycopeptide-functionalized PBIs underwent 1D self-assembly in water and showed modest IRI activity, which could be tuned through substitution of the PBI core. This work presents essential proof-of-principle for the development of novel IRIs as potential supramolecular cryoprotectants and glycoprotein mimics.

Structural and rheological changes of lamellar liquid crystals as a result of compositional changes and added silica nanoparticles.

Lamellar liquid crystals comprising oil, water and surfactant(s) were formulated and analysed in order to examine how these materials responded to the inclusion of inorganic nanoparticles, in terms of their structural and rheological characteristics. Lamellar phases were formed from mixtures of water, para-xylene and Triton X-100, and analysis was performed via small-angle neutron scattering (SANS), polarising light microscopy (PLM), and amplitude and viscosity sweeps. The partial replacement of Triton X-100 with oleic acid appeared to cause an increase in bilayer thickness, attributed to less efficient packing of the different molecules. Addition of oleic acid also appeared to cause both a loss in lamellar repeat ordering, attributed to heterogeneity of the bilayers, and a rise in long range order, potentially caused by the stiffer bilayers. Adding silica nanoparticles of different size and surface chemistry caused a stiffening of the samples at the expense of a longer-range lamellar repeat order. This strengthening is attributed to aggregation at the domain boundaries, and it was found that hydrophobic particles tended to form stronger aggregates while for larger particles (20 nm as opposed to 10 nm) aggregation was apparently reversible. These results give a more comprehensive understanding of how to reliably control the structural and rheological properties of lamellar liquid crystals, and emphasise the importance of the size and surface chemistry of any inclusions, for applications in cosmetics, drug delivery, and microfluidics.

Controlled Growth of Monocrystalline Organo-Lead Halide Perovskite and Its Application in Photonic Devices.

Organo-lead halide perovskites (OHPs) have recently emerged as a new class of exceptional optoelectronic materials, which may find use in many applications, including solar cells, light emitting diodes, and photodetectors. More complex applications, such as lasers and electro-optic modulators, require the use of monocrystalline perovskite materials to reach their ultimate performance levels. Conventional methods for forming single crystals of OHPs like methylammonium lead bromide (MAPbBr3 ) afford limited control over the product morphology, rendering the assembly of defined microcavity nanostructures difficult. We overcame this by synthesizing for the first time (MA)[PbBr3 ]⋅DMF (1), and demonstrating its facile transformation into monocrystalline MAPbBr3 microplatelets. The MAPbBr3 microplatelets were tailored into waveguide based photonic devices, of which an ultra-low propagation loss of 0.04 dB µm-1 for a propagation distance of 100 µm was demonstrated. An efficient active electro-optical modulator (AEOM) consisting of a MAPbBr3 non-linear arc waveguide was demonstrated, exhibiting a 98.4 % PL intensity modulation with an external voltage of 45 V. This novel synthetic approach, as well as the demonstration of effective waveguiding, will pave the way for developing a wide range of photonic devices based on organo-lead halide perovskites.

Local determination of thin liquid film profiles using colour interferometry.

We explore theoretically the interference of white light between two interfaces as a function of the optical conditions, using separately: a) idealised conditions where the light is composed of three discrete wavelengths; b) a more typically experimentally realisable case where light comprises a sum of three Gaussian wavelength distributions; and c) unfiltered white light from a broadband source comprising a broad distribution of wavelengths. It is demonstrated that the latter case is not only optically simple to arrange, but also provides unambiguous absolute separation information over the range 0-1µm --a useful range in studies of cell adhesion, thin liquid films and lubrication-- when coupled to detection using a typical colour camera. The utility of this technique is verified experimentally by exploring the air film between a cylinder and surface, as well as arbitrary liquid films beneath air bubbles that are interacting with solid surfaces.

Unexpected photoluminescence of fluorinated naphthalene diimides.

Two new amino core-substituted naphthalene diimides (cNDIs) bearing fluorinated side chains have been synthesised. Steady-state and time-resolved fluorescence spectroscopy reveals unprecedented optical properties for the cNDIs with high quantum yields of ∼0.8 and fluorescence lifetimes of ∼13 ns in a range of solvents. These properties are apparent at the level of single molecules, where the compounds also show exceptional photostability under pulsed-laser excitation. Photon emission is remarkably consistent with very few long timescale (millisecond or longer) interruptions with molecules regularly undergoing >10(7) cycles of excitation and emission. Intermittencies owing to triplet-state formation occur on a sub-millisecond timescale with a low yield of 1-2%, indicating that the presence of the fluorine atoms does not lead to a significant triplet yield through the heavy-atom effect. These properties make the compounds excellent candidates for single-molecule labelling applications.

Tuning the Photoluminescence Anisotropy of Semiconductor Nanocrystals.

Semiconductor nanocrystals are promising optoelectronic materials. Understanding their anisotropic photoluminescence is fundamental for developing quantum-dot-based devices such as light-emitting diodes, solar cells, and polarized single-photon sources. In this study, we experimentally and theoretically investigate the photoluminescence anisotropy of CdSe semiconductor nanocrystals with various shapes, including plates, rods, and spheres, with either wurtzite or zincblende structures. We use defocused wide-field microscopy to visualize the emission dipole orientation and find that spheres, rods, and plates exhibit the optical properties of 2D, 1D, and 2D emission dipoles, respectively. We rationalize the seemingly counterintuitive observation that despite having similar aspect ratios (width/length), rods and long nanoplatelets exhibit different defocused emission patterns by considering valence band structures calculated using multiband effective mass theory and the dielectric effect. The principles are extended to provide general relationships that can be used to tune the emission dipole orientation for different materials, crystalline structures, and shapes.

Live-Cell SOFI Correlation with SMLM and AFM Imaging.

Standard optical imaging is diffraction-limited and lacks the resolving power to visualize many of the organelles and proteins found within the cell. The advent of super-resolution techniques overcame this barrier, enabling observation of subcellular structures down to tens of nanometers in size; however these techniques require or are typically applied to fixed samples. This raises the question of how well a fixed-cell image represents the system prior to fixation. Here we present the addition of live-cell Super-Resolution Optical Fluctuation Imaging (SOFI) to a previously reported correlative process using Single Molecule Localization Microscopy (SMLM) and Atomic Force Microscopy (AFM). SOFI was used with fluorescent proteins and low laser power to observe cellular ultrastructure in live COS-7 cells. SOFI-SMLM-AFM of microtubules showed minimal changes to the microtubule network in the 20 min between live-cell SOFI and fixation. Microtubule diameters were also analyzed through all microscopies; SOFI found diameters of 249 ± 68 nm and SMLM was 71 ± 33 nm. AFM height measurements found microtubules to protrude 26 ± 13 nm above the surrounding cellular material. The correlation of SMLM and AFM was extended to two-color SMLM to image both microtubules and actin. Two target SOFI was performed with various fluorescent protein combinations. rsGreen1-rsKAME, rsGreen1-Dronpa, and ffDronpaF-rsKAME fluorescent protein combinations were determined to be suitable for two target SOFI imaging. This correlative application of super-resolution live-cell and fixed-cell imaging revealed minimal artifacts created for the imaged target structures through the sample preparation procedure and emphasizes the power of correlative microscopy.

Design of Polarity-Dependent Immunosensors Based on the Structural Analysis of Engineered Antibodies.

"Reagentless" immunosensors are emerging to address the challenge of practical and sensitive detection of important biomarkers in real biological samples without the need for multistep assays and user intervention, with applications ranging from research tools to point-of-care diagnostics. Selective target binding to an affinity reagent is detected and reported in one step without the need for washing or additional reporters. In this study, we used a structure-guided approach to identify a mutation site in an antibody fragment for the polarity-dependent fluorophore, Anap, such that upon binding of the protein target cardiac troponin I, the Anap-labeled antibody would produce a detectable and dose-dependent shift in emission wavelength. We observed a significant emission wavelength shift of the Anap-labeled anti-cTnI mutant, with a blue shift of up to 37 nm, upon binding to the cTnI protein. Key differences in the resulting emission spectra between target peptides in comparison to whole proteins were also found; however, the affinity and binding characteristics remained unaffected when compared to the wild-type antibody. We also highlighted the potential flexibility of the approach by incorporating a near-infrared dye, IRDye800CW, into the same mutation site, which also resulted in a dose-dependent wavelength shift upon target incubation. These reagents can be used in experiments and devices to create simpler and more efficient biosensors across a range of research, medical laboratory, and point-of-care platforms.

Pediatric glioma histone H3.3 K27M/G34R mutations drive abnormalities in PML nuclear bodies.

BACKGROUND: Point mutations in histone variant H3.3 (H3.3K27M, H3.3G34R) and the H3.3-specific ATRX/DAXX chaperone complex are frequent events in pediatric gliomas. These H3.3 point mutations affect many chromatin modifications but the exact oncogenic mechanisms are currently unclear. Histone H3.3 is known to localize to nuclear compartments known as promyelocytic leukemia (PML) nuclear bodies, which are frequently mutated and confirmed as oncogenic drivers in acute promyelocytic leukemia. RESULTS: We find that the pediatric glioma-associated H3.3 point mutations disrupt the formation of PML nuclear bodies and this prevents differentiation down glial lineages. Similar to leukemias driven by PML mutations, H3.3-mutated glioma cells are sensitive to drugs that target PML bodies. We also find that point mutations in IDH1/2-which are common events in adult gliomas and myeloid leukemias-also disrupt the formation of PML bodies. CONCLUSIONS: We identify PML as a contributor to oncogenesis in a subset of gliomas and show that targeting PML bodies is effective in treating these H3.3-mutated pediatric gliomas.

3D Single Molecule Super-Resolution Microscopy of Whole Nuclear Lamina.

Single molecule (SM) super-resolution microscopies bypass the diffraction limit of conventional optical techniques and provide excellent spatial resolutions in the tens of nanometers without overly complex microscope hardware. SM imaging using optical astigmatism is an efficient strategy for visualizing subcellular features in 3D with a z-range of up to ∼1 µm per acquisition. This approach however, places high demands on fluorophore brightness and photoswitching resilience meaning that imaging entire cell volumes in 3D using SM super-resolution remains challenging. Here we employ SM astigmatism together with multiplane acquisition to visualize the whole nuclear lamina of COS-7 and T cells in 3D. Nuclear lamina provides structural support to the nuclear envelope and participates in vital nuclear functions including internuclear transport, chromatin organization and gene regulation. Its position at the periphery of the nucleus provides a visible reference of the nuclear boundary and can be used to quantify the spatial distribution of intranuclear components such as histone modifications and transcription factors. We found Alexa Fluor 647, a popular photoswitchable fluorophore, remained viable for over an hour of continuous high laser power exposure, and provided sufficient brightness detectable up to 8 µm deep into a cell, allowing us to capture the entire nuclear lamina in 3D. Our approach provides sufficient super-resolution detail of nuclear lamina morphology to enable quantification of overall nuclear dimensions and local membrane features.