A Novel Interaction Between RAD23A/B and Y-family DNA Polymerases.

The Y-family DNA polymerases - Pol ι, Pol η, Pol κ and Rev1 - are most well-known for their roles in the DNA damage tolerance pathway of translesion synthesis (TLS). They function to overcome replication barriers by bypassing DNA damage lesions that cannot be normally replicated, allowing replication forks to continue without stalling. In this work, we demonstrate a novel interaction between each Y-family polymerase and the nucleotide excision repair (NER) proteins, RAD23A and RAD23B. We initially focus on the interaction between RAD23A and Pol ι, and through a series of biochemical, cell-based, and structural assays, find that the RAD23A ubiquitin-binding domains (UBA1 and UBA2) interact with separate sites within the Pol ι catalytic domain. While this interaction involves the ubiquitin-binding cleft of UBA2, Pol ι interacts with a distinct surface on UBA1. We further find that mutating or deleting either UBA domain disrupts the RAD23A-Pol ι interaction, demonstrating that both interactions are necessary for stable binding. We also provide evidence that both RAD23 proteins interact with Pol ι in a similar manner, as well as with each of the Y-family polymerases. These results shed light on the interplay between the different functions of the RAD23 proteins and reveal novel binding partners for the Y-family TLS polymerases.

Conformational exchange at a C2H2 zinc-binding site facilitates redox sensing by the PML protein.

The promyelocytic leukemia protein, PML, plays a vital role in the cellular response to oxidative stress; however, the molecular mechanism of its action remains poorly understood. Here, we identify redox-sensitive sites of PML. A molecule of PML is cysteine-rich and contains three zinc-binding domains including RING, B-box1, and B-box2. Using in vitro assays, we have compared the sensitivity of the isolated RING and B-box1 domains and shown that B-box1 is more sensitive to oxidation. NMR studies of PML dynamics showed that one of the Zn-coordination sites within the B-box1 undergoes significant conformational exchange, revealing a hotspot for exposure of reactive cysteines. In agreement with the in vitro data, enhancement of the B-box1 Zn-coordination dynamics led to more efficient recruitment of PML into PML nuclear bodies in cells. Overall, our results suggest that the increased sensitivity of B-box1 to oxidative stress makes this domain an important redox-sensing component of PML.

Linear and Nonlinear Elastic Properties of Polystyrene-Based Nanocomposites with Allotropic Carbon Fillers and Binary Mixtures.

We report measurements of linear and nonlinear elastic properties of polystyrene-based nanocomposites with six types of nanofillers, including single and binary mixtures of allotropic carbon nanoparticles. Composite samples were fabricated by the same technology and contained the same filler concentration (5% wt.), which allowed for a direct comparison of their properties. It was shown that the most significant variations of linear and nonlinear elastic properties occur in different nanocomposites. In particular, the most pronounced enhancements of linear elastic moduli (in about 50%) obtained in tensile and flexural tests and in dynamic mechanical analysis were recorded in the sample filled with spherical fullerene nanoparticles. While the most profound rise of absolute values of nonlinear elastic moduli (tens of times) was obtained in the sample filled with the mixture of carbon nanotubes and graphene. The observed tendencies demonstrated the synergistic effect of fillers of different dimensionality on the elastic properties of nanocomposites.

The B-box1 domain of PML mediates SUMO E2-E3 complex formation through an atypical interaction with UBC9.

The small, ubiquitin-like modifier SUMO is covalently attached to substrates by the enzyme UBC9. SUMO conjugation of substrates often requires an E3 ligase, which ensures substrate specificity by simultaneously binding UBC9 and the substrate. E3 SUMO ligases commonly use a RING domain to engage UBC9. The Promyelocytic Leukemia protein (PML) has been implicated as a probable SUMO ligase. Although PML does contain a RING domain, which is expected to recruit UBC9, we demonstrate that PML RING does not bind UBC9 in vitro. Instead, we show that isolated PML B-box1 possesses UBC9-binding activity and map the B-box1 binding site on UBC9. This site also binds the upstream E1 enzyme that transfers SUMO to UBC9. The overlap of these two binding sites suggests that UBC9 cannot interact with its E1 and E3 partners simultaneously. Furthermore, we present a model of the PML dimer that supports the accessibility of B-box1 for UBC9 binding in the context of the full-length PML.

Machine Learning Assisted Classification of Cell Lines and Cell States on Quantitative Phase Images.

In this report, we present implementation and validation of machine-learning classifiers for distinguishing between cell types (HeLa, A549, 3T3 cell lines) and states (live, necrosis, apoptosis) based on the analysis of optical parameters derived from cell phase images. Validation of the developed classifier shows the accuracy for distinguishing between the three cell types of about 93% and between different cell states of the same cell line of about 89%. In the field test of the developed algorithm, we demonstrate successful evaluation of the temporal dynamics of relative amounts of live, apoptotic and necrotic cells after photodynamic treatment at different doses.

Relaxation dynamics of alkyl derivatives of fluorescein MitoFluo and C8-Fl in solutions with liposomes.

We present results of experimental and theoretical studies of excited state dynamics in two alkyl derivatives of fluorescein, MitoFluo and C8-Fl in solutions with liposomes. The liposomes DOPC and soybeanPC + 20% Cardiolipin (Azo-Cl), modelling cellular and inner mitochondrial membranes, respectively, were used in experiments. Both types of liposomes were shown to reduce significantly the fluorescence quantum yield as compared to that of pure fluorescein derivatives in solutions, while DOPC liposomes also caused a noticeable (ca 10 nm) red shift of fluorescence maximum. The study of fluorescence polarization decay has been carried out where important fluorescence parameters: polarization anisotropy, fluorescence lifetimes, and rotational diffusion times have been determined. It was shown that the isotropic fluorescence decay of C8-Fl in liposome containing solutions was single-exponential and the anisotropic decay was double-exponential for both types of lyposomes. In the case of MitoFluo both isotropic and anisotropic fluorescence decays were fitted satisfactory only with double-exponential functions. The interpretation of the experimental data obtained was supported by ab initio calculations of the structure and excitation properties of MitoFluo and C8-Fl in aqueous solution. The analysis of anisotropic fluorescence decay allowed for isolation of the contributions of fluorescein derivatives free in solution from those embedded in liposomes. Also, the experimental data suggest that MitoFluo interacts with liposomes more effectively than C8-Fl. Basing on the experimental and theoretical results obtained we conclude that free C8-Fl and MitoFluo molecules in solution were mostly in their dimer forms.

Application of digital holographic tomography in antitumor effect of cantharides complex on 4T1 breast cancer cells.

The study focuses on a methodology providing noninvasive monitoring and evaluation of the antitumor effect of traditional Chinese medicine, cantharides complex (canth), on 4T1 breast tumor cells. Digital holographic tomography (DHT) and developed data post-processing algorithms were used for quantitative estimation of changes in optical and morphological parameters of cells. We calculated and compared data on the refractive index, thickness, and projected area of 4T1 breast tumor cells in control untreated specimens and those treated with doxorubicin hydrochloride (DOX), canth, and their combinations. Post-treatment changes in cellular morphology recorded by DHT demonstrated that the two drugs led to noticeably different morphological changes in cells that can be presumably associated with different pathways of their death, apoptosis, or necrosis. The effect of combined treatment with these two drugs strongly depended on their relative concentrations and could lead to changes characteristic either for DOX or for canth; however, being more profound than those obtained when using each drug solely. The results obtained by DHT are in a good correspondence with commonly used cell viability analysis and immunofluorescent analysis of changes in cellular cytoskeleton.

Polystyrene-Based Nanocomposites with Different Fillers: Fabrication and Mechanical Properties.

The paper presents a comprehensive analysis of the elastic properties of polystyrene-based nanocomposites filled with different types of inclusions: small spherical particles (SiO2 and Al2O3), alumosilicates (montmorillonite, halloysite natural tubules and mica), and carbon nanofillers (carbon black and multi-walled carbon nanotubes). Block samples of composites with different filler concentrations were fabricated by melt technology, and their linear and non-linear elastic properties were studied. The introduction of more rigid particles led to a more profound increase in the elastic modulus of a composite, with the highest rise of about 80% obtained with carbon fillers. Non-linear elastic moduli of composites were shown to be more sensitive to addition of filler particles to the polymer matrix than linear ones. A non-linearity modulus ßs comprising the combination of linear and non-linear elastic moduli of a material demonstrated considerable changes correlating with those of the Young's modulus. The changes in non-linear elasticity of fabricated composites were compared with parameters of bulk non-linear strain waves propagating in them. Variations of wave velocity and decay decrement correlated with the observed enhancement of materials' non-linearity.

Protein kinase A, which regulates intracellular transport, forms complexes with molecular motors on organelles.

Major signaling cascades have been shown to play a role in the regulation of intracellular organelle transport . Aggregation and dispersion of pigment granules in melanophores are regulated by the second messenger cAMP through the protein kinase A (PKA) signaling pathway ; however, the exact mechanisms of this regulation are poorly understood. To study the role of signaling molecules in the regulation of pigment transport in melanophores, we have asked the question whether the components of the cAMP-signaling pathway are bound to pigment granules and whether they interact with molecular motors to regulate the granule movement throughout the cytoplasm. We found that purified pigment granules contain PKA and scaffolding proteins and that PKA associates with pigment granules in cells. Furthermore, we found that the PKA regulatory subunit forms two separate complexes, one with cytoplasmic dynein ("aggregation complex") and one with kinesin II and myosin V ("dispersion complex"), and that the removal of PKA from granules causes dissociation of dynein and disruption of dynein-dependent pigment aggregation. We conclude that cytoplasmic organelles contain protein complexes that include motor proteins and signaling molecules involved in different components of intracellular transport. We propose to call such complexes 'regulated motor units' (RMU).