Development of New Models of Oral Mucosa to Investigate the Impact of the Structure of Transmembrane Mucin-1 on the Mucosal Pellicle Formation and Its Physicochemical Properties.

The mucosal pellicle (MP) is a biological film protecting the oral mucosa. It is composed of bounded salivary proteins and transmembrane mucin MUC1 expressed by oral epithelial cells. Previous research indicates that MUC1 expression enhances the binding of the main salivary protein forming the MP, MUC5B. This study investigated the influence of MUC1 structure on MP formation. A TR146 cell line, which does not express MUC1 natively, was stably transfected with genes coding for three MUC1 isoforms differing in the structure of the two main extracellular domains: the VNTR domain, exhibiting a variable number of tandem repeats, and the SEA domain, maintaining the two bound subunits of MUC1. Semi-quantification of MUC1 using dot blot chemiluminescence showed comparable expression levels in all transfected cell lines. Semi-quantification of MUC5B by immunostaining after incubation with saliva revealed that MUC1 expression significantly increased MUC5B adsorption. Neither the VNTR domain nor the SEA domain was influenced MUC5B anchoring, suggesting the key role of the MUC1 N-terminal domain. AFM-IR nanospectroscopy revealed discernible shifts indicative of changes in the chemical properties at the cell surface due to the expression of the MUC1 isoform. Furthermore, the observed chemical shifts suggest the involvement of hydrophobic effects in the interaction between MUC1 and salivary proteins.

Structural analysis of the Candida albicans mitochondrial DNA maintenance factor Gcf1p reveals a dynamic DNA-bridging mechanism.

The compaction of mitochondrial DNA (mtDNA) is regulated by architectural HMG-box proteins whose limited cross-species similarity suggests diverse underlying mechanisms. Viability of Candida albicans, a human antibiotic-resistant mucosal pathogen, is compromised by altering mtDNA regulators. Among them, there is the mtDNA maintenance factor Gcf1p, which differs in sequence and structure from its human and Saccharomyces cerevisiae counterparts, TFAM and Abf2p. Our crystallographic, biophysical, biochemical and computational analysis showed that Gcf1p forms dynamic protein/DNA multimers by a combined action of an N-terminal unstructured tail and a long helix. Furthermore, an HMG-box domain canonically binds the minor groove and dramatically bends the DNA while, unprecedentedly, a second HMG-box binds the major groove without imposing distortions. This architectural protein thus uses its multiple domains to bridge co-aligned DNA segments without altering the DNA topology, revealing a new mechanism of mtDNA condensation.

Impact of Increased Sonication-Induced Dispersion of Sepiolite on Its Interaction with Biological Macromolecules and Toxicity/Proliferation in Human Cells.

Sepiolite is a natural clay silicate that is widely used, including biomedical applications; notably sepiolite shows promising features for the transfer of biological macromolecules into mammalian cells. However, before its use, such an approach should address the efficiency of binding to biological macromolecules and cell toxicity. Because sepiolite spontaneously forms aggregates, its disaggregation can represent an important challenge for improving the suspension performance and the assembly with biological species. However, this can also influence the toxicity of sepiolite in mammalian cells. Here, a very pure commercial sepiolite (Pangel S9), which is present as a partially defibrillated clay mineral, is used to study the consequences of additional deagglomeration/dispersion through sonication. We analyzed the impact of extra sonication on the dispersion of sepiolite aggregates. Factors such as sonication time, sonicator power, and temperature are taken into account. With increasing sonication time, a decrease in aggregation is observed, as well as a decrease in the length of the nanofibers monitored by atomic force microscopy. Changes in the temperature and pH of the solution are also observed during the sonication process. Moreover, although the adsorption capacity of bovine serum albumin (BSA) protein on sepiolite is increased with sonication time, the DNA adsorption efficiency remains unaffected. Finally, sonication of sepiolite decreases the hemolytic activity in blood cells and the toxicity in two different human cell lines. These data show that extra sonication of deagglomerated sepiolite can further favor its interaction with some biomacromolecules (e.g., BSA), and, in parallel, decrease sepiolite toxicity in mammalian cells. Therefore, sonication represents an alluring procedure for future biomedical applications of sepiolite, even when using commercial defibrillated particles.

Infrared nanospectroscopic imaging of DNA molecules on mica surface.

Significant efforts have been done in last two decades to develop nanoscale spectroscopy techniques owning to their great potential for single-molecule structural detection and in addition, to resolve open questions in heterogeneous biological systems, such as protein-DNA complexes. Applying IR-AFM technique has become a powerful leverage for obtaining simultaneous absorption spectra with a nanoscale spatial resolution for studied proteins, however the AFM-IR investigation of DNA molecules on surface, as a benchmark for a nucleoprotein complexes nanocharacterization, has remained elusive. Herein, we demonstrate methodological approach for acquisition of AFM-IR mapping modalities with corresponding absorption spectra based on two different DNA deposition protocols on spermidine and Ni2+ pretreated mica surface. The nanoscale IR absorbance of distinctly formed DNA morphologies on mica are demonstrated through series of AFM-IR absorption maps with corresponding IR spectrum. Our results thus demonstrate the sensitivity of AFM-IR nanospectroscopy for a nucleic acid research with an open potential to be employed in further investigation of nucleoprotein complexes.

Core-shell polygalacturonate magnetic iron oxide nanoparticles: Synthesis, characterization, and functionalities.

This work aims to synthesize polygalacturonate-based magnetic iron oxide nanoparticles (INP-polyGalA). The synthesis consists of the diffusion of both Fe2+ and Fe3+ at a molar ratio of 1:2 through polyGalA solution followed by the addition of an alkaline solution. To form individual nanoparticle materials, the polyGalA concentration needs to be below its overlapping concentration (C*). The synthesized materials (INP-polyGalA) contain about 45% of organic compound (polyGalA), and they have an average particle size ranging from 10 to 50 nm as estimated by several techniques (DLS, TEM and AFM) and their surfaces are negatively charged in pH range 2 to 7. The synthesized NPs showed magnetic characteristics, thanks to the formation of magnetite (Fe3O4) as confirmed by X-ray diffractions (XRD). Moreover, AFM combined with Infra-red mapping allowed us to conclude that polyGalA is located in the core of the nanoparticles but also on their surfaces. More specially, both carboxylate (COO-) and carboxylic (COOH) groups of polyGalA are observed on the NPs surfaces. The presence of such functional groups allowed the synthesized material to (i) bind through the electrostatic interactions methylene blue (MB) which may have a great potential for r pollution control or (ii) to form hydrogel beads (ionotropic gelation) by using calcium as a crosslinking agent which can be used to encapsulate active molecules and target their release by using an external stimulus (magnetic field).

RNA Nanostructure Molecular Imaging.

Atomic force and transmission electron microscopies (AFM/TEM) are powerful tools to analyze RNA-based nanostructures. While cryo-TEM analysis allows the determination of near-atomic resolution structures of large RNA complexes, this chapter intends to present how RNA nanostructures can be analyzed at room temperature on surfaces. Indeed, TEM and AFM analyses permit the conformation of a large population of individual molecular structures to be observed, providing a statistical basis for the variability of these nanostructures within the population. Nevertheless, if double-stranded DNA molecular imaging has been described extensively, only a few investigations of single-stranded DNA and RNA filaments have been conducted so far. Indeed, technique for spreading and adsorption of ss-molecules on AFM surfaces or TEM grids is a crucial step to avoid disturbing RNA conformation on the surface. In this chapter, we present a specific method to analyze RNA assemblies and RNA-protein complexes for molecular microscopies.

Recombinases and Related Proteins in the Context of Homologous Recombination Analyzed by Molecular Microscopy.

Transmission electron microscopy (TEM) and atomic force microscopy (AFM) are powerful tools to study the behavior of various actors in homologous recombination including molecular motors such as recombinases and helicases/translocases. Here we present specific approaches developed in terms of sample preparation and imaging methods to contribute to the understanding of homologous recombination process and its regulation focusing on the interplay between recombinases and other related proteins such as mediators or antirecombinase actors.Homologous recombination (HR) is a high-fidelity DNA repair pathway since it uses a homologous DNA as template. Recombinases such as RecA in bacteria, RadA in archaea, and Rad51 in eukaryotes are key proteins in the HR pathway: HR is initiated with formation of an ssDNA overhang on which recombinases polymerize and form a dynamic active nucleoprotein filament able to search for homology and to exchange DNA strand in an ATP-dependent manner. We provide practical methods to analyze presynaptic filament formation on ssDNA, its composition and regulation in presence of mediator partners, antirecombinase activity of translocase, and chromatin remodeling events.

The role of the N-terminal domain of human apurinic/apyrimidinic endonuclease 1, APE1, in DNA glycosylase stimulation.

The base excision repair (BER) pathway consists of sequential action of DNA glycosylase and apurinic/apyrimidinic (AP) endonuclease necessary to remove a damaged base and generate a single-strand break in duplex DNA. Human multifunctional AP endonuclease 1 (APE1, a.k.a. APEX1, HAP-1, or Ref-1) plays essential roles in BER by acting downstream of DNA glycosylases to incise a DNA duplex at AP sites and remove 3'-blocking sugar moieties at DNA strand breaks. Human 8-oxoguanine-DNA glycosylase (OGG1), methyl-CpG-binding domain 4 (MBD4, a.k.a. MED1), and alkyl-N-purine-DNA glycosylase (ANPG, a.k.a. Aag or MPG) excise a variety of damaged bases from DNA. Here we demonstrated that the redox-deficient truncated APE1 protein lacking the first N-terminal 61 amino acid residues (APE1-NΔ61) cannot stimulate DNA glycosylase activities of OGG1, MBD4, and ANPG on duplex DNA substrates. Electron microscopy imaging of APE1-DNA complexes revealed oligomerization of APE1 along the DNA duplex and APE1-mediated DNA bridging followed by DNA aggregation. APE1 polymerizes on both undamaged and damaged DNA in cooperative mode. Association of APE1 with undamaged DNA may enable scanning for damage; however, this event reduces effective concentration of the enzyme and subsequently decreases APE1-catalyzed cleavage rates on long DNA substrates. We propose that APE1 oligomers on DNA induce helix distortions thereby enhancing molecular recognition of DNA lesions by DNA glycosylases via a conformational proofreading/selection mechanism. Thus, APE1-mediated structural deformations of the DNA helix stabilize the enzyme-substrate complex and promote dissociation of human DNA glycosylases from the AP site with a subsequent increase in their turnover rate. SIGNIFICANCE STATEMENT: The major human apurinic/apyrimidinic (AP) endonuclease, APE1, stimulates DNA glycosylases by increasing their turnover rate on duplex DNA substrates. At present, the mechanism of the stimulation remains unclear. We report that the redox domain of APE1 is necessary for the active mode of stimulation of DNA glycosylases. Electron microscopy revealed that full-length APE1 oligomerizes on DNA possibly via cooperative binding to DNA. Consequently, APE1 shows DNA length dependence with preferential repair of short DNA duplexes. We propose that APE1-catalyzed oligomerization along DNA induces helix distortions, which in turn enable conformational selection and stimulation of DNA glycosylases. This new biochemical property of APE1 sheds light on the mechanism of redox function and its role in DNA repair.

Sepiolite as a New Nanocarrier for DNA Transfer into Mammalian Cells: Proof of Concept, Issues and Perspectives.

Sepiolite is a nanofibrous natural silicate that can be used as a nanocarrier for DNA transfer thanks to its strong interaction with DNA molecules and its ability to be naturally internalized into mammalian cells through both non-endocytic and endocytic pathways. Sepiolite, due to its ability to bind various biomolecules, could be a good candidate for use as a nanocarrier for the simultaneous vectorization of diverse biological molecules. In this paper, we review our recent work, issued from a starting collaboration with Prof. Ruiz-Hitzky, that includes diverse aspects on the characterization and main features of sepiolite/DNA nanohybrids, and we present an outlook for the further development of sepiolite for DNA transfer.

Cellular uptake pathways of sepiolite nanofibers and DNA transfection improvement.

Sepiolite is a nanofibrous natural silicate that can be used as a nanocarrier because it can be naturally internalized into mammalian cells, due to its nano-size dimension. Therefore, deciphering the mechanisms of sepiolite cell internalization constitutes a question interesting biotechnology, for the use of sepiolite as nanocarrier, as well as environmental and public health concerns. Though it is low, the perfectly stable and natural intrinsic fluorescence of sepiolite nanofibers allows to follow their fate into cells by specifically sensitive technics. By combining fluorescence microscopy (including confocal analysis), time-lapse video microscopy, fluorescence activated cell sorting and transmission electron microscopy, we show that sepiolite can be spontaneously internalized into mammalian cells through both non-endocytic and endocytic pathways, macropinocytosis being one of the main pathways. Interestingly, exposure of the cells to endocytosis inhibitors, such as chloroquine, two-fold increase the efficiency of sepiolite-mediated gene transfer, in addition to the 100-fold increased resulting from sepiolite sonomechanical treatment. As sepiolite is able to bind various biological molecules, this nanoparticulate silicate could be a good candidate as a nanocarrier for simultaneous vectorization of diverse biological molecules.

Physical interactions between DNA and sepiolite nanofibers, and potential application for DNA transfer into mammalian cells.

Nanofibers of sepiolite, a natural silicate belonging to the clay minerals family, might constitute a potential promising nanocarrier for the non-viral transfer of bio-molecules. We show here that sepiolite nanofibers efficiently bind different types of DNA molecules through electrostatic interactions, hydrogen bonding, cation bridges, and van der Waals forces. Moreover, Fourier-transform infrared spectroscopy identified the external silanol groups as the main sites of interaction with the DNA. Furthermore, as a proof of concept, we show that sepiolite is able to stably transfer plasmid DNA into mammalian cells and that the efficiency can be optimized. Indeed, sonication of sepiolite 100-fold stimulated DNA transfection efficiency. These results open the way to the use of sepiolite-based biohybrids as a novel class of nanoplatform for gene transfer with potential clinical applications.

αTAT1 controls longitudinal spreading of acetylation marks from open microtubules extremities.

Acetylation of the lysine 40 of α-tubulin (K40) is a post-translational modification occurring in the lumen of microtubules (MTs) and is controlled by the α-tubulin acetyl-transferase αTAT1. How αTAT1 accesses the lumen and acetylates α-tubulin there has been an open question. Here, we report that acetylation starts at open ends of MTs and progressively spreads longitudinally from there. We observed acetylation marks at the open ends of in vivo MTs re-growing after a Nocodazole block, and acetylated segments growing in length with time. Bias for MTs extremities was even more pronounced when using non-dynamic MTs extracted from HeLa cells. In contrast, K40 acetylation was mostly uniform along the length of MTs reconstituted from purified tubulin in vitro. Quantitative modelling of luminal diffusion of αTAT1 suggested that the uniform acetylation pattern observed in vitro is consistent with defects in the MT lattice providing lateral access to the lumen. Indeed, we observed that in vitro MTs are permeable to macromolecules along their shaft while cellular MTs are not. Our results demonstrate αTAT1 enters the lumen from open extremities and spreads K40 acetylation marks longitudinally along cellular MTs. This mode of tip-directed microtubule acetylation may allow for selective acetylation of subsets of microtubules.

Role of tau in the spatial organization of axonal microtubules: keeping parallel microtubules evenly distributed despite macromolecular crowding.

Opposing views have been proposed regarding the role of tau, the principal microtubule-associated protein in axons. On the one hand, tau forms cross-bridges at the interface between microtubules and induces microtubule bundling in neurons. On the other hand, tau is also considered a polymer brush which efficiently separates microtubules. In mature axons, microtubules are indeed arranged in parallel arrays and are well separated from each other. To reconcile these views, we developed a mechanistic model based on in vitro and cellular approaches combined to analytical and numerical analyses. The results indicate that tau forms long-range cross-bridges between microtubules under macromolecular crowding conditions. Tau cross-bridges prevent the redistribution of tau away from the interface between microtubules, which would have occurred in the polymer brush model. Consequently, the short-range attractive force between microtubules induced by macromolecular crowding is avoided and thus microtubules remain well separated from each other. Interestingly, in this unified model, tau diffusion on microtubules enables to keep microtubules evenly distributed in axonal sections at low tau levels.

C-terminal region of bacterial Ku controls DNA bridging, DNA threading and recruitment of DNA ligase D for double strand breaks repair.

Non-homologous end joining is a ligation process repairing DNA double strand breaks in eukaryotes and many prokaryotes. The ring structured eukaryotic Ku binds DNA ends and recruits other factors which can access DNA ends through the threading of Ku inward the DNA, making this protein a key ingredient for the scaffolding of the NHEJ machinery. However, this threading ability seems unevenly conserved among bacterial Ku. As bacterial Ku differ mainly by their C-terminus, we evaluate the role of this region in the loading and the threading abilities of Bacillus subtilis Ku and the stimulation of the DNA ligase LigD. We identify two distinct sub-regions: a ubiquitous minimal C-terminal region and a frequent basic C-terminal extension. We show that truncation of one or both of these sub-regions in Bacillus subtilis Ku impairs the stimulation of the LigD end joining activity in vitro. We further demonstrate that the minimal C-terminus is required for the Ku-LigD interaction, whereas the basic extension controls the threading and DNA bridging abilities of Ku. We propose that the Ku basic C-terminal extension increases the concentration of Ku near DNA ends, favoring the recruitment of LigD at the break, thanks to the minimal C-terminal sub-region.

Free mRNA in excess upon polysome dissociation is a scaffold for protein multimerization to form stress granules.

The sequence of events leading to stress granule assembly in stressed cells remains elusive. We show here, using isotope labeling and ion microprobe, that proportionally more RNA than proteins are present in stress granules than in surrounding cytoplasm. We further demonstrate that the delivery of single strand polynucleotides, mRNA and ssDNA, to the cytoplasm can trigger stress granule assembly. On the other hand, increasing the cytoplasmic level of mRNA-binding proteins like YB-1 can directly prevent the aggregation of mRNA by forming isolated mRNPs, as evidenced by atomic force microscopy. Interestingly, we also discovered that enucleated cells do form stress granules, demonstrating that the translocation to the cytoplasm of nuclear prion-like RNA-binding proteins like TIA-1 is dispensable for stress granule assembly. The results lead to an alternative view on stress granule formation based on the following sequence of events: after the massive dissociation of polysomes during stress, mRNA-stabilizing proteins like YB-1 are outnumbered by the burst of nonpolysomal mRNA. mRNA freed of ribosomes thus becomes accessible to mRNA-binding aggregation-prone proteins or misfolded proteins, which induces stress granule formation. Within the frame of this model, the shuttling of nuclear mRNA-stabilizing proteins to the cytoplasm could dissociate stress granules or prevent their assembly.

Synthesis of a multibranched porphyrin-oligonucleotide scaffold for the construction of DNA-based nano-architectures.

The interest in the functionalization of oligonucleotides with organic molecules has grown considerably over the last decade. In this work, we report on the synthesis and characterization of porphyrin-oligonucleotide hybrids containing one to four DNA strands (P1-P4). The hybrid P4, which inserts one porphyrin and four DNA fragments, was combined with gold nanoparticles and imaged by transmission electron microscopy.

Guided assembly of tetramolecular G-quadruplexes.

Nucleic acids are finding applications in nanotechnology as nanomaterials, mechanical devices, templates, and biosensors. G-quadruplex DNA, formed by π-π stacking of guanine (G) quartets, is an attractive alternative to regular B-DNA because of the kinetic and thermodynamic stability of quadruplexes. However, they suffer from a fatal flaw: the rules of recognition, i.e., the formation of a G-quartet in which four identical bases are paired, prevent the controlled assembly between different strands, leading to complex mixtures. In this report, we present the solution to this recognition problem. The proposed design combines two DNA elements: parallel-stranded duplexes and a quadruplex core. Parallel-stranded duplexes direct controlled assembly of the quadruplex core, and their strands present convenient points of attachments for potential modifiers. The exceptional stability of the quadruplex core provides integrity to the entire structure, which could be used as a building block for nucleic acid-based nanomaterials. As a proof of principle for the design's versatility, we assembled quadruplex-based 1D structures and visualized them using atomic force and transmission electron microscopy. Our findings pave the way to broader utilization of G-quadruplex DNA in structural DNA nanomaterials.

Effect of Rap1 binding on DNA distortion and potassium permanganate hypersensitivity.

Repressor activator protein 1 (Rap1) is an essential factor involved in transcription and telomere stability in the budding yeast Saccharomyces cerevisiae. Its interaction with DNA causes hypersensitivity to potassium permanganate, suggesting local DNA melting and/or distortion. In this study, various Rap1-DNA crystal forms were obtained using specifically designed crystal screens. Analysis of the DNA conformation showed that its distortion was not sufficient to explain the permanganate reactivity. However, anomalous data collected at the Mn edge using a Rap1-DNA crystal soaked in potassium permanganate solution indicated that the DNA conformation in the crystal was compatible with interaction with permanganate ions. Sequence-conservation analysis revealed that double-Myb-containing Rap1 proteins all carry a fully conserved Arg580 at a position that may favour interaction with permanganate ions, although it is not involved in the hypersensitive cytosine distortion. Permanganate reactivity assays with wild-type Rap1 and the Rap1[R580A] mutant demonstrated that Arg580 is essential for hypersensitivity. AFM experiments showed that wild-type Rap1 and the Rap1[R580A] mutant interact with DNA over 16 successive binding sites, leading to local DNA stiffening but not to accumulation of the observed local distortion. Therefore, Rap1 may cause permanganate hypersensitivity of DNA by forming a pocket between the reactive cytosine and Arg580, driving the permanganate ion towards the C5-C6 bond of the cytosine.

ATP-independent cooperative binding of yeast Isw1a to bare and nucleosomal DNA.

Among chromatin remodeling factors, the ISWI family displays a nucleosome-enhanced ATPase activity coupled to DNA translocation. While these enzymes are known to bind to DNA, their activity has not been fully characterized. Here we use TEM imaging and single molecule manipulation to investigate the interaction between DNA and yeast Isw1a. We show that Isw1a displays a highly cooperative ATP-independent binding to and bridging between DNA segments. Under appropriate tension, rare single nucleation events can sometimes be observed and loop DNA with a regular step. These nucleation events are often followed by binding of successive complexes bridging between nearby DNA segments in a zipper-like fashion, as confirmed by TEM observations. On nucleosomal substrates, we show that the specific ATP-dependent remodeling activity occurs in the context of cooperative Isw1a complexes bridging extranucleosomal DNA. Our results are interpreted in the context of the recently published partial structure of Isw1a and support its acting as a "protein ruler" (with possibly more than one tick).

Structural characterization of filaments formed by human Xrcc4-Cernunnos/XLF complex involved in nonhomologous DNA end-joining.

Cernunnos/XLF is a core protein of the nonhomologous DNA end-joining (NHEJ) pathway that processes the majority of DNA double-strand breaks in mammals. Cernunnos stimulates the final ligation step catalyzed by the complex between DNA ligase IV and Xrcc4 (X4). Here we present the crystal structure of the X4(1-157)-Cernunnos(1-224) complex at 5.5-Å resolution and identify the relative positions of the two factors and their binding sites. The X-ray structure reveals a filament arrangement for X4(1-157) and Cernunnos(1-224) homodimers mediated by repeated interactions through their N-terminal head domains. A filament arrangement of the X4-Cernunnos complex was confirmed by transmission electron microscopy analyses both with truncated and full-length proteins. We further modeled the interface and used structure-based site-directed mutagenesis and calorimetry to characterize the roles of various residues at the X4-Cernunnos interface. We identified four X4 residues (Glu(55), Asp(58), Met(61), and Phe(106)) essential for the interaction with Cernunnos. These findings provide new insights into the molecular bases for stimulatory and bridging roles of Cernunnos in the final DNA ligation step.