Identification and Detection of a Peptide Biomarker and Its Enantiomer by Nanopore.

Until now, no fast, low-cost, and direct technique exists to identify and detect protein/peptide enantiomers, because their mass and charge are identical. They are essential since l- and d-protein enantiomers have different biological activities due to their unique conformations. Enantiomers have potential for diagnostic purposes for several diseases or normal bodily functions but have yet to be utilized. This work uses an aerolysin nanopore and electrical detection to identify vasopressin enantiomers, l-AVP and d-AVP, associated with different biological processes and pathologies. We show their identification according to their conformations, in either native or reducing conditions, using their specific electrical signature. To improve their identification, we used a principal component analysis approach to define the most relevant electrical parameters for their identification. Finally, we used the Monte Carlo prediction to assign each event type to a specific l- or d-AVP enantiomer.

Using Adhesive Micropatterns and AFM to Assess Cancer Cell Morphology and Mechanics.

The mechanical properties of living cells reflect their physiological and pathological state. In particular, cancer cells undergo cytoskeletal modifications that typically make them softer than healthy cells, a property that could be used as a diagnostic tool. However, this is challenging because cells are complex structures displaying a broad range of morphologies when cultured in standard 2D culture dishes. Here, we use adhesive micropatterns to impose the cell geometry and thus standardize the mechanics and morphologies of cancer cells, which we measure by atomic force microscopy (AFM), mechanical nanomapping, and membrane nanotube pulling. We show that micropatterning cancer cells leads to distinct morphological and mechanical changes for different cell lines. Micropatterns did not systematically lower the variability in cell elastic modulus distribution. These effects emerge from a variable cell spreading rate associated with differences in the organization of the cytoskeleton, thus providing detailed insights into the structure-mechanics relationship of cancer cells cultured on micropatterns. Combining AFM with micropatterns reveals new mechanical and morphological observables applicable to cancer cells and possibly other cell types.

Circulating Tumor Cells in Cancer Diagnostics and Prognostics by Single-Molecule and Single-Cell Characterization.

Circulating tumor cells (CTCs) represent an interesting source of biomarkers for diagnosis, prognosis, and the prediction of cancer recurrence, yet while they are extensively studied in oncobiology research, their diagnostic utility has not yet been demonstrated and validated. Their scarcity in human biological fluids impedes the identification of dangerous CTC subpopulations that may promote metastatic dissemination. In this Perspective, we discuss promising techniques that could be used for the identification of these metastatic cells. We first describe methods for isolating patient-derived CTCs and then the use of 3D biomimetic matrixes in their amplification and analysis, followed by methods for further CTC analyses at the single-cell and single-molecule levels. Finally, we discuss how the elucidation of mechanical and morphological properties using techniques such as atomic force microscopy and molecular biomarker identification using nanopore-based detection could be combined in the future to provide patients and their healthcare providers with a more accurate diagnosis.

Porous yet dense matrices: using ice to shape collagen 3D cell culture systems with increased physiological relevance.

Standard in vitro cell cultures are one of the pillars of biomedical sciences. However, there is increasing evidence that 2D systems provide biological responses that are often in disagreement with in vivo observations, partially due to limitations in reproducing the native cellular microenvironment. 3D materials that are able to mimic the native cellular microenvironment to a greater extent tackle these limitations. Here, we report Porous yet Dense (PyD) type I collagen materials obtained by ice-templating followed by topotactic fibrillogenesis. These materials combine extensive macroporosity, favouring the cell migration and nutrient exchange, as well as dense collagen walls, which mimic locally the extracellular matrix. When seeded with Normal Human Dermal Fibroblasts (NHDFs), PyD matrices allow for faster and more extensive colonisation when compared with equivalent non-porous matrices. The textural properties of the PyD materials also impact cytoskeletal and nuclear 3D morphometric parameters. Due to the effectiveness in creating a biomimetic 3D environment for NHDFs and the ability to promote cell culture for more than 28 days without subculture, we anticipate that PyD materials could configure an important step towards in vitro systems applicable to other cell types and with higher physiological relevance.

Versatile cyclodextrin nanotube synthesis with functional anchors for efficient ion channel formation: design, characterization and ion conductance.

Biomimetic ion channels with different materials have been extensively designed to study the dynamics in a confined medium. These channels allow the development of several applications, such as ultra-fast sequencing and biomarker detection. When considering their synthesis, the use of cheap, non-cytotoxic and readily available materials is an increasing priority. Cyclodextrins, in supramolecular architectures, are widely utilized for pharmaceutical and biotechnological applications. Recent work has shown that short nanotubes (NTs) based on alpha-cyclodextrin (α-CD) assemble transient ion channels into membranes without cytotoxicity. In this study, we probe the influence of new cyclodextrin NT structural parameters and chemical modifications on channel formation, stability and electrical conductance. We report the successful synthesis of ß- and γ-cyclodextrin nanotubes (ß-CDNTs and γ-CDNTs), as evidenced by mass-spectrometry and high-resolution transmission electron microscopy. CDNTs were characterized by their length, diameter and number of CDs. Two hydrophobic groups, silylated or vinylated, were attached along the γ-CDNTs, improving the insertion time into the membrane. All NTs synthesized form spontaneous biomimetic ion channels. The hydrophobic NTs exhibit higher stability in membranes. Electrophysiological measurements show that ion transport is the main contribution of NT conductance and that the ion energy penalty for the entry into these NTs is similar to that of biological channels.

Biomimetic ion channels formation by emulsion based on chemically modified cyclodextrin nanotubes.

Biomimetic ion channels can be made to display the high sensitivity of natural protein nanopores and to develop new properties as a function of the material used. How to design the best future biomimetic channels? The main challenges are to control their sensitivity, as well as their syntheses, chemical modifications, insertion and lifetime in a lipid membrane. To address these challenges, we have recently designed short cyclodextrin nanotubes characterized by mass spectrometry and high-resolution transmission electron microscopy. They form non-permanent ion channels in lipid bilayers. Here we show how to improve the nanotube insertion in order to limit multiple insertions, how to stabilize biomimetic channels into the membrane, and how to understand the ion dynamics in confined medium scale.

Dynamics of a polyelectrolyte through aerolysin channel as a function of applied voltage and concentration⋆.

We describe the behaviour of a polyelectrolyte in confined geometry. The transport of a polyelectrolyte, dextran sulfate, through a recombinant protein channel, aerolysin, inserted into a planar lipid bilayer is studied as a function of applied voltage and polyelectrolyte concentration and chain length. The aerolysin pore has a weak geometry asymmetry, a high number of charged residues and the polyelectrolyte is strongly negatively charged. The resulting current blockades were characterized by short and long dwelling times. Their frequency varies exponentially as a function of applied voltage and linearly as a function of polyelectrolyte concentration. The long blockade duration decreases exponentially when the electrical force increases. The ratio of the population of short events to the one of long events decreases when the applied voltage increases and displays an exponential variation. The long residence time increases with the polyelectrolyte chain length. We measure a reduction of the effective charge of the polyelectrolyte at the pore entry and inside the channel. For a fixed applied voltage, + / - 100 mV, at both sides of the protein pore entrance, the events frequency is similar as a function of dextran sulfate concentration. The mean blockade durations are independent of polyelectrolyte concentration and are similar for both entrances of the pore and remain constant as a function of the electrical force.

Elasticity, Adhesion, and Tether Extrusion on Breast Cancer Cells Provide a Signature of Their Invasive Potential.

We use single-cell force spectroscopy to compare elasticity, adhesion, and tether extrusion on four breast cancer cell lines with an increasing invasive potential. We perform cell attachment/detachment experiments either on fibronectin or on another cell using an atomic force microscope. Our study on the membrane tether formation from cancer cells show that they are easier to extrude from aggressive invasive cells. Measured elastic modulus values confirm that more invasive cells are softer. Moreover, the adhesion force increases with the invasive potential. Our results provide a mechanical signature of breast cancer cells that correlates with their invasivity.

Biomimetic Nanotubes Based on Cyclodextrins for Ion-Channel Applications.

Biomimetic membrane channels offer a great potential for fundamental studies and applications. Here, we report the fabrication and characterization of short cyclodextrin nanotubes, their insertion into membranes, and cytotoxicity assay. Mass spectrometry and high-resolution transmission electron microscopy were used to confirm the synthesis pathway leading to the formation of short nanotubes and to describe their structural parameters in terms of length, diameter, and number of cyclodextrins. Our results show the control of the number of cyclodextrins threaded on the polyrotaxane leading to nanotube synthesis. Structural parameters obtained by electron microscopy are consistent with the distribution of the number of cyclodextrins evaluated by mass spectrometry from the initial polymer distribution. An electrophysiological study at single molecule level demonstrates the ion channel formation into lipid bilayers, and the energy penalty for the entry of ions into the confined nanotube. In the presence of nanotubes, the cell physiology is not altered.

Mapping the conformational stability of maltose binding protein at the residue scale using nuclear magnetic resonance hydrogen exchange experiments.

Being able to differentiate local fluctuations from global folding-unfolding dynamics of a protein is of major interest for improving our understanding of structure-function determinants. The maltose binding protein (MBP), a protein that belongs to the maltose transport system, has a structure composed of two globular domains separated by a rigid-body "hinge bending". Here we determined, by using hydrogen exchange (HX) nuclear magnetic resonance experiments, the apparent stabilization free energies of 101 residues of MBP bound to ß-cyclodextrin (MBP-ßCD) under native conditions. We observed that the last helix of MBP (helix α14) has a lower protection factor than the rest of the protein. Further, HX experiments were performed using guanidine hydrochloride under subdenaturing conditions to discriminate between local fluctuations and global unfolding events and to determine the MBP-ßCD energy landscape. The results show that helix α4 and a part of helices α5 and α6 are clearly grouped into a subdenaturing folding unit and represent a partially folded intermediate under native conditions. In addition, we observed that amide protons located in the hinge between the two globular domains share similar ΔG(gu)(app) and m values and should unfold simultaneously. These observations provide new points of view for improving our understanding of the thermodynamic stability and the mechanisms that drive folding-unfolding dynamics of proteins.

Dynamics of unfolded protein transport through an aerolysin pore.

Protein export is an essential mechanism in living cells and exported proteins are usually translocated through a protein-conducting channel in an unfolded state. Here we analyze, by electrical detection, the entry and transport of unfolded proteins, at the single molecule level, with different stabilities through an aerolysin pore, as a function of the applied voltage and protein concentration. The frequency of ionic current blockades varies exponentially as a function of the applied voltage and linearly as a function of protein concentration. The transport time of unfolded proteins decreases exponentially when the applied voltage increases. We prove that the ionic current blockade duration of a double-sized protein is longer than that assessed for a single protein supporting the transport phenomenon. Our results fit with the theory of confined polyelectrolyte and with some experimental results about DNA or synthetic polyelectrolyte translocation through protein channels as a function of applied voltage. We discuss the potential of the aerolysin nanopore as a tool for protein folding studies as it has already been done for α-hemolysin.

Polyelectrolyte and unfolded protein pore entrance depends on the pore geometry.

We determined the ability of Maltose Binding Protein and the polyelectrolyte dextran sulfate to enter into and interact with channels formed by Staphylococcus aureus alpha-hemolysin. The entry of either macromolecule in the channel pore causes transient, but well-defined decreases in the single-channel ionic current. The protein and polyelectrolyte were more likely to enter the pore mouth at the channel's cap domain than at the stem side. When the cap domain was denatured in the presence of 4 M urea, the probability that either the denatured protein or polyelectrolyte entered the pore from the cap-domain side decreased. For channels in their native conformation, the polyelectrolyte-induced current blockades were characterized by two mean residence times that were independent of the side of entry. For channels with a denaturated cap domain, the mean polyelectrolyte residence times for relatively long-lived blockades decreased, while that for short-lived blockades were unchanged. For denatured protein, we also observed 2 characteristic residence times that were relatively fast. Only the relatively short-lived blockades were observed with native channels. When the alpha-hemolysin monomers in aqueous solution were incubated in 4 M urea before channel formation, the two characteristic residence times were greater than those for pre-formed pores that were subsequently perturbed by urea. These times might correspond to the interactions between the unfolded protein and the partially unfolded channel.

Polyelectrolyte entry and transport through an asymmetric alpha-hemolysin channel.

We study the entry and transport of a polyelectrolyte, dextran sulfate (DS), through an asymmetric alpha-hemolysin protein channel inserted into a planar lipid bilayer. We compare the dynamics of the DS chains as they enter the channel at the opposite stem or vestibule sides. Experiments are performed at the single-molecule level by using an electrical method. The frequency of current blockades varies exponentially as a function of applied voltage. This frequency is smaller for the stem entrance than for the vestibule one, due to a smaller coupling with the electric field and a larger activation energy for entry. The value of the activation energy is quantitatively interpreted as an entropic effect of chain confinement. The translocation time decreases when the applied voltage increases and displays an exponential variation which is independent of the stem or vestibule sides.

Urea denaturation of alpha-hemolysin pore inserted in planar lipid bilayer detected by single nanopore recording: loss of structural asymmetry.

The aim of this work is to study pore protein denaturation inside a lipid bilayer and to probe current asymmetry as a function of the channel conformation. We describe the urea denaturation of alpha-hemolysin channel and the channel formation of alpha-hemolysin monomer incubated with urea prior to insertion into a lipid bilayer. Analysis of single-channel recordings of current traces reveals a sigmoid curve of current intensity as a function of urea concentration. The normalized current asymmetry at 29+/-4% is observed between 0 and 3.56M concentrations and vanishes abruptly down to 0 concentration exceeds 4M. The loss of current asymmetry through alpha-hemolysin is due to the denaturation of the channel's cap. We also show that the alpha-hemolysin pore inserted into a lipid bilayer is much more resistant to urea denaturation than the alpha-hemolysin monomer in solution: The pore remains in the lipid bilayer up to 7.2M urea. The pore formation is possible up to 4.66M urea when protein monomers were previously incubated in urea.

Involvement of alphavbeta 3 integrin and disruption of endothelial fibronectin network during the adhesion of the human ovarian adenocarcinoma cell line IGROV1 on the human umbilical vein cell extracellular matrix.

Like the majority of tumor cells, ovarian cancer cell growth is critically dependent on their neovascularization. Adhesion molecules and cellular events that lead to ovarian tumor cell interactions with endothelial extracellular matrix surrounding the vasculature are poorly identified. To understand the role of alphavbeta3 integrin and its ligand fibronectin in this process, we used in vitro coculture models with IGROV1 human ovarian adenocarcinoma cell line and human umbilical vein endothelial cells (HUVEC). Adhesion assays revealed a strong ability of IGROV1 cells to adhere to HUVEC-ECM. alphavbeta3 is mainly implicated and seems to cooperate with alpha5beta1 integrin in this event. Immunofluorescence staining revealed the presence of alphavbeta3 and alpha5beta1 in IGROV1 cells adhering on HUVEC-ECM at regions of cell sub-stratum contacts. Furthermore, our data showed the absence of fibronectin staining in IGROV1 cells and the disruption of the HUVEC-ECM fibrillar fibronectin network under IGROV1 cell influence. In situ experiments in ovarian neoplastic tissue corroborated the absence of fibronectin in the tumor and its strong detection in vasculature. These findings suggest the active participation of alphavbeta3 and alpha5beta1 integrins and the reorganization of endothelial fibronectin during the adhesion of IGROV1 cells to HUVEC-ECM whereas IGROV1 cells seem to be unable to synthesize fibronectin. Thus, fibronectin integrin receptors expressed by ovarian tumor cells and endothelial fibronectin may be of importance in ovarian carcinoma neovascularization and during tumor-vasculature interactions.