Publications by authors named "Giovanni Dietler"

99 Publications

Environmental Control of Amyloid Polymorphism by Modulation of Hydrodynamic Stress.

ACS Nano 2021 01 22;15(1):944-953. Epub 2020 Dec 22.

Laboratory of Physics of Living Matter, École Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland.

The phenomenon of amyloid polymorphism is a key feature of protein aggregation. Unravelling this phenomenon is of great significance for understanding the underlying molecular mechanisms associated with neurodegenerative diseases and for the development of amyloid-based functional biomaterials. However, the understanding of the molecular origins and the physicochemical factors modulating amyloid polymorphs remains challenging. Herein, we demonstrate an association between amyloid polymorphism and environmental stress in solution, induced by an air/water interface in motion. Our results reveal that low-stress environments produce heterogeneous amyloid polymorphs, including twisted, helical, and rod-like fibrils, whereas high-stress conditions generate only homogeneous rod-like fibrils. Moreover, high environmental stress converts twisted fibrils into rod-like fibrils both in-pathway and after the completion of mature amyloid formation. These results enrich our understanding of the environmental origin of polymorphism of pathological amyloids and shed light on the potential of environmentally controlled fabrication of homogeneous amyloid biomaterials for biotechnological applications.
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http://dx.doi.org/10.1021/acsnano.0c07570DOI Listing
January 2021

Single yeast cell nanomotions correlate with cellular activity.

Sci Adv 2020 Jun 24;6(26):eaba3139. Epub 2020 Jun 24.

International Joint Research Group BioNanotechnology & NanoMedicine (NANO), Vrije Universiteit Brussel-Ecole Polytechnique de Lausanne (EPFL), B-1050 Brussels, Belgium-B-1015 Lausanne, Switzerland.

Living single yeast cells show a specific cellular motion at the nanometer scale with a magnitude that is proportional to the cellular activity of the cell. We characterized this cellular nanomotion pattern of nonattached single yeast cells using classical optical microscopy. The distribution of the cellular displacements over a short time period is distinct from random motion. The range and shape of such nanomotion displacement distributions change substantially according to the metabolic state of the cell. The analysis of the nanomotion frequency pattern demonstrated that single living yeast cells oscillate at relatively low frequencies of around 2 hertz. The simplicity of the technique should open the way to numerous applications among which antifungal susceptibility tests seem the most straightforward.
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http://dx.doi.org/10.1126/sciadv.aba3139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314535PMC
June 2020

Fiber-Tip Polymer Microcantilever for Fast and Highly Sensitive Hydrogen Measurement.

ACS Appl Mater Interfaces 2020 Jul 17;12(29):33163-33172. Epub 2020 Jun 17.

Guangdong and Hong Kong Joint Research Centre for Optical Fiber Sensors, Shenzhen University, Shenzhen 518060, China.

Hydrogen as an antioxidant gas has been widely used in the medical and biological fields for preventing cancer or treating inflammation. However, controlling the hydrogen concentration is crucial for practical use due to its explosive property when its volume concentration in air reaches the explosive limit (4%). In this work, a polymer-based microcantilever (μ-cantilever) hydrogen sensor located at the end of a fiber tip is proposed to detect the hydrogen concentration in medical and biological applications. The proposed sensor was developed using femtosecond laser-induced two-photon polymerization (TPP) to print the polymer μ-cantilever and magnetron sputtering to coat a palladium (Pd) film on the upper surface of the μ-cantilever. Such a device exhibits a high sensitivity, roughly -2 nm % when the hydrogen concentration rises from 0% to 4.5% (v/v) and a short response time, around 13.5 s at 4% (v/v), making it suitable for medical and environmental applications. In addition to providing an ultracompact optical solution for fast and highly sensitive hydrogen measurement, the polymer μ-cantilever fiber sensor can be used for diverse medical and biological sensing applications by replacing Pd with other functional materials.
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http://dx.doi.org/10.1021/acsami.0c06179DOI Listing
July 2020

Filamentous and step-like behavior of gelling coarse fibrin networks revealed by high-frequency microrheology.

Soft Matter 2020 May 16;16(17):4234-4242. Epub 2020 Apr 16.

Dep. Física Interdisciplinar, Universidad Nacional de Educación a Distancia (UNED), Madrid 28040, Spain.

By a micro-experimental methodology, we study the ongoing molecular process inside coarse fibrin networks by means of microrheology. We made these networks gelate around a probe microbead, allowing us to observe a temporal evolution compatible with the well-known molecular formation of fibrin networks in four steps: monomer, protofibril, fiber and network. Thanks to the access that optical-trapping interferometry provides to the short-time scale on the bead's Brownian motion, we observe a Kelvin-Voigt mechanical behavior from low to high frequencies, range not available in conventional rheometry. We exploit that mechanical model for obtaining the characteristic lengths of the filamentous structures composing these fibrin networks, whose obtained values are compatible with a non-affine behavior characterized by bending modes. At very long gelation times, a ω power-law is observed in the loss modulus, theoretically related with the longitudinal response of the molecular structures.
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http://dx.doi.org/10.1039/c9sm02228gDOI Listing
May 2020

A perspective view on the nanomotion detection of living organisms and its features.

J Mol Recognit 2020 12 30;33(12):e2849. Epub 2020 Mar 30.

Laboratoire de Physique de la Matière Vivante, Institut de Physique, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

The insurgence of newly arising, rapidly developing health threats, such as drug-resistant bacteria and cancers, is one of the most urgent public-health issues of modern times. This menace calls for the development of sensitive and reliable diagnostic tools to monitor the response of single cells to chemical or pharmaceutical stimuli. Recently, it has been demonstrated that all living organisms oscillate at a nanometric scale and that these oscillations stop as soon as the organisms die. These nanometric scale oscillations can be detected by depositing living cells onto a micro-fabricated cantilever and by monitoring its displacements with an atomic force microscope-based electronics. Such devices, named nanomotion sensors, have been employed to determine the resistance profiles of life-threatening bacteria within minutes, to evaluate, among others, the effect of chemicals on yeast, neurons, and cancer cells. The data obtained so far demonstrate the advantages of nanomotion sensing devices in rapidly characterizing microorganism susceptibility to pharmaceutical agents. Here, we review the key aspects of this technique, presenting its major applications. and detailing its working protocols.
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http://dx.doi.org/10.1002/jmr.2849DOI Listing
December 2020

Effects of sedimentation, microgravity, hydrodynamic mixing and air-water interface on α-synuclein amyloid formation.

Chem Sci 2020 Mar 10;11(14):3687-3693. Epub 2020 Mar 10.

Laboratory of Physics of Living Matter, École Polytechnique Fédérale de Lausanne (EPFL) CH-1015 Lausanne Switzerland

The formation of amyloid fibrils is a characterizing feature of a range of protein misfolding diseases, including Parkinson's disease. The propensity of native proteins to form such amyloid fibril, both and , is highly sensitive to the surrounding environment, which can alter the aggregation kinetics and fibrillization mechanisms. Here, we investigate systematically the influence of several representative environmental stimuli on α-synuclein aggregation, including hydrodynamic mixing, the presence of an air-water interface and sedimentation. Our results show that hydrodynamic mixing and interfacial effects are critical in promoting several microscopic steps of α-synuclein aggregation and amyloid fibril formation. The presence of an air-water interface under agitation significantly promoted primary nucleation. Secondary processes were facilitated by hydrodynamic mixing, produced by 3D rotation and shaking either in the presence or in the absence of an air-water interface. Effects of sedimentation, as investigated in a microgravity incubator, of α-synuclein lead only to minor changes on the aggregation kinetics rates in comparison to static conditions. These results forward the understanding of α-synuclein fibrillization, paving the way for the development of high-throughput assays for the screening of pharmacological approaches targeting Parkinson's disease.
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http://dx.doi.org/10.1039/d0sc00281jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8152616PMC
March 2020

Electrical Excitation of Long-Range Surface Plasmons in PC/OLED Structure with Two Metal Nanolayers.

Nanomicro Lett 2020 Jan 22;12(1):35. Epub 2020 Jan 22.

Laboratoire de Physique de La Matière Vivante, IPHYS, Ecole Polytechnique Fédérale de Lausanne, 1015, Lausanne, Switzerland.

A current-driven source of long-range surface plasmons (LRSPs) on a duplex metal nanolayer is reported. Electrical excitation of LRSPs was experimentally observed in a planar structure, where an organic light-emitting film was sandwiched between two metal nanolayers that served as electrodes. To achieve the LRSP propagation in these metal nanolayers at the interface with air, the light-emitting structure was bordered by a one-dimensional photonic crystal (PC) on the other side. The dispersion of the light emitted by such a hybrid PC/organic-light-emitting-diode structure (PC/OLED) comprising two thin metal electrodes was obtained, with a clearly identified LRSP resonance peak.
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http://dx.doi.org/10.1007/s40820-020-0369-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7770686PMC
January 2020

Structural and DNA binding properties of mycobacterial integration host factor mIHF.

J Struct Biol 2020 03 14;209(3):107434. Epub 2019 Dec 14.

École Polytechnique Fédérale de Lausanne, School of Life Sciences, Station 19, 1015 Lausanne, Switzerland. Electronic address:

In bacteria, nucleoid associated proteins (NAPs) take part in active chromosome organization by supercoil management, three-dimensional DNA looping and direct transcriptional control. Mycobacterial integration host factor (mIHF, rv1388) is a NAP restricted to Actinobacteria and essential for survival of the human pathogen Mycobacterium tuberculosis. We show in vitro that DNA binding by mIHF strongly stabilizes the protein and increases its melting temperature. The structure obtained by Nuclear Magnetic Resonance (NMR) spectroscopy characterizes mIHF as a globular protein with a protruding alpha helix and a disordered N-terminus, similar to Streptomyces coelicolor IHF (sIHF). NMR revealed no residues of high flexibility, suggesting that mIHF is a rigid protein overall that does not undergo structural rearrangements. We show that mIHF only binds to double stranded DNA in solution, through two DNA binding sites (DBSs) similar to those identified in the X-ray structure of sIHF. According to Atomic Force Microscopy, mIHF is able to introduce left-handed loops of ca. 100 nm size (~300 bp) in supercoiled cosmids, thereby unwinding and relaxing the DNA.
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http://dx.doi.org/10.1016/j.jsb.2019.107434DOI Listing
March 2020

The role of xanthophylls in the supramolecular organization of the photosynthetic complex LHCII in lipid membranes studied by high-resolution imaging and nanospectroscopy.

Biochim Biophys Acta Bioenerg 2020 02 14;1861(2):148117. Epub 2019 Nov 14.

Department of Biophysics, Institute of Physics, Maria Curie-Sklodowska University, 20-031 Lublin, Poland. Electronic address:

The xanthophyll cycle is a regulatory mechanism operating in the photosynthetic apparatus of plants. It consists of the conversion of the xanthophyll pigment violaxanthin to zeaxanthin, and vice versa, in response to light intensity. According to the current understanding, one of the modes of regulatory activity of the cycle is associated with the influence on a molecular organization of pigment-protein complexes. In the present work, we analyzed the effect of violaxanthin and zeaxanthin on the molecular organization of the LHCII complex, in the environment of membranes formed with chloroplast lipids. Nanoscale imaging based on atomic force microscopy (AFM) showed that the presence of exogenous xanthophylls promotes the formation of the protein supramolecular structures. Nanoscale infrared (IR) absorption analysis based on AFM-IR nanospectroscopy suggests that zeaxanthin promotes the formation of LHCII supramolecular structures by forming inter-molecular β-structures. Meanwhile, the molecules of violaxanthin act as "molecular spacers" preventing self-aggregation of the protein, potentially leading to uncontrolled dissipation of excitation energy in the complex. This latter mechanism was demonstrated with the application of fluorescence lifetime imaging microscopy. The intensity-averaged chlorophyll a fluorescence lifetime determined in the LHCII samples without exogenous xanthophylls at the level of 0.72 ns was longer in the samples containing exogenous violaxanthin (2.14 ns), but shorter under the presence of zeaxanthin (0.49 ns) thus suggesting a role of this xanthophyll in promotion of the formation of structures characterized by effective excitation quenching. This mechanism can be considered as a representation of the overall photoprotective activity of the xanthophyll cycle.
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http://dx.doi.org/10.1016/j.bbabio.2019.148117DOI Listing
February 2020

Gap-Plasmon-Enhanced High-Spatial-Resolution Imaging by Photothermal-Induced Resonance in the Visible Range.

Nano Lett 2019 11 30;19(11):8278-8286. Epub 2019 Oct 30.

Laboratory of Physics of Living Matter , IPHYS, Ecole Polytechnique Fédérale de Lausanne (EPFL) , CH-1015 Lausanne , Switzerland.

Chemical characterization at the nanoscale is of significant importance for many applications in physics, analytical chemistry, material science, and biology. Despite the intensive studies in the infrared range, high-spatial-resolution and high-sensitivity imaging for compositional identification in the visible range is rarely exploited. In this work, we present a gap-plasmon-enhanced imaging approach based on photothermal-induced resonance (PTIR) for nanoscale chemical identification. With this approach, we experimentally obtained a high spatial resolution of ∼5 nm for rhodamine nanohill characterization and achieved monolayer sensitivity for mapping the single-layer chlorophyll-a islands with the thickness of only 1.9 nm. We also successfully characterized amyloid fibrils stained with methylene blue dye, indicating that this methodology can be also utilized for identification of the radiation-insensitive macromolecules. We believe that our proposed high-performance visible PTIR system can be used to broaden the applications of nanoscale chemical identification ranging from nanomaterial to life science areas.
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http://dx.doi.org/10.1021/acs.nanolett.9b03844DOI Listing
November 2019

Infrared nanospectroscopic mapping of a single metaphase chromosome.

Nucleic Acids Res 2019 10;47(18):e108

Institute of Nuclear Physics, Polish Academy of Sciences, PL-31342 Krakow, Poland.

The integrity of the chromatin structure is essential to every process occurring within eukaryotic nuclei. However, there are no reliable tools to decipher the molecular composition of metaphase chromosomes. Here, we have applied infrared nanospectroscopy (AFM-IR) to demonstrate molecular difference between eu- and heterochromatin and generate infrared maps of single metaphase chromosomes revealing detailed information on their molecular composition, with nanometric lateral spatial resolution. AFM-IR coupled with principal component analysis has confirmed that chromosome areas containing euchromatin and heterochromatin are distinguishable based on differences in the degree of methylation. AFM-IR distribution of eu- and heterochromatin was compared to standard fluorescent staining. We demonstrate the ability of our methodology to locate spatially the presence of anticancer drug sites in metaphase chromosomes and cellular nuclei. We show that the anticancer 'rule breaker' platinum compound [Pt[N(p-HC6F4)CH2]2py2] preferentially binds to heterochromatin, forming localized discrete foci due to condensation of DNA interacting with the drug. Given the importance of DNA methylation in the development of nearly all types of cancer, there is potential for infrared nanospectroscopy to be used to detect gene expression/suppression sites in the whole genome and to become an early screening tool for malignancy.
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http://dx.doi.org/10.1093/nar/gkz630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6765102PMC
October 2019

Universal Soft Robotic Microgripper.

Small 2019 01 28;15(4):e1803870. Epub 2018 Nov 28.

Institute of Mechanical Engineering, Ecole Polytechnique Fédérale de Lausanne, CH-1015, Lausanne, Switzerland.

Here, a soft robotic microgripper is presented that consists of a smart actuated microgel connected to a spatially photopatterned multifunctional base. When pressed onto a target object, the microgel component conforms to its shape, thus providing a simple and adaptive solution for versatile micromanipulation. Without the need for active visual or force feedback, objects of widely varying mechanical and surface properties are reliably gripped through a combination of geometrical interlocking mechanisms instantiated by reversible shape-memory and thermal responsive swelling of the microgel. The gripper applies holding forces exceeding 400 µN, which is high enough to lift loads 1000 times heavier than the microgel. An untethered version of the gripper is developed by remotely controlling the position using magnetic actuation and the contractile state of the microgel using plasmonic absorption. Gentle yet stable robotic manipulation of biological samples under physiological conditions opens up possibilities for high-throughput interrogation and minimally invasive interventions.
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http://dx.doi.org/10.1002/smll.201803870DOI Listing
January 2019

Periodic Motion of Sedimenting Flexible Knots.

Phys Rev Lett 2018 Sep;121(12):127801

Institute of Theoretical Physics, Faculty of Physics, University of Warsaw, Pasteura 5, 02-093 Warsaw, Poland.

We study the dynamics of knotted deformable closed chains sedimenting in a viscous fluid. We show experimentally that trefoil and other torus knots often attain a remarkably regular horizontal toroidal structure while sedimenting, with a number of intertwined loops, oscillating periodically around each other. We then recover this motion numerically and find out that it is accompanied by a very slow rotation around the vertical symmetry axis. We analyze the dependence of the characteristic timescales on the chain flexibility and aspect ratio. It is observed in the experiments that this oscillating mode of the dynamics can spontaneously form even when starting from a qualitatively different initial configuration. In numerical simulations, the oscillating modes are usually present as transients or final stages of the evolution, depending on chain aspect ratio and flexibility, and the number of loops.
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http://dx.doi.org/10.1103/PhysRevLett.121.127801DOI Listing
September 2018

Identification of Oxidative Stress in Red Blood Cells with Nanoscale Chemical Resolution by Infrared Nanospectroscopy.

Int J Mol Sci 2018 Aug 30;19(9). Epub 2018 Aug 30.

Department of Chemistry, Cambridge University, Cambridge CB21EW, UK.

During their lifespan, Red blood cells (RBC), due to their inability to self-replicate, undergo an ageing degradation phenomenon. This pathway, both in vitro and in vivo, consists of a series of chemical and morphological modifications, which include deviation from the biconcave cellular shape, oxidative stress, membrane peroxidation, lipid content decrease and uncoupling of the membrane-skeleton from the lipid bilayer. Here, we use the capabilities of atomic force microscopy based infrared nanospectroscopy (AFM-IR) to study and correlate, with nanoscale resolution, the morphological and chemical modifications that occur during the natural degradation of RBCs at the subcellular level. By using the tip of an AFM to detect the photothermal expansion of RBCs, it is possible to obtain nearly two orders of magnitude higher spatial resolution IR spectra, and absorbance images than can be obtained on diffraction-limited commercial Fourier-transform Infrared (FT-IR) microscopes. Using this approach, we demonstrate that we can identify localized sites of oxidative stress and membrane peroxidation on individual RBC, before the occurrence of neat morphological changes in the cellular shape.
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http://dx.doi.org/10.3390/ijms19092582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6163177PMC
August 2018

N-terminal Huntingtin (Htt) phosphorylation is a molecular switch regulating Htt aggregation, helical conformation, internalization, and nuclear targeting.

J Biol Chem 2018 11 5;293(48):18540-18558. Epub 2018 Sep 5.

From the Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, École Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland and

Huntington's disease is a fatal neurodegenerative disorder resulting from a CAG repeat expansion in the first exon of the gene encoding the Huntingtin protein (Htt). Phosphorylation of this protein region (Httex1) has been shown to play important roles in regulating the structure, toxicity, and cellular properties of N-terminal fragments and full-length Htt. However, increasing evidence suggests that phosphomimetic substitutions in Htt result in inconsistent findings and do not reproduce all aspects of true phosphorylation. Here, we investigated the effects of phosphorylation at Ser-13 or Ser-16 on the structure, aggregation, membrane binding, and subcellular properties of the Httex1-Q18A variant and compared these effects with those of phosphomimetic substitutions. We show that phosphorylation at either Ser-13 and/or Ser-16 or phosphomimetic substitutions at both these residues inhibit the aggregation of mutant Httex1, but that only phosphorylation strongly disrupts the amphipathic α-helix of the N terminus and prompts the internalization and nuclear targeting of preformed Httex1 aggregates. In synthetic peptides, phosphorylation at Ser-13, Ser-16, or both residues strongly disrupted the amphipathic α-helix of the N-terminal 17 residues (Nt17) of Httex1 and Nt17 membrane binding. Experiments with peptides bearing different combinations of phosphorylation sites within Nt17 revealed a phosphorylation-dependent switch that regulates the Httex1 structure, involving cross-talk between phosphorylation at Thr-3 and Ser-13 or Ser-16. Our results provide crucial insights into the role of phosphorylation in regulating Httex1 structure and function, and underscore the critical importance of identifying the enzymes responsible for regulating Htt phosphorylation, and their potential as therapeutic targets for managing Huntington's disease.
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http://dx.doi.org/10.1074/jbc.RA118.004621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6290154PMC
November 2018

Nanoplasmonic mid-infrared biosensor for protein secondary structure detection.

Light Sci Appl 2017 Aug 25;6(8):e17029. Epub 2017 Aug 25.

Bionanophotonic Systems Laboratory, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne CH-1015, Switzerland.

Plasmonic nanoantennas offer new applications in mid-infrared (mid-IR) absorption spectroscopy with ultrasensitive detection of structural signatures of biomolecules, such as proteins, due to their strong resonant near-fields. The amide I fingerprint of a protein contains conformational information that is greatly important for understanding its function in health and disease. Here, we introduce a non-invasive, label-free mid-IR nanoantenna-array sensor for secondary structure identification of nanometer-thin protein layers in aqueous solution by resolving the content of plasmonically enhanced amide I signatures. We successfully detect random coil to cross β-sheet conformational changes associated with α-synuclein protein aggregation, a detrimental process in many neurodegenerative disorders. Notably, our experimental results demonstrate high conformational sensitivity by differentiating subtle secondary-structural variations in a native β-sheet protein monolayer from those of cross β-sheets, which are characteristic of pathological aggregates. Our nanoplasmonic biosensor is a highly promising and versatile tool for structural analysis of thin protein layers.
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http://dx.doi.org/10.1038/lsa.2017.29DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6062318PMC
August 2017

Identification and nanomechanical characterization of the fundamental single-strand protofilaments of amyloid α-synuclein fibrils.

Proc Natl Acad Sci U S A 2018 07 25;115(28):7230-7235. Epub 2018 Jun 25.

Laboratory of Physics of Living Matter, École Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland;

The formation and spreading of amyloid aggregates from the presynaptic protein α-synuclein in the brain play central roles in the pathogenesis of Parkinson's disease. Here, we use high-resolution atomic force microscopy to investigate the early oligomerization events of α-synuclein with single monomer angstrom resolution. We identify, visualize, and characterize directly the smallest elementary unit in the hierarchical assembly of amyloid fibrils, termed here single-strand protofilaments. We show that protofilaments form from the direct molecular assembly of unfolded monomeric α-synuclein polypeptide chains. To unravel protofilaments' internal structure and elastic properties, we manipulated nanomechanically these species by atomic force spectroscopy. The single-molecule scale identification and characterization of the fundamental unit of amyloid assemblies provide insights into early events underlying their formation and shed light on opportunities for therapeutic intervention at the early stages of aberrant protein self-assembly.
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http://dx.doi.org/10.1073/pnas.1721220115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048494PMC
July 2018

Nanoscale Investigation into the Cellular Response of Glioblastoma Cells Exposed to Protons.

Anal Chem 2018 06 6;90(12):7644-7650. Epub 2018 Jun 6.

Institute of Physics, Laboratory of Physics of Living Matter , Ecole Polytechnique Fédérale de Lausanne (EPFL) , CH-1015 Lausanne , Switzerland.

Exposure to ionizing radiation can induce cellular defense mechanisms including cell activation and rapid proliferation prior to metastasis and in extreme cases can result in cell death. Herewith we apply infrared nano- and microspectroscopy combined with multidimensional data analysis to characterize the effect of ionizing radiation on single glioblastoma nuclei isolated from cells treated with 10 Gy of X-rays or 1 and 10 Gy of protons. We observed chromatin fragmentation related to the formation of apoptotic bodies following X-ray exposure. Following proton irradiation we detected evidence of a DNA conformational change (B-DNA to A-DNA transition) related to DNA repair and accompanied by an increase in protein content related to the synthesis of peptide enzymes involved in DNA repair. We also show that proton exposure can increase cholesterol and sterol ester synthesis, which are important lipids involved in the metastatic process changing the fluidity of the cellular membrane in preparation for rapid proliferation.
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http://dx.doi.org/10.1021/acs.analchem.8b01497DOI Listing
June 2018

Author Correction: Nanomechanics of multidomain neuronal cell adhesion protein contactin revealed by single molecule AFM and SMD.

Sci Rep 2018 Mar 6;8(1):4291. Epub 2018 Mar 6.

Institute of Physics, Faculty of Physics, Astronomy and Applied Informatics, Nicolaus Copernicus University, Grudziadzka 5, 87-100, Torun, Poland.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-018-21746-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5840351PMC
March 2018

Nanomotion Detection Method for Testing Antibiotic Resistance and Susceptibility of Slow-Growing Bacteria.

Small 2018 01 4;14(4). Epub 2017 Dec 4.

Laboratoire de Physique de la Matière Vivante, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015, Lausanne, Switzerland.

Infectious diseases are caused by pathogenic microorganisms and are often severe. Time to fully characterize an infectious agent after sampling and to find the right antibiotic and dose are important factors in the overall success of a patient's treatment. Previous results suggest that a nanomotion detection method could be a convenient tool for reducing antibiotic sensitivity characterization time to several hours. Here, the application of the method for slow-growing bacteria is demonstrated, taking Bordetella pertussis strains as a model. A low-cost nanomotion device is able to characterize B. pertussis sensitivity against specific antibiotics within several hours, instead of days, as it is still the case with conventional growth-based techniques. It can discriminate between resistant and susceptible B. pertussis strains, based on the changes of the sensor's signal before and after the antibiotic addition. Furthermore, minimum inhibitory and bactericidal concentrations of clinically applied antibiotics are compared using both techniques and the suggested similarity is discussed.
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http://dx.doi.org/10.1002/smll.201702671DOI Listing
January 2018

Amyloid single-cell cytotoxicity assays by nanomotion detection.

Cell Death Discov 2017 21;3:17053. Epub 2017 Aug 21.

Institute of Physics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne CH-1015, Switzerland.

Cells are extremely complex systems able to actively modify their metabolism and behavior in response to environmental conditions and stimuli such as pathogenic agents or drugs. The comprehension of these responses is central to understand the molecular bases of human pathologies, including amyloid misfolding diseases. Conventional bulk biological assays are limited by intrinsic cellular heterogeneity in gene, protein and metabolite expression, and can investigate only indirectly cellular reactions in non-physiological conditions. Here we employ a label-free nanomotion sensor to study single neuroblastoma cells exposed to extracellular monomeric and amyloid -synuclein species in real-time and in physiological conditions. Combining this technique with fluorescence microscopy, we demonstrate multispecies cooperative cytotoxic effect of amyloids and aggregate-induced loss of cellular membrane integrity. Notably, the method can study cellular reactions and cytotoxicity an order of magnitude faster, and using 100-fold smaller volume of reagents when compared to conventional bulk analyses. This rapidity and sensitivity will allow testing novel pharmacological approaches to stop or delay a wide range of human diseases.
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http://dx.doi.org/10.1038/cddiscovery.2017.53DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5564330PMC
August 2017

Nanomechanics of multidomain neuronal cell adhesion protein contactin revealed by single molecule AFM and SMD.

Sci Rep 2017 08 18;7(1):8852. Epub 2017 Aug 18.

Institute of Physics, Faculty of Physics, Astronomy and Applied Informatics, Nicolaus Copernicus University, Grudziadzka 5, 87-100, Torun, Poland.

Contactin-4 (CNTN4) is a complex cell adhesion molecule (CAM) localized at neuronal membranes, playing a key role in maintaining the mechanical integrity and signaling properties of the synapse. CNTN4 consists of six immunoglobulin C2 type (IgC2) domains and four fibronectin type III (FnIII) domains that are shared with many other CAMs. Mutations in CNTN4 gene have been linked to various psychiatric disorders. Toward elucidating the response of this modular protein to mechanical stress, we studied its force-induced unfolding using single molecule atomic force microscopy (smAFM) and steered molecular dynamics (SMD) simulations. Extensive smAFM and SMD data both indicate the distinctive mechanical behavior of the two types of modules distinguished by unique force-extension signatures. The data also reveal the heterogeneity of the response of the individual FNIII and IgC2 modules, which presumably plays a role in the adaptability of CNTN4 to maintaining cell-cell communication and adhesion properties under different conditions. Results show that extensive sampling of force spectra, facilitated by robot-enhanced AFM, can help reveal the existence of weak stabilizing interactions between the domains of multidomain proteins, and provide insights into the nanomechanics of such multidomain or heteromeric proteins.
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http://dx.doi.org/10.1038/s41598-017-09482-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5562865PMC
August 2017

Mutant Exon1 Huntingtin Aggregation is Regulated by T3 Phosphorylation-Induced Structural Changes and Crosstalk between T3 Phosphorylation and Acetylation at K6.

Angew Chem Int Ed Engl 2017 05 23;56(19):5202-5207. Epub 2017 Mar 23.

Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Institute of Physics of Biological Systems, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-, 1015, Lausanne, Switzerland.

Herein, we used protein semisynthesis to investigate, for the first time, the effect of lysine acetylation and phosphorylation, as well as the crosstalk between these modifications on the structure and aggregation of mutant huntingtin exon1 (Httex1). Our results demonstrate that phosphorylation at T3 stabilizes the α-helical conformation of the N-terminal 17 amino acids (Nt17) and significantly inhibits the aggregation of mutant Httex1. Acetylation of single lysine residues, K6, K9 or K15, had no effect on Httex1 aggregation. Interestingly, acetylation at K6, but not at K9 or K15, reversed the inhibitory effect of T3 phosphorylation. Together, our results provide novel insight into the role of Nt17 post-translational modifications in regulating the structure and aggregation of Httex1 and suggest that its aggregation and possibly its function(s) are controlled by regulatory mechanisms involving crosstalk between different PTMs.
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http://dx.doi.org/10.1002/anie.201611750DOI Listing
May 2017

Spatial organization of DNA sequences directs the assembly of bacterial chromatin by a nucleoid-associated protein.

J Biol Chem 2017 05 18;292(18):7607-7618. Epub 2017 Mar 18.

Jacobs University, D-28759 Bremen, Germany,

Structural differentiation of bacterial chromatin depends on cooperative binding of abundant nucleoid-associated proteins at numerous genomic DNA sites and stabilization of distinct long-range nucleoprotein structures. Histone-like nucleoid-structuring protein (H-NS) is an abundant DNA-bridging, nucleoid-associated protein that binds to an AT-rich conserved DNA sequence motif and regulates both the shape and the genetic expression of the bacterial chromosome. Although there is ample evidence that the mode of H-NS binding depends on environmental conditions, the role of the spatial organization of H-NS-binding sequences in the assembly of long-range nucleoprotein structures remains unknown. In this study, by using high-resolution atomic force microscopy combined with biochemical assays, we explored the formation of H-NS nucleoprotein complexes on circular DNA molecules having different arrangements of identical sequences containing high-affinity H-NS-binding sites. We provide the first experimental evidence that variable sequence arrangements result in various three-dimensional nucleoprotein structures that differ in their shape and the capacity to constrain supercoils and compact the DNA. We believe that the DNA sequence-directed versatile assembly of periodic higher-order structures reveals a general organizational principle that can be exploited for knowledge-based design of long-range nucleoprotein complexes and purposeful manipulation of the bacterial chromatin architecture.
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http://dx.doi.org/10.1074/jbc.M117.780239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418058PMC
May 2017

Spatial confinement induces hairpins in nicked circular DNA.

Nucleic Acids Res 2017 05;45(8):4905-4914

Laboratory of Physics of Living Matter, EPFL, 1015 Lausanne, Switzerland.

In living cells, DNA is highly confined in space with the help of condensing agents, DNA binding proteins and high levels of supercoiling. Due to challenges associated with experimentally studying DNA under confinement, little is known about the impact of spatial confinement on the local structure of the DNA. Here, we have used well characterized slits of different sizes to collect high resolution atomic force microscopy images of confined circular DNA with the aim of assessing the impact of the spatial confinement on global and local conformational properties of DNA. Our findings, supported by numerical simulations, indicate that confinement imposes a large mechanical stress on the DNA as evidenced by a pronounced anisotropy and tangent-tangent correlation function with respect to non-constrained DNA. For the strongest confinement we observed nanometer sized hairpins and interwound structures associated with the nicked sites in the DNA sequence. Based on these findings, we propose that spatial DNA confinement in vivo can promote the formation of localized defects at mechanically weak sites that could be co-opted for biological regulatory functions.
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http://dx.doi.org/10.1093/nar/gkx098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605231PMC
May 2017

Hyperplectonemes: A Higher Order Compact and Dynamic DNA Self-Organization.

Nano Lett 2017 03 16;17(3):1938-1948. Epub 2017 Feb 16.

Laboratory of Physics of Living Matter, Ecole Polytechnique Fédérale de Lausanne , 1015 Lausanne, Switzerland.

Bacterial chromosome has a compact structure that dynamically changes its shape in response to bacterial growth rate and growth phase. Determining how chromatin remains accessible to DNA binding proteins, and transcription machinery is crucial to understand the link between genetic regulation, DNA structure, and topology. Here, we study very large supercoiled dsDNA using high-resolution characterization, theoretical modeling, and molecular dynamics calculations. We unveil a new type of highly ordered DNA organization forming in the presence of attractive DNA-DNA interactions, which we call hyperplectonemes. We demonstrate that their formation depends on DNA size, supercoiling, and bacterial physiology. We compare structural, nanomechanic, and dynamic properties of hyperplectonemes bound by three highly abundant nucleoid-associated proteins (FIS, H-NS, and HU). In all these cases, the negative supercoiling of DNA determines molecular dynamics, modulating their 3D shape. Overall, our findings provide a mechanistic insight into the critical role of DNA topology in genetic regulation.
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http://dx.doi.org/10.1021/acs.nanolett.6b05294DOI Listing
March 2017

Micro- and nanoscale hierarchical structure of core-shell protein microgels.

J Mater Chem B 2016 Dec 25;4(48):7989-7999. Epub 2016 Nov 25.

Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW, UK.

Protein nanofibrils were first discovered in the context of misfolding and neurodegenerative diseases but have recently been found in naturally occurring functional materials including algal adhesives, bacterial coatings, and even mammalian melanosomes. These physiologically beneficial roles have led to the exploration of their use as the basis for artificial protein-based functional materials for a range of applications as bioscaffolds and carrier agents. In this work, we fabricate core-shell protein microgels stabilized by protein fibrillation with hierarchical structuring on scales ranging from a few nanometers to tens of microns. With the aid of droplet microfluidics, we exploit fibrillar protein self-assembly together with the aqueous phase separation of a polysaccharide and polyethylene glycol to control the internal structure of the microgels on the micro- and nanoscales. We further elucidate the local composition, morphology, and structural characteristics of the microgels and demonstrate a potential application of core-shell protein microgels for controlling the storage and sequential release of small drug-like molecules. The controlled self-assembly of protein nanofibrils into hierarchical structures can be used in this manner to generate a class of nanomaterials with a range of potential functions and applications.
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http://dx.doi.org/10.1039/c6tb02683dDOI Listing
December 2016

Kinetics of Antibody Binding to Membranes of Living Bacteria Measured by a Photonic Crystal-Based Biosensor.

Biosensors (Basel) 2016 Oct 11;6(4). Epub 2016 Oct 11.

Laboratoire de Physique de la Matière Vivante, IPHYS, École Polytechnique Fédérale de Lausanne (EPFL); Rte de la Sorge, 1015 Lausanne, Switzerland.

Optical biosensors based on photonic crystal surface waves (PC SWs) offer a possibility to study binding interactions with living cells, overcoming the limitation of rather small evanescent field penetration depth into a sample medium that is characteristic for typical optical biosensors. Besides this, simultaneous excitation of - and -polarized surface waves with different penetration depths is realized here, permitting unambiguous separation of surface and volume contributions to the measured signal. PC-based biosensors do not require a bulk signal correction, compared to widely used surface plasmon resonance-based devices. We developed a chitosan-based protocol of PC chip functionalization for bacterial attachment and performed experiments on antibody binding to living bacteria measured in real time by the PCSW-based biosensor. Data analysis reveals specific binding and gives the value of the dissociation constant for monoclonal antibodies (IgG2b) against bacterial lipopolysaccharides equal to = 6.2 ± 3.4 nM. To our knowledge, this is a first demonstration of antibody-binding kinetics to living bacteria by a label-free optical biosensor.
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http://dx.doi.org/10.3390/bios6040052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5192372PMC
October 2016

Correction: The persistence length of adsorbed dendronized polymers.

Nanoscale 2016 10;8(39):17383

Department of Inorganic and Analytical Chemistry, University of Geneva, Sciences II, 30 Quai Ernest-Ansermet, 1205 Geneva, Switzerland.

Correction for 'The persistence length of adsorbed dendronized polymers' by Lucie Grebikova, et al., Nanoscale, 2016, 8, 13498-13506.
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http://dx.doi.org/10.1039/c6nr90204aDOI Listing
October 2016

Can Dissipative Properties of Single Molecules Be Extracted from a Force Spectroscopy Experiment?

Biophys J 2016 Sep;111(6):1163-1172

Laboratoire de Physique de la Matière Vivante, IPHYS, BSP, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland. Electronic address:

We performed dynamic force spectroscopy of single dextran and titin I27 molecules using small-amplitude and low-frequency (40-240 Hz) dithering of an atomic force microscope tip excited by a sine wave voltage fed onto the tip-carrying piezo. We show that for such low-frequency dithering experiments, recorded phase information can be unambiguously interpreted within the framework of a transparent theoretical model that starts from a well-known partial differential equation to describe the dithering of an atomic force microscope cantilever and a single molecule attached to its end system, uses an appropriate set of initial and boundary conditions, and does not exploit any implicit suggestions. We conclude that the observed phase (dissipation) signal is due completely to the dissipation related to the dithering of the cantilever itself (i.e., to the change of boundary conditions in the course of stretching). For both cases, only the upper bound of the dissipation of a single molecule has been established as not exceeding 3⋅10(-7)kg/s. We compare our results with previously reported measurements of the viscoelastic properties of single molecules, and we emphasize that extreme caution must be taken in distinguishing between the dissipation related to the stretched molecule and the dissipation that originates from the viscous damping of the dithered cantilever. We also present the results of an amplitude channel data analysis, which reveal that the typical values of the spring constant of a I27 molecule at the moment of module unfolding are equal to 4±1.5mN/m, and the typical values of the spring constant of dextran at the moment of chair-boat transition are equal to 30-50mN/m.
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http://dx.doi.org/10.1016/j.bpj.2016.08.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5034718PMC
September 2016
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