Poster Session I
Wednesday, 15 September
17:00-18:00 PM (CEST)
Dr. Iuliana Stoica
Dr. Andreea Irina Barzic
Disha Mohan Bangalore
Prof. Maria Starodubtseva
Dr. Paolo Moretti
Dr. Cai Wanhao
Extracellular Vesicles and Atomic Force Microscopy
Probing the ring motion along the polymer track
Pretesting laser-patterned polyimide films as substrates for flexible electronic devices
A new texturing approach of a polyimide shielding cover for enhanced light propagation in photovoltaic devices
Automated AFM analysis of DNA bending reveals initial lesion sensing strategies of DNA glycosylases
Biomolecular Interactions EVs-antibodies by AFM
Visualization of the cellular uptake of extracellular vesicles (EVs) of Trypanosoma cruzi by AFM
AFM-based detection of microparticle interaction with lipid bilayers
Application of machine learning methods to the analysis of multidimensional AFM images of the endothelial cell surface
Guanosine hydrogel: understanding nanodimensional nets
Effect of x-rays on nanomechanical properties of red blood cells of rats fed a cholesterol-rich diet
Angle-dependent Force Spectroscopy shows Direction Dependence of Single-molecule Mechanics
Characterization of multivalent interactions using multifunctional polymers
Multiple receptors mediated Ebola virus adhesion to cells
AFM characterization of sensory filiform mechanosensory hair on cockroach cercus
Pietro Parisse: "Extracellular Vesicles and Atomic Force Microscopy"
Italian National Research Council
Keywords: Extracellular vesicles, biomechanical properties, atomic force microscopy, supported lipid bilayers
Extracellular vesicles (EVs) are tiny lipid-bilayers enclosed containers sending messages through their cargo
and surface markers from one cell to another. Initially considered as “trash”, they are getting more and more
recognized as intercellular mediators and actual players in the intercellular communications and extracellular
matrix remodeling . Due to their heterogeneity in size (from 30 nm to micrometer scale) and in origin
(endosomal or from cell membrane budding) their isolation and characterization are not yet standardized
hampering their actual use in diagnosis and/or therapy . Among the different characterization techniques
of EVs, Atomic Force Microscopy starts to be a quite common tool for the direct visualization of single vesicles
and for the evaluation of size distribution . Moreover combined with force-spectroscopy can provide
nanomechanical characterization helping in distinguishing vesicles from other co-isolated particles and in
defining peculiar properties of specific subpopulations of EVs . Here together with the classical study of
EVs immobilized on surfaces (left panel of Figure 1) we will show how AFM can turn useful also for the
investigation of the interaction of EVs with the plasma membrane , giving morphological and structural
insights on the fusion mechanisms (central panel of Figure 1) and also for the study of the functional role of
EVs in modifying the recipient cells properties. In fact, the EVs can induce a biomechanical rearrangement of
the recipient cell altering its mechanical response that can be recorded by force spectroscopy measurements
(right panel of Figure 1).
Figure 1: Schematic of the different experiments where Atomic Force Microscopy is employed to study different
properties of EVs. (Left panel) Three-dimensional 100nmx100nmx30nm Atomic Force Microscopy micrograph of EVs deposited on oxygen-plasma cleaned glass. Measurements performed in PBS buffer in AC mode (Olympus BL-AC40TS cantilever, spring constant: 0.1 N/m, 10 nm radius of curvature). Central panel: Three-dimensional 2μmx1μmx15nm Atomic Force Microscopy micrograph of EVs interacting with a DOPC supported lipid bilayer on mica. Measurements performed in PBS buffer in AC mode (Olympus BL-AC40TS cantilever, spring constant: 0.1 N/m, 10 nm radius of curvature). Right panel: Force-distance curves of MDA-MB-231 cells before (blue line) and after (red line) EV exposure. Measurements performed in PBS buffer with a CSG01-NTMDT tipless cantilever (spring constant: 0.004 N/m) with a glued silicon bead of 10 μm in diameter.
References:  M. Mathieu, et al. Nat Cell Biol 2019, 21, 9 ;  F. Royo, et al. Cells 2020, 9, 1955.  P. Parisse et al. Eur. Biophys. 2017, 46, 813, N. Sebaihi, et al. Meas. Sci. Technol. 2017, 28, 034006  R. Sorkin, et al. Small 2018, 14, 1801650. A Ridolfi, et al, Analytical chemistry 92 (2020) 10274  F. Perissinotto et al. , Nanoscale 13 (2021) 5224
Narangerel Ganbaatar: "Probing the ring motion along the polymer track of a macrorotaxane at the single molecule level"
University of Liege, Belgium
Molecular machines play an important role in a variety of key biological and cellular activities such as muscle contraction and intracellular vesicle transport. Many reports have been published on designing fully synthetic molecular machines and some successful examples show that they are able to use biased Brownian motion to perform work or collectively induce movement of much larger objects. For more than a decade, much of the information about how biological machines operate has been gleaned from force measurements made on single molecules. In contrast, few single-molecule force spectroscopy investigations have been successively realized on synthetic molecular machines. The rarity of such studies comes from the difficulty of developing proper tools and preparing appropriate molecules that can be interfaced with these techniques, especially when one wants to probe sub-molecular motions. Here, we report on the study of macrorotaxanes, i.e. rotaxanes made of a macrocycle threaded onto a polymer chain, by AFM-based single-molecule force spectroscopy. Specific monomers and functions have been included on the polymer thread, such as 2,6-pyridine dicarboxamide, to assess the ability of macrorotaxanes to act as single molecule chemical sequencers. The macrocycle is initially hold into place on the thread by a 3+1 Pd(II) complex between a tridendate pyridine bisamide ligand (macrocycle) and a monodendate pyridine ligand (thread). The bulky stopper near the macrocycle is equipped with a functional group allowing its chemisorption on a surface. A poly(ethylene oxide) chain is attached to the macrocycle to act as tether to link the macrocycle with the AFM tip. Force measurements indicate that the macrocycle does travel along the thread after being caught by the AFM tip and detaches from the binding station. Characteristic force profiles show the signature of the interaction between the macrocycle and functions present on the polymer thread, demonstrating the sequencing ability of the system.
Dr. Iuliana Stoica: "Pretesting laser-patterned polyimide films as substrates for flexible electronic devices"
Iuliana Stoica1, Ion Sava1, Elena Gabriela Hitruc1, Alexandru Florentin Trandabat2
1 “Petru Poni” Institute of Macromolecular Chemistry, 41A Grigore Ghica Voda Alley, 700487 Iasi, Romania
2 S.C. INTELECTRO IASI S.R.L, Iancu Bacalu Street, no. 3, 700029, Iasi, Romania
The proposed research is focused on 3D patterning high-performance azo-polyimide materials, with special properties, such as thermal stability, high glass transition temperature, low susceptibility to laser damage, low dielectric constant value, photochromic behavior, appropriate morphology, good piezoelectric response and not lastly high flexibility. The three-dimensional micro- and nanostructures thus obtained on these polymeric films optimized for optoelectronic and biomedical applications and characterized by atomic force microscopy are pretested as alternative to conventional support materials used in current flexible electronic devices. This research direction is a topical one, the studies and investigations involved can bring important benefits in surface science and nanotechnology in the future, by manipulating the various structures at the micro- and nanometric scale.
This work was funded by the national fellowship program L’Oreal – Unesco “For Women in Science”.
Dr. Andreea Irina Barzic: "A new texturing approach of a polyimide shielding cover for enhanced light propagation in photovoltaic devices"
Andreea Irina Barzic, Iuliana Stoica, Raluca Marinica Albu
“Petru Poni” Institute of Macromolecular Chemistry, 41A Grigore Ghica Voda Alley, 700487 Iasi, Romania
Among engineering polymers, polyimides (PIs) are widely known for their remarkable mechanical and thermal resistance. For photovoltaic applications, the PI shielding cover must have high optical clarity (no light absorption), but it also must possess specific surface features for achieving proper light management in the device. In this work, a novel strategy of surface texturing of PI layer is proposed, consisting in multi-directional rubbing, which could lead to a better light propagation. Atomic Force Microscopy (AFM) was employed to study the outcome of this potentially advanced light-trapping scheme. Since it is not clear yet if random or periodic surface morphology determines better light trapping in solar cells, the developed texturing approach creates a balance between these two aspects. Successive surface roughening with sand paper on several directions of PI film indicates important changes not only in surface topography, but also on the level of anisotropy. Such morphological analyses help to identify relevant light-trapping design routes that contribute to an enhanced conversion efficiency of photovoltaic devices.
Acknowledgements: This work was supported by a grant of the Ministry of Research, Innovation and Digitization, CNCS/CCCDI – UEFISCDI, project number TE 83/1.09.2020 within PNCDI III (code PN-III-P1-1.1-TE-2019-1878).
Disha Mohan Bangalore: "Automated AFM analysis of DNA bending reveals initial lesion sensing strategies of DNA glycosylases "
Julius Maximilians University, Wuerzburg, Germany
Base excision repair (BER) is the dominant DNA repair pathway of chemical modifications such as deamination, oxidation, or alkylation of DNA bases, which endanger genome integrity due to their high mutagenic potential. Detection and excision of the damaged base in BER is achieved by DNA glycosylases in a highly efficient manner. We developed an unbiased, high-throughput automated analysis approach of single molecule atomic force microscopy (AFM) imaging for the resolution of subtly different conformational states of several glycosylases (with different target lesions) during DNA lesion search. Our results lend support to a model of initial lesion detection that proposes preferential occupancy of target lesion sites by glycosylase search complexes based on altered mechanical properties at lesions. Furthermore, our data reveal dynamic intermediates in BER search and interrogation strategies on undamaged DNA. Since the presented approach is largely versatile, other applications with similar challenges and constraints will benefit, taking the power of single molecule AFM analyses of protein-DNA interactions to a new level.
Fatima Linares: "Biomolecular Interactions EVs-antibodies by AFM "
University of Granada, Spain
Atomic force microscopy (AFM) is nowadays frequently applied to determine interaction forces between biological molecules. Starting with the detection of the first discrete unbinding forces between ligands and receptors by AFM only several years ago, measurements have become more and more quantitative. At the same time, theories have been developed to describe and understand the dynamics of the unbinding process and experimental techniques have been refined to verify this theory. In addition, the detection of molecular recognition forces has been exploited to map and image the location of binding sites.
In this work we discuss the results for the interaction between extracellular exovesicles secreted by T. cruzi trypomastigotes Pan 4 and anti-Trans-sialidase (mAb39) antibodies. In addition, we emphasize the potential of chemically well-defined surface modification techniques to further improve reproducible AFM measurements.
The experimental part has consisted of four well-differentiated stages:
1.- Immobilization of trypomastigote EVs on the flat surface of mica sheets for using in the study of molecular recognition.
2.- Development a protocol for the immobilization of the antibody in the AFM tips for use in force measurements and to check its reproducibility.
3.- To study for the first time the process of molecular recognition through the measurement of the breakdown forces between the anti-Transialidase antibody and the afore mentioned exovesicles, as well as the work of adhesion in said process.
4.- To determine the specificity of the bond break events obtained with dynamic force spectroscopy.
Oscar H. Willemsen et al. Biophysical Journal, Volume 79, 2000, 3267–328. Hinterdorfer et al., Single Molecules, Volume 1, Issue 2, 2000, p. 99-103.
Ebner A. et al, J. of Chemical Physics and Physical Chemistry , 6, 2005, 897-900.
Alexa Prescilla: "Visualization of the cellular uptake of extracellular vesicles (EVs) of Trypanosoma cruzi by AFM "
University of Granada, Spain
Extracellular vesicles (EV) are nanovesicles secreted by all cell types that perform different functions in cell biology, especially in intercellular communication. Its size and structure and biology will depend on the cell type of origin and its "Cargo" of proteins, nucleic acids, and lipids. Due to its small size, different methods have been used for its characterization; Morphological analyzes have been performed almost exclusively by transmission electron microscopy. In this work, we have used atomic force microscopy (AFM) to characterize the mechanisms of interaction of the EVs of Trypanosoma cruzi, the agent of Chagas disease, with cells.
Some studies point to different mechanisms of interaction, internalization, and uptake, among which are the induction of endocytosis by the recipient cell, and the membrane-cell membrane fusion of EV. In macrophages, it is carried out by phagocytosis. In the present work, using Atomic Force Microscopy, we have studied the internalization process in non-phagocytic Vero cells at different observation times 5, 7.5, and 10 min to observe the evolution of the EVs uptake process.
Parisse, P. et al, 2017, European Biophysics Journal 46(8):813–20.
Xiao, L. et al., 2013, Analytical Methods, 5(4), pp.874–879.
Rebecca Schlatterer: "AFM-based detection of microparticle interaction with lipid bilayers"
Albert-Ludwigs-Universität Freiburg, Germany
Plastic is practical, versatile and we can hardly imagine our modern life without it. But plastic can also be harmful, both to the environment and our health. Few threats to today’s nature are as visible as the burden of plastic waste, the impact on the environment through pollution is easily recognizable. The impact on our health on the other side is harder to quantify. One question for example is, how far plastic particles can penetrate the body, especially since plastic has long been proven to be part of the food chain and is used for biomedical applications. Experimental data is needed to shed light into the interaction of polymeric materials with living organisms, in particular with cell membranes, at different scales.[2-4]
Here, we study the interaction of beads of different sizes and materials with supported lipid bilayers as model systems for microparticles interacting with lipid membranes. First, a combination of AFM-based imaging and fluorescence microscopy is used to examine the quality of the supported lipid bilayers. Subsequently, AFM-based force spectroscopy is used to investigate the interaction mechanism of beads with these bilayers. Finally, single polymers are used to better understand the molecular mechanism for the entry of micro to nano sized objects into lipid bilayers. This will serve for assessing the impact of plastics in the environment to biological systems and might lead to the design of more environment friendly types of polymeric materials in the future.
 D. M. Mitrano, P. Wick, B. Nowack, Nat. Nanotechnol. 2021, 16, 491–500.
 B. Jing, R. C. T. Abot, Y. Zhu, The Journal of Physical Chemistry B 2014, 118,
 G. Rossi, J. Barnoud, L. Monticelli, Journal of Physical Chemistry Letters 2014, 5,
 M. Schulz, A. Olubummo, W. H. Binder, Soft Matter 2012, 8, 4849–4864.
Prof. Maria Starodubtseva: "Application of machine learning methods to the analysis of multidimensional AFM images of the endothelial cell surface"
Gomel State Medical University, Belarus
Machine learning methods are widely used and have already proven their effectiveness for practical purposes in medicine and biology. Atomic force microscopy (AFM) of the cell surface provides the researcher with information about a complex of structural and mechanical properties recorded simultaneously in different channels. The multidimensionality of the data obtained by AFM scanning (for example, when using modes such as PeakForce QNM™ (Bruker), Hybrid mode™ (NT-MDT), PinPoint™ (Park Systems) is an obvious advantage of the AFM method, but multidimensional data is rarely used for complex analysis of the properties and state of the cell surface. The work aims to demonstrate the effectiveness of using machine learning methods to determine the difference in the states of endothelial cell surface areas depending on the cell zones and the treatment of cells with antibodies.
Endothelial cells (HUVEC) were isolated from the human umbilical vein and cultivated during 2-4 passages. The cell monolayer was treated with antibodies (IgG and monoclonal antibodies against CD109 antigen of cell surface), fixed with 2% glutaraldehyde, washed with phosphate-buffered saline, and distilled water, and dried at room temperature. Scanning in air was performed using a Bruker BioScope Resolve AFM and a Scanasyst-Air probe (Bruker) in MIROview mode. Classification, accuracy estimation, division into training (80%) and testing (20%) samples, and estimation of the coefficient of determination (R2) were implemented in Python using the scikit-learn, numpy, pandas, and xgboost libraries. We have analyzed the sets of endothelial cell surface AFM images (1 µm x 1 µm, 256 × 256 pixels) recorded in channels: adhesion, height sensor, Log(DMT Modulus), and deformation. AFM image samples included three 'cell treatment' sets (control, processing of IgG cells, and IgG+CD109 AB) each of that contained three 'cell surface zone' sub-sets (nuclear, perinuclear, and peripheral zones). We carried out the classification of a complex cell surface profile composed of four profiles of different AFM images corresponding to a certain position and containing 1024 (4×256) points in each using three algorithms of machine learning: xgboost (decision trees gradient boosting), k-nearest neighbors, decision trees. Our findings showed that the worst classification accuracy was when using the decision trees model, the best when using xgboost. The results indicated the difference in the complex profiles of the spatial distribution of cell surface structural and mechanical properties (1) for different cell surface zones, (2) for one zone type of cells without or after the cell treatment with different antibodies.
Conclusion: Machine learning-based classification of complex profiles of the spatial distribution of four AFM parameters predicted the difference in the cell surface layer state for endothelial cells in different cell zones and after cell treatment with anti-endothelial cells antibodies.
The work was carried out within the framework of the project of the Belarusian Republican Foundation for Fundamental Research (M20KИ-026, 2020-2021, Belarus).
Dr. Paolo Moretti: "Guanosine hydrogel: understanding nanodimensional nets"
Università Politecnica delle Marche, Italy
Supramolecular hydrogels are a new class of biomaterials with potential applications in tissue engineering and drug-delivery because of their unique properties such as biocompatibility, biostability, self-assembling, self-healing and external stimuli-responsiveness .
The case of guanosine is particularly relevant . Guanosine 5'-monophosphate (GMP) in water self-assembles in supramolecular, columnar helicoidal structures (G-quadruplexs), made by stacked GMP planar tetramers (G-quartets). If the hydrophobic guanosine (Gua) is also present in solution, stable and transparent hydrogels, made by a quadruplex 3D network, occur at high hydration (up to 99% w/w of water) and over a large temperature range: in fact, GMP helps to solubilize the insoluble guanosine (that enters in the formation of G-quartets) and the insolubility of Gua promotes the gelation at low concentrations, probably because of the reduction of the number of charges-per-unit-length in the G-quadruplexes. Quadruplex interactions, flexibility and chirality seem to play a unique role in the hydrogel formation and stability.
Here, an extended Atomic Force Microscopy study, combined with Small Angle X-ray Scattering and Dynamic Light Scattering experiments on hydrogels prepared at different Gua/GMP molar ratios (1:2; 1:4) as well as in the presence of additional molecules (ThT, DAPI) mimicking intercalating anti-cancer drugs, will be presented. The discussion on the structural properties of G-quadruplexes will focus on the composition effects on the quadruplex pitch, effects of ThT and DAPI binding on the network stiffness and on the quadruplex (un)winding and on the interplay between knotting and quadruplex flexibility.
1. Dong et al, Biomater. Sci., 2015,3,7
2. Kopeček J et al, Chem Int Ed Engl. 2012, 51, 7396
3. F. Carducci et al, Soft Matter,2018,14,2938.
Nastassia Shkliarava: "Effect of x-rays on nanomechanical properties of red blood cells of rats fed a cholesterol-rich diet"
Institute of Radiobiology of the NAS of Belarus, Gomel, Belarus
Atomic force microscopy (AFM) allows investigating the cell membrane mechanical properties that determine the cell's ability to migrate, divide, maintain morphological structure, polarity, and others. The state of the cell surface can change under pathological conditions including ionizing radiation. Cholesterol belongs to the main fraction of membrane lipids and plays a crucial role in maintaining the physical properties of the membrane through interaction with both other lipids and proteins. Changes in the saturation of the cell membrane with cholesterol can affect its lipid organization and, as a result, mechanical properties. Our work aims to reveal the effects of a cholesterol-rich diet and X-rays on the structural and mechanical properties of the rat erythrocyte membrane using the AFM nanomechanical mapping mode.
The blood of 9 month rats fed a cholesterol-rich diet (2 months) was irradiated by X-rays with dose of 1Gy and 100Gy in vitro. Erythrocyte samples was prepared using 1% glutaraldehyde fixation, washing in phosphate-buffered saline, placing on the glass slides with an adhesive coating and drying at room temperature. The cell samples were scanned in air using AFM Bruker Bioscope Resolve (USA) in PFQNM in Air mode using a SCANASYST-AIR probe (Bruker). Probe calibration was performed at 2 and 0.5 kHz with drive amplitude 150 nm and ramp setpoint 0.2 V. Scan size was 250 nm × 250nm; feedback gain 0.8 and PFS 500 pN. To perform ANOVA test to the raw AFM data using R Studio, the "calico" method was used, in which every third line of the AFM image was removed from the sample to be analyzed. For analyzing the median values of the AFM images of different types (adhesion force map, DMT modulus map, topography map) the on-line One-way ANOVA on ranks calculator was used (https://www.statskingdom.com). We found that 1 Gy X-rays enhanced the stiffness and 100 Gy X-rays returned the stiffness of the erythrocyte surface at the nanoscale to approximately control values (ANOVA, p<0.00002). At the same time, there were no significant changes in the averaged adhesion force and topographic roughness at the nanoscale under the studied conditions. However, using power spectral density analysis of the maps of the adhesion force, we revealed the significant change in the averaged spatial period of the maps that pointed to a decrease at 1 Gy and return to control values at 100 Gy of the cell size of the membrane skeleton network (ANOVA, p<0.0071).
Conclusion: X-rays in the dose range between 1 Gy and 100 Gy cause the membrane skeleton rearrangements that lead to changes in the stiffness of the erythrocyte surface at the nanoscale in rats fed a cholesterol-rich diet.
Financial support from State Research Program “Natural Resources and the Environment” (task 3.1.2, 2021-2025; Belarus) is gratefully acknowledged.
Cai Wanhao: "Angle-dependent Force Spectroscopy shows Direction Dependence of Single-molecule Mechanics"
Università Politecnica delle Marche, Italy
Single molecule mechanics is of fundamental importance in physical and biological systems, because it lays the base of the macroscopic structure and behaviors. The elastic response of several types of single polymer chains and the rupture of various bonds have been studied by atomic force microscopy-based single molecule force spectroscopy (SMFS) using vertical pulling [1-3]. However, the mechanic response along different pulling directions still remains unclear. Herein, by utilizing angle-dependent SMFS, a single polyethylene glycol (PEG) chain with a S-Si bond acting as an anchor to the surface is studied under various pulling angles and velocities (force loading rates). Then the chain elasticity and anchor bond strength are reconstructed along the pulling direction. Surprisingly, we find different effects of pulling direction on the elastic response compared to bond rupture (anchoring strength). The chain elasticity is virtually constant while the bond rupture is severely affected by the pulling angle. Our results shed new light on the interplay between friction and adhesion at the single molecule level. Moreover, they may guide the bottom-up design of force-responsive materials.
1. Cai, W.; Xu, D.; Qian, L.; Wei, J.; Xiao, C.; Qian, L.; Lu, Z.-Y.; Cui, S. Force-induced Transition of π-π Stacking in a Single Polystyrene Chain. J. Am. Chem. Soc. 2019, 141, 9500-9503.
2. Kolberg, A.; Wenzel, C.; Hackenstrass, K.; Schwarzl, R.; Rüttiger, C.; Hugel, T.; Gallei, M.; Netz, R. R.; Balzer, B. N. Opposing temperature dependence of the stretching response of single PEG and PNiPAM polymers. J. Am. Chem. Soc. 2019, 141, 11603-11613.
3. Xue, Y.; Li, X.; Li, H.; Zhang, W. Quantifying thiol–gold interactions towards the efficient strength control. Nat. Commun., 2014, 5, 1-9.
Max Lallemang: "Characterization of multivalent interactions using multifunctional polymers"
University of Freiburg, Italy
Multivalent interactions describe the additivity of weak non-covalent interactions and are essential in biological and chemical systems. [1, 2] Compared to monovalent interactions, they show increased strength and selectivity, but due to their complexity, they are much less understood. Here, we use atomic force microscopy (AFM)-based single molecule force spectroscopy (SMFS) of catechol-based multifunctional polymers, compromising azide, alkyne, quinone and amine groups, on non-crosslinked and crosslinked polymer films of the same to study the strength of multivalent bonds. We were able to detect single molecule rupture events in force-extension curves with forces of up to several nanonewton and could correlate these forces to the presence of different types of bonds. Thus, we could obtain force-extension signatures of multivalent physical and chemical bonds. Hence, we could show how multivalent interactions serve to create stable polymer layers using multifunctional polymers. Our findings strongly support the idea to use complex polymers with different chemical moieties for future polymer coatings.
 C. Fasting, C.A. Schalley, M. Weber, O. Seitz, S. Hecht, B. Koksch, J. Dernedde, C. Graf, E.W. Knapp, R. Haag, Angewandte Chemie International Edition, 51 (2012) 10472-10498.
 M.l.W. Kulka, I.S. Donskyi, N. Wurzler, D. Salz, O.z. Özcan, W.E. Unger, R. Haag, ACS Applied Bio Materials, 2 (2019) 5749-5759.
Qingrong Zhang: "Multiple receptors mediated Ebola virus adhesion to cells"
Université catholique de Louvain, Belgium
Ebola virus (EboV) is a dangerous and fatal virus affecting humans and other primates1. EboV infects a wide range of mammalian cells (from immune cells to epithelial cells) by binding a variety of cell surface receptors, including the T-cell Ig and mucin domain 1 (TIM-1) and C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin receptor (DC-SIGNR). TIM1 does not only directly interact with the phosphatidylserine (PS) on the surface of the virion lipid membrane, but possibly also binds to the viral glycoprotein (GP). On the opposite, DC-SIGNR has been identified to specifically binds to EboV GP. However, the mechanism as well as the kinetic and thermodynamic parameters describing the early binding step of EboV to cell surface remain unclear, pointing out the need to clearly identify the role of each receptors during early step of EboV infection.
Here, we analyze the interactions between single EboV particles and DC-SIGNR or TIM1 receptors using force-distance curve-based atomic force microscopy (FD-based AFM)2. We found that the EboV specifically binds to DC-SIGNR or TIM1 by viral GP. The kinetics and thermodynamics parameters of the interactions show the binding of EboV-receptors is high-affinity in vitro. Using the FD-based AFM combined fluorescent microscopy, we showed that the binding capacity of EboV-receptors is identical on living cells. Interestingly, since TIM1 can directly interact with GP and PS at the single-molecule level, we also confirmed the fact that TIM1 acts as dual-function receptors of EboV.
Figure: Probing EboV binding to receptors. (a) overlay images of GFP and DIC channel, showing the confluent layer of mock HEK 293T and HEK 293T-TIM1-coGFP and the AFM tip placed above. FD-based AFM height image (b) and adhesion map (c). (d) the EboV high-affinity bind to DC-SIGNR or TIM1 by viral GP, PS on the surface of the virion lipid membrane directly binds to TIM1 after GP cleaved by an enzyme (TACE).
1. Jacob, S. T. et al. Ebola virus disease. Nat. Rev. Dis. Primers, 6, 13 (2020).
2. Alsteens, D. et al. Nanomechanical mapping of first binding steps of a virus to animal cells. Nat. Nanotechnol., 12, 177–183 (2017).
Janusu Strzelecki: "AFM characterization of sensory filiform mechanosensory hair on cockroach cercus"
Nicolaus Copernicus University, Poland
Invertebrates can sense even minute air currents with filiform mechanosensory hairs covering various places of their bodies. In cockroaches these hairs can be found mostly on a pair of horn-like abdominal appendages, called cerci. In P. americana cockroach the ventral surface of each cercus contains about 200 filiform hairs. Movement of only one such hair the hair in one direction along its plane induces a depolarization in the receptor cell and a burst of action potentials (APs) in the sensory neuron. With such highly sensitive response to low velocity air currents filiform hairs of cockroaches are of great interest to neuroscientist and engineers alike. However, only limited structural and biomechanical characterization of those hairs was made. Atomic Force Microscope (AFM) is an ideal tool for this task, as it allows both high resolution imaging and force spectroscopy mechanical measurements. We report results of contact mode imaging of filiform hair surface showing intricate anatomical details. Callibrated AFM cantilever was also used to deform single hairs and an average value of 2GPa stiffness was obtained. A comparative analysis with results obtained for non-sensory scopula hairs was also made.