181 results found
Sato K, Sato F, Kumano M, et al., 2021, Electrochemical Quantitative Evaluation of the Surface Charge of a Poly(1-vinylimidazole) Multilayer Film and Application to Nanopore pH Sensor, ELECTROANALYSIS, ISSN: 1040-0397
Maynard SA, Pchelintseva E, Zwi-Dantsis L, et al., 2021, IL-1β mediated nanoscale surface clustering of integrin α5β1 regulates the adhesion of mesenchymal stem cells, Scientific Reports, Vol: 11, Pages: 1-14, ISSN: 2045-2322
Clinical use of human mesenchymal stem cells (hMSCs) is limited due to their rapid clearance, reducing their therapeutic efficacy. The inflammatory cytokine IL-1β activates hMSCs and is known to enhance their engraftment. Consequently, understanding the molecular mechanism of this inflammation-triggered adhesion is of great clinical interest to improving hMSC retention at sites of tissue damage. Integrins are cell–matrix adhesion receptors, and clustering of integrins at the nanoscale underlies cell adhesion. Here, we found that IL-1β enhances adhesion of hMSCs via increased focal adhesion contacts in an α5β1 integrin-specific manner. Further, through quantitative super-resolution imaging we elucidated that IL-1β specifically increases nanoscale integrin α5β1 availability and clustering at the plasma membrane, whilst conserving cluster area. Taken together, these results demonstrate that hMSC adhesion via IL-1β stimulation is partly regulated through integrin α5β1 spatial organization at the cell surface. These results provide new insight into integrin clustering in inflammation and provide a rational basis for design of therapies directed at improving hMSC engraftment.
Kolmogorov VS, Erofeev AS, Woodcock E, et al., 2021, Mapping mechanical properties of living cells at nanoscale using intrinsic nanopipette-sample force interactions dagger, Nanoscale, Vol: 13, Pages: 6558-6568, ISSN: 2040-3364
Mechanical properties of living cells determined by cytoskeletal elements play a crucial role in a wide range of biological functions. However, low-stress mapping of mechanical properties with nanoscale resolution but with a minimal effect on the fragile structure of cells remains difficult. Scanning Ion-Conductance Microscopy (SICM) for quantitative nanomechanical mapping (QNM) is based on intrinsic force interactions between nanopipettes and samples and has been previously suggested as a promising alternative to conventional techniques. In this work, we have provided an alternative estimation of intrinsic force and stress and demonstrated the possibility to perform qualitative and quantitative analysis of cell nanomechanical properties of a variety of living cells. Force estimation on decane droplets with well-known elastic properties, similar to living cells, revealed that the forces applied using a nanopipette are much smaller than in the case using atomic force microscopy. We have shown that we can perform nanoscale topography and QNM using a scanning procedure with no detectable effect on live cells, allowing long-term QNM as well as detection of nanomechanical properties under drug-induced alterations of actin filaments and microtubulin.
Savin N, Kolmogorov V, Iakovlev A, et al., 2021, Antimicrobial Activity of Antifungal Drugs on Candida Parapsilosis Studied by Scanning Ionconductance Microscopy (SICM), Publisher: CELL PRESS, Pages: 361A-361A, ISSN: 0006-3495
Kolmogorov V, Vaneev A, Efremov YM, et al., 2021, Studying the Local Young's Modulus of PC-3 Cells Via Scanning Ion-Conductance Microscopy, Publisher: CELL PRESS, Pages: 162A-162A, ISSN: 0006-3495
Vaneev A, Timoshenko R, Kolmogorov V, et al., 2021, Electrochemical Method for Real-Time ROS Measurements in Single Cells, Publisher: CELL PRESS, Pages: 365A-365A, ISSN: 0006-3495
Ren R, Wang X, Cai S, et al., 2020, Selective sensing of proteins using aptamer functionalised nanopore extended field-effect transistors, Small Methods, Vol: 4, Pages: 1-8, ISSN: 2366-9608
The ability to sense proteins and protein‐related interactions at the single‐molecule level is becoming of increasing importance to understand biological processes and diseases better. Single‐molecule sensors, such as nanopores have shown substantial promise for the label‐free detection of proteins; however, challenges remain due to the lack of selectivity and the need for relatively high analyte concentrations. An aptamer‐functionalized nanopore extended field‐effect transistor (nexFET) sensor is reported here, where protein transport can be controlled via the gate voltage that in turn improves single‐molecule sensitivity and analyte capture rates. Importantly, these sensors allow for selective detection, based on the choice of aptamer chemistry, and can provide a valuable addition to the existing methods for the analysis of proteins and biomarkers in biological fluids.
Bednarska J, Novak P, Korchev Y, et al., 2020, Release of insulin granules by simultaneous, high-speed correlative SICM-FCM, Journal of Microscopy, Vol: 282, Pages: 21-29, ISSN: 0022-2720
Exocytosis of peptides and steroids stored in a dense core vesicular (DCV) form is the final step of every secretory pathway, indispensable for the function of nervous, endocrine and immune systems. The lack of live imaging techniques capable of direct, label‐free visualisation of DCV release makes many aspects of the exocytotic process inaccessible to investigation. We describe the application of correlative scanning ion conductance and fluorescence confocal microscopy (SICM‐FCM) to study the exocytosis of individual granules of insulin from the top, nonadherent, surface of pancreatic β‐cells. Using SICM‐FCM, we were first to directly follow the topographical changes associated with physiologically induced release of insulin DCVs. This allowed us to report the kinetics of the full fusion of the insulin vesicle as well as the subsequent solubilisation of the released insulin crystal.
Anand U, Jones B, Korchev Y, et al., 2020, CBD effects on TRPV1 signaling pathways in cultured DRG neurons, Journal of Pain Research, Vol: 2020, Pages: 2269-2278, ISSN: 1178-7090
Introduction: Cannabidiol (CBD) is reported to produce pain relief, but the clinically relevant cellular and molecular mechanisms remain uncertain. The TRPV1 receptor integrates noxious stimuli and plays a key role in pain signaling. Hence, we conducted in vitro studies, to elucidate the efficacy and mechanisms of CBD for inhibiting neuronal hypersensitivity in cultured rat sensory neurons, following activation of TRPV1. Methods: Adult rat dorsal root ganglion (DRG) neurons were cultured, and supplemented with the neurotrophic factors NGF and GDNF, in an established model of neuronal hypersensitivity. 48 h after plating, neurons were stimulated with CBD (Adven 150, EMMAC Life Sciences) at 1, 10, 100 nMol/L and 1, 10 and 50 µMol/L. In separate experiments, DRG neurons were also stimulated with capsaicin with or without CBD (1 nMol/L to10 µMol/L), in a functional calcium imaging assay. The effects of the adenylyl cyclase activator forskolin and the calcineurin inhibitor cyclosporin were determined. We also measured forskolin-stimulated cAMP levels, without and after treatment with CBD, using a homogenous time resolved fluorescence (HTRF) assay. The results were analysed using Student’s t-test. Results: DRG neurons treated with 10 and 50 µMol/L CBD showed calcium influx, but not at lower doses. Neurons treated with capsaicin demonstrated robust calcium influx, which was dose-dependently reduced in the presence of low dose CBD (IC50 = 100 nMol/L). The inhibition or desensitization by CBD was reversed in the presence of forskolin and cyclosporin. Forskolin stimulated cAMP levels were significantly reduced in CBD treated neurons.Conclusions: CBD at low doses corresponding to plasma concentrations observed physiologically, inhibits or desensitizes neuronal TRPV1 signalling by inhibiting the adenylyl cyclase – cAMP pathway, which is essential for maintaining TRPV1 phosphorylation and sensitization. CBD also facilitated calcineurin-med
Bednarska J, Pelchen-Matthews A, Novak P, et al., 2020, Rapid formation of human immunodeficiency virus-like particles., Proceedings of the National Academy of Sciences of USA, Vol: 117, Pages: 21637-21646, ISSN: 0027-8424
Understanding the molecular mechanisms involved in the assembly of viruses is essential for discerning how viruses transmit from cell to cell and host to host. Although molecular aspects of assembly have been studied for many viruses, we still have little information about these events in real time. Enveloped viruses such as HIV that assemble at, and bud from, the plasma membrane have been studied in some detail using live cell fluorescence imaging techniques; however, these approaches provide little information about the real-time morphological changes that take place as viral components come together to form individual virus particles. Here we used correlative scanning ion conductance microscopy and fluorescence confocal microscopy to measure the topological changes, together with the recruitment of fluorescently labeled viral proteins such as Gag and Vpr, during the assembly and release of individual HIV virus-like particles (VLPs) from the top, nonadherent surfaces of living cells. We show that 1) labeling of viral proteins with green fluorescent protein affects particle formation, 2) the kinetics of particle assembly on different plasma membrane domains can vary, possibly as a consequence of differences in membrane biophysical properties, and 3) VLPs budding from the top, unimpeded surface of cells can reach full size in 20 s and disappear from the budding site in 0.5 to 3 min from the moment curvature is initially detected, significantly faster than has been previously reported.
Swiatlowska P, Sanchez-Alonso JL, Mansfield C, et al., 2020, Short-term angiotensin II treatment regulates cardiac nanomechanics via microtubule modifications., Nanoscale, Vol: 12, Pages: 16315-16329, ISSN: 2040-3364
Mechanical properties of single myocytes contribute to the whole heart performance, but the measurement of mechanics in living cells at high resolution with minimal force interaction remains challenging. Angiotensin II (AngII) is a peptide hormone that regulates a number of physiological functions, including heart performance. It has also been shown to contribute to cell mechanics by inducing cell stiffening. Using non-contact high-resolution Scanning Ion Conductance Microscopy (SICM), we determine simultaneously cell topography and membrane transverse Young's modulus (YM) by a constant pressure application through a nanopipette. While applying pressure, the vertical position is recorded and a deformation map is generated from which YM can be calculated and corrected for the uneven geometry. High resolution of this method also allows studying specific membrane subdomains, such as Z-grooves and crests. We found that short-term AngII treatment reduces the transversal YM in isolated adult rat cardiomyocytes acting via an AT1 receptor. Blocking either a TGF-β1 receptor or Rho kinase abolishes this effect. Analysis of the cytoskeleton showed that AngII depletes microtubules by decreasing long-lived detyrosinated and acetylated microtubule populations. Interestingly, in the failing cardiomyocytes, which are stiffer than controls, the short-term AngII treatment also reduces the YM, thus normalizing the mechanical state of cells. This suggests that the short-term softening effect of AngII on cardiac cells is opposite to the well-characterized long-term hypertrophic effect. In conclusion, we generate a precise nanoscale indication map of location-specific transverse cortical YM within the cell and this can substantially advance our understanding of cellular mechanics in a physiological environment, for example in isolated cardiac myocytes.
Vaneev AN, Gorelkin PV, Garanina AS, et al., 2020, In Vitro and In Vivo Electrochemical Measurement of Reactive Oxygen Species After Treatment with Anticancer Drugs, ANALYTICAL CHEMISTRY, Vol: 92, Pages: 8010-8014, ISSN: 0003-2700
Takahashi Y, Kobayashi Y, Wang Z, et al., 2020, High-Resolution Electrochemical Mapping of the Hydrogen Evolution Reaction on Transition-Metal Dichalcogenide Nanosheets, ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, Vol: 59, Pages: 3601-3608, ISSN: 1433-7851
Takahashi Y, Zhou Y, Miyamoto T, et al., 2020, High-Speed SICM for the Visualization of Nanoscale Dynamic Structural Changes in Hippocampal Neurons, ANALYTICAL CHEMISTRY, Vol: 92, Pages: 2159-2167, ISSN: 0003-2700
Alova A, Erofeev A, Gorelkin P, et al., 2020, Prolonged oxygen depletion in microwounded cells of Chara corallina detected with novel oxygen nanosensors, JOURNAL OF EXPERIMENTAL BOTANY, Vol: 71, Pages: 386-398, ISSN: 0022-0957
Gorelkin P, Erofeev A, Kolmogorov V, et al., 2020, Scanning Ion Conductance Microscopy (SICM) for Low-stress Directly Examining of Cellular Mechanics, ISSN: 1431-9276
Zhang Y, Takahashi Y, Hong SP, et al., 2019, High-resolution label-free 3D mapping of extracellular pH of single living cells, Nature Communications, Vol: 10, Pages: 1-9, ISSN: 2041-1723
Dynamic mapping of extracellular pH (pHe) at the single-cell level is critical for understanding the role of H+ in cellular and subcellular processes, with particular importance in cancer. While several pHe sensing techniques have been developed, accessing this information at the single-cell level requires improvement in sensitivity, spatial and temporal resolution. We report on a zwitterionic label-free pH nanoprobe that addresses these long-standing challenges. The probe has a sensitivity >0.01 units, 2 ms response time, and 50 nm spatial resolution. The technology was incorporated into a double-barrel nanoprobe integrating pH sensing with feedback-controlled distance sensing via Scanning Ion Conductance Microscopy. This allows for the simultaneous 3D topographical imaging and pHe monitoring of living cancer cells. These classes of nanoprobes were used for real-time high spatiotemporal resolution pHe mapping at the subcellular level and revealed tumour heterogeneity of the peri-cellular environments of melanoma and breast cancer cells.
Anand U, Korchev Y, Anand P, 2019, The role of urea in neuronal degeneration and sensitization: an in vitro model of uremic neuropathy, Molecular Pain, Vol: 15, ISSN: 1744-8069
Background: Uremic neuropathy commonly affects patients with chronic kidney disease (CKD), with painful sensations in the feet, followed by numbness and weakness in the legs and hands. The symptoms usually resolve following kidney transplantation, but the mechanisms of uremic neuropathy and associated pain symptoms remain unknown. As blood urea levels are elevated inpatients with CKD, we examined the morphological and functional effects of clinically observed levels of urea on sensory neurons. Methods: Rat DRG neurons were treated with 10or50 mMol/L urea for 48 hours, fixed and immunostained for PGP9.5 and βIII tubulin immunofluorescence, ,. Neurons were also immunostained for TRPV1, TRPM8 and Gap43 expression, and the capsaic insensitivity of urea or vehicle treated neurons was determined.Results: Urea treated neurons had degenerating neurites with diminished PGP9.5 immunofluorescence,and swollen, retracted growth cones. βIII tubulin appeared clumped after urea treatment. Neurite lengths were significantly reduced to 60 ± 2.6%(10 mMol/L, **P<0.01), and to 56.2± 3.3 %, (50 mMol/L, **P<0.01),urea treatmentfor 48 hours, compared with control neurons. Fewer neurons survived urea treatment,with 70.08 ± 13.3% remaining after 10 mMol/L (*P<0.05), and 61.49 ± 7.4 % after 50 mMol/L ureatreatment (**P<0.01), compared with controls. The proportion of neurons expressing TRPV1 wasreduced after urea treatment, but not TRPM8 expressing neurons. In functional studies, treatment with urea resulted in dose-dependent neuronal sensitization.Capsaicinresponses were significantly increased to 115.29 ± 3.4%(10 mMol/L, **P<0.01) and 125.3 ± 4.2%(50 mMol/L,**P<0.01), compared with controls. Sensitization due to urea was eliminated in the presence of the TRPV1 inhibitor SB705498, the MEKinhibitor PD98059,the PI3 kinase inhibitor LY294002, and the TRPM8 inhibitor AMTB. ConclusionNeurite degenerationandsensitization a
Erofeev A, Gorelkin P, Edwards C, et al., 2019, Novel Biophysics Methodologies for Single Cell Analysis with Electrochemical Nanoprobes, Joint 12th EBSA European Biophysics Congress / 10th IUPAP International Conference on Biological Physics (ICBP), Publisher: SPRINGER, Pages: S243-S243, ISSN: 0175-7571
Kolmogorov V, Alova A, Yudina A, et al., 2019, Effect of therapeutic agents on tumor cell compartments studied by local measurement of cell stiffness via Scanning Ion-Conductance Microscopy, Joint 12th EBSA European Biophysics Congress / 10th IUPAP International Conference on Biological Physics (ICBP), Publisher: SPRINGER, Pages: S101-S101, ISSN: 0175-7571
Gorelkin P, Erofeev A, Kolmogorov V, et al., 2019, Nanoscale Bioimaging with Scanning Ion Conductance Microscopy, Joint 12th EBSA European Biophysics Congress / 10th IUPAP International Conference on Biological Physics (ICBP), Publisher: SPRINGER, Pages: S157-S157, ISSN: 0175-7571
Vaneev A, Lopatuhina E, Alova A, et al., 2019, A new in vitro screening system for evaluating of anticancer drugs effect, Joint 12th EBSA European Biophysics Congress / 10th IUPAP International Conference on Biological Physics (ICBP), Publisher: SPRINGER, Pages: S241-S241, ISSN: 0175-7571
Kolmogorov V, Alova A, Yudina A, et al., 2019, Drug-induced alterations in cancer cell compartments studied by local stiffness measurement via Scanning Ion-Conductance Microscopy, Publisher: WILEY, Pages: 168-169, ISSN: 2211-5463
Ali T, Bednarska J, Vassilopoulos S, et al., 2019, Correlative SICM-FCM reveals changes in morphology and kinetics of endocytic pits induced by disease-associated mutations in dynamin, The FASEB Journal, Vol: 33, Pages: 8504-8518, ISSN: 0892-6638
Dynamin 2 (DNM2) is a GTP-binding protein that controls endocytic vesicle scission and defines a wholeclass of dynamin-dependent endocytosis, including clathrin-mediated endocytosis bycaveoli. It has been suggestedthat mutations in theDNM2gene, associated with 3 inherited diseases, disrupt endocytosis. However, how exactlymutations affect the nanoscale morphology of endocytic machinery has never been studied. In this paper, we used livecorrelative scanning ion conductance microscopy (SICM) and fluorescence confocal microscopy (FCM) to study howdisease-associated mutations affect the morphology and kinetics of clathrin-coated pits (CCPs) by directly followingtheir dynamics of formation, maturation, and internalizationinskinfibroblastsfrompatients with centronuclearmyopathy (CNM) and in Cos-7 cells expressing corresponding dynamin mutants. Using SICM-FCM, which we havedeveloped, we show how p.R465W mutation disrupts pit structure, preventing its maturation and internalization, andsignificantly increases the lifetime of CCPs. Differently,p.R522H slows down the formation of CCPs without affectingtheir internalization. We also found that CNM mutations inDNM2affect the distribution of caveoli and reduce dorsalruffling in human skin fibroblasts. Collectively, our SICM-FCM findings at single CCP level, backed up by electronmicroscopy data,argue for the impairment of several forms of endocytosis inDNM2-linked CNM.—Ali,T.,Bednarska,J.,Vassilopoulos,S.,Tran,M.,Diakonov,I.A.,Ziyadeh-Isleem,A.,Guicheney,P.,Gorelik,J.,Korchev,Y.E.,Reilly,M.M.,Bitoun,M.,Shevchuk,A.CorrelativeSICM-FCMreveals changes in morphology and kinetics of endocytic pitsinduced by disease-associated mutations in dynamin.
Garanina A, Vaneev A, Erofeev A, et al., 2019, Novel method for intravital investigation of anticancer drug pharmacokinetics and metabolite detection in mice tumors, Publisher: WILEY, Pages: 361-361, ISSN: 2211-5463
Erofeev A, Gorelkin P, Shevchuck A, et al., 2019, Novel electrochemical nanoprobes for single cell analysis, Publisher: WILEY, Pages: 169-169, ISSN: 2211-5463
Vaneev A, Lopatuhina E, Alova A, et al., 2019, Novel rapid method for evaluating of anticancer drugs effect on tumor cells, Publisher: WILEY, Pages: 169-170, ISSN: 2211-5463
Akasov R, Vaneev A, Alova A, et al., 2019, Electrochemical nanoprobe for label-free and real-time analyses of 3D tumor spheroids, Publisher: WILEY, Pages: 387-387, ISSN: 2211-5463
Gorelkin P, Erofeev A, Shevchuk A, et al., 2019, Scanning Ion Conductance Microscope as a new tool for bionanotechnology, Publisher: WILEY, Pages: 169-169, ISSN: 2211-5463
Gopal S, Chiappini C, Penders J, et al., 2019, Porous silicon nanoneedles modulate endocytosis to deliver biological payloads, Advanced Materials, Vol: 31, ISSN: 0935-9648
Owing to their ability to efficiently deliver biological cargo and sense the intracellular milieu, vertical arrays of high aspect ratio nanostructures, known as nanoneedles, are being developed as minimally invasive tools for cell manipulation. However, little is known of the mechanisms of cargo transfer across the cell membrane‐nanoneedle interface. In particular, the contributions of membrane piercing, modulation of membrane permeability and endocytosis to cargo transfer remain largely unexplored. Here, combining state‐of‐the‐art electron and scanning ion conductance microscopy with molecular biology techniques, it is shown that porous silicon nanoneedle arrays concurrently stimulate independent endocytic pathways which contribute to enhanced biomolecule delivery into human mesenchymal stem cells. Electron microscopy of the cell membrane at nanoneedle sites shows an intact lipid bilayer, accompanied by an accumulation of clathrin‐coated pits and caveolae. Nanoneedles enhance the internalization of biomolecular markers of endocytosis, highlighting the concurrent activation of caveolae‐ and clathrin‐mediated endocytosis, alongside macropinocytosis. These events contribute to the nanoneedle‐mediated delivery (nanoinjection) of nucleic acids into human stem cells, which distribute across the cytosol and the endolysosomal system. This data extends the understanding of how nanoneedles modulate biological processes to mediate interaction with the intracellular space, providing indications for the rational design of improved cell‐manipulation technologies.
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