Imperial College London

Dr Kuimova

Faculty of Natural SciencesDepartment of Chemistry

Reader in Chemical Physics
 
 
 
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Contact

 

+44 (0)20 7594 8558m.kuimova

 
 
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Location

 

207BMolecular Sciences Research HubWhite City Campus

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Summary

 

Publications

Publication Type
Year
to

126 results found

Sherin PS, Vysniauskas A, Lopez-Duarte I, Ogilby PR, Kuimova MKet al., 2021, Visualising UV-A light-induced damage to plasma membranes of eye lens, Journal of Photochemistry and Photobiology, B: Biology, Vol: 225, Pages: 1-10, ISSN: 1011-1344

An eye lens is constantly exposed to the solar UV radiation, which is considered the most important external source of age-related changes to eye lens constituents. The accumulation of modifications of proteins and lipids with age can eventually lead to the development of progressive lens opacifications, such as cataracts. Though the impact of solar UV radiation on the structure and function of proteins is actively studied, little is known about the effect of photodamage on plasma membranes of lens cells. In this work we exploit Fluorescence Lifetime Imaging Microscopy (FLIM), together with viscosity-sensitive fluorophores termed molecular rotors, to study the changes in viscosity of plasma membranes of porcine eye lens resulting from two different types of photodamage: Type I (electron transfer) and Type II (singlet oxygen) reactions. We demonstrate that these two types of photodamage result in clearly distinct changes in viscosity – a decrease in the case of Type I damage and an increase in the case of Type II processes. Finally, to simulate age-related changes that occur in vivo, we expose an intact eye lens to UV-A light under anaerobic conditions. The observed decrease in viscosity within plasma membranes is consistent with the ability of eye lens constituents to sensitize Type I photodamage under natural irradiation conditions. These changes are likely to alter the transport of metabolites and predispose the whole tissue to the development of pathological processes such as cataracts.

Journal article

Vilar R, Priessner M, Summers PA, Lewis B, Sastre M, Ying L, Kuimova MKet al., 2021, Selective detection of Cu+ ions in live cells via fluorescence lifetime imaging microscopy., Angewandte Chemie International Edition, Vol: 60, Pages: 23148-23153, ISSN: 1433-7851

Copper is an essential trace element in living organisms with its levels and localisation being carefully managed by the cellular machinery. However, if misregulated, deficiency or excess of copper ions can lead to several diseases. Therefore, it is important to have reliable methods to detect, monitor and visualise this metal in cells. Herein we report a new optical probe based on BODIPY, which shows a switch-on in its fluorescence intensity upon binding to copper(I), but not in the presence of high concentration of other physiologically relevant metal ions. More interestingly, binding to copper(I) leads to significant changes in the fluorescence lifetime of the new probe, which can be used to visualize copper(I) pools in lysosomes of live cells via fluorescence lifetime imaging microscopy (FLIM).

Journal article

Summers PA, Thomas AP, Kench T, Vannier J-B, Kuimova MK, Vilar Ret al., 2021, Cationic helicenes as selective G4 DNA binders and optical probes for cellular imaging, CHEMICAL SCIENCE, Vol: 12, Pages: 14624-14634, ISSN: 2041-6520

Journal article

Shimolina L, Lukina M, Shcheslavskiy V, Elagin V, Dudenkova V, Ignatova N, Kuimova MK, Shirmanova Met al., 2021, Probing metabolism and viscosity of cancer cells using fluorescence lifetime imaging microscopy, Jove-Journal of Visualized Experiments, Pages: 1-22, ISSN: 1940-087X

Viscosity is an important physical property of a biological membrane, as it is one of the key parameters for the regulation of morphological and physiological state of living cells. Plasma membranes of tumor cells are known to have significant alterations in their composition, structure, and functional characteristics. Along with dysregulated metabolism of glucose and lipids, these specific membrane properties help tumor cells to adapt to the hostile microenvironment and develop resistance to drug therapies. Here, we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to sequentially image cellular metabolism and plasma membrane viscosity in live cancer cell culture. Metabolic assessments are performed by detecting fluorescence of endogenous metabolic cofactors, such as reduced nicotinamide adenine dinucleotide NAD(P)H and oxidized flavins. Viscosity is measured using a fluorescent molecular rotor, a synthetic viscosity-sensitive dye, with a strong fluorescence lifetime dependence on the viscosity of the immediate environment. In combination, these techniques enable us to better understand the links between membrane state and metabolic profile of cancer cells and to visualize the changes induced by chemotherapy.

Journal article

Vysniauskas A, Cornell B, Sherin P, Maleckaitė K, Kubankova M, Izquierdo MA, Vu TT, Volkova Y, Budynina E, Molteni C, Kuimova Met al., 2021, Cyclopropyl substituents transform the viscosity-sensitive BODIPY molecular rotor into a temperature sensor, ACS Sensors, Vol: 6, Pages: 2158-2167, ISSN: 2379-3694

A quantitative fluorescent probe that responds to changes in temperature is highly desirable for studies of biological environments, particularly in cellulo. Here, we report new cell-permeable fluorescence probes based on the BODIPY moiety that respond to environmental temperature. The new probes were developed on the basis of a well-established BODIPY-based viscosity probe by functionalization with cyclopropyl substituents at α and β positions of the BODIPY core. In contrast to the parent BODIPY fluorophore, α-cyclopropyl-substituted fluorophore displays temperature-dependent time-resolved fluorescence decays showing greatly diminished viscosity dependence, making it an attractive sensor to be used with fluorescence lifetime imaging microscopy (FLIM). We performed theoretical calculations that help rationalize the effect of the cyclopropyl substituents on the photophysical behavior of the new BODIPYs. In summary, we designed an attractive new quantitative FLIM-based temperature probe that can be used for temperature sensing in live cells.

Journal article

Reyes JB, Kuimova MK, Vilar R, 2021, Metal complexes as optical probes for DNA sensing and imaging, CURRENT OPINION IN CHEMICAL BIOLOGY, Vol: 61, Pages: 179-190, ISSN: 1367-5931

Journal article

Paez-Perez M, Lopez-Duarte I, Vysniauskas A, Brooks NJ, Kuimova MKet al., 2021, Imaging non-classical mechanical responses of lipid membranes using molecular rotors, Chemical Science, Vol: 12, Pages: 2604-2613, ISSN: 2041-6520

Lipid packing in cellular membranes has a direct effect on membrane tension and microviscosity, and plays a central role in cellular adaptation, homeostasis and disease. According to conventional mechanical descriptions, viscosity and tension are directly interconnected, with increased tension leading to decreased membrane microviscosity. However, the intricate molecular interactions that combine to build the structure and function of a cell membrane suggest a more complex relationship between these parameters. In this work, a viscosity-sensitive fluorophore (‘molecular rotor’) is used to map changes in microviscosity in model membranes under conditions of osmotic stress. Our results suggest that the relationship between membrane tension and microviscosity is strongly influenced by the bilayer's lipid composition. In particular, we show that the effects of increasing tension are minimised for membranes that exhibit liquid disordered (Ld) – liquid ordered (Lo) phase coexistence; while, surprisingly, membranes in pure gel and Lo phases exhibit a negative compressibility behaviour, i.e. they soften upon compression.

Journal article

Kench T, Summers PA, Kuimova MK, Lewis JEM, Vilar Ret al., 2021, Rotaxanes as cages to control DNA binding, cytotoxicity, and cellular uptake of a small molecule, Angewandte Chemie International Edition, Vol: 60, Pages: 1-1, ISSN: 1433-7851

The efficacy of many drugs can be limited by undesirable properties, such as poor aqueous solubility, low bioavailability, and “off‐target” interactions. To combat this, various drug carriers have been investigated to enhance the pharmacological profile of therapeutic agents. In this work, we demonstrate the use of mechanical protection to “cage” a DNA‐targeting metallodrug within a photodegradable rotaxane. More specifically, we report the synthesis of rotaxanes incorporating as a stoppering unit a known G‐quadruplex DNA binder, namely a PtII‐salphen complex. This compound cannot interact with DNA when it is part of the mechanically interlocked assembly. The second rotaxane stopper can be cleaved by either light or an esterase, releasing the PtII‐salphen complex. This system shows enhanced cell permeability and limited cytotoxicity within osteosarcoma cells compared to the free drug. Light activation leads to a dramatic increase in cytotoxicity, arising from the translocation of PtII‐salphen to the nucleus and its binding to DNA.

Journal article

Vilar R, Lewis BW, Bisballe N, Santella M, Summers PA, Vannier J-B, Kuimova MK, Laursen BWet al., 2021, Assessing the key photophysical properties of triangulenium dyes for DNA binding by alteration of the fluorescent core, Chemistry: A European Journal, Vol: 27, Pages: 2523-2536, ISSN: 0947-6539

Four-stranded G-quadruplex (G4) DNA is a non-canonical DNA topology that has been proposed to form in cells and play key roles in how the genome is read and used by the cellular machinery. Previously, a fluorescent triangulenium probe (DAOTA-M2) was used to visualise G4s in cellulo, thanks to its distinct fluorescence lifetimes when bound to different DNA topologies. Herein, we expand the library of available triangulenium probes to explore how modifications to the fluorescent core of the molecule affect its photophysical characteristics, interaction with DNA and cellular localisation. The benzo-bridged and isopropyl-bridged diazatriangulenium dyes, BDATA-M2 and CDATA-M2 respectively, featuring ethyl-morpholino substituents, were synthesised and characterised. The interactions of these molecules with different DNA topologies were studied to determine their binding affinity, fluorescence enhancement and fluorescence lifetime response. Finally, the cellular uptake and localisation of these optical probes were investigated. Whilst structural modifications to the triangulenium core only slightly alter the binding affinity to DNA, BDATA-M2 and CDATA-M2 cannot distinguish between DNA topologies through their fluorescence lifetime. This work presents valuable new evidence into the critical role of PET quenching when using the fluorescence lifetime of triangulenium dyes to discriminate G4 DNA from duplex DNA, highlighting the importance of fine tuning redox and spectral properties when developing new triangulenium-based G4 probes.

Journal article

Vilar Compte R, Summers P, Lewis B, Gonzalez-Garcia J, Porreca RM, Lim A, Cadinu P, Martin-Pintado N, Mann D, Edel J, Vannier JB, Kuimova M, Vilar Compte Ret al., 2021, Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy, Nature Communications, Vol: 12, ISSN: 2041-1723

Guanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4s). Increasing evidence suggests that these G4 structures form in vivo and play a crucial role in cellular processes. However, their direct observation in live cells remains a challenge. Here we demonstrate that a fluorescent probe (DAOTA-M2) in conjunction with fluorescence lifetime imaging microscopy (FLIM) can identify G4s within nuclei of live and fixed cells. We present a FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4s and apply it to a wide range of drug candidates. We also demonstrate that DAOTA-M2 can be used to study G4 stability in live cells. Reduction of FancJ and RTEL1 expression in mammalian cells increases the DAOTA-M2 lifetime and therefore suggests an increased number of G4s in these cells, implying that FancJ and RTEL1 play a role in resolving G4 structures in cellulo.

Journal article

Shimolina LE, Gulin AA, Paez-Perez M, Lopez-Duarte I, Druzhkova IN, Lukina MM, Gubina M, Brooks NJ, Zagaynova E, Kuimova MK, Shirmanova Met al., 2020, Mapping cisplatin-induced viscosity alterations in cancer cells using molecular rotor and fluorescence lifetime imaging microscopy, Journal of Biomedical Optics, Vol: 25, Pages: 1-16, ISSN: 1083-3668

Significance: Despite the importance of the cell membrane in regulation of drug activity, the influence of drug treatments on its physical properties is still poorly understood. The combination of fluorescence lifetime imaging microscopy (FLIM) with specific viscosity-sensitive fluorescent molecular rotors allows the quantification of membrane viscosity with high spatiotemporal resolution, down to the individual cell organelles.Aim: The aim of our work was to analyze microviscosity of the plasma membrane of living cancer cells during chemotherapy with cisplatin using FLIM and correlate the observed changes with lipid composition and cell’s response to treatment.Approach: FLIM together with viscosity-sensitive boron dipyrromethene-based fluorescent molecular rotor was used to map the fluidity of the cell’s membrane. Chemical analysis of membrane lipid composition was performed with time-of-flight secondary ion mass spectrometry (ToF-SIMS).Results: We detected a significant steady increase in membrane viscosity in viable cancer cells, both in cell monolayers and tumor spheroids, upon prolonged treatment with cisplatin, as well as in cisplatin-adapted cell line. ToF-SIMS revealed correlative changes in lipid profile of cisplatin-treated cells.Conclusions: These results suggest an involvement of membrane viscosity in the cell adaptation to the drug and in the acquisition of drug resistance.

Journal article

Robson JA, Kubánková M, Bond T, Hendley RA, White AJP, Kuimova MK, WiltonEly JDETet al., 2020, Simultaneous detection of carbon monoxide and viscosity changes in cells, Angewandte Chemie, Vol: 132, Pages: 21615-21619, ISSN: 0044-8249

A new family of robust, non‐toxic, water‐compatible ruthenium(II) vinyl probes allows the rapid, selective and sensitive detection of endogenous carbon monoxide (CO) in live mammalian cells under normoxic and hypoxic conditions. Uniquely, these probes incorporate a viscosity‐sensitive BODIPY fluorophore that allows the measurement of microscopic viscosity in live cells via fluorescence lifetime imaging microscopy (FLIM) while also monitoring CO levels. This is the first example of a probe that can simultaneously detect CO alongside small viscosity changes in organelles of live cells.

Journal article

Robson J, Kubankova M, Bond T, Hendley R, White A, Kuimova M, Wilton-Ely Jet al., 2020, Simultaneous detection of carbon monoxide and viscosity changes in cells, Angewandte Chemie International Edition, Vol: 59, Pages: 21431-21435, ISSN: 1433-7851

A new family of robust, non‐toxic, water‐compatible ruthenium(II) vinyl probes allows the rapid, selective and sensitive detection of endogenous carbon monoxide (CO) in live mammalian cells under normoxic and hypoxic conditions. Uniquely, these probes incorporate a viscosity‐sensitive BODIPY fluorophore that allows the measurement of microscopic viscosity in live cells via Fluorescence Lifetime Imaging Microscopy (FLIM) in conjunction with measuring CO. This is the first example of a probe that can simultaneously detect CO alongside small viscosity changes in organelles of live cells.

Journal article

Vysniauskas A, Kuimova M, 2020, Microviscosity and temperature sensors: the twists and turns of the photophysics of conjugated porphyrin dimers — a SPP/JPP Young Investigator Award paper, Jornal of Porphyrins and Phthalocyanines, Vol: 24, Pages: 1372-1386, ISSN: 1088-4246

Conjugated porphyrin dimers have captured the imagination of scientists due to a set of unique spectroscopic features such as remarkable nonlinear-optical properties, high yields of singlet oxygen sensitization and the absorption and emission in the far-red region of the visible spectrum. Here we review a range of newly emerged applications of porphyrin dimers as sensors of their microenvironment such as viscosity and temperature. We discuss the sensing mechanism based on the known conformational flexibility of the dimer structure and describe possible applications of these unique sensors, from detecting viscosity increase during photoinduced cell death to structural responses of polymers and artificial lipid membranes, to temperature changes, and to mechanical deformation.

Journal article

Shi Y, Summers PA, Kuimova MK, Azevedo HSet al., 2020, Unravelling the Enzymatic Degradation Mechanism of Supramolecular Peptide Nanofibers and Its Correlation with Their Internal Viscosity, NANO LETTERS, Vol: 20, Pages: 7375-7381, ISSN: 1530-6984

Journal article

Ameer-Beg S, Suhling K, Kuimova M, 2020, Special issue on fluorescence lifetime imaging (FLIM): from fundamentals to applications, METHODS AND APPLICATIONS IN FLUORESCENCE, Vol: 8, ISSN: 2050-6120

Journal article

Maurice J, Lett A, Skinner C, Lim A, Richardson M, Painadath Thomas A, Summers P, Vyas K, Tadbier A, Vilar R, Kuimova M, Miodragovic S, Vergis N, Kelly P, Cordeiro M, Hoare J, Darzi A, Goldin R, Thursz M, Thompson Aet al., 2020, Transcutaneous fluorescence spectroscopy as a tool for non-invasive monitoring of gut function: first clinical experiences, Scientific Reports, Vol: 10, ISSN: 2045-2322

Gastro-intestinal function plays a vital role in conditions ranging from inflammatory bowel disease and HIV through to sepsis and malnutrition. However, the techniques that are currently used to assess gut function are either highly invasive or unreliable. Here we present an alternative, non-invasive sensing modality for assessment of gut function based on fluorescence spectroscopy. In this approach, patients receive an oral dose of a fluorescent contrast agent and a fibre-optic probe is used to make fluorescence measurements through the skin. This provides a readout of the degree to which fluorescent dyes have permeated from the gut into the blood stream. We present preliminary results from our first measurements in human volunteers demonstrating the potential of the technique for non-invasive monitoring of multiple aspects of gastro-intestinal health.

Journal article

Bednarska J, Pelchen-Matthews A, Novak P, Burden JJ, Summers PA, Kuimova MK, Korchev Y, Marsh M, Shevchuk Aet 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.

Journal article

Kuimova M, Kashirina A, Lopez Duarte I, Kubankova M, Gulin A, Dudenkova V, Rodimova S, Torgomyan H, Zagaynova E, Meleshina Aet al., 2020, Monitoring membrane viscosity in differentiating stem cells using BODIPY-based molecular rotors and FLIM, Scientific Reports, Vol: 10, ISSN: 2045-2322

Membrane fluidity plays an important role in many cell functions such as cell adhesion, and migration. In stem cell lines membrane fluidity may play a role in differentiation. Here we report the use of viscosity-sensitive fluorophores based on a BODIPY core, termed “molecular rotors”, in combination with Fluorescence Lifetime Imaging Microscopy, for monitoring of plasma membrane viscosity changes in mesenchymal stem cells (MSCs) during osteogenic and chondrogenic differentiation. In order to correlate the viscosity values with membrane lipid composition, the detailed analysis of the corresponding membrane lipid composition of differentiated cells was performed by time-of-flight secondary ion mass spectrometry. Our results directly demonstrate for the first time that differentiation of MSCs results in distinct membrane viscosities, that reflect the change in lipidome of the cells following differentiation.

Journal article

Clark R, Nawawi MA, Dobre A, Pugh D, Liu Q, Ivanov AP, White AJP, Edel JB, Kuimova MK, McIntosh AJS, Welton Tet al., 2020, The effect of structural heterogeneity upon the microviscosity of ionic liquids, Chemical Science, Vol: 11, Pages: 6121-6133, ISSN: 2041-6520

The behaviour of two molecular rotors, one charged – 3,3′-diethylthiacarbocyanine iodide (Cy3) and one neutral – 8-[4-decyloxyphenyl]-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY-C10), have been studied in various ionic liquids. The fluorescent decay lifetime has been used to elucidate the structure of the immediate region around the rotor. The neutral BODIPY-C10 was found to prefer the non-polar alkyl chain environment, leading to two trends in the lifetime of the dye: one when it was fully partitioned into the non-polar domain, and one when it also sampled polar moieties. The positively charged Cy3 dye showed a complex relationship between the bulk viscosity of the ionic liquid and lifetime of the molecular rotor. This was attributed to a combination of polarity related spectral changes, changes in anion cages around the dye, and temperature dependent fluorescent lifetimes alongside the dependence of the rotor upon the viscosity.

Journal article

Davidson NM, Gallimore PJ, Bateman B, Ward AD, Botchway SW, Kalberer M, Kuimova MK, Pope FDet al., 2020, Measurement of the fluorescence lifetime of GFP in high refractive index levitated droplets using FLIM., Physical Chemistry Chemical Physics, Vol: 22, Pages: 14704-14711, ISSN: 1463-9076

Green fluorescent protein (GFP) is a widely used fluorescent probe in the life sciences and biosciences due to its high quantum yield and extinction coefficient, and its ability to bind to biological systems of interest. This study measures the fluorescence lifetime of GFP in sucrose/water solutions of known molarity in order to determine the refractive index dependent lifetime of GFP. A range of refractive indices from 1.43-1.53 were probed by levitating micron sized droplets composed of water/sucrose/GFP in an optical trap under well-constrained conditions of relative humidity. This setup allows for the first reported measurements of the fluorescence lifetime of GFP at refractive indices greater than 1.46. The results obtained at refractive indices less than 1.46 show good agreement with previous studies. Further experiments that trapped droplets of deionised water containing GFP allowed the hygroscopic properties of GFP to be measured. GFP is found to be mildly hygroscopic by mass, but the high ratio of molecular masses of GFP to water (ca. 1500 : 1) signifies that water uptake is large on a per-mole basis. Hygroscopic properties are verified using brightfield microscope imaging, of GFP droplets at low and high relative humidity, by measuring the humidity dependent droplet size. In addition, this experiment allowed the refractive index of pure GFP to be estimated for the first time (1.72 ± 0.07). This work provides reference data for future experiments involving GFP, especially for those conducted in high refractive index media. The work also demonstrates that GFP can be used as a probe for aerosol studies, which require determination of the refractive index of the aerosol of any shape.

Journal article

Shimolina LE, Shirmanova MV, Kuimova MK, Lukina MM, Ignatova NI, Druzhkova IN, Zagaynova EVet al., 2020, Imaging plasma membrane microviscosity in cancer cells during chemotherapy, Symposium on Multiphoton Microscopy in the Biomedical Sciences XX held at SPIE BiOS Conference, Publisher: SPIE-INT SOC OPTICAL ENGINEERING, ISSN: 0277-786X

Conference paper

Kuimova M, Kubankova M, Chambers J, Marciniak S, Bond P, Huber Ret al., 2019, Linker length affects photostability of protein-targeted sensor of cellular microviscosity, Methods and Applications in Fluorescence, Vol: 7, ISSN: 2050-6120

Viscosity sensitive fluorophores termed ‘molecular rotors’ represent a convenient and quantitative tool for measuring intracellular viscosity via Fluorescence Lifetime Imaging Microscopy (FLIM). We compare the FLIM performance of two BODIPY-based molecular rotors bound to HaloTag protein expressed in different subcellular locations. While both rotors are able to penetrate live cells and specifically label the desired intracellular location, we found that the rotor with a longer HaloTag protein recognition motif was significantly affected by photo-induced damage when bound to the HaloTag protein, while the other dye showed no changes upon irradiation. Molecular dynamics modelling indicates that the irradiation-induced electron transfer between the BODIPY moiety and the HaloTag protein is a plausible explanation for these photostability issues. Our results demonstrate that binding to the targeted protein may significantly alter the photophysical behaviour of a fluorescent probe and therefore its thorough characterisation in the protein bound form is essential prior to any in vitro and in cellulo applications.

Journal article

Kubankova M, Summers P, Lopez-Duarte I, Kiryushko D, Kuimova MKet al., 2019, Microscopic viscosity of neuronal plasma membranes measured using fluorescent molecular rotors: effects of oxidative stress and neuroprotection., ACS Applied Materials and Interfaces, Vol: 11, Pages: 36307-36315, ISSN: 1944-8244

Molecular mobility in neuronal plasma membranes is a crucial factor in brain function. Microscopic viscosity is an important parameter that determines molecular mobility. This study presents the first direct measurements of the microviscosity of plasma membranes of live neurons. Microviscosity maps were obtained using fluorescence lifetime imaging of environment-sensing dyes termed 'molecular rotors'. Neurons were investigated both in the basal state and following common neurodegenerative stimuli, excitotoxicity or oxidative stress. Both types of neurotoxic challenges induced microviscosity decrease in cultured neurons, and the oxidant-induced membrane fluidification was counteracted by the wide-spectrum neuroprotectant, the H3 peptide. These results provide new insights into molecular mobility in neuronal membranes, paramount for basic brain function, and suggest that preservation of membrane stability may be an important aspect of neuroprotection in brain insults and neurodegenerative disorders.

Journal article

Perez MP, Kuimova MK, Brooks NJ, 2019, Reorganization of liquid ordered lipid domains dampens changes in membrane tension, Joint 12th EBSA European Biophysics Congress / 10th IUPAP International Conference on Biological Physics (ICBP), Publisher: SPRINGER, Pages: S232-S232, ISSN: 0175-7571

Conference paper

Kubánková M, Lin X, Albrecht T, Edel JB, Kuimova MKet al., 2019, Rapid fragmentation during seeded lysozyme aggregation revealed at the single molecule level, Analytical Chemistry, Vol: 91, Pages: 6880-6886, ISSN: 0003-2700

Protein aggregation is associated with neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. The poorly understood pathogenic mechanism of amyloid diseases makes early stage diagnostics or therapeutic intervention a challenge. Seeded polymerization that reduces the duration of the lag phase and accelerates fibril growth is a widespread model to study amyloid formation. Seeding effects are hypothesized to be important in the "infectivity" of amyloids and are linked to the development of systemic amyloidosis in vivo. The exact mechanism of seeding is unclear yet critical to illuminating the propagation of amyloids. Here we report on the lateral and axial fragmentation of seed fibrils in the presence of lysozyme monomers at short time scales, followed by the generation of oligomers and growth of fibrils.

Journal article

Woodcock EM, Girvan P, Eckert J, Lopez Duarte I, Kubankova M, van Loon J, Brooks N, Kuimova Met al., 2019, Measuring intracellular viscosity in conditions of hypergravity, Biophysical Journal, Vol: 116, Pages: 1984-1993, ISSN: 0006-3495

Gravity-sensitive cellular responses are regularly observed in both specialised and non-specialised cells. One potential mechanism for this sensitivity is a changing viscosity of the intracellular organelles. Here, we report a novel viscosity-sensitive molecular rotor based on meso-substituted boron-dipyrrin (BODIPY) used to investigate the response of viscosity of cellular membranes to hypergravity conditions created at the Large Diameter Centrifuge at the European Space Agency Technology Centre ESA-ESTEC. Mouse osteoblastic (MC3T3-E1) and endothelial (HUVEC) cell lines were tested and an increase in viscosity was found with increasing hypergravity loading. This response is thought to be primarily biologically driven, with the potential for a small, instantaneous physical mechanism also contributing to the observed effect. This work provides the first quantitative data for cellular viscosity changes under hypergravity, up to 15 g.

Journal article

Kuimova M, Kubankova M, Kiryushko D, Lopez Duarte Iet al., 2018, Molecular rotors report on changes of live cell plasma membrane microviscosity upon interaction with beta-amyloid aggregates, Soft Matter, Vol: 14, Pages: 9466-9474, ISSN: 1744-683X

Amyloid deposits of aggregated beta-amyloid Aβ(1–42) peptides are a pathological hallmark of Alzheimer's disease. Aβ(1–42) aggregates are known to induce biophysical alterations in cells, including disruption of plasma membranes. We investigated the microviscosity of plasma membranes upon interaction with oligomeric and fibrillar forms of Aβ(1–42). Viscosity-sensing fluorophores termed molecular rotors were utilised to directly measure the microviscosities of giant plasma membrane vesicles (GPMVs) and plasma membranes of live SH-SY5Y and HeLa cells. The fluorescence lifetimes of membrane-inserting BODIPY-based molecular rotors revealed a decrease in bilayer microviscosity upon incubation with Aβ(1–42) oligomers, while fibrillar Aβ(1–42) did not significantly affect the microviscosity of the bilayer. In addition, we demonstrate that the neuroprotective peptide H3 counteracts the microviscosity change induced by Aβ(1–42) oligomers, suggesting the utility of H3 as a neuroprotective therapeutic agent in neurodegenerative disorders and indicating that ligand-induced membrane stabilisation may be a possible mechanism of neuroprotection during neurodegenerative disorders such as Alzheimer's disease.

Journal article

Vysniauskas A, Kuimova MK, 2018, A twisted tale: measuring viscosity and temperature of microenvironments using molecular rotors, International Reviews in Physical Chemistry, Vol: 37, Pages: 259-285, ISSN: 0144-235X

Measuring viscosity and temperature on the microscale is a challening yet very important task, in materials sciences and in biology alike. In this perpsective we review and discuss fluorescent microviscosity sensors, termed ‘molecular rotors’, that offer a convenient way of measuring microscopic viscosity and sometimes may even be used to measure microscopic temperature in addition to viscosity. We discuss how temperature in combination with various solvent properties can affect microviscosity measurements and we review possible action mechanisms that make molecular rotors sensitive to multiple parameters of their environment. Overall, we reveal a complicated, yet exciting, behaviour of molecular rotors at different viscosity, temperature and solvent properties on the microscale and how this behaviour can be explained and exploited.

Journal article

Chambers JE, Kubánková M, Huber RG, López-Duarte I, Avezov E, Bond PJ, Marciniak SJ, Kuimova MKet al., 2018, An optical technique for mapping microviscosity dynamics in cellular organelles, ACS Nano, Vol: 12, Pages: 4398-4407, ISSN: 1936-0851

Microscopic viscosity (microviscosity) is a key determinant of diffusion in the cell and defines the rate of biological processes occurring at the nanoscale, including enzyme-driven metabolism and protein folding. Here we establish a rotor-based organelle viscosity imaging (ROVI) methodology that enables real-time quantitative mapping of cell microviscosity. This approach uses environment-sensitive dyes termed molecular rotors, covalently linked to genetically encoded probes to provide compartment-specific microviscosity measurements via fluorescence lifetime imaging. ROVI visualized spatial and temporal dynamics of microviscosity with suborganellar resolution, reporting on a microviscosity difference of nearly an order of magnitude between subcellular compartments. In the mitochondrial matrix, ROVI revealed several striking findings: a broad heterogeneity of microviscosity among individual mitochondria, unparalleled resilience to osmotic stress, and real-time changes in microviscosity during mitochondrial depolarization. These findings demonstrate the use of ROVI to explore the biophysical mechanisms underlying cell biological processes.

Journal article

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