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Structure of Biomimetic Materials
A large number of natural and synthetic hydrogels are currently used for tissue engineering and regenerative medicine. Over the last decade, there has been an increasing awareness of the role of material properties of the substrates in guiding cellular behaviour. This has inspired chemists to create a new generation of materials with mechanical properties closed to that of natural occurring biopolymer networks. Recently, the groups of Prof. Alan Rowan (Queens University, Australia) and Prof. Paul Kouwer (Radboud University of Nijmegen, The Netherlands) were able to develop a fully synthetic material that mimics in all aspects the gels prepared from cellular filaments. These synthetics gels are prepared from polyisocyanopeptides (PICs) grafted with oligo(ethylene glycol) chains and share structural features of biopolymers: their helical structure renders the polymer molecules relatively stiff while the interaction between the side chains enable the formation of bundles or fibrils of defined dimensions. The triethylene glycol side chains attached to the polymer backbone render the material thermo-responsive (it will gel upon heating beyond 20 °C and become liquid again upon cooling). Despite being characterized extensively in bulk, the fundamental dynamics and the relation between the macroscopic properties and the microscopic structure at cellular length scales of PIC-based hydrogels remains obscure.
Classically, structural characterization of materials is performed with electron microscopy or scanning probe microscopy. Despite the high spatial resolution achievable with these techniques, they are unable to measure dynamics ‘in situ’ and sample preparation can be a laborious process. In contrast, optical microscopy has the potential to unravel the dynamics in complex heterogeneous systems but has been limited to a spatial resolution of ca. 200 nm. In the past 10 years fluorescence imaging has been revolutionized by the successful development of sub-diffraction (super-resolution) microscopy modalities which can achieve resolutions down to tens of nanometers (see Molecular Organization at the Nanoscale).The various possibilities of fluorescence microscopy to probe dynamics and heterogeneities, with molecular resolution, for a wide range of time scales makes it an ideal tool to address many topics of polymer science. In this project we are using STED to image the polymer network at the nanometer scale.
For more information on PIC-based hydrogels:
- Kouwer P.H.J., et al. (2013) Responsive biomimetic networks from polyisocyanopeptide hydrogels, Nature, 493, pages 651–655 (article can be found here)
- Jasper M., et al. (2014) Ultra-responsive soft matter from strain-stiffening hydrogels, Nature Communications, 5, 5808 (article can be found here)
- Jasper M., et al. (2016) Bundle Formation in Biomimetic Hydrogels, Macromolecules, 17(8), pages 2642–2649 (article can be found here)
Polymer reptation in 3D
Our current theoretical understanding of entangled polymer chain dynamics is based on the reptation model. First proposed by Doi and Edwards, and further expanded by de Gennes, the reptation model assumes that a polymer chain is confined by the surrounding matrix and is therefore forced to move inside an imaginary tube defined by the transient network of entangled neighboring chains. Intuitively this motion resembles that of a snake or worm. The reptation model predicts five dynamical regimes for segment diffusion, summarized in the figure below. These regimes are as follows: (0) sub-segmental processes (“glassy dynamics”) at very short times (microseconds), (I) small motion subject only to chain connectivity, (II) “local reptation”: short-distance motion within the constraints imposed by the surrounding chains (“tube”), (III) “reptation”: diffusive motion along the curvilinear tube over distances larger than the polymer size, and (IV) free diffusion.
Rheology at the micrometer scale
Due to the crucial role of physical cues in regulating cell behaviour, the mechanical properties of hydrogels are a key design parameter in tissue engineering applications. The shear elastic properties of viscoelastic materials are commonly measured by mechanical rheometers. Storage and loss moduli of a material can be measured by application of strain while measuring stress or vice versa. In contrast, recently developed optical micro-rheology techniques use nanometer- or micrometer-sized particles embedded in the material to obtain the viscoelastic response parameters. Thermal or passive micro-rheology for viscoelastic materials is based on an extension of the concepts of Brownian motion of particles in simple liquids. The movement of the embedded particles can be monitored using particle tracking. Initially developed to investigate the rheological properties of uniform complex fluids, particle tracking micro-rheology (PTM) is becoming a popular technique to analyze polymer blends and gels, as well as the deformability and elasticity within cells. However, if the beads locally modify the structure of the gel or are contained in a pore in an inhomogeneous matrix, the bulk rheological properties will not be retrieved. A solution is to use the cross-correlated thermal fluctuation of pairs of tracer particles, ‘two-point micro-rheology’. This method provides a better agreement between micro and macro-rheology, even in complex micro-structured fluids. However, technical constrains limit the wide application of this technique. One of the major limitations of two-point micro-rheology is the reduced number of trajectories that can be used for analysis. During particle tracking micro-rheology, the length of the calculated trajectories is limited by the time spent by the tracers in the field of view (x,y) and depth of focus (z). Consequently, mechanical characterization of complex polymer matrixes at the micrometer scale would benefit greatly of a new method for (fast) tracking in 3D. We are developing a new method for fast tracking of (fluorescent) beads in 3D using a multi-plane wide field microscope. This will allow a better mechanical characterization of soft materials, at the microscale.
Cellular adhesion in 3D matrices
Cells sense physical forces and the mechanical properties of the microenvironment via several distinct mechanisms and cellular components. The first step of cellular adhesion to the ECM occurs via transmembrane heterodimers of the integrin family. Once integrin molecules adhere to the ECM, they are activated and form clusters. As the number of bound molecules increases, some of the focal complexes evolve from small (0.5-1µm in diameter) transient ‘dot-like’ contacts to elongated structures (3-10µm) which couple with actin and associated proteins. The mechanical coupling between the ECM and the cell cytoskeleton is controlled by the dynamics of the focal adhesion complexes (assembly, disassembly and turnover).
Protein-Protein Interactions
Protein-protein interactions (PPIs) are intrinsic to all cellular processes, driving both metabolic and regulatory pathways. Despite the numerous techniques available, detection of transient short-lived PPIs remains challenging4. The main fluorescence microscopic techniques developed for visualizing PPIs in a cellular context are based on Föster resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC)5. Both techniques detect the interaction between a pair of labeled molecules. Although highly informative, they require fine positioning of the labels and in the majority of the applications the spatial resolution achieved is limited by the diffraction of light to about 200 nm. More information concerning the use of FRET to detect PPIs can be found at Cellular Signalling).
We have use a single molecule localization based super-resolution technique to detect and map PPIs at the cell membrane. This new variant of PAINT that enables mapping of short-lived transient interactions between cytosolic and membrane-bound proteins inside living mammalian cells, at the nanometer scale. In this method the protein of interest is labeled with a light-controllable fluorescent protein and imaged under TIRF illumination, which leads to the selective activation and subsequent detection of molecules in close proximity with the plasma membrane. Interacting molecules are discriminated using a stringent fitting of the fluorescence signal recorded for every single molecule.
Organoids: models for cell communication
Nowadays, human organoids are becoming a highly promising tool to model organ development, function and especially human diseases in vitro. In general, organoids are miniature, simplified organs that can easily propagate in vitro originating from one or a few cells, typically stem cells.
Single cell manipulation by endoscopy
Nanowire-based endoscopy has attracted interest due to its ability to manipulate cells at the single-cell level with minimal cellular perturbation. High-density, vertically aligned nanowire arrays have been used as an efficient gene delivery system. Despite the high transfection rates, culturing the cells on nanowire arrays might have other influences on the cellular behaviour. For example, stem cells cultured on silicon nanowires show significantly different adhesion, proliferation and differentiation, compared with flat silicon or other control substrates. Furthermore, such arrays are not location-specific and require optimization of the nanowire density and dimension for the different the cell types. In collaboration with the group of Prof. Hiroshi Uji-i we are developing a method to delivery genetic material using a single nanowire. In contrast to the existing methods, this approach can be applied to any cell type and is extremely specific: it can target a single cell and it can deliver the genetic material exactly at the desired position, such as inside of the nucleus, with no damage to the cell. Since gene editing is a stochastic event occurring in only a fraction of the cells, the transfer of genetic material (or proteins) is of crucial importance in genome editing methods, where the nucleases must be efficiently delivered. The duration and magnitude of the nuclease expression are critical parameters for the level of both on-target and off-target nuclease activity. Additionally, the dose of donor template DNA is important to ensure efficient homologous recombination. The proposed method offers the possibility to deliver different molecules at different times, in synchronization with the cell cycle. The lab of Prof. Uji-i is one of the first (and few) groups worldwide to have developed and optimized a novel nanoscopic technique using 1D nanowires, with a diameter of less than 100 nm, for SERS endoscopic studies. It has been already proven by us that the thin diameter and 1D structure of the NW greatly reduces the damage induced to a live cell during probe insertion. Although designed for a different purpose, this nanoprobe is ideal as a starting point to develop a new NW-based gene delivery system.
New Drug delivery systems
In this decade, the pharmacology field has been intensively exploring different approaches to deliver multiple drugs with a single drug nano-carrier, such as liposomes, polymer nanoparticles, and inorganic nanoparticles. The advantage of nanoparticle based drug delivery is the ability to unify pharmacokinetics by simultaneous delivery of multiple drugs to specific target cells.
Ever since first reported in 2001, mesoporous silica nanoparticles (MSNPs) have manifested themselves as highly potential candidates for targeted drug delivery. They owe their popularity to their high drug load capacity, chemical stability, biocompatibility and easy functionalization. Since the diameter of the nanoparticles (100 to 200 nm) is tunable, one can obtain a size suitable for passive targeting through the hyperpermeable tumor vasculature, thereby promoting accumulation of the nanoparticles in tumor tissue due to the enhanced permeability and retention effect (EPR). Additionally, functionalization of the nanoparticles with ligands which have a high affinity for tumor cell specific surface receptors promotes more specific internalization in cancer cells. For example, hyaluronic acid (HA) has been extensively used as a targeting ligand due to its affinity for CD44, a transmembrane glycoprotein receptor that plays a critical role in malignant cell activities and, most importantly, it is overexpressed in many solid tumor cells, in metastasis and cancer stem cells.
Correlative AFM and Fluorescence Microscopy
Biological processes are often carried out in the context of macromolecular assemblies. In addition, arrangements of these complexes can be dynamic, resulting in a heterogeneous ensemble. Single molecule techniques can resolve distinct populations in heterogeneous systems, in contrast to bulk experiments where heterogeneity is averaged out. In turn, mechanistic details of bio-macromolecular interactions can be uncovered. Atomic force microscopy (AFM) is a technique that can generate 3D reconstructions of individual biomolecules and complexes thereof in a label-free fashion, and with ~ nm resolution. To this end a very sharp tip, mounted on a flexible cantilever, scans a sample surface in a raster pattern using a piezo-scanner, while keeping the interaction force between sample and tip constant. In every pixel (x,y) of the scanned area, the z-position is recorded. Consequently, a 3D representation of the surface topography can be reconstructed. An alternative way to study single molecules is by fluorescence microscopy. The molecule of interest is labeled with a fluorescent tag providing high contrast. Emission of the tag after excitation, is detected through an optical system. Due to the wave character of light, the emitted light is spread out on the detector described by the point spread function (PSF) of the optical system. This effect limits the resolution achieved with optical microscopy, referred to as the diffraction limit. However, when the signal of a single molecule is detected, the position of this molecule can be determined by fitting of the recorded fluorescence signal with a mathematical approximation of the PSF such as a two-dimensional Gaussian function. This principle underlies single molecule localization microscopy (SMLM). AFM and SMLM are highly complementary technologies: AFM can provide insight in topographic features at a nanometer resolution while SMLM is sensitive towards specifically labelled molecules in complex samples. Integrated setups combining both technologies can therefore provide orthogonal information at the single-molecule level.
Cell signalling: probes and methods
Cell signaling involves the sensing of an extracellular signal by a cell surface receptor, which then transduces this signal to an intracellular response. Despite the numerous studies performed on signaling pathways and mechanisms, little is known about the initial steps occurring at the plasma membrane: receptor pre-assembly at the molecular level and potential reorganization after ligand activation. Traditionally crystallography is used to investigate receptor multimerization. However, the crystallized state might not represent the biochemically active form due to the harsh preparation conditions and the absence of the cellular environment. Other approaches include macroscopic biochemical or biophysical methods, such as chemical cross-linking, ion-channel gating, immunoprecipitation or binding assays. Nowadays, established fluorescence imaging and spectroscopic techniques offer a versatile toolbox to study membrane receptor organization in (living) cells.
In the lab we are using fluorescence fluctuation spectroscopy to quantify physicochemical processes (mobility, binding affinity, stoichiometry, absolute concentration) occurring on a micro-to-millisecond time scale. Fluorescence experiments down to picoseconds are also commonly possible with methods such as time-correlated single photon counting (TCSPC), that allow, e.g., measuring fluorescence lifetimes and molecular tumbling. Additionally, spatially resolved microscopy with high temporal resolution also has clear benefits. For example, combined with confocal laser scanning microscopy (LSM), TCSPC allows protein-protein interactions (PPIs) to be imaged via Förster resonance energy transfer (FRET) based fluorescence lifetime imaging microscopy (FLIM). Imaging based FCS methods such as raster (RICS), number and brightness analysis (N&B) or (spatio-) temporal image correlation spectroscopy [(S)TICS] combine the quantitative analytical power of fluctuation methods with spatial information to map, among many other things, mobility and stoichiometry inside living systems. Simultaneous dual-color fluorescence imaging is possible when fast alternating excitation (alias pulsed interleaved excitation, PIE) is employed. PIE renders analysis of dual-color point FCS experiments considerably more straightforward. The combination of PIE with fluctuation imaging (PIE-FI) allows extracting the maximum amount of molecular information (mobility, stoichiometry, interactions…) from each species present in dual-color LSM images.
For more information on these methods:
- Hendrix J., Lamb D.C. (2014) Implementation and Application of Pulsed Interleaved Excitation for Dual-Color FCS and RICS. In: Engelborghs Y., Visser A. (eds) Fluorescence Spectroscopy and Microscopy. Methods in Molecular Biology (Methods and Protocols), vol 1076. Humana Press, Totowa, NJ (chapter can be found here)
- Hendrix J., Schrimpf W., Höller M., Lamb D.C. (2013) Pulsed Interleaved Excitation Fluctuation Imaging, Biophysical Journal, 105(4), 848-861 (article can be found here)
Imaging single HIV virions
Viruses are simple agents exhibiting complex reproductive mechanisms. Decades of research have provided crucial basic insights, antiviral medication and moderately successful gene therapy trials. The most infectious viral particle is, however, not always the most abundant one in a population, questioning the utility of classic ensemble-averaging virology. Indeed, viral replication is often not particularly efficient, prone to errors or containing parallel routes. In collaboration with Prof. Zeger Debeyser (KU Leuven) and Prof Hendrix (UHasselt) we have applied different single-molecule sensitive fluorescence methods to investigate viruses, one-by-one. While this collaboration is still ongoing, there is already several publications that show-case the potential of imaging single virions.
publications
DNA-Endonuclease Complex Dynamics by Simultaneous FRET and Fluorophore Intensity in Evanescent Field
Abstract
The single-molecule Förster resonance energy transfer (FRET) is a powerful tool to study interactions and conformational changes of biological molecules in the distance range from a few to 10 nm. In this study, we demonstrate a method to augment this range with longer distances. The method is based on the intensity changes of a tethered fluorophore, diffusing in the exponentially decaying evanescent excitation field. In combination with FRET it allowed us to reveal and characterize the dynamics of what had been inaccessible conformations of the DNA-protein complex. Our model system, restriction enzyme Ecl18kI, interacts with a FRET pair-labeled DNA fragment to form two different DNA loop conformations. The DNA-protein interaction geometry is such that the efficient FRET is expected for one of these conformations—“antiparallel” loop. In the alternative “parallel” loop, the expected distance between the dyes is outside the range accessible by FRET. Therefore, “antiparallel” looping is observed in a single-molecule time trajectory as discrete transitions to a state of high FRET efficiency. At the same time, transitions to a high-intensity state of the directly excited acceptor fluorophore on a DNA tether are due to a change of its average position in the evanescent field of excitation and can be associated with a loop of either “parallel” or “antiparallel” configuration. Simultaneous analysis of FRET and acceptor intensity trajectories then allows us to discriminate different DNA loop conformations and access the average lifetimes of different states.
Published in Biophysical Journal, 2017
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Probing the dynamics of restriction endonuclease NgoMIV-DNA interaction by single-molecule FRET
Abstract
Many type II restriction endonucleases require two copies of their recognition sequence for optimal activity. Concomitant binding of two DNA sites by such an enzyme produces a DNA loop. Here we exploit single-molecule Förster resonance energy transfer (smFRET) of surface-immobilized DNA fragments to study the dynamics of DNA looping induced by tetrameric endonuclease NgoMIV. We have employed a DNA fragment with two NgoMIV recognition sites and a FRET dye pair such that upon protein-induced DNA looping the dyes are brought to close proximity resulting in a FRET signal. The dynamics of DNA-NgoMIV interactions proved to be heterogeneous, with individual smFRET trajectories exhibiting broadly different average looped state durations. Distinct types of the dynamics were attributed to different types of DNA-protein complexes, mediated either by one NgoMIV tetramer simultaneously bound to two specific sites (“slow” trajectories) or by semi-specific interactions of two DNA-bound NgoMIV tetramers (“fast” trajectories), as well as to conformational heterogeneity of individual NgoMIV molecules.
Published in Biopolymers, 2017
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Influence of the Carotenoid Composition on the Conformational Dynamics of Photosynthetic Light-Harvesting Complexes
Abstract
Nonphotochemical quenching (NPQ) is the major self-regulatory mechanism of green plants, performed on a molecular level to protect them from an overexcitation during the direct sunlight. It is believed that NPQ becomes available due to conformational dynamics of the light-harvesting photosynthetic complexes and involves a direct participation of carotenoids. In this work, we perform a single-molecule microscopy on major light-harvesting complexes (LHCII) from different Arabidopsis thaliana mutants exhibiting various carotenoid composition. We show how the distinct carotenoids affect the dynamics of the conformational switching between multiple coexisting light-emitting states of LHCII and demonstrate that properties of the quenched conformation are not influenced by the particular carotenoids available in LHCII. We also discuss the possible origin of different conformational states and relate them to the fluorescence decay kinetics observed during the bulk measurements.
Published in Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2017
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Fluorescence Microscopy of Single Liposomes with Incorporated Pigment–Proteins
Abstract
Reconstitution of transmembrane proteins into liposomes is a widely used method to study their behavior under conditions closely resembling the natural ones. However, this approach does not allow precise control of the liposome size, reconstitution efficiency, and the actual protein-to-lipid ratio in the formed proteoliposomes, which might be critical for some applications and/or interpretation of data acquired during the spectroscopic measurements. Here, we present a novel strategy employing methods of proteoliposome preparation, fluorescent labeling, purification, and surface immobilization that allow us to quantify these properties using fluorescence microscopy at the single-liposome level and for the first time apply it to study photosynthetic pigment–protein complexes LHCII. We show that LHCII proteoliposome samples, even after purification with a density gradient, always contain a fraction of nonreconstituted protein and are extremely heterogeneous in both protein density and liposome sizes. This strategy enables quantitative analysis of the reconstitution efficiency of different protocols and precise fluorescence spectroscopic study of various transmembrane proteins in a controlled nativelike environment.
Published in Langmuir, 2018
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Fixed DNA Molecule Arrays for High-Throughput Single DNA–Protein Interaction Studies
Abstract
The DNA Curtains assay is a recently developed experimental platform for protein–DNA interaction studies at the single-molecule level that is based on anchoring and alignment of DNA fragments. The DNA Curtains so far have been made by using chromium barriers and fluid lipid bilayer membranes, which makes such a specialized assay technically challenging and relatively unstable. Herein, we report on an alternative strategy for DNA arraying for analysis of individual DNA–protein interactions. It relies on stable DNA tethering onto nanopatterned protein templates via high affinity molecular recognition. We describe fabrication of streptavidin templates (line features as narrow as 200 nm) onto modified glass coverslips by combining surface chemistry, atomic force microscopy (AFM), and soft lithography techniques with affinity-driven assembly. We have employed such chips for arraying single- and double-tethered DNA strands, and we characterized the obtained molecular architecture: we evaluated the structural characteristics and specific versus nonspecific binding of fluorescence-labeled DNA using AFM and total internal reflection fluorescence microscopy. We demonstrate the feasibility of our DNA molecule arrays for short single-tethered as well as for lambda single- and double-tethered DNA. The latter type of arrays proved very suitable for localization of single DNA–protein interactions employing restriction endonucleases. The presented molecular architecture and facile method of fabrication of our nanoscale platform does not require clean room equipment, and it offers advanced functional studies of DNA machineries and the development of future nanodevices.
Published in Langmuir, 2019
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Single-molecule microscopy studies of LHCII enriched in Vio or Zea
Abstract
Plants have developed multiple self-regulatory mechanisms to efficiently function under varying sunlight conditions. At high light intensities, non-photochemical quenching (NPQ) is activated on a molecular level, safely dissipating an excess excitation as heat. The exact molecular mechanism for NPQ is still under debate, but it is widely agreed that the direct participation of the carotenoid pigments is involved, one of the proposed candidate being the zeaxanthin. In this work, we performed fluorescence measurements of violaxanthin- and zeaxanthin-enriched major light-harvesting complexes (LHCII), in ensemble and at the single pigment–protein complex level, where aggregation is prevented by immobilization of LHCIIs onto a surface. We show that a selective enrichment of LHCII with violaxanthin or zeaxanthin affects neither the ability of LHCII to switch into a dissipative conformation nor the maximal level of induced quenching. However, the kinetics of the fluorescence decrease due to aggregation on the timescale of seconds are different, prompting towards a modulatory effect of zeaxanthin in the dynamics of quenching.
Published in Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2019
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Aggregation-Related Nonphotochemical Quenching in the Photosynthetic Membrane
Abstract
The photosynthetic apparatus of plants is a robust self-adjustable molecular system, able to function efficiently under varying environmental conditions. Under strong sunlight, it switches into photoprotective mode to avoid overexcitation by safely dissipating the excess absorbed light energy via nonphotochemical quenching (NPQ). Unfortunately, heterogeneous organization and simultaneous occurrence of multiple processes within the thylakoid membrane impede the study of natural NPQ under in vivo conditions; thus, usually artificially prepared antennae have been studied instead. However, it has never been shown directly that the origin of fluorescence quenching observed in these artificial systems underlies natural NPQ. Here we report the time-resolved fluorescence measurements of the dark-adapted and preilluminated—to induce NPQ—intact chloroplasts, performed over a broad temperature range. We show that their spectral response matches that observed in the LHCII aggregates, thus demonstrating explicitly for the first time that the latter in vitro system preserves essential properties of natural photoprotection.
Published in J. Phys. Chem. Lett., 2019
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Oriented Soft DNA Curtains for Single-Molecule Imaging
Abstract
Over the past 20 years, single-molecule methods have become extremely important for biophysical studies. These methods, in combination with new nanotechnological platforms, can significantly facilitate experimental design and enable faster data acquisition. A nanotechnological platform, which utilizes a flow-stretch of immobilized DNA molecules, called DNA Curtains, is one of the best examples of such combinations. Here, we employed new strategies to fabricate a flow-stretch assay of stably immobilized and oriented DNA molecules using a protein template-directed assembly. In our assay, a protein template patterned on a glass coverslip served for directional assembly of biotinylated DNA molecules. In these arrays, DNA molecules were oriented to one another and maintained extended by either single- or both-end immobilization to the protein templates. For oriented both-end DNA immobilization, we employed heterologous DNA labeling and protein template coverage with the antidigoxigenin antibody. In contrast to single-end immobilization, both-end immobilization does not require constant buffer flow for keeping DNAs in an extended configuration, allowing us to study protein–DNA interactions at more controllable reaction conditions. Additionally, we increased the immobilization stability of the biotinylated DNA molecules using protein templates fabricated from traptavidin. Finally, we demonstrated that double-tethered Soft DNA Curtains can be used in nucleic acid-interacting protein (e.g., CRISPR-Cas9) binding assay that monitors the binding location and position of individual fluorescently labeled proteins on DNA.
Published in Langmuir, 2021
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Aggregation-related quenching of LHCII fluorescence in liposomes revealed by single-molecule spectroscopy
Abstract
Incorporation of membrane proteins into reconstituted lipid membranes is a common approach for studying their structure and function relationship in a native-like environment. In this work, we investigated fluorescence properties of liposome-reconstituted major light-harvesting complexes of plants (LHCII). By utilizing liposome labelling with the fluorescent dye molecules and single-molecule microscopy techniques, we were able to study truly liposome-reconstituted LHCII and compare them with bulk measurements and liposome-free LHCII aggregates bound to the surface. Our results showed that fluorescence lifetime obtained in bulk and in single liposome measurements were correlated. The fluorescence lifetimes of LHCII were shorter for liposome-free LHCII than for reconstituted LHCII. In the case of liposome-reconstituted LHCII, fluorescence lifetime showed dependence on the protein density reminiscent to concentration quenching. The dependence of fluorescence lifetime of LHCII on the liposome size was not significant. Our results demonstrated that fluorescence quenching can be induced by LHCII – LHCII interactions in reconstituted membranes, most likely occurring via the same mechanism as photoprotective non-photochemical quenching in vivo.
Published in Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2021
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Microscopy made to order
Abstract
The proliferation of versatile open software and hardware for microscopy is helping to democratize biological imaging for both current and aspiring scientists.
Published in Nat Methods, 2021
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DNA Flow-Stretch Assays for Studies of Protein-DNA Interactions at the Single-Molecule Level
Abstract
Protein-DNA interactions are the core of the cells molecular machinery. For a long time, conventional biochemical methods served as a powerful investigatory basis of protein-DNA interactions and target search mechanisms. Currently single-molecule (SM) techniques have emerged as a complementary tool for studying these interactions and have revealed plenty of previously obscured mechanistic details. In comparison to the traditional ones, SM methods allow direct monitoring of individual biomolecules. Therefore, SM methods reveal reactions that are otherwise hidden by the ensemble averaging observed in conventional bulk-type methods. SM biophysical techniques employing various nanobiotechnology methods for immobilization of studied molecules grant the possibility to monitor individual reaction trajectories of biomolecules. Next-generation in vitro SM biophysics approaches enabling high-throughput studies are characterized by much greater complexity than the ones developed previously. Currently, several high-throughput DNA flow-stretch assays have been published and have shown many benefits for mechanistic target search studies of various DNA-binding proteins, such as CRISPR-Cas, Argonaute, various ATP-fueled helicases and translocases, and others. This review focuses on SM techniques employing surfaceimmobilized and relatively long DNA molecules for studying protein-DNA interaction mechanisms
Published in Applied Nano, 2022
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TGFβ-induced changes in membrane curvature influence Ras oncoprotein membrane localization
Abstract
In the course of cancer progression tumor cells undergo morphological changes that lead to increased motility and invasiveness thus promoting formation of metastases. This process called epithelial to mesenchymal transition (EMT) is triggered by transforming growth factor (TGFβ) but for gaining the full invasive potential an interplay between signaling of TGFβ and Ras GTPases is required. Ras proteins possess a lipidated domain that mediates Ras association with the plasma membrane, which is essential for Ras biological functions. Type and number of the lipid anchors are the main difference among three Ras variants—H-ras, N-ras and K-ras. The lipid anchors determine membrane partitioning of lipidated proteins into membrane areas of specific physico-chemical properties and curvature. In this study, we investigated the effect of TGFβ treatment on the subcellular localization of H-ras and K-ras. We show that TGFβ increases positive plasma membrane curvature, which is subsequently sensed by H-ras, leading to its elevated plasma membrane localization and activation. This observation suggests the existence of a novel positive feedback loop whereby the increased level of plasma membrane curvature during TGFβ induced EMT attracts more Ras molecules to the plasma membrane resulting in increased Ras activity which in turn promotes further EMT and thus ultimately enables the acquisition of full invasive potential.
Published in Scientific reports, 2022
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Disarming of type IF CRISPR-Cas surveillance complex by anti-CRISPR proteins AcrIF6 and AcrIF9
Abstract
CRISPR-Cas systems are prokaryotic adaptive immune systems that protect against phages and other invading nucleic acids. The evolutionary arms race between prokaryotes and phages gave rise to phage anti-CRISPR (Acr) proteins that act as a counter defence against CRISPR-Cas systems by inhibiting the effector complex. Here, we used a combination of bulk biochemical experiments, X-ray crystallography and single-molecule techniques to explore the inhibitory activity of AcrIF6 and AcrIF9 proteins against the type I-F CRISPR-Cas system from Aggregatibacter actinomycetemcomitans (Aa). We showed that AcrIF6 and AcrIF9 proteins hinder Aa-Cascade complex binding to target DNA. We solved a crystal structure of Aa1-AcrIF9 protein, which differ from other known AcrIF9 proteins by an additional structurally important loop presumably involved in the interaction with Cascade. We revealed that AcrIF9 association with Aa-Cascade promotes its binding to off-target DNA sites, which facilitates inhibition of CRISPR-Cas protection.
Published in Scientific reports, 2022
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The miEye: Bench-top super-resolution microscope with cost-effective equipment
Abstract
Commercial super-resolution (SR) imaging systems require a high budget, while current more affordable open source microscopy systems lack modularity and sometimes are too complex or lack reliability. We present miEye a cost-effective microscope designed for high-resolution wide-field fluorescence imaging. The build is constructed using a CNC milled aluminum microscope body and commercially available optomechanics, with open-source Python-based microscope control, data visualization, and analysis software integration. The data acquisition software works robustly with commonly used industrial-grade complementary metal oxide semiconductor (iCMOS) cameras, performs IR beam back-reflection-based automatic focus stabilization, and allows for laser control via an Arduino-based laser relay. The open-source nature of the design is aimed to facilitate adaptation by the community. The build can be constructed for a cost of roughly 50 k €. It contains SM-fiber and MM-fiber excitation paths that are easy to interchange and an adaptable emission path. Also, it ensures less than 5 nm per min stability of the sample on all axes, and allows achieving less than 30 nm lateral resolution for dSTORM and DNA-PAINT single-molecule localization microscopy (SMLM) experiments. Thus it serves as a cost-effective and adaptable addition to the open source microscopy community and potentially will allow high-quality SR imaging even for limited-budget research groups.
Published in HardwareX, 2022
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Advancing Understanding of DNA-BfiI Restriction Endonuclease Cis and Trans Interactions through smFRET Technology
Abstract
Monitoring of DNA-protein interactions is essential in understanding many biological processes. Proteins must find their target site on a DNA molecule to perform their function, and the mechanisms for target search differ across proteins. Revealing temporal interactions with two target sites, both in Cis and in Trans, is crucial in target search mechanisms studies. Here, we present two single-molecule Förster resonance energy transfer (smFRET)-based assays to study BfiI-DNA interactions. The first assay, smFRET-based DNA looping assay, detects both “Phi” and “U”-shaped DNA looping events. We modified it to only allow in Trans BfiI-target DNA interactions to improve specificity and reduce limitations in the observation time. Our TIRF microscopy measurements directly observe the on- and off-target binding events and characterize BfiI binding events. Our results show that BfiI binding events last longer on target sites and that the BfiI rarely changes conformations during binding. This newly developed assay could be useful for other two-targets-binding DNA-interacting proteins and could be employed for dsDNA substrate BfiI-PAINT, which is useful for DNA stretch-assays and other super-resolution fluorescence microscopy studies.
Published in bioRxiv, 2023
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smFRET Detection of Cis and Trans DNA Interactions by the BfiI Restriction Endonuclease
Abstract
Protein–DNA interactions are fundamental to many biological processes. Proteins must find their target site on a DNA molecule to perform their function, and mechanisms for target search differ across proteins. Especially challenging phenomena to monitor and understand are transient binding events that occur across two DNA target sites, whether occurring in cis or trans. Type IIS restriction endonucleases rely on such interactions. They play a crucial role in safeguarding bacteria against foreign DNA, including viral genetic material. BfiI, a type IIS restriction endonuclease, acts upon a specific asymmetric sequence, 5-ACTGGG-3, and precisely cuts both upper and lower DNA strands at fixed locations downstream of this sequence. Here, we present two single-molecule Förster resonance energy-transfer-based assays to study such interactions in a BfiI–DNA system. The first assay focuses on DNA looping, detecting both “Phi”- and “U”-shaped DNA looping events. The second assay only allows in trans BfiI–target DNA interactions, improving the specificity and reducing the limits on observation time. With total internal reflection fluorescence microscopy, we directly observe on- and off-target binding events and characterize BfiI binding events. Our results show that BfiI binds longer to target sites and that BfiI rarely changes conformations during binding. This newly developed assay could be employed for other DNA-interacting proteins that bind two targets and for the dsDNA substrate BfiI-PAINT, a useful strategy for DNA stretch assays and other super-resolution fluorescence microscopy studies.
Published in J. Phys. Chem. B, 2023
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Probing Activation and Conformational Dynamics of the Vesicle-Reconstituted β2 Adrenergic Receptor at the Single-Molecule Level
Abstract
G-protein-coupled receptors (GPCRs) are structurally flexible membrane proteins that mediate a host of physiological responses to extracellular ligands like hormones and neurotransmitters. Fine features of their dynamic structural behavior are hypothesized to encode the functional plasticity seen in GPCR activity, where ligands with different efficacies can direct the same receptor toward different signaling phenotypes. Although the number of GPCR crystal structures is increasing, the receptors are characterized by complex and poorly understood conformational landscapes. Therefore, we employed a fluorescence microscopy assay to monitor conformational dynamics of single β2 adrenergic receptors (β2ARs). To increase the biological relevance of our findings, we decided not to reconstitute the receptor in detergent micelles but rather lipid membranes as proteoliposomes. The conformational dynamics were monitored by changes in the intensity of an environmentally sensitive boron-dipyrromethene (BODIPY 493/503) fluorophore conjugated to an endogenous cysteine (located at the cytoplasmic end of the sixth transmembrane helix of the receptor). Using total internal reflection fluorescence microscopy (TIRFM) and a single small unilamellar liposome assay that we previously developed, we followed the real-time dynamic properties of hundreds of single β2ARs reconstituted in a native-like environment─lipid membranes. Our results showed that β2AR-BODIPY fluctuates between several states of different intensity on a time scale of seconds, compared to BODIPY-lipid conjugates that show almost entirely stable fluorescence emission in the absence and presence of the full agonist BI-167107. Agonist stimulation changes the β2AR dynamics, increasing the population of states with higher intensities and prolonging their durations, consistent with bulk experiments. The transition density plot demonstrates that β2AR-BODIPY, in the absence of the full agonist, interconverts between states of low and moderate intensity, while the full agonist renders transitions between moderate and high-intensity states more probable. This redistribution is consistent with a mechanism of conformational selection and is a promising first step toward characterizing the conformational dynamics of GPCRs embedded in a lipid bilayer.
Published in J. Phys. Chem. B, 2024
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Fluorescence quenching in aggregates of fucoxanthin–chlorophyll protein complexes: Interplay of fluorescing and dark states
Abstract
Diatoms, a major group of algae, account for about a quarter of the global primary production on Earth. These photosynthetic organisms face significant challenges due to light intensity variations in their underwater habitat. To avoid photodamage, they have developed very efficient non-photochemical quenching (NPQ) mechanisms. These mechanisms originate in their light-harvesting antenna – the fucoxanthin–chlorophyll protein (FCP) complexes. Spectroscopic studies of NPQ in vivo are often hindered by strongly overlapping signals from the photosystems and their antennae. Fortunately, in vitro FCP aggregates constitute a useful model system to study fluorescence (FL) quenching in diatoms. In this work, we present streak-camera FL measurements on FCPa and FCPb complexes, isolated from a centric diatom Cyclotella meneghiniana, and their aggregates. We find that spectra of non-aggregated FCP are dominated by a single fluorescing species, but the FL spectra of FCP aggregates additionally contain contributions from a redshifted emissive state. We relate this red state to a charge transfer state between chlorophyll c and chlorophyll a molecules. The FL quenching, on the other hand, is due to an additional dark state that involves incoherent energy transfer to the fucoxanthin carotenoids. Overall, the global picture of energy transfer and quenching in FCP aggregates is very similar to that of major light-harvesting complexes in higher plants (LHCII), but microscopic details between FCPs and LHCIIs differ significantly.
Published in Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2024
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Formation of Calprotectin Inhibits Amyloid Aggregation of S100A8 and S100A9 Proteins
Abstract
Calcium-binding S100A8 and S100A9 proteins play a significant role in various disorders due to their pro-inflammatory functions. Substantially, they are also relevant in neurodegenerative disorders via the delivery of signals for the immune response. However, at the same time, they can aggregate and accelerate the progression of diseases. Natively, S100A8 and S100A9 exist as homo- and heterodimers, but upon aggregation, they form amyloid-like oligomers, fibrils, or amorphous aggregates. In this study, we aimed to elucidate the aggregation propensities of S100A8, S100A9, and their heterodimer calprotectin by investigating aggregation kinetics, secondary structures, and morphologies of the aggregates. For the first time, we followed the in vitro aggregation of S100A8, which formed spherical aggregates, unlike the fibrillar structures of S100A9 under the same conditions. The aggregates were sensitive to amyloid-specific ThT and ThS dyes and had a secondary structure composed of β-sheets. Similarly to S100A9, S100A8 protein was stabilized by calcium ions, resulting in aggregation inhibition. Finally, the formation of S100A8 and S100A9 heterodimers stabilized the proteins in the absence of calcium ions and prevented their aggregation.
Published in ACS Chemical Neuroscience, 2024
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Liquid-liquid phase separation of alpha-synuclein increases the structural variability of fibrils formed during amyloid aggregation
Abstract
Protein liquid–liquid phase separation (LLPS) is a rapidly emerging field of study on biomolecular condensate formation. In recent years, this phenomenon has been implicated in the process of amyloid fibril formation, serving as an intermediate step between the native protein transition into their aggregated state. The formation of fibrils via LLPS has been demonstrated for a number of proteins related to neurodegenerative disorders, as well as other amyloidoses. Despite the surge in amyloid-related LLPS studies, the influence of protein condensate formation on the end-point fibril characteristics is still far from fully understood. In this work, we compare alpha-synuclein aggregation under different conditions, which promote or negate its LLPS and examine the differences between the formed aggregates. We show that alpha-synuclein phase separation generates a wide variety of assemblies with distinct secondary structures and morphologies. The LLPS-induced structures also possess higher levels of toxicity to cells, indicating that biomolecular condensate formation may be a critical step in the appearance of disease-related fibril variants.
Published in The FEBS Journal, 2024
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research
Open microscopy
Published:
talks
teaching
Teaching experience 1
This is a description of a teaching experience. You can use markdown like any other post.
Undergraduate course, University 1, Department, 2014
Teaching experience 2
This is a description of a teaching experience. You can use markdown like any other post.
Workshop, University 1, Department, 2015
team
Marijonas Tutkus
About Marijonas
Marijonas Tutkus during undergraduate studies at Dr Ramūnas Valiokas laboratory (FTMC) gained experience with the fluorescence microscopy, DNA biochips and DNA methylation. Later during graduate studies and research assistant work in prof. Dimitrios Stamou laboratory (Copenhagen University) he gained experience with SM TIRF microscopy, confocal microscopy, transmembrane proteins, liposomes, SM signal acquisition and analysis. Next, in Lithuania, he joined the Biophysical Research Laboratory (FTMC) where he in 2019 obtained a PhD degree in Physics. His PhD studies were focused on SM level fluorescence microscopy of DNA-protein interactions and ultrafast spectroscopy of light-harvesting antenna proteins. Right after finishing PhD studies he got a postdoc grant (Supervisor Dr. Mindaugas Zaremba) dedicated to SM level studies of prokaryotic Argonaute proteins’ target search mechanism in vitro and in vivo. Almost at the same time he got a grant as PI from Research Council of Lithuania for studying target search mechanisms of CRISPR-Cas proteins’ using in vitro assays such as his developed Soft DNA Curtains, and in vivo SM tracking methods. Recently he got an EMBL fellowship grant for studying DNA double-strand break repair inside living cells.
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MT Team
About TEAM
MT team at the 2022 annual department trip to Puvociai.
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MT Team
About TEAM
MT team in front of Life Sciences Center (Vilnius) of Vilnius University, 2023 September. This summer Aina and Monika joined our team. Ayush join the team last November.
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Tomas Marčiulionis
About Tomas
Tomas joined our team during his bachelor studies and worked on DNA-protein interaction studies using smFRET. He worked in our team from 2015 summer till 2016 autumn.
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Ignas Čiplys
About Ignas
Ignas joined our team during his bachelor studies and worked on light-harvesting antenna proteins from plants. He worked in our team from 2017 summer till 2018 autumn.
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Oskaras Venckus
About Oskaras
Oskaras joined our team during his bachelor studies and worked on DNA Curtains and light-harvesting antenna proteins from plants. He worked in our team from 2017 summer till 2019 spring.
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Šarūnė Ivanovaitė
About Šarūnė
Šarūnė joined our team during her bachelor studies and worked on DNA Curtains. She worked in our team with some few breaks from 2017 summer till 2022 summer.
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Ayush Ganguli
About Ayush
Ayush joined our team just recenty in the begining of 2023. He is joint PhD student of me and Algirdas Toleikis. Ayush is working with Soft DNA Curtains and single-molecule fluorescence microscopy for DNA-protein interaction studies. He is focusing on helicases and other translocating proteins.
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Aurimas Kopūstas
About Aurimas
Aurimas joined our team during his bachelor studies, obtained BSc. degree in Biochemistry (VU) and MSc. degree in Biochemistry (VU). Aurimas Kopūstas currently is a 2nd-year Biochemistry PhD student at VU. Since 2017 AK gained vast experience by developing assays for DNA-protein interaction studies and employing SM fluorescence microscopy. Currently he is focusing on in vivo and in vitro SM level studies of different bacteria antiviral defense systems. AK possesses an interdisciplinary and versatile skill set: SM fluorescence microscopy, sample preparation, genetic engineering and biochemical characterization of NA-binding proteins.
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Justė Paksaitė
About Justė
Justė joined our team during her bachelor studies, obtained BSc. degree in Molecular Biology (VU). Currently she is studying in MSc. Molecular biology programme (VU). Justė Paksaitė focuses on protein-DNA interaction studies at SM level, and also is highly skilled in protein expression, purification, genetic engineering, and cell work. During the MSc studies, Justė concentrated on researching Cas9 interactions with DNA in vivo.
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Simonas Ašmontas
About Simonas
Simonas joined our team during his MSc studies, and obtained MSc. degree in Molecular Biology (VU). He worked on pAgo studies using DNA Curtains and single-particle-tracking methods. He worked in our team till 2022 autumn.
Research themes
Publications with our group
| Disarming of type IF CRISPR-Cas surveillance complex by anti-CRISPR proteins AcrIF6 and AcrIF9 Egle Kupcinskaite, Marijonas Tutkus, Aurimas Kopūstas, Simonas Ašmontas, Marija Jankunec, Mindaugas Zaremba, Giedre Tamulaitiene, Tomas Sinkunas Published in Scientific reports, September 2022 (see publication ) |
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Meda Jurevičiūtė
About Meda
Meda joined our team during her bachelor studies, obtained BSc. degree in Molecular Biology (VU). And Currently is studying in MSc. Molecular biology programme (VU). Meda Jurevičiūtė is a first year MSc student in Molecular Biology at VU. In her BSc degree, she was working both with bacteria and mammalian cell lines, cell sample preparation for microscopy, and developed skills in cloning for bacterial and eukaryotic protein expression systems. Also, she has essential skills in SM fluorescence microscopy, data analysis, labelling of biomolecules and protein purification.
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Mohammad Nour Alsamsam
About Nour
Mohammad Nour Alsamsam joined our team during his bachelor studies, obtained BSc. degree in Physics (VU). Currently Nour is a 2nd-year photonics and nanotechnology MSc student and holds a cum laude light engineering bachelor’s degree from VU. He also has experience in software development, mechatronics engineering, and prototyping. He offers the skills required for adjusting and maintaining the existing microscopy optical setup to support different imaging modularities. His contribution so far includes prototyping the miEye SMLM microscope and providing the Python microEye package for hardware control, data acquisition, analysis, and visualization. On 2023 spring Nour obtained his physics Masters degre, and recently started with his PhD studies.
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Monika Roliūtė
About Meda
Monika joined our team right after her BSc thesis defense. Currently is studying in MSc. Molecular biology programme (VU). She is working both with bacteria and mammalian cell lines, cell sample preparation for microscopy, and cloning. Also, she is working on SM fluorescence microscopy, data analysis, labelling of biomolecules and protein purification.
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Aina El Ravvas
About Aina
Aina joined our research team on 2023 summer. She is BSc student in Neuro-biophysics study programme at Vilnius University. Aina is working with DNA-protein interaction studies at the single-molecule level and developing platforms for such a studies.
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Guoda Taraškevičiūtė
About Guoda
Guoda joined our research team for a few months practice on 2022.
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