PhD. Gražvydas Lukinavičius, Department of NanoBiophotinics, Max Plank Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Gottingen, Germany

Live-cell compatible fluorescent probes for chromatin nanoscopy

One of the most intriguing and challenging structures to image in the cell is chromatin – information carrying biopolymer, composed of DNA, RNA and proteins. Our probes consist of DNA binder – Hoechst conjugated to a series of fluorescent dyes compatible with live-cell fluorescence nanoscopy methods. A minimal change in the attachment point of the dye has dramatic effects on the properties of the final probe. All tested 6′-carboxyl dye-containing probes exhibited dual-mode binding to DNA and formed a dimmer complex at high DNA concentrations. The 5′-carboxyl dye-containing probes exhibited single-mode binding to DNA which translated into increased brightness and lower cytotoxicity. Up to 10-fold brighter nuclear staining by the newly developed probes allowed acquisition of stimulated emission depletion (STED) nanoscopy images of outstanding quality in living and fixed cells. Therefore we were able to estimate a diameter of ∼155 nm of the heterochromatin exclusion zones in the nuclear pore region in living cells. Finally, we have extended this approach to hydroxymethyl silicon-rhodamine and Hoechst conjugates which allowed single molecule localization microscopy imaging of heterochromatin in living cells at sub-30 nm resolution.

The recording of the talk:

PhD Kazimierz Trebacz
Department of Plant Physiology and Biophysics, Institute of Biological Sciences Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland
 kazimierz.trebacz@poczta.umcs.lublin.pl

Long-distance signaling in plants: electrophysiological and molecular approach

Plants as sessile organisms have developed a sophisticated system of adequate responses to environmental stimuli like changes in illumination, temperature, humidity etc., and many biotic and abiotic stressors causing partial plant damage. The long-distance signaling system, in addition to the chemical one based mainly on volatiles, consists of electrical signals: action potentials APs and variation potentials VPs. The ability to generate APs evolved already in algae and continued in Bryophyte after plant terrestrialization. In addition to APs, vascular plants, i.e. the next step in plant evolution, generate VPs in response to severe damaging stimuli like wounding or burning. The mechanism of APs and VPs has been examined for years with application of electrophysiological methods: extra- and intracellular potential measurements, voltage- and patch-clamp, ion-selective microelectrodes, MIFE, etc. The recent progress in molecular biology has resulted in genetic identification of potential players in AP/VP electrogenesis – ion channels. The development of optical methods of cytoplasmic Ca2+ imaging, especially GCaMP, allows observation of calcium waves accompanying APs or VPs. A combination of electrophysiological techniques, Ca2+ imaging, and experiments on ion channel mutants accelerate the process of detailed recognition of the molecular mechanism of AP and VP generation and their physiological consequences.

PhD student Dominyka Dapkutė
Biomedical Physics Laboratory in National Cancer Institute, Vilnius, Lithuania

The winner of L’Oréal Baltic-UNESCO Programme ‘For Women in Science’

Hitching a ride: stem cell-based delivery of theranostic nanoparticles

Nanomedicine and stem cell therapy are current scientific hot-topics. A synergy of both has potential application in oncology, especially in eradication of the most threatening cancer stem cells. Nanotechnology raises big hopes in treatment of cancer – a single nanoparticle can incorporate multiple functions and act as theranostic (combining both diagnostics and therapy) tool. However, the unwanted accumulation of nanoparticles in healthy tissues led to the search of their new more advanced delivery methods. Unique cells in our body called mesenchymal stem cells are responsible for tissue regeneration, also migrate specifically to cancerous tissue and therefore can be used as Trojan horses transporting cancer-killing nanoparticles. Delivery of antitumor agents using these cells is biologically compatible, clinically safe and showed tremendous success in pre-clinical studies. This project aims to address the current limitations of existing cancer diagnostics and therapeutics by investigating an elegant marriage between smartly-designed theranostic tool and stem cell-based nanoparticle delivery method. Hopefully, this intrinsically interdisciplinary research will constitute a significant leap towards the end of never-ending battle with cancer as well as bring innovative insights from a more fundamental point of view.

The recording of the talk:

PhD. Rima Budvydytė
Life Sciencs Center, University of Vilnius, Vilnius, Lithuania

The winner of L’Oréal Baltic-UNESCO Programme ‘For Women in Science’

Interactions of misfolded proteins with lipid membranes: Implications for neurodegeneration

A central event in pathogenesis of Alzhaimer’s diseases are thought to be intracellular and extracellular accumulation, aggregation and misfolding of low molecular mass peptides such as -amyloid (A1-42), tau protein (Tau) and s100A9.  Small size aggregates-oligomers were found to be extremely neurotoxic in vitro and in vivo with the ability to disrupt the major neuron membranes and lead to synaptic dysfunction, mithochondrial dysfunction, neuronal apoptosis and brain damage.

In this work different sizes of soluble recombinant A1-42 and s100A9 aggregates were used to investigate their interaction with tethered phospholipid membranes (tBLM) as well as their toxicity to rat cerebellar granule cells (CGC) [6].  The morphology and size of misfolded protein aggregates was monitored by dynamic light scattering (DLS) and atomic force microscopy (AFM). However, differently sized amyloid oligomers exhibited different levels of neurotoxicity in CGC toxicity tests.

These protein aggregates exhibited the different membrane damaging properties as probed by the electrochemical impedance spectroscopy (EIS). The function and morphology of s100A9 and amyloid beta aggregates were depending on different oligomerisation conditions: temperature and time. The interaction between amyloid beta, s100A9 and tBLM was monitored by EIS time series measurements. The observed lag phase of this interaction were significantly decreased at s100A9 aggregates concentration level. Membrane composition was found to be one of the important factors affecting the interaction of the misfolded oligomers to phospholipid membranes.

A link to the publication:

https://link.springer.com/content/pdf/10.1007%2Fs00249-017-1222-x.pdf

The recording of the talk:

Prof. Dr. Med. Sc. Ago Rinken
Institute of Chemistry, University of Tartu, Ravila 14a, 50411 Tartu, Estonia ago.rinken@ut.ee

Characterization of ligand binding to membrane proteins by using fluorescence anisotropy and TIRF microscopy-based assays.

The utilization of budded baculoviruses that express membrane proteins of interest on their surfaces has opened new possibilities for the characterization of ligand binding [1]. The fluorescence anisotropy (FA) -based assay allows on-line monitoring of ligand binding in solution and obtain valuable kinetic data, which enables to solve even complex binding mechanisms [2]. However, there are still numerous systems where FA assays are not applicable due to ligands’ size, binding affinity, or fluorophores’ properties. Therefore the total internal reflection fluorescence (TIRF) microscopy-based method was developed to study additional aspects of ligand binding. The novel method combines membrane protein display in budded baculovirus particles and the immobilization of these particles to a functionalized coverslip. To scale the system for more routine ligand binding assays, we developed both open-source multiwell systems and image analysis software SPOTNIC (http://gpcr.ut.ee/aparecium.html) for flexible assay design. The immobilization selectivity was determined, and ligand-binding assays were validated using budded baculovirus particles displaying neuropeptide Y Y1 receptors and high-affinity TAMRA labeled fluorescent ligand UR-MC026. The TIRFM based system could be extended to various advanced assays involving super-resolution methods, enabling the investigation and description of the ligand-binding processes of membrane proteins at the single-molecule level.

  1. Link R., Veiksina S., Tahk M.J., Laasfeld T., Paiste P., Kopanchuk S., Rinken A. (2020) The constitutive activity of melanocortin 4 receptors is allosterically modulated by zinc and copper ions. J. Neurochem. 153, 346-361. https://doi.org/10.1111/JNC.14933
  2. Rinken A., Lavogina D., Kopanchuk S. (2018) Assays with Detection of Fluorescence Anisotropy: Challenges and Possibilities to Characterize Ligand Binding to GPCRs. Trends Pharmacol. Sci. 39, 187-199. http://dx.doi.org/10.1016/j.tips.2017.10.004

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The recording of the talk:

Department of Physics, Department of Chemical and Physical Sciences, University of Toronto, Canada
E-mail: virgis.barzda@utoronto.ca

Laser Research Center, Faculty of Physics, Vilnius University Lithuania

Deciphering collagen structure and function with advanced nonlinear microscopy

KEY WORDS:  Nonlinear microscopy, polarimetric microscopy, Second-harmonic generation,
Stokes-Mueller polarimetry, Second-order Susceptibility, Collagen

Collagen is the main constituent of the extracellular matrix in biological tissues. The collagen is piezoelectric, and therefore its polarity and organization strongly influence the mechanical and electrical properties of the tissue. Being piezoelectric, collagen generates strong second harmonic response, which is used in nonlinear microscopy to visualize the structure of collagen fibers. The polarimetric second-harmonic generation (SHG) microscopy is a powerful imaging modality that enables to characterize nanoscopic structural organizations of biological materials beyond the diffraction limited resolution. The ultrastructural organization within individual voxels of the images is characterized by the nonlinear susceptibility tensor, which can be obtained using double Stokes-Mueller polarimetry of SHG signal recorded with a laser scanning microscope. In our studies, the double Stokes-Mueller polarimetric microscopy has been applied for investigations of collagen in the cardiac conduction system, and the extracellular matrix of malignant and normal tissues of various organs. The method can be readily used for histopathology investigations. Together with the Bgold standard H&E stained images, SHG polarimetric microscopy images can aid pathologist in cancer diagnostics of various tissue types.

Research profile

Dr. Virginijus Barzda is a Professor at the Departments of Physics, and Chemical and Physical Sciences, University of Toronto, and a Project Leader at the Laser Research Center, Physics Faculty, Vilnius University. His research focuses on the development of novel imaging modalities for non-linear optical microscopy. He developed a wide range of microscopy techniques that employ second harmonic generation, third harmonic generation and multiphoton excitation fluorescence image contrasts for non-invasive label-free imaging of biological structures, live cells and subcellular organelles. He also focuses on the ultrastructural characterization of biological tissue with nonlinear polarimetric microscopy methods. The ultrastructural imaging is applied to study organization of collagen in cancer tissue and to investigate contractility mechanisms in cardiac and skeletal muscles. In addition, he develops novel labels for nonlinear microscopy, and studies photosynthetic structures.

The recording of the talk:

Prof. Rytis Prekeris
Director of Molecular Biology Graduate Program Department of Cell and Developmental Biology School of medicine. Anshutz Medical Campus, University of Colorado

How to make invadopodia: the role of cytoskeleton dynamics and localized protein degradation during cancer cell metastasis

One of the most fundamental challenges in cell biology is understanding how cells migrate in three-dimensional extracellular matrices at specific times and to specific locations. Cell migration helps shape all tissues and organs during development and the disruption of mechanisms that normally control migration dramatically enhance the lethality of cancers. Recent studies have demonstrated that coordinated remodeling of actin cytoskeleton and endocytic membrane traffic is a key step in cell migration during normal development, as well as during cancer metastasis. In this presentation I will describe the most recent work from our laboratory on understanding the molecular machinery governing migration and invasion of cancer cells.

The recording of the talk: