Paweł Hermanowicz

AtomicJ: An open source software for analysis of force curves

We present an open source Java application for analysis of force curves and images recorded with the Atomic Force Microscope. AtomicJ supports a wide range of contact mechanics models and implements procedures that reduce the influence of deviations from the contact model. It generates maps of mechanical properties, including maps of Youngs modulus, adhesion force, and sample height. It can also calculate stacks, which reveal how samples response to deformation changes with indentation depth. AtomicJ analyzes force curves concurrently on multiple threads, which allows for high speed of analysis. It runs on all popular operating systems, including Windows, Linux, and Macintosh.

The impact of temperature on blue light induced chloroplast movements in Arabidopsis thaliana.

Chloroplast movements in Arabidopsis thaliana are controlled by two blue light photoreceptors, phototropin1 and phototropin2. Under weak blue light chloroplasts gather at cell walls perpendicular to the direction of incident light. This response, called chloroplast accumulation, is redundantly regulated by both phototropins. Under strong blue light chloroplasts move to cell walls parallel to the direction of incident light, this avoidance response being solely dependent on phototropin2. Temperature is an important factor in modulating chloroplast relocations. Here we focus on temperature effects in Arabidopsis leaves. At room temperature, under medium blue light chloroplasts start to move to cell walls parallel to the light direction and undergo a partial avoidance response. In the same conditions, at low temperatures the avoidance response is strongly enhanced-chloroplasts behave as if they were responding to strong light. Higher sensitivity of avoidance response is correlated with changes in gene expression. After cold treatment, in darkness, the expression of phototropin1 is down-regulated, while phototropin2 levels are up-regulated. The motile system of chloroplasts in Arabidopsis is more sensitive to blue light at low temperatures, similar to other species studied before. The physiological role of the cold-enhancement of the avoidance response is explained in the context of phototropin levels, photochemical activities and signaling in the cell.

Cell elasticity is an important indicator of the metastatic phenotype of melanoma cells.

The relationship between melanin pigmentation and metastatic phenotype of melanoma cells is an intricate issue, which needs to be unambiguously determined to fully understand the process of metastasis of malignant melanoma. Despite significant research efforts undertaken to solve this problem, the outcomes are far from being satisfying. Importantly, none of the proposed explanations takes into consideration biophysical aspects of the phenomenon such as cell elasticity. Recently, we have demonstrated that melanin granules dramatically modify elastic properties of pigmented melanoma cells. This prompted us to examine the mechanical effects of melanosomes on the transmigration abilities of melanoma cells. Here, we show for the first time that melanin granules inhibit transmigration abilities of melanoma cells in a number of granules dependent manner. Moreover, we demonstrate that the inhibitory effect of melanosomes is mechanical in nature. Results obtained in this study demonstrate that cell elasticity may play a key role in the efficiency of melanoma cells spread in vivo. Our findings may also contribute to better understanding of the process of metastasis of malignant melanoma.

Undifferentiated Bronchial Fibroblasts Derived from Asthmatic Patients Display Higher Elastic Modulus than Their Non-Asthmatic Counterparts

During asthma development, differentiation of epithelial cells and fibroblasts towards the contractile phenotype is associated with bronchial wall remodeling and airway constriction. Pathological fibroblast-to-myofibroblast transition (FMT) can be triggered by local inflammation of bronchial walls. Recently, we have demonstrated that human bronchial fibroblasts (HBFs) derived from asthmatic patients display some inherent features which facilitate their FMT in vitro. In spite of intensive research efforts, these properties remain unknown. Importantly, the role of undifferentiated HBFs in the asthmatic process was systematically omitted. Specifically, biomechanical properties of undifferentiated HBFs have not been considered in either FMT or airway remodeling in vivo. Here, we combine atomic force spectroscopy with fluorescence microscopy to compare mechanical properties and actin cytoskeleton architecture of HBFs derived from asthmatic patients and non-asthmatic donors. Our results demonstrate that asthmatic HBFs form thick and aligned ‘ventral’ stress fibers accompanied by enlarged focal adhesions. The differences in cytoskeleton architecture between asthmatic and non-asthmatic cells correlate with higher elastic modulus of asthmatic HBFs and their increased predilection to TGF-β-induced FMT. Due to the obvious links between cytoskeleton architecture and mechanical equilibrium, our observations indicate that HBFs derived from asthmatic bronchi can develop considerably higher static tension than non-asthmatic HBFs. This previously unexplored property of asthmatic HBFs may be potentially important for their myofibroblastic differentiation and bronchial wall remodeling during asthma development.

Mobilization of storage materials during light-induced germination of tomato (Solanum lycopersicum) seeds.

The aim of this study was to analyze the metabolism of storage materials in germinating tomato (Solanum lycopersicum) seeds and to determine whether it is regulated by light via phytochromes. Wild type, single and multiple phytochrome A, B1 and B2 mutants were investigated. Imbibed seeds were briefly irradiated with far-red or far-red followed by red light, and germinated in darkness. Triacylglycerols and starch were quantified using biochemical assays in germinating seeds and seedlings during the first 5 days of growth. To investigate the process of fat-carbohydrate transformation, the activity of the glyoxylate cycle was assessed. Our results confirm the role of phytochrome in the control of tomato seed germination. Phytochromes A and B2 were shown to play specific roles, acting antagonistically in far-red light. While the breakdown of triacylglycerols proceeded independently of light, phytochrome control was visible in the next stages of the lipid-carbohydrate transformation. The key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, were regulated by phytochrome(s). This was reflected in a greater increase of starch content during seedling growth in response to additional red light treatment. This study is the first attempt to build a comprehensive image of storage material metabolism regulation by light in germinating dicotyledonous seeds.

Blue light-dependent changes in loosely bound calcium in Arabidopsis mesophyll cells: an X-ray microanalysis study

Highlight Localization of loosely bound calcium in Arabidopsis mesophyll changes under strong blue light in the wild type, but not in phot2 and phot1phot2 mutants. This indicates that phot2 is involved in calcium homeostasis.

Inhibition of DNA replication by an anti-PCNA aptamer/PCNA complex

Abstract Proliferating cell nuclear antigen (PCNA) is a multifunctional protein present in the nuclei of eukaryotic cells that plays an important role as a component of the DNA replication machinery, as well as DNA repair systems. PCNA was recently proposed as a potential non-oncogenic target for anti-cancer therapy. In this study, using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method, we developed a short DNA aptamer that binds human PCNA. In the presence of PCNA, the anti-PCNA aptamer inhibited the activity of human DNA polymerase δ and ϵ at nM concentrations. Moreover, PCNA protected the anti-PCNA aptamer against the exonucleolytic activity of these DNA polymerases. Investigation of the mechanism of anti-PCNA aptamer-dependent inhibition of DNA replication revealed that the aptamer did not block formation, but was a component of PCNA/DNA polymerase δ or ϵ complexes. Additionally, the anti-PCNA aptamer competed with the primer-template DNA for binding to the PCNA/DNA polymerase δ or ϵ complex. Based on the observations, a model of anti-PCNA aptamer/PCNA complex-dependent inhibition of DNA replication was proposed.

Selection and analysis of a DNA aptamer binding α-amanitin from Amanita phalloides

Mushroom foraging is very popular in some regions of the world. Sometimes toxic and edible mushrooms are mistaken by mushroom collectors, leading to serious human poisoning. The group of mushrooms highly dangerous for human health includes Amanita phalloides. This mushroom produces a toxic octapeptide called α-amanitin which is an inhibitor of nuclear RNA polymerase II. The inhibition of this polymerase results in the abortion of mRNA synthesis. The ingestion of A. phalloides causes liver failure due to the fact that most of the toxin is uptaken by hepatocytes. The hospitalization of poisoned patients involves the removal of the toxin from the digestive tract, its dilution in the circulatory system and the administration of therapeutic adjuvants. Since there is no effective antidote against amanitin poisoning, in this study we developed a DNA aptamer exhibiting specific binding to α-amanitin. This aptamer was selected using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method. Next, its ability of toxin removal from aqueous solution was confirmed by pull-down assay. The aptamer region sufficient for α-amanitin binding was determined. Finally, the dissociation constant of the α-amanitin/DNA aptamer complex was calculated.

Fine tuning chloroplast movements through physical interactions between phototropins.

Highlight Physical interactions between phototropin molecules can alter their signaling outcomes, providing the means for the plant to fine-tune its blue light responses.

6,4–PP Photolyase Encoded by AtUVR3 is Localized in Nuclei, Chloroplasts and Mitochondria and its Expression is Down-Regulated by Light in a Photosynthesis-Dependent Manner

Pyrimidine dimers are the most important DNA lesions induced by UVB irradiation. They can be repaired directly by photoreactivation or indirectly by the excision repair pathways. Photoreactivation is carried out by photolyases, enzymes which bind to the dimers and use the energy of blue light or UVA to split bonds between adjacent pyrimidines. Arabidopsis thaliana has three known photolyases: AtPHR1, AtCRY3 and AtUVR3. Little is known about the cellular localization and regulation of AtUVR3 expression. We have found that its transcript level is down-regulated by light (red, blue or white) in a photosynthesis-dependent manner. The down-regulatory effect of red light is absent in mature leaves of the phyB mutant, but present in leaves of phyAphyB. UVB irradiation does not increase AtUVR3 expression in leaves. Transiently expressed AtUVR3-green fluorescent protein (GFP) is found in the nuclei, chloroplasts and mitochondria of Nicotiana benthamiana epidermal cells. In the nucleoplasm, AtUVR3-GFP is distributed uniformly, while in the nucleolus it forms speckles. Truncated AtUVR3 and muteins were used to identify the sequences responsible for its subcellular localization. Mitochondrial and chloroplast localization of AtUVR3 is independent of its N-terminal sequence. Amino acids located at the C-terminal loop of the protein are involved in its transport into chloroplasts and its retention inside the nucleolus.