Research - Laboratories of Core Facilities - Laboratory of Cellular Imaging

Aladina KALAC
ITC student

Ferhan AYAYDIN
senior research associate

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Ildikó VALKONYNÉ KELEMEN laboratory assistant

CELLULAR IMAGING LABORATORY

At the Cellular Imaging Laboratory, we are strongly committed to the development and application of advanced imaging, fluorescent labeling and biochemical techniques and approaches that will enable us and others to understand the complex organization within and between cells. Our recent achievements include the application of the fast and robust ethynyldeoxyuridine-based replication assay for plant cells, discovery of novel blue fluorescent dyes for in vivo detection and 3D analysis of lipid droplets and high efficiency oligonucleotide-directed gene editing monitored by mutant GFP and fluorescence imaging methods.


Our modern imaging center is equipped with state-of-the-art confocal laser scanning microscopes, fluorescence and stereo microscopes, two photon microscope, laser microdissection microscope and powerful image analysis computers with imaging software. With these modern microscopes we can perform protein localization and mobility analyses, three dimensional, time course dynamic analyses of live cells, tissues and organisms. Thanks to the new imaging techniques and the development of new fluorescent dyes and proteins, today’s biological and medical research has increasingly become dependent on microscopy and image analysis. It is now possible to specifically label virtually any molecule and directly probe its function in live cells by light microscopy.


This ability to visualize the dynamics of proteins in vesicles, organelles, cells and tissue has begun to provide new insights into how cells function in health and disease. Such work yields unique mechanistic insight by directly illustrating the complex spatial-temporal dynamics of fundamental cellular processes such as mitosis, morphogenesis, polarization, embryonic development, membrane trafficking and cytoskeleton dynamics. Many of these processes are highly dynamic and are challenging to image by traditional means. In this aim, we are strongly committed to the development and application of optical imaging, fluorescent labeling and biochemical techniques that will enable us and others to understand the complex organization within and between cells.

Microscopy and cytometry application of a new cell proliferation assay

Labeling, detection and quantification of cells in the S-phase (DNA synthesis) of cell cycle progression are crucial in characterizing the cellular responses to various treatments and genetic modifications. Bromo-deoxyuridine (BrdU) labeling of cells followed by antibody staining is the standard method for detecting cells in the S-phase. Antibody detection of BrdU involves harsh treatments or nuclease digestion to facilitate epitope access. Moreover in plants cells, cell wall digestion is also necessary. These steps could interfere with cellular morphology and are time-consuming. We have optimized the recently developed ethynyl-deoxyuridine (EdU) method on plant cell cultures and seed-derived roots as well as on isolated plant nuclei using confocal laser scanning microscopy and flow cytometry (Kotogany et al., 2010, Ayaydin et al., 2011, Kuntam and Ayaydin, 2015)


Microscopy and cytometry application of EdU-based replication assay


Novel fluorescent dyes for plant lipid droplet imaging

Plant lipid droplets (LDs) similar to their yeast and mammalian counterparts, are highly dynamic. Their proteomic analysis has revealed that a host of proteins reside on their surface with varying composition under different conditions. But a lot still remains to be uncovered in the area of LD protein and lipid composition as well as LD transport, mechanism of protein targeting, assembly and regulation. Live cell analysis is thus required to unravel the dynamic regulation of this important organelle. We have recently reported the development and characterization of novel fluorochromes as markers for LDs in living plant cells which emit in the blue range hence presenting flexibility during multicolor imaging. (Kuntam et al., 2015)


Discovery of novel fluorescent dyes for plant lipid droplet imaging


High efficiency oligonucleotide-directed gene editing

Targeted genome editing has been developed as an alternative to classical mutation breeding and transgenic (GMO) methods to improve crop plants. The Oligonucleotide Directed Mutagenesis (ODM) as Targeted Nucleotide Exchange (TNE) by single stranded DNA oligonucleotides (SDOs) attracts special attention for use in both basic science and plant breeding. On the other hand, one of the major limitations of this technique is the low frequency of TNE events. Using a mutant version of green fluorescent protein and chromatin modifying agents, recently, in collaboration with Prof. Dénes Dudits (Institute of Plant Biology, BRC, HAS, Szeged), we have achieved significantly increased frequency of TNE on cultured maize cells. We predict our results will increase the use and applicability of this new gene editing technique which in turn may lead to crops with improved characteristics not imparted by introduction of foreign DNA sequences. (Tiricz et al, 2015)


Restoration of GFP fluorescence by targeted nucleotide exchange

Selected publications

Ayaydin, F., Vissi, E., Meszaros, T., Miskolczi, P., Kovacs, I., Feher, A., Dombradi, V., Erdodi, F., Gergely, P. and Dudits, D. (2000). Inhibition of serine/threonine-specific protein phosphatases causes premature activation of cdc2MsF kinase at G2/M transition and early mitotic microtubule organization. Plant J. 23(1):85-96.

Ayaydin, F. and Dasso, M. (2004). Distinct in vivo dynamics of vertebrate SUMO paralogues. Mol. Biol. Cell 15(12):5208-5218.

Kotogány, E., Dudits, D., Horváth, V.G. and Ayaydin F. (2010). A rapid and robust assay for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine. Plant Methods (6:5)

Ayaydin, F., Kotogány E., Ábrahám E and Horváth V.G. (2011). Synchronization of Medicago sativa Cell Suspension Culture. Methods Mol Biol. 761:227-238.

Kuntam S., Puskás L.G., Ayaydin F. (2015). Characterization of a new class of blue-fluorescent lipid droplet markers for live-cell imaging in plants. Plant Cell Rep. 34:655-665.

Ayaydin F., Biro J., Domoki M., Ferenc G., Feher A. (2015) Arabidopsis NAP-related proteins (NRPs) are soluble nuclear proteins immobilized by heat. Acta Phy. Plant 37: Paper 3. 8 p.

Kuntam S. and Ayaydin F. Detection of S-phase of cell division cycle in plant cells and tissues by using 5-ethynyl-2’-deoxyuridine (EdU) (2015) in Plant Microtechniques: Methods and Protocols. Eds. Yeung C.T.E., Stasolla C, Sumner M.J., Huang B.Q. Springer, pp. 311-322.

Dudits D., Török K., Cseri A., Paul K., Nagy A.V., Nagy B., Sass L., Ferenc G., Vankova R., Dobrev P., Vass I., Ayaydin F. (2016) Response of Organ Structure and Physiology to Autotetraploidization in Early Development of Energy Willow Salix viminalis. Plant Physiol. 170:1504-1523.

Tiricz H., Nagy B., Ferenc G., Török K., Nagy I., Dudits D. and Ayaydin F. (2017, submitted) Relaxed chromatin induced by histone deacetylase inhibitors improves the oligonucleotide-directed gene editing in plant cells