Long-term fluorescence live-cell imaging experiments have long been limited by the effects of exc... more Long-term fluorescence live-cell imaging experiments have long been limited by the effects of excitation-induced phototoxicity. The advent of light-sheet microscopy now allows users to overcome this limitation by restricting excitation to a narrow illumination plane. In addition, light-sheet imaging allows for high-speed image acquisition with uniform illumination of samples composed of multiple cell layers. The majority of studies conducted thus far have used custom-built platforms with specialized hardware and software, along with specific sample handling approaches. The first versatile commercially available light-sheet microscope, Lightsheet Z.1, offers a number of innovative solutions, but it requires specific strategies for sample handling during long-term imaging experiments. There are currently no standard procedures describing the preparation of plant specimens for imaging with the Lightsheet Z.1. Here we describe a detailed protocol to prepare plant specimens for light-sheet microscopy, in which Arabidopsis seeds or seedlings are placed in solid medium within glass capillaries or fluorinated ethylene propylene tubes. Preparation of plant material for imaging may be completed within one working day.
Methods in molecular biology (Clifton, N.J.), 2014
Reorganization of microtubules during cell cycle depends on the modulation of activity of microtu... more Reorganization of microtubules during cell cycle depends on the modulation of activity of microtubule-associated proteins. MAP65 is one of the main microtubule structural proteins in plants responsible for the formation of bundles of parallel and antiparallel microtubules. A member of MAP65 protein family, MAP65-1, binds to microtubules of preprophase band during early stages of cell division and later to the midzone of anaphase spindle and the phragmoplast, but exhibits no or reduced microtubule binding during metaphase. Artificially induced interaction of MAP65-1 with microtubules during metaphase promotes excessive formation of pole-to-pole microtubule bundles and causes delay of anaphase onset. The exact mechanism of this delay is not known, but it was suggested that microtubule bundles induced by MAP65 impose spatial constraints on the chromosome movement obstructing their alignment in the metaphase plate. Interaction of MAP65-1 with microtubules is controlled by phosphorylatio...
Methods in molecular biology (Clifton, N.J.), 2008
Plant embryogenesis requires a tight balance between cell proliferation and differentiation. In a... more Plant embryogenesis requires a tight balance between cell proliferation and differentiation. In animals, embryogenesis is dependent on cell migrations, which is in contrast to plant embryogenesis where the rigid cell wall precludes migration. Therefore, plants have to position cells correctly by defining the direction of the division plane during proliferation and control cell shape by local cell expansion. Both these processes are reliant on the organization and dynamics of the cytoskeleton-actin filaments and microtubules. In previous work (7), we have shown that differentiation of the embryo suspensor is accompanied by reorientation of microtubules from random to transverse and reorganization of actin filaments from a fine filamentous network to bundled longitudinal cables. Here, we describe the technique for visualization of cytoskeletal components including actin filaments, microtubules and their associated proteins during the development of plant embryos in whole-mount specimens.
Vacuolar programmed cell death (PCD) is indispensable for plant development and is accompanied by... more Vacuolar programmed cell death (PCD) is indispensable for plant development and is accompanied by a dramatic growth of lytic vacuoles, which gradually digest cytoplasmic content leading to self-clearance of dying cells. Our recent data demonstrate that vacuolar PCD critically requires autophagy and its upstream regulator, a caspase-fold protease metacaspase. Furthermore, both components lie downstream of the point of no return in the cell-death pathway. Here we consider the possibilities that i) autophagy could have both cytotoxic and cytoprotective roles in the vacuolar PCD, and ii) metacaspase could augment autophagic flux through targeting an as yet unknown autophagy repressor.
Reorganization of the actin and microtubule networks is known to occur in targeted vascular paren... more Reorganization of the actin and microtubule networks is known to occur in targeted vascular parenchymal root cells upon infection with the nematode Meloidogyne incognita. Here, we show that actin-depolymerizing factor (ADF) is upregulated in the giant feeding cells of Arabidopsis thaliana that develop upon nematode infection and that knockdown of a specific ADF isotype inhibits nematode proliferation. Analysis of the levels of transcript and the localization of seven ADF genes shows that five are upregulated in galls that result from the infection and that ADF2 expression is particularly increased between 14 and 21 d after nematode inoculation. Further analysis of ADF2 function in inducible RNA interference lines designed to knock down ADF2 expression reveals that this protein is required for normal cell growth and plant development. The net effect of decreased levels of ADF2 is F-actin stabilization in cells, resulting from decreased F-actin turnover. In nematode-infected plants wi...
Long-term fluorescence live-cell imaging experiments have long been limited by the effects of exc... more Long-term fluorescence live-cell imaging experiments have long been limited by the effects of excitation-induced phototoxicity. The advent of light-sheet microscopy now allows users to overcome this limitation by restricting excitation to a narrow illumination plane. In addition, light-sheet imaging allows for high-speed image acquisition with uniform illumination of samples composed of multiple cell layers. The majority of studies conducted thus far have used custom-built platforms with specialized hardware and software, along with specific sample handling approaches. The first versatile commercially available light-sheet microscope, Lightsheet Z.1, offers a number of innovative solutions, but it requires specific strategies for sample handling during long-term imaging experiments. There are currently no standard procedures describing the preparation of plant specimens for imaging with the Lightsheet Z.1. Here we describe a detailed protocol to prepare plant specimens for light-sheet microscopy, in which Arabidopsis seeds or seedlings are placed in solid medium within glass capillaries or fluorinated ethylene propylene tubes. Preparation of plant material for imaging may be completed within one working day.
Methods in molecular biology (Clifton, N.J.), 2014
Reorganization of microtubules during cell cycle depends on the modulation of activity of microtu... more Reorganization of microtubules during cell cycle depends on the modulation of activity of microtubule-associated proteins. MAP65 is one of the main microtubule structural proteins in plants responsible for the formation of bundles of parallel and antiparallel microtubules. A member of MAP65 protein family, MAP65-1, binds to microtubules of preprophase band during early stages of cell division and later to the midzone of anaphase spindle and the phragmoplast, but exhibits no or reduced microtubule binding during metaphase. Artificially induced interaction of MAP65-1 with microtubules during metaphase promotes excessive formation of pole-to-pole microtubule bundles and causes delay of anaphase onset. The exact mechanism of this delay is not known, but it was suggested that microtubule bundles induced by MAP65 impose spatial constraints on the chromosome movement obstructing their alignment in the metaphase plate. Interaction of MAP65-1 with microtubules is controlled by phosphorylatio...
Methods in molecular biology (Clifton, N.J.), 2008
Plant embryogenesis requires a tight balance between cell proliferation and differentiation. In a... more Plant embryogenesis requires a tight balance between cell proliferation and differentiation. In animals, embryogenesis is dependent on cell migrations, which is in contrast to plant embryogenesis where the rigid cell wall precludes migration. Therefore, plants have to position cells correctly by defining the direction of the division plane during proliferation and control cell shape by local cell expansion. Both these processes are reliant on the organization and dynamics of the cytoskeleton-actin filaments and microtubules. In previous work (7), we have shown that differentiation of the embryo suspensor is accompanied by reorientation of microtubules from random to transverse and reorganization of actin filaments from a fine filamentous network to bundled longitudinal cables. Here, we describe the technique for visualization of cytoskeletal components including actin filaments, microtubules and their associated proteins during the development of plant embryos in whole-mount specimens.
Vacuolar programmed cell death (PCD) is indispensable for plant development and is accompanied by... more Vacuolar programmed cell death (PCD) is indispensable for plant development and is accompanied by a dramatic growth of lytic vacuoles, which gradually digest cytoplasmic content leading to self-clearance of dying cells. Our recent data demonstrate that vacuolar PCD critically requires autophagy and its upstream regulator, a caspase-fold protease metacaspase. Furthermore, both components lie downstream of the point of no return in the cell-death pathway. Here we consider the possibilities that i) autophagy could have both cytotoxic and cytoprotective roles in the vacuolar PCD, and ii) metacaspase could augment autophagic flux through targeting an as yet unknown autophagy repressor.
Reorganization of the actin and microtubule networks is known to occur in targeted vascular paren... more Reorganization of the actin and microtubule networks is known to occur in targeted vascular parenchymal root cells upon infection with the nematode Meloidogyne incognita. Here, we show that actin-depolymerizing factor (ADF) is upregulated in the giant feeding cells of Arabidopsis thaliana that develop upon nematode infection and that knockdown of a specific ADF isotype inhibits nematode proliferation. Analysis of the levels of transcript and the localization of seven ADF genes shows that five are upregulated in galls that result from the infection and that ADF2 expression is particularly increased between 14 and 21 d after nematode inoculation. Further analysis of ADF2 function in inducible RNA interference lines designed to knock down ADF2 expression reveals that this protein is required for normal cell growth and plant development. The net effect of decreased levels of ADF2 is F-actin stabilization in cells, resulting from decreased F-actin turnover. In nematode-infected plants wi...
Conference Paper: 73rd Annual Scientific Conference of Univ. Latvia, Section: FOTONIKA-LV FP7-REGPOT-CT-2011-285912 Project - The Third Year Scientific Outcomes, 6-7 Feb 2015, Riga, Latvia, Book of Abstracts, Ed. A. Atvars, V. Beldavs, A. Ubelis, Riga Photonics Centre, Univ. Latvia, Feb 6, 2015
Possible approaches to bio-sensing and diagnosis of environmental stress-induced responses in mit... more Possible approaches to bio-sensing and diagnosis of environmental stress-induced responses in mitochondrial subcellular structures by biomarkers using (i) fluorescent optical microscopy and flow cytometry or (ii) bioelectronic sensors based on field-effect transistors with semiconductor nanowires and microwave whispering-gallery-mode dielectric resonators with microfluidic channels are discussed and analyzes in terms of their use in biological studies. References [1] N. Kandaurova, et al., Peculiarities of optical imaging of mitochondrial organelles in biological studies, in Optics and High Technology Material Science, Taras Shevchenko Nat. Univ. Kyiv, DP.7 p.213 (2013) [2] M. Landrum M. et al., BODIPY probes to study peroxisome dynamics in vivo, Plant J., Vol.62, pp.529-538 (2010) [3] A. Poghossian, M. J. Schoning, Label-free sensing of biomolecules with field-effect devices for clinical applications, Electroanalysis, Vol.26, pp.1197-1213 (2014)
Conference Paper: 15th Ukrainian Conference on Space Research, 24-28 Aug 2015, Odessa, Ukraine, Book of Abstracts, Ed. O. Fedorov, T. Skorokhod, K.: SRI NASU, Aug 1, 2015
Possible approaches to biosensing and diagnosis of UV stress-induced reactions in plants using re... more Possible approaches to biosensing and diagnosis of UV stress-induced reactions in plants using reactive oxygen species (ROS) in the cell organelles: mitochondria, peroxisomes and chloroplasts as biomarkers to study the plant cells ability to adapt and defend from supraphysiological levels of solar radiation in extreme environments to understand the mechanisms of bioprotection and predict the chance of survival in case of catastrophic changes in UV climate are discussed and analyzes in terms of their use in space biological studies.
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Papers by Andrei Smertenko
References
[1] N. Kandaurova, et al., Peculiarities of optical imaging of mitochondrial organelles in biological studies, in Optics and High Technology Material Science, Taras Shevchenko Nat. Univ. Kyiv, DP.7 p.213 (2013)
[2] M. Landrum M. et al., BODIPY probes to study peroxisome dynamics in vivo, Plant J., Vol.62, pp.529-538 (2010)
[3] A. Poghossian, M. J. Schoning, Label-free sensing of biomolecules with field-effect devices for clinical applications, Electroanalysis, Vol.26, pp.1197-1213 (2014)