Scanning Confocal Microscope Detection of Nuclear Actin Using Green Fluorecent Protein (GFP)


17. září 2010 v 14:34 | Cell Biology

„Průkaz jaderného aktinu skenovacím konfokálním mikroskopem pomocí zeleně fluoreskujího proteinu GFP“
(Abstract), in Czech.
P. Kufner, L. Hájková J. Reischig, M. Salášek
Charles Univ. in Praque, Faculty of Medicine in Pilsen, Institute of Biology.

Transgenic Drosophila larvae were studied with respect to the expression and distribution pattern of GFP-coupled proteins. Larval organs were isolated and the fluorescent signal scrutinized using laser scanning confocal microscopy. Cytoplasms, nuclei, and non-cellular backgrounds were submitted to spectral analysis and fluorescence intensity measurements. The cell line expressing GFP-actin exhibited both cytoplasmic and nuclear staining, which was not the case in the remaining two cell lines, whose staining was exclusively cytoplasmic, and which can in this respect be considered as negative controls. The presence of actin in the eukaryotic nucleus has not been unequivocally accepted by the scientific community and the observations remained an enigma until the very recent years when several examples of nuclear functions of actin have been demonstrated. Our findings may represent a significant contribution to the growing understanding of the role of actin in the nucleus.

Melatonin protects mitochondrial transmembrane potential from the effect of antimycin A in normal as well as cancer cell lines


Melatonin protects mitochondrial transmembrane potential from the effect of antimycin A in normal as well as cancer cell lines 6. listopadu 2008 v 11:42 | MUDr. Petr Kufner, MUDr. Lucie Hájková, Ph.D., Doc. MUDr. Josef Reischig, CSc. | Cell Biology Melatonin (N-acetyl-5-metoxytryptamin) has been reported to have beneficial effects in an ever growing number of … Celý příspěvek

Melatonin and mitochondrial transmembrane potential


Melatonin protects mitochondrial transmembrane potential from the effect of antimycin A in normal as well as cancer cell lines

6. listopadu 2008 v 11:42 | MUDr. Petr Kufner, MUDr. Lucie Hájková, Ph.D., Doc. MUDr. Josef Reischig, CSc. | Cell Biology
Melatonin (N-acetyl-5-metoxytryptamin) has been reported to have beneficial effects in an ever growing number of pathological conditions. This hormone produced predominantly by the pineal gland is well-known for its direct (ROS scavenger), as well as many indirect antioxidative effects, and is therefore a promising candidate for a therapeutic agent possibly with a minimal level of inauspicious side effects. Deeper understanding is required before its routine clinical use, however.
Ischemia-reperfusion injury (IRI), a typical phenomenon in various ischemia-related pathological states including organ transplantations, is caused by an overproduction of reactive oxygen species (ROS) in the reperfused tissue. The ROS cause cellular oxidative stress and tissue damage.
The aim of our efforts has been to design a model for monitoring and treatment of hypoxia-related processes. We have established an assay for a study of cellular oxidative stress using cultured mammalian cells. As a measure of the oxidative state of the cells, mitochondrial transmembrane potential (ΔΨm) was chosen. The potential was monitored in live cells stained with the fluorescent probe JC-1, whose transition between the monomeric state and JC-1 aggregates, accompanied by a shift of the emission wavelengths (from λ = 529nm to λ = 590nm), is dependent on the value of the mitochondrial transmembrane potential. Live adherent cells of various cell lines (cancerous as well as normal, of mammalian origin including human), grown on the bottom glass of a Bachofer perfusion chamber, were observed in the Olympus Fluoview1000 laser scanning confocal microscope, using fluorescent as well as DIC microscopy.
Obrázek 1:
The oxidative cellular stress was induced chemically by addition of antimycin A, a specific inhibitor of complex III of the respiratory chain, an activity corresponding to that of nitric oxide.
The effect of antimycin A (inner mitochondrial membrane depolarization) was measured as a ratio of average relative fluorescence intensities in the green (IF(G)) and red (IF(R)) channelsrespectively, (IF(G))/(IF(R)).
Detection and quantification of the fluorescent signals were optimized through exciting both the JC-1 monomers and JC-1 aggregates by 488nm laser, and submitting the emissions to a strict spectral separation to eliminate any overlap of the signals in the area between the emission peaks. A minimum of ten view fields per every sample were scanned and quantified.
This model was thereafter employed to examine the
potential cytoprotective effect of melatonin against the consequences of the oxidative challenge.
Obrázek 2
Pretreatment of the cells with melatonin before their exposure to antimycin A lead to a drop in the previously observed antimycin-induced decrease of mitochondrial transmembrane potential, thus implicating an antioxidative cytoprotective effect of melatonin in this system. A statistically significant decrease in the IF(G)IF(R) ratio was observed. Hence, melatonin stabilizes the mitochondrial transmembrane potential in the studied cell lines and protects the cells against oxidative stress.
The above appears to be a suitable method for estimating and comparing the protective effects of antioxidants with a therapeutic potential.