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Study of mechanical properties and dynamics of biomembranes and single molecules with the use of laser beam - an overview
Jonáš, Alexandr ; Zemánek, Pavel
A micron-sized dielectric particle confined in a laser trap can be employed as a probe for the measurement of forces in the range from piconewtons to hundreds of piconewtons. Thus, if a biological system of interest (e.g. DNA molecule, single myosin or kinesin molecule, cell membrane) is attached to such a probe, its mechanical and dynamical properties (elasticity, viscosty, forces associated with movement) can be studied during its interaction with the environment with unprecedented resolution. This article introduces the basic principles of the laser trapping and force measurement and illustrates on several examples the great potential of the light-based force transducer for exploiting non-invasively the dynamics of basic biological systems.
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Dynamika pohybu a umlčování transgenních lokusů HP1 v živých buňkách
Ondřej, Vladan ; Kozubek, Stanislav ; Lukášová, Emilie ; Matula, Pa. ; Matula, Pe. ; Kozubek, Michal
Intranuclear localization and mobility of Cy3 labelled transgene loci were studied in living cells. Observations showed occurrence of several transgene copies in each cell nucleus. The majority of copies were localized in the centromeric heterochromatin defined by HPlbeta-GFP fusion protein, the minority of copies in euchromatin. The tracking of loci showed restricted diffusive motion of these in the short-time range. During long-time observation of HPlbeta domains and transgene loci, we have found that in most cases the proximity of these objects decreased within time. The changes of positions of HPl domains were very small but transgene loci displayed directional movement towards the relevant HPl domain. Our data records support the idea that the cell nucleus consists of several separated higher-order compartments. The genes relocate within these compartments, which is finally connected with changes of their expression status.
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Využití vysokorozlišovací cytometrie ke studiu živých buněk
Vařecha, M. ; Amrichová, J. ; Ondřej, Vladan ; Lukášová, Emilie ; Kozubek, Stanislav ; Matula, Pa. ; Matula, Pe. ; Kozubek, Michal
The fluorescent proteins are important innovation in the field of cell biology and in vivo experiments that use fluorescent proteins bring new interesting results from a different point of view than in vitro and in situ experiments. This presentation will introduce our automated 2D/3D high-resolution time-lapse image acquisition and analysis of living cells transfected with plasmids encoding fusion proteins or dyed with fluorescence probes. One of our in vivo projects, we will mention, is focused on the study of mitochondrial and nuclear apoptotic processes in living cells. Another in vivo project concentrates on the study of topography of telomeres and incidence of telomere-association phenomenon in various types of human tumor and healthy cells. In our experiments, cells are stably or transiently transfected with plasmid DNAs coding mitochondrial apoptogenic or telomere-binding proteins with fluorescent proteins.
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Methylace histonu H3K9 v lidských krevních buňkách a v granulocytech pacientů s myeloidní leukemií
Lukášová, Emilie ; Falk, Martin ; Kořistek, Z. ; Kozubek, Stanislav ; Grigoryev, S. ; Kozubek, Michal ; Ondřej, Vladan ; Kroupová, I.
Common heterochromatin antigenic protein markers while present in human blood progenitor CD34+ cells, differentiated lymphocytes and monocytes are absent in neutrophile granulocytes and, to large extent, in eosinophiles. In acute CML and AML, strong methylation of H3K9 and all isoformes of HP1 are detected. In chronic forms of CML, no strong correlations among the level of histone methylation, disease progression and modality of treatment were observed. Reprogramming of leukemia HL60 cells to terminal differentiation by retinoic acid does not eliminate histone H3K9 methylation and the presence of HP1 isoformes from differentiated granulocytes. Our study shows for the firs time that histone H3 methylation may be dramatically changed during normal cell differentiation.
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Využití opticky zachycené sondy pro studium velmi slabých interakcí na molekulární úrovni - přehled
Zemánek, Pavel ; Jonáš, Alexandr
A micron-sized dielectric particle confined in a laser trap can be employed as a probe for the measurement of forces in the range from piconewtons to hundreds of piconewtons. Thus, if a biological system of interest (e.g. DNA molecule, single myosin or kinesin molecule, cell membrane) is attached to such a probe, its mechanical and dynamical properties (elasticity, viscosity, forces associated with movement) can be studied during its interaction with the environment with unprecedented resolution. This article introduces the basic principles of the laser trapping and force measurement and illustrates on several examples the great potential of the light-based force transducer for exploiting non-invasively the dynamics of molecular systems
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