The plasma membrane, as a highly complex cell organelle, serves as a crucial platform for a multitude of cellular processes. Its collective biophysical properties are largely determined by the structural diversity of the different lipid species it accommodates. Therefore, a detailed investigation of biophysical properties of the plasma membrane is of utmost importance for a comprehensive understanding of biological processes occurring therein. During the past two decades, several environment-sensitive probes have been developed and become popular tools to investigate membrane properties. Although these probes are assumed to report on membrane order in similar ways, their individual mechanisms remain to be elucidated. In this study, using model membrane systems, we characterized the probes Pro12A, NR12S and NR12A in depth and examined their sensitivity to parameters with potential biological implications, such as the degree of lipid saturation, double bond position and configuration (cis versus trans), phospholipid headgroup and cholesterol content. Applying spectral imaging together with atomistic molecular dynamics simulations and time-dependent fluorescent shift analyses, we unravelled individual sensitivities of these probes to different biophysical properties, their distinct localizations and specific relaxation processes in membranes. Overall, Pro12A, NR12S and NR12A serve together as a toolbox with a wide range of applications allowing to select the most appropriate probe for each specific research question.
The influence of experimental conditions on chromatographic behaviour of promising oligodeoxynucleotide double-labelled molecular probes containing an azaphthalocyanine macrocycle as a perspective dark quencher was studied. A recently introduced new stationary phase based on styrene-divinylbenzene copolymer was tested. The planar and hydrophobic structure of the azaphthalocyanine is considerably different from those of currently used fluorophores and quenchers. Thus, the most challenging issue was the separation of the double-labelled probe from its main impurity represented by a mono-labelled probe, containing only the azaphthalocyanine macrocycle. The absorbance measurement cannot simply determine this impurity, and its presence fundamentally compromises the biological assay. The commonly used gradient elution was not suitable and isocratic conditions seemed to be more appropriate. The azaphthalocyanine moiety influences the properties of the modified oligodeoxynucleotides substantially, and thus their chromatographic behaviour was determined predominantly by this quencher. Acetonitrile was the preferred organic solvent for the analysis of probes containing the azaphthalocyanine quencher and the effect of ion-pairing reagents was dependent on the probe structure. The temperature seemed to be an effective parameter for fine-tuning of the separation and mass transfer improvement. Generally, our findings could be helpful in method development for purity evaluation of double-labelled oligodeoxynucleotide probes and semipreparative methods.
- MeSH
- acetonitrily chemie MeSH
- aza sloučeniny * analýza chemie MeSH
- fluorescenční barviva * analýza chemie MeSH
- hydrofobní a hydrofilní interakce MeSH
- molekulární sondy * analýza chemie MeSH
- oligodeoxyribonukleotidy * analýza chemie MeSH
- rozpouštědla MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
Nestr.
Amyloidosis is a group of diseases resulting from misfolded protein deposits. Despite better diagnostics and therapy and also covering up in research activities in last decades, examination of patients with suspected disease is not yet straight-forward process. Apart from clinical check-up, a biopsy with proof of deposits is crucial. The subsequent correct amyloid typing is obligate due to disease-bound different attitude to patients and is mainly aimed to antigen-antibody based methods. To avoid frequent non-specific immuno-protein detection, the new methodology based on mass spectrometry seems to be highly specific and sensitive with direct protein recognition in a small amount of tissue. Therefore and in the frame of the proposed project we have decided to improve amyloidosis diagnostics opportunities, to identify new potential proteomic biomarkers and specific fluorescent labels. To facilitate mutual collaboration for the patient benefit and maximise number of analysed samples we have established consortium where four Czech University hospital centres are involved.
Amyloidóza zahrnuje skupinu onemocnění, pro než jsou typická depozita chybně složeného proteinového materiálu v různých tkáních. Stanovení diagnózy není snadné, a to navzdory zlepšení diagnostiky, terapie i zvýšeného zájmu vědců v posledních dekádách. Kromě klinického vyšetření je nezbytná biopsie tkáně s průkazem amyloidu. Přesné určení typu amyloidu je stěžejní pro volbu vhodné strategie péče o pacienta, která se mezi jednotlivými typy nemoci podstatně liší. V současnosti dominují metody založené na reakci antigen-protilátka, avšak nové postupy využívající hmotnostní spektrometrii odstranily rizika spojená s imunologickou detekcí proteinu a jsou vysoce specifické a sensitivní při přímém průkazu proteinu z malého množství tkáně. V tomto kontextu jsme se rozhodli zlepšit možnosti diagnostiky amyloidózy a identifikovat nové potencionální proteomické biomarkery a specifická fluorescenční barviva. Zapojením center při fakultních nemocnicích vznikla pracovní skupina, jejímž cílem je maximalizace počtu vyšetřených vzorků a usnadnění vzájemné spolupráce s výrazným benefitem pro pacienta.
- MeSH
- amyloid chemie MeSH
- amyloidóza diagnostické zobrazování MeSH
- biologické markery chemie MeSH
- fluorescenční barviva analýza MeSH
- imunohistochemie metody MeSH
- lidé MeSH
- optické zobrazování metody MeSH
- proteomika metody MeSH
- Check Tag
- lidé MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- vnitřní lékařství
- experimentální medicína
- biochemie
- NLK Publikační typ
- závěrečné zprávy o řešení grantu AZV MZ ČR
Precise and efficient endocytosis is critical for sustained neurotransmission during continuous neuronal activity. Endocytosis is a prerequisite for maintaining the auditory function. However, the differences between the patterns of endocytosis in cochlear inner hair cells (IHCs) and outer hair cells (OHCs) remain unclear. Both IHCs and OHCs were obtained from adult C57 mice. Patterns of endocytosis in cells were estimated by analyzing the uptake of FM1-43, a fluorescent. The observations were made using live confocal imaging, fluorescence intensities were calculated statistically. Results revealed the details about following phenomenon, i) sites of entry: the FM1-43 dye was found to enter IHC at the apical area initially, the additional sites of entry were then found at basolateral membrane of the cells, The entry of the dye into OHCs initially appeared to be occurring around whole apical membranes area, which then diffused towards the other membrane surface of the cells, ii) capacity of endocytosis: fluorescence intensity in IHCs showed significantly higher than that of OHCs (P<0.01). We have found different patterns of endocytosis between IHCs and OHCs, this indicated functional distinctions between them. Moreover, FM1-43 dye can be potentially used as an indicator of the functional loss or repair of cochlear hair cells.
- MeSH
- biologický transport fyziologie MeSH
- endocytóza fyziologie MeSH
- fluorescenční barviva analýza metabolismus MeSH
- kvartérní amoniové sloučeniny analýza metabolismus MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- orgánové kultury - kultivační techniky MeSH
- pyridinové sloučeniny analýza metabolismus MeSH
- sluchové evokované potenciály fyziologie MeSH
- vnější vláskové buňky chemie metabolismus MeSH
- vnitřní vláskové buňky chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Oleaginous microbes, which contain over 20% intracellular lipid, predominantly triacylglycerols (TG), by dry weight, have been discovered to have high oil content by many different protocols, ranging from simple staining to more complex chromatographic methods. In our laboratory, a methodical process was implemented to identify high oil yeasts, designed to minimize labor while optimizing success in identifying high oil strains among thousands of candidates. First, criteria were developed to select candidate yeast strains for analysis. These included observation of buoyancy of the yeast cell mass in 20% glycerol, and phylogenetic placement near known oleaginous species. A low-labor, semiquantitative Nile red staining protocol was implemented to screen numerous yeast cultures for high oil content in 96-well plates. Then, promising candidates were selected for more quantitative analysis. A more labor-intensive and quantitative gravimetric assay was implemented that gave consistent values for intracellular oil content for a broad range of yeast species. Finally, an LC-MS protocol was utilized to quantify and identify yeast triacylglycerols. This progressive approach was successful in identifying 30 new oleaginous yeast species, out of over 1000 species represented in the Phaff Yeast Culture Collection.
- MeSH
- barvení a značení metody MeSH
- chromatografie kapalinová metody MeSH
- fluorescenční barviva analýza MeSH
- fluorescenční mikroskopie metody MeSH
- hmotnostní spektrometrie metody MeSH
- kvasinky chemie MeSH
- lipidomika metody MeSH
- lipidy analýza MeSH
- oxaziny analýza MeSH
- triglyceridy analýza MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Fluorescence proteins changing spectral properties after exposure to light with a specific wavelength have recently become outstanding aids in the study of intracellular protein dynamics. Herein we show using Arabidopsis SYNAPTOTAGMIN 1 as a model protein that the Dendra2 green to red photoconvertible protein tag in combination with confocal scanning laser microscopy is a useful tool to study membrane protein intracellular dynamics.
- MeSH
- Arabidopsis chemie cytologie ultrastruktura MeSH
- fluorescenční barviva analýza MeSH
- intracelulární membrány chemie ultrastruktura MeSH
- konfokální mikroskopie metody MeSH
- kořeny rostlin chemie ultrastruktura MeSH
- luminescentní proteiny analýza MeSH
- proteiny huseníčku analýza MeSH
- světlo MeSH
- synaptotagmin I analýza MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Anionic phospholipids represent only minor fraction of cell membranes lipids but they are critically important for many membrane-related processes, including membrane identity, charge, shape, the generation of second messengers, and the recruitment of peripheral proteins. The main anionic phospholipids of the plasma membrane are phosphoinositides phosphatidylinositol 4-phosphate (PI4P), phosphatidylinositol 4,5-bisphosphate (PI4,5P2), phosphatidylserine (PS), and phosphatidic acid (PA). Recent insights in the understanding of the nature of protein-phospholipid interactions enabled the design of genetically encoded fluorescent molecular probes that can interact with various phospholipids in a specific manner allowing their imaging in live cells. Here, we describe the use of transiently transformed plant cells to study phospholipid-dependent membrane recruitment.
- MeSH
- exprese genu MeSH
- fluorescenční barviva analýza metabolismus MeSH
- fluorescenční mikroskopie metody MeSH
- fosfatidylinositoly analýza metabolismus MeSH
- fosfolipidy analýza metabolismus MeSH
- konfokální mikroskopie metody MeSH
- luminescentní proteiny analýza genetika MeSH
- pyl chemie genetika MeSH
- rostlinné buňky chemie metabolismus MeSH
- tabák chemie cytologie genetika MeSH
- transformace genetická MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
FM (Fei-Mao) styryl dyes are commonly used for the fluorescence imaging of plasma membrane (PM) and endocytosis in vivo. Thanks to their amphiphilic character, these dyes are incorporated in the outer leaflet of the PM lipid bilayer and emit fluorescence in its hydrophobic environment. The endocytic pathway of FM dye uptake starts with rapid PM staining and continues in PM invaginations and membrane vesicles during endocytosis, followed by staining of trans-Golgi network (TGN) and ending in tonoplast (vacuolar membrane). FM dyes do not stain endoplasmic reticulum and nuclear membrane. The time-lapse fluorescence microscopy could track endocytic vesicles and characterize the rate of endocytosis in vivo. On the other hand, fixable FM dyes (FX) can be used for the visualization of particular steps in the FM dye uptake in situ. Staining with FM dyes and subsequent microscopic observations could be performed on both tissue and cellular level. Here, we describe simple procedures for the effective FM dye staining and destaining in root tip of Arabidopsis thaliana seedlings and suspension-cultured tobacco cells.
- MeSH
- Arabidopsis cytologie ultrastruktura MeSH
- buněčná membrána ultrastruktura MeSH
- endocytóza * MeSH
- fluorescenční barviva analýza MeSH
- fluorescenční mikroskopie metody MeSH
- kořeny rostlin cytologie ultrastruktura MeSH
- optické zobrazování metody MeSH
- tabák cytologie ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The scope of this two-part review is to summarize publications dealing with CE and nanomaterials together. This topic can be viewed from two broad perspectives, and this article is trying to highlight these two approaches: (i) CE of nanomaterials, and (ii) nanomaterials in CE. The second part aims at summarization of publications dealing with application of nanomaterials for enhancement of CE performance either in terms of increasing the separation resolution or for improvement of the detection. To increase the resolution, nanomaterials are employed as either surface modification of the capillary wall forming open tubular column or as additives to the separation electrolyte resulting in a pseudostationary phase. Moreover, nanomaterials have proven to be very beneficial for increasing also the sensitivity of detection employed in CE or even they enable the detection (e.g., fluorescent tags of nonfluorescent molecules).
- MeSH
- dendrimery chemie MeSH
- elektroforéza kapilární přístrojové vybavení metody MeSH
- elektrolyty chemie MeSH
- fluorescenční barviva analýza MeSH
- liposomy chemie MeSH
- nanostruktury chemie MeSH
- nanotrubičky uhlíkové chemie MeSH
- povrchové vlastnosti MeSH
- senzitivita a specificita MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
BACKGROUND INFORMATION: Macarpine (MA) is a quaternary benzophenanthridine plant alkaloid isolated from Macleaya microcarpa or Stylophorum lasiocarpum. Benzophenanthridine alkaloids are interesting natural products that display antiproliferative, antimicrobial, antifungal and anti-inflammatory activities, and also fluorescence properties. In a previous study, we demonstrated that thanks to its ability to interact with DNA and its spectral properties MA could be used as a supravital DNA probe for fluorescence microscopy and flow cytometry including analyses of the cell cycle. In this study, we evaluated the suitability of MA as a DNA dye for time-lapse microscopy and flow-cytometric cell sorting. RESULTS: Living A-375 and MEF cells stained with MA were monitored by time-lapse microscopy for 24 h. Mitoses were observed at MA concentrations up to 0.5 μg/ml during the first 2-3 h. After this period of time, cells treated with MA at concentrations of 0.75 and 0.5 μg/ml underwent apoptosis. Cells cultivated with MA at concentration of 0.25 μg/ml or lower survived throughout the 24 h period. Toxicity of MA was dependent on light wavelength and frequency of image capturing. The intensity of MA fluorescence decreased during the incubation. MA concentration of 0.1 μg/ml was identified as the most suitable for live cell imaging with respect to fluorescence intensity and toxicity. MA at the concentration 10 μg/ml was used for sorting of enhanced green fluorescent protein (EGFP)-labelled neurons and fibroblasts yielding profiles similar to those obtained with DRAQ5. Contrary to DRAQ5, MA-stained cells survived in culture, and the sorted cells lost the MA signal suggesting reversible binding of the dye to the DNA. CONCLUSION: The results proved that MA may readily be used for chromosomes depicting and mitosis monitoring by time-lapse microscopy. In addition, MA has shown to be a suitable probe for sorting of EGFP-labelled cells, including neurons, that survived the labelling process. SIGNIFICANCE: In consideration of the results, we highly anticipate an onward use of MA in a broad range of applications based on live cell sorting and imaging, for example, cell synchronisation and monitoring of proliferation as an important experimental and/or diagnostic utility.
- MeSH
- benzofenantridiny analýza MeSH
- buněčné kultury MeSH
- buněčný cyklus fyziologie MeSH
- DNA analýza MeSH
- fluorescenční barviva analýza MeSH
- fluorescenční mikroskopie metody MeSH
- lidé MeSH
- průtoková cytometrie * metody MeSH
- separace buněk metody MeSH
- viabilita buněk MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
