A number of fluorescence microscopy techniques are described to study dynamics of fluorescently labeled proteins, lipids, nucleic acids, and whole organelles. However, for studies of plant plasma membrane (PM) proteins, the number of these techniques is still limited because of the high complexity of processes that determine the dynamics of PM proteins and the existence of cell wall. Here, we report on the usage of raster image correlation spectroscopy (RICS) for studies of integral PM proteins in suspension-cultured tobacco cells and show its potential in comparison with the more widely used fluorescence recovery after photobleaching method. For RICS, a set of microscopy images is obtained by single-photon confocal laser scanning microscopy (CLSM). Fluorescence fluctuations are subsequently correlated between individual pixels and the information on protein mobility are extracted using a model that considers processes generating the fluctuations such as diffusion and chemical binding reactions. As we show here using an example of two integral PM transporters of the plant hormone auxin, RICS uncovered their distinct short-distance lateral mobility within the PM that is dependent on cytoskeleton and sterol composition of the PM. RICS, which is routinely accessible on modern CLSM instruments, thus represents a valuable approach for studies of dynamics of PM proteins in plants.

• Klíčová slova
• auxin efflux, auxin influx, fluorescence recovery after photobleaching, plasma membrane, raster image correlation spectroscopy,
• MeSH
• buněčná membrána chemie   MeSH
• konfokální mikroskopie metody   MeSH
• membránové proteiny analýza   MeSH
• počítačové zpracování obrazu metody   MeSH
• rostlinné buňky chemie   MeSH
• spektrální analýza metody   MeSH
• tabák chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové proteiny MeSH

Dynamic processes such as diffusion and binding/unbinding of macromolecules (e.g. growth factors or nutrients) are crucial parameters for the design and application of effective artificial tissue materials. Here, dynamics of selected macromolecules were studied in two different composite tissue engineering scaffolds containing an electrospun nanofiber mesh (polycaprolactone or hydrophobically plasma modified polyvinylalcohol-chitosan) encapsulated in agarose hydrogels by a conventional approach fluorescence recovery after photobleaching (FRAP) and a novel technique, raster image correlation spectroscopy (RICS). The two approaches are compared, and it is shown that FRAP is unable to determine processes occurring at low molecular concentrations, especially accurately separating binding/unbinding from diffusion, and its results depend on the concentration of the studied molecules. RICS measures processes of single molecules and, because of its multiple adjustable timescales, can distinguish whether diffusion or binding controls molecular movement and separates fast diffusion from slow transient binding. In addition, RICS provides a robust read-out parameter quantifying binding affinity. Finally, the combination of FRAP and RICS helps to characterize diffusion and binding of macromolecules in tested artificial tissues better, and therefore predicts the behavior of biologically active molecules in these materials for medical applications.

• MeSH
• difuze MeSH
• nanovlákna * MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cellular membranes are composed of lipids typically organized in a double-leaflet structure. Interactions between these two leaflets - often referred to as interleaflet coupling - play a crucial role in various cellular processes. Despite extensive study, the mechanisms governing such interactions remain incompletely understood. Here, we investigate the effects of interleaflet coupling from a specific point of view, i.e. by comparing diffusive dynamics in bilayers and monolayers, focusing on potential lipid-specific interactions between opposing leaflets. Through quantitative fluorescence microscopy techniques, we characterize lipid diffusion and mean molecular area in monolayers and bilayers composed of different lipids. Our results suggest that the observed decrease in bilayer lipid diffusion compared to monolayers depends on lipid identity. Furthermore, our analysis suggests that lipid acyl chain structure and spatial configuration at the bilayer may strongly influence interleaflet interactions and dynamics in bilayers. These findings provide insights into the role of lipid structure in mediating interleaflet coupling and underscore the need for further experimental investigations to elucidate the underlying mechanisms.

• Klíčová slova
• Bilayer, Fluorescence correlation spectroscopy, Interleaflet coupling, Lipids, Monolayer, Raster image correlation spectroscopy,
• MeSH
• buněčná membrána chemie   metabolismus   MeSH
• difuze MeSH
• fluorescenční mikroskopie MeSH
• fosfatidylcholiny chemie   MeSH
• lipidové dvojvrstvy * chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• fosfatidylcholiny MeSH
• lipidové dvojvrstvy * MeSH

Cells continuously communicate with the surrounding environment employing variety of signaling molecules and pathways to integrate and transport the information in the cell. An example of signaling initiation is binding of extracellular ligand to its receptor at the plasma membrane. This initializes enzymatic reactions leading to the formation of bi- or multimolecular signaling complexes responsible for the regulation or progress of signal transduction. Here, we describe three imaging techniques enabling detection of individual signaling molecules, their complexes, and clusters in human cells. Described imaging techniques require only basic microscopy systems available in the majority of current biomedical research centers but apply advanced data processing. First, total internal reflection fluorescence microscopy (TIRFM) variant of wide-field fluorescence microscopy for imaging highly dynamic clusters is described. Second, superresolution localization microscopy techniques-photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM)-recently enabled to achieve higher resolution with precision limit of about 20 nm in fixed samples. The developments toward live cell superresolution imaging are indicated. Third, raster image correlation spectroscopy (RICS) employed for molecular diffusion and binding analysis explains the advantages and hurdles of this novel method. Presented techniques provide a new level of detail one can learn about higher organization of signaling events in human cells.

• MeSH
• buněčná membrána metabolismus   MeSH
• buněčný tracking metody   MeSH
• fluorescenční barviva * MeSH
• fluorescenční mikroskopie metody   MeSH
• interpretace obrazu počítačem metody   MeSH
• Jurkat buňky MeSH
• lidé MeSH
• ligandy MeSH
• mezibuněčná komunikace MeSH
• signální transdukce MeSH
• zelené fluorescenční proteiny * MeSH
• zobrazování trojrozměrné MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fluorescenční barviva * MeSH
• ligandy MeSH
• zelené fluorescenční proteiny * MeSH

UNLABELLED: Here we investigated the effect of disruption of plasma membrane integrity by cholesterol depletion on thyrotropin-releasing hormone receptor (TRH-R) surface mobility in HEK293 cells stably expressing TRH-R-eGFP fusion protein (VTGP cells). Detailed analysis by fluorescence recovery after photobleaching (FRAP) in bleached spots of different sizes indicated that cholesterol depletion did not result in statistically significant alteration of mobile fraction of receptor molecules (Mf). The apparent diffusion coefficient (Dapp) was decreased, but this decrease was detectable only under the special conditions of screening and calculation of FRAP data. Analysis of mobility of receptor molecules by raster image correlation spectroscopy (RICS) did not indicate any significant difference between control and cholesterol-depleted cells. Results of our FRAP and RICS experiments may be collectively interpreted in terms of a "membrane fence" model which regards the plasma membrane of living cells as compartmentalized plane where lateral diffusion of membrane proteins is limited to restricted areas by cytoskeleton constraints. Hydrophobic interior of plasma membrane, studied by steady-state and time-resolved fluorescence anisotropy of hydrophobic membrane probe DPH, became substantially more "fluid" and chaotically organized in cholesterol-depleted cells. Decrease of cholesterol level impaired the functional coupling between the receptor and the cognate G proteins of Gq/G11 family. IN CONCLUSION: the presence of an unaltered level of cholesterol in the plasma membrane represents an obligatory condition for an optimum functioning of TRH-R signaling cascade. The decreased order and increased fluidity of hydrophobic membrane interior suggest an important role of this membrane area in TRH-R-Gq/G11α protein coupling.

• Klíčová slova
• Cholesterol, DPH fluorescence, FRAP, G protein coupling, RICS, TRH-R-eGFP mobility,
• MeSH
• algoritmy MeSH
• buněčná membrána chemie   metabolismus   MeSH
• cholesterol metabolismus   MeSH
• difenylhexatrien chemie   metabolismus   MeSH
• difuze MeSH
• fluorescenční polarizace MeSH
• FRAP MeSH
• HEK293 buňky MeSH
• kinetika MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• proteiny vázající GTP - alfa-podjednotky Gq-G11 metabolismus   MeSH
• receptory thyroliberinu chemie   genetika   metabolismus   MeSH
• transport proteinů MeSH
• vazba proteinů MeSH
• zelené fluorescenční proteiny chemie   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cholesterol MeSH
• difenylhexatrien MeSH
• enhanced green fluorescent protein MeSH Prohlížeč
• proteiny vázající GTP - alfa-podjednotky Gq-G11 MeSH
• receptory thyroliberinu MeSH
• zelené fluorescenční proteiny MeSH