Quantitative approaches for characterizing molecular organization of cell membrane molecules under physiological and pathological conditions profit from recently developed super-resolution imaging techniques. Current tools employ statistical algorithms to determine clusters of molecules based on single-molecule localization microscopy (SMLM) data. These approaches are limited by the ability of SMLM techniques to identify and localize molecules in densely populated areas and experimental conditions of sample preparation and image acquisition. We have developed a robust, model-free, quantitative clustering analysis to determine the distribution of membrane molecules that excels in densely labeled areas and is tolerant to various experimental conditions, i.e. multiple-blinking or high blinking rates. The method is based on a TIRF microscope followed by a super-resolution optical fluctuation imaging (SOFI) analysis. The effectiveness and robustness of the method is validated using simulated and experimental data investigating nanoscale distribution of CD4 glycoprotein mutants in the plasma membrane of T cells.

• MeSH
• algoritmy MeSH
• antigeny CD4 genetika   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• fluorescenční barviva MeSH
• fluorescenční mikroskopie metody   statistika a číselné údaje   MeSH
• Jurkat buňky MeSH
• lidé MeSH
• membránové proteiny genetika   metabolismus   MeSH
• mutantní proteiny genetika   metabolismus   MeSH
• optické zobrazování metody   statistika a číselné údaje   MeSH
• shluková analýza MeSH
• T-lymfocyty imunologie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• validační studie MeSH
• Názvy látek
• antigeny CD4 MeSH
• fluorescenční barviva MeSH
• membránové proteiny MeSH
• mutantní proteiny MeSH

Ever since technologies enabled the characterization of eukaryotic plasma membranes, heterogeneities in the distributions of its constituents were observed. Over the years this led to the proposal of various models describing the plasma membrane organization such as lipid shells, picket-and-fences, lipid rafts, or protein islands, as addressed in numerous publications and reviews. Instead of emphasizing on one model we in this review give a brief overview over current models and highlight how current experimental work in one or the other way do not support the existence of a single overarching model. Instead, we highlight the vast variety of membrane properties and components, their influences and impacts. We believe that highlighting such controversial discoveries will stimulate unbiased research on plasma membrane organization and functionality, leading to a better understanding of this essential cellular structure.

• Klíčová slova
• heterogenous distribution, membrane organization models, membrane physical properties, nanodomains, plasma membrane,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Brd2 is a member of the bromodomain extra terminal (BET) protein family, which consists of four chromatin-interacting proteins that regulate gene expression. Each BET protein contains two N-terminal bromodomains, which recognize acetylated histones, and the C-terminal protein-protein interaction domain. Using a genome-wide screen, we identify 1450 genes whose transcription is regulated by Brd2. In addition, almost 290 genes change their alternative splicing pattern upon Brd2 depletion. Brd2 is specifically localized at promoters of target genes, and our data show that Brd2 interaction with chromatin cannot be explained solely by histone acetylation. Using coimmunoprecipitation and live-cell imaging, we show that the C-terminal part is crucial for Brd2 association with chromatin. Live-cell microscopy also allows us to map the average binding time of Brd2 to chromatin and quantify the contributions of individual Brd2 domains to the interaction with chromatin. Finally, we show that bromodomains and the C-terminal domain are equally important for transcription and splicing regulation, which correlates with the role of these domains in Brd2 binding to chromatin.

• MeSH
• alternativní sestřih MeSH
• chromatin metabolismus   MeSH
• genetická transkripce MeSH
• genom lidský * MeSH
• HeLa buňky MeSH
• histony genetika   metabolismus   MeSH
• lidé MeSH
• promotorové oblasti (genetika) MeSH
• protein-serin-threoninkinasy genetika   metabolismus   MeSH
• regulace genové exprese * MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• signální transdukce MeSH
• terciární struktura proteinů MeSH
• transkripční faktory MeSH
• vazba proteinů MeSH
• videomikroskopie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• BRD2 protein, human MeSH Prohlížeč
• chromatin MeSH
• histony MeSH
• protein-serin-threoninkinasy MeSH
• rekombinantní fúzní proteiny MeSH
• transkripční faktory MeSH

Fluorescence correlation spectroscopy (FCS) is a single molecule technique used mainly for determination of mobility and local concentration of molecules. This review describes the specific problems of FCS in planar systems and reviews the state of the art experimental approaches such as 2-focus, Z-scan or scanning FCS, which overcome most of the artefacts and limitations of standard FCS. We focus on diffusion measurements of lipids and proteins in planar lipid membranes and review the contributions of FCS to elucidating membrane dynamics and the factors influencing it, such as membrane composition, ionic strength, presence of membrane proteins or frictional coupling with solid support.

• Klíčová slova
• biomembranes, confocal microscopy, fluorescence fluctuation spectroscopy, giant unilamellar vesicles, lateral diffusion, supported lipid bilayers,
• MeSH
• difuze MeSH
• fluorescenční spektrometrie * MeSH
• konfokální mikroskopie MeSH
• lipidové dvojvrstvy chemie   MeSH
• membránové lipidy chemie   MeSH
• unilamelární lipozómy chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• lipidové dvojvrstvy MeSH
• membránové lipidy MeSH
• unilamelární lipozómy MeSH