Artykuły w czasopismach: „Living Cells - Fluorescence Correlation Spectroscopy”

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Kim, Sally A., Katrin G. Heinze i Petra Schwille. "Fluorescence correlation spectroscopy in living cells". Nature Methods 4, nr 11 (30.10.2007): 963–73. http://dx.doi.org/10.1038/nmeth1104.

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Bacia, Kirsten, Sally A. Kim i Petra Schwille. "Fluorescence cross-correlation spectroscopy in living cells". Nature Methods 3, nr 2 (23.01.2006): 83–89. http://dx.doi.org/10.1038/nmeth822.

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Kinjo, M., H. Sakata i S. Mikuni. "First Steps for Fluorescence Correlation Spectroscopy of Living Cells". Cold Spring Harbor Protocols 2011, nr 10 (1.10.2011): pdb.top065920. http://dx.doi.org/10.1101/pdb.top065920.

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Unsay, Joseph D., i Ana J. Garcia-Saez. "Scanning Fluorescence Correlation Spectroscopy in Mitochondria of Living Cells". Biophysical Journal 106, nr 2 (styczeń 2014): 196a. http://dx.doi.org/10.1016/j.bpj.2013.11.1160.

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Ho Hur, Kwang, John Kohler i Joachim D. Mueller. "Unbiased Fluorescence Correlation Spectroscopy of Diffusive Processes in Living Cells". Biophysical Journal 120, nr 3 (luty 2021): 357a. http://dx.doi.org/10.1016/j.bpj.2020.11.2210.

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Weiss, Matthias. "Probing the Interior of Living Cells with Fluorescence Correlation Spectroscopy". Annals of the New York Academy of Sciences 1130, nr 1 (maj 2008): 21–27. http://dx.doi.org/10.1196/annals.1430.002.

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Markiewicz, Roksana, Jagoda Litowczenko, Jacek Gapiński, Anna Woźniak, Stefan Jurga i Adam Patkowski. "Nanomolar Nitric Oxide Concentrations in Living Cells Measured by Means of Fluorescence Correlation Spectroscopy". Molecules 27, nr 3 (2.02.2022): 1010. http://dx.doi.org/10.3390/molecules27031010.

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Measurement of the nitric oxide (NO) concentration in living cells in the physiological nanomolar range is crucial in understanding NO biochemical functions, as well as in characterizing the efficiency and kinetics of NO delivery by NO-releasing drugs. Here, we show that fluorescence correlation spectroscopy (FCS) is perfectly suited for these purposes, due to its sensitivity, selectivity, and spatial resolution. Using the fluorescent indicators, diaminofluoresceins (DAFs), and FCS, we measured the NO concentrations in NO-producing living human primary endothelial cells, as well as NO delivery kinetics, by an external NO donor to the immortal human epithelial living cells. Due to the high spatial resolution of FCS, the NO concentration in different parts of the cells were also measured. The detection of nitric oxide by means of diaminofluoresceins is much more efficient and faster in living cells than in PBS solutions, even though the conversion to the fluorescent form is a multi-step reaction.

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Engelke, Hanna, Doris Heinrich i Joachim O. Rädler. "Probing GFP-actin diffusion in living cells using fluorescence correlation spectroscopy". Physical Biology 7, nr 4 (1.12.2010): 046014. http://dx.doi.org/10.1088/1478-3975/7/4/046014.

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Martinez, Michelle M., Randall D. Reif i Dimitri Pappas. "Early detection of apoptosis in living cells by fluorescence correlation spectroscopy". Analytical and Bioanalytical Chemistry 396, nr 3 (25.11.2009): 1177–85. http://dx.doi.org/10.1007/s00216-009-3298-3.

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Gao, Xinwei, Yanfeng Liu, Jia Zhang, Luwei Wang, Yong Guo, Yinru Zhu, Zhigang Yang, Wei Yan i Junle Qu. "Nanodrug Transmembrane Transport Research Based on Fluorescence Correlation Spectroscopy". Membranes 11, nr 11 (19.11.2021): 891. http://dx.doi.org/10.3390/membranes11110891.

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Although conventional fluorescence intensity imaging can be used to qualitatively study the drug toxicity of nanodrug carrier systems at the single-cell level, it has limitations for studying nanodrug transport across membranes. Fluorescence correlation spectroscopy (FCS) can provide quantitative information on nanodrug concentration and diffusion in a small area of the cell membrane; thus, it is an ideal tool for studying drug transport across the membrane. In this paper, the FCS method was used to measure the diffusion coefficients and concentrations of carbon dots (CDs), doxorubicin (DOX) and CDs-DOX composites in living cells (COS7 and U2OS) for the first time. The drug concentration and diffusion coefficient in living cells determined by FCS measurements indicated that the CDs-DOX composite distinctively improved the transmembrane efficiency and rate of drug molecules, in accordance with the conclusions drawn from the fluorescence imaging results. Furthermore, the effects of pH values and ATP concentrations on drug transport across the membrane were also studied. Compared with free DOX under acidic conditions, the CDs-DOX complex has higher cellular uptake and better transmembrane efficacy in U2OS cells. Additionally, high concentrations of ATP will cause negative changes in cell membrane permeability, which will hinder the transmembrane transport of CDs and DOX and delay the rapid diffusion of CDs-DOX. The results of this study show that the FCS method can be utilized as a powerful tool for studying the expansion and transport of nanodrugs in living cells, and might provide a new drug exploitation strategy for cancer treatment in vivo.

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Guan, Yinghua, Matthias Meurer, Sarada Raghavan, Aleksander Rebane, Jake R. Lindquist, Sofia Santos, Ilia Kats i in. "Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein". Molecular Biology of the Cell 26, nr 11 (czerwiec 2015): 2054–66. http://dx.doi.org/10.1091/mbc.e14-10-1473.

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We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5. The increased intracellular brightness and large Stokes shift (∼180 nm) make it an excellent partner with teal fluorescent protein (mTFP1) for multiphoton, multicolor applications. Excitation of this pair by a single multiphoton excitation wavelength (MPE, 850 nm) yields well-separable emission peaks (∼120-nm separation). Using this pair, we measure homo- and hetero-oligomerization interactions in living cells via multiphoton excitation fluorescence correlation spectroscopy (MPE-FCS). Using tandem dimer proteins and small-molecule inducible dimerization domains, we demonstrate robust and quantitative detection of intracellular protein–protein interactions. We also use MPE-FCCS to detect drug–protein interactions in the intracellular environment using a Coumarin 343 (C343)-conjugated drug and hmKeima8.5 as a fluorescence pair. The mTFP1/hmKeima8.5 and C343/hmKeima8.5 combinations, together with our calibration constructs, provide a practical and broadly applicable toolbox for the investigation of molecular interactions in the cytoplasm of living cells.

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OHSUGI, Y., i MASATAKA KINJO. "ANALYSIS OF MEMBRANE-BINDING PROTEIN MOBILITY IN LIVING CELLS USING TOTAL INTERNAL REFLECTION FLUORESCENCE CORRELATION SPECTROSCOPY". Biophysical Reviews and Letters 01, nr 03 (lipiec 2006): 293–99. http://dx.doi.org/10.1142/s1793048006000227.

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Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) is an appropriate method for measuring diffusion constants and the number of fluorescent molecules very close to the coverglass surface. Recently, we have reported the application of TIR-FCS to cell biology, measuring membrane-binding farnesylated green fluorescent proteins (EGFP-F) in living cells. In this research, we measured the signal transduction molecule, protein kinase C (PKC), fused with EGFP in living HeLa cells by using TIR-FCS. We observed two different diffusional mobilities of PKCβII-EGFP, three-dimensional faster diffusion near the plasma membrane and slower lateral diffusion on the plasma membrane after adinosine tri phosphate (ATP) activation. These results indicate that it is possible to use TIR-FCS in the study of molecular dynamics and interactions of signal transduction proteins on the plasma membrane of the living cell.

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Aoki, Kazuhiro. "Quantification of dissociation constant in living cells by fluorescence cross-correlation spectroscopy". Folia Pharmacologica Japonica 147, nr 2 (2016): 74–79. http://dx.doi.org/10.1254/fpj.147.74.

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Weiss, Matthias, Hitoshi Hashimoto i Tommy Nilsson. "Anomalous Protein Diffusion in Living Cells as Seen by Fluorescence Correlation Spectroscopy". Biophysical Journal 84, nr 6 (czerwiec 2003): 4043–52. http://dx.doi.org/10.1016/s0006-3495(03)75130-3.

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Karpińska, Aneta, Marta Pilz, Joanna Buczkowska, Paweł J. Żuk, Karolina Kucharska, Gaweł Magiera, Karina Kwapiszewska i Robert Hołyst. "Quantitative analysis of biochemical processes in living cells at a single-molecule level: a case of olaparib–PARP1 (DNA repair protein) interactions". Analyst 146, nr 23 (2021): 7131–43. http://dx.doi.org/10.1039/d1an01769a.

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Wachsmuth, Malte, Christian Conrad, Jutta Bulkescher, Birgit Koch, Robert Mahen, Mayumi Isokane, Rainer Pepperkok i Jan Ellenberg. "High-throughput fluorescence correlation spectroscopy enables analysis of proteome dynamics in living cells". Nature Biotechnology 33, nr 4 (16.03.2015): 384–89. http://dx.doi.org/10.1038/nbt.3146.

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Hui, Yuen Yung, Bailin Zhang, Yuan-Chang Chang, Cheng-Chun Chang, Huan-Cheng Chang, Jui-Hung Hsu, Karen Chang i Fu-Hsiung Chang. "Two-photon fluorescence correlation spectroscopy of lipid-encapsulated fluorescent nanodiamonds in living cells". Optics Express 18, nr 6 (10.03.2010): 5896. http://dx.doi.org/10.1364/oe.18.005896.

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Spiegel, Evan T., P. Lee, L. Toth i W. R. Zipfel. "Studying Fluorescent Proteins in Living Cells: An Application for Segmented Fluorescence Correlation Spectroscopy". Biophysical Journal 98, nr 3 (styczeń 2010): 584a. http://dx.doi.org/10.1016/j.bpj.2009.12.3176.

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Fujita, Hirotaka, Ryota Oikawa, Mayu Hayakawa, Fumiaki Tomoike, Yasuaki Kimura, Hiroyuki Okuno, Yoshiki Hatashita i in. "Quantification of native mRNA dynamics in living neurons using fluorescence correlation spectroscopy and reduction-triggered fluorescent probes". Journal of Biological Chemistry 295, nr 23 (27.04.2020): 7923–40. http://dx.doi.org/10.1074/jbc.ra119.010921.

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RNA localization in subcellular compartments is essential for spatial and temporal regulation of protein expression in neurons. Several techniques have been developed to visualize mRNAs inside cells, but the study of the behavior of endogenous and nonengineered mRNAs in living neurons has just started. In this study, we combined reduction-triggered fluorescent (RETF) probes and fluorescence correlation spectroscopy (FCS) to investigate the diffusion properties of activity-regulated cytoskeleton-associated protein (Arc) and inositol 1,4,5-trisphosphate receptor type 1 (Ip3r1) mRNAs. This approach enabled us to discriminate between RNA-bound and unbound fluorescent probes and to quantify mRNA diffusion parameters and concentrations in living rat primary hippocampal neurons. Specifically, we detected the induction of Arc mRNA production after neuronal activation in real time. Results from computer simulations with mRNA diffusion coefficients obtained in these analyses supported the idea that free diffusion is incapable of transporting mRNA of sizes close to those of Arc or Ip3r1 to distal dendrites. In conclusion, the combined RETF-FCS approach reported here enables analyses of the dynamics of endogenous, unmodified mRNAs in living neurons, affording a glimpse into the intracellular dynamics of RNA in live cells.

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Petrich, Annett, Amit Koikkarah Aji, Valentin Dunsing i Salvatore Chiantia. "Benchmarking of novel green fluorescent proteins for the quantification of protein oligomerization in living cells". PLOS ONE 18, nr 8 (3.08.2023): e0285486. http://dx.doi.org/10.1371/journal.pone.0285486.

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Protein-protein-interactions play an important role in many cellular functions. Quantitative non-invasive techniques are applied in living cells to evaluate such interactions, thereby providing a broader understanding of complex biological processes. Fluorescence fluctuation spectroscopy describes a group of quantitative microscopy approaches for the characterization of molecular interactions at single cell resolution. Through the obtained molecular brightness, it is possible to determine the oligomeric state of proteins. This is usually achieved by fusing fluorescent proteins (FPs) to the protein of interest. Recently, the number of novel green FPs has increased, with consequent improvements to the quality of fluctuation-based measurements. The photophysical behavior of FPs is influenced by multiple factors (including photobleaching, protonation-induced “blinking” and long-lived dark states). Assessing these factors is critical for selecting the appropriate fluorescent tag for live cell imaging applications. In this work, we focus on novel green FPs that are extensively used in live cell imaging. A systematic performance comparison of several green FPs in living cells under different pH conditions using Number & Brightness (N&B) analysis and scanning fluorescence correlation spectroscopy was performed. Our results show that the new FP Gamillus exhibits higher brightness at the cost of lower photostability and fluorescence probability (pf), especially at lower pH. mGreenLantern, on the other hand, thanks to a very high pf, is best suited for multimerization quantification at neutral pH. At lower pH, mEGFP remains apparently the best choice for multimerization investigation. These guidelines provide the information needed to plan quantitative fluorescence microscopy involving these FPs, both for general imaging or for protein-protein-interactions quantification via fluorescence fluctuation-based methods.

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Yao, Jun, Xiangyi Huang i Jicun Ren. "In situ determination of secretory kinase Fam20C from living cells using fluorescence correlation spectroscopy". Talanta 232 (wrzesień 2021): 122473. http://dx.doi.org/10.1016/j.talanta.2021.122473.

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Malchus, Nina, i Matthias Weiss. "Elucidating Anomalous Protein Diffusion in Living Cells with Fluorescence Correlation Spectroscopy—Facts and Pitfalls". Journal of Fluorescence 20, nr 1 (7.07.2009): 19–26. http://dx.doi.org/10.1007/s10895-009-0517-4.

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Braet, Christophe, Holger Stephan, Ian M. Dobbie, Denisio M. Togashi, Alan G. Ryder, Zeno Földes-Papp, Noel Lowndes i Heinz Peter Nasheuer. "Mobility and distribution of replication protein A in living cells using fluorescence correlation spectroscopy". Experimental and Molecular Pathology 82, nr 2 (kwiecień 2007): 156–62. http://dx.doi.org/10.1016/j.yexmp.2006.12.008.

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Nederveen-Schippers, Laura M., Pragya Pathak, Ineke Keizer-Gunnink, Adrie H. Westphal, Peter J. M. van Haastert, Jan Willem Borst, Arjan Kortholt i Victor Skakun. "Combined FCS and PCH Analysis to Quantify Protein Dimerization in Living Cells". International Journal of Molecular Sciences 22, nr 14 (7.07.2021): 7300. http://dx.doi.org/10.3390/ijms22147300.

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Protein dimerization plays a crucial role in the regulation of numerous biological processes. However, detecting protein dimers in a cellular environment is still a challenge. Here we present a methodology to measure the extent of dimerization of GFP-tagged proteins in living cells, using a combination of fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis of single-color fluorescence fluctuation data. We named this analysis method brightness and diffusion global analysis (BDGA) and adapted it for biological purposes. Using cell lysates containing different ratios of GFP and tandem-dimer GFP (diGFP), we show that the average brightness per particle is proportional to the fraction of dimer present. We further adapted this methodology for its application in living cells, and we were able to distinguish GFP, diGFP, as well as ligand-induced dimerization of FKBP12 (FK506 binding protein 12)-GFP. While other analysis methods have only sporadically been used to study dimerization in living cells and may be prone to errors, this paper provides a robust approach for the investigation of any cytosolic protein using single-color fluorescence fluctuation spectroscopy.

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Deng, Liyun, Xiangyi Huang, Chaoqing Dong i Jicun Ren. "Simultaneously monitoring endogenous MAPK members in single living cells by multi-channel fluorescence correlation spectroscopy". Analyst 146, nr 8 (2021): 2581–90. http://dx.doi.org/10.1039/d1an00090j.

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Politz, J. C., E. S. Browne, D. E. Wolf i T. Pederson. "Intranuclear diffusion and hybridization state of oligonucleotides measured by fluorescence correlation spectroscopy in living cells". Proceedings of the National Academy of Sciences 95, nr 11 (26.05.1998): 6043–48. http://dx.doi.org/10.1073/pnas.95.11.6043.

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Neugart, Felix, Andrea Zappe, Deborah M. Buk, Inna Ziegler, Steffen Steinert, Monika Schumacher, Eva Schopf i in. "Detection of ligand-induced CNTF receptor dimers in living cells by fluorescence cross correlation spectroscopy". Biochimica et Biophysica Acta (BBA) - Biomembranes 1788, nr 9 (wrzesień 2009): 1890–900. http://dx.doi.org/10.1016/j.bbamem.2009.05.013.

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Weidemann, Thomas, Malte Wachsmuth, Tobias A. Knoch, Gabriele Müller, Waldemar Waldeck i Jörg Langowski. "Counting Nucleosomes in Living Cells with a Combination of Fluorescence Correlation Spectroscopy and Confocal Imaging". Journal of Molecular Biology 334, nr 2 (listopad 2003): 229–40. http://dx.doi.org/10.1016/j.jmb.2003.08.063.

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Paulson, Bjorn, Yeonhee Shin, Akimitsu Okamoto, Yeon-Mok Oh, Jun Ki Kim i Chan-Gi Pack. "Poly(A)+ Sensing of Hybridization-Sensitive Fluorescent Oligonucleotide Probe Characterized by Fluorescence Correlation Methods". International Journal of Molecular Sciences 22, nr 12 (16.06.2021): 6433. http://dx.doi.org/10.3390/ijms22126433.

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Ribonucleic acid (RNA) plays an important role in many cellular processes. Thus, visualizing and quantifying the molecular dynamics of RNA directly in living cells is essential to uncovering their role in RNA metabolism. Among the wide variety of fluorescent probes available for RNA visualization, exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes are useful because of their low fluorescence background. In this study, we apply fluorescence correlation methods to ECHO probes targeting the poly(A) tail of mRNA. In this way, we demonstrate not only the visualization but also the quantification of the interaction between the probe and the target, as well as of the change in the fluorescence brightness and the diffusion coefficient caused by the binding. In particular, the uptake of ECHO probes to detect mRNA is demonstrated in HeLa cells. These results are expected to provide new insights that help us better understand the metabolism of intracellular mRNA.

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Tiwari, Manisha, Shintaro Mikuni i Masataka Kinjo. "2P293 Determination of dissociation constants of NFκB p50/p65 heterodimer using fluorescence cross-correlation spectroscopy in the living cell(27. Bioimaging,Poster)". Seibutsu Butsuri 53, supplement1-2 (2013): S207. http://dx.doi.org/10.2142/biophys.53.s207_4.

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Larson, Daniel R., Yu May Ma, Volker M. Vogt i Watt W. Webb. "Direct measurement of Gag–Gag interaction during retrovirus assembly with FRET and fluorescence correlation spectroscopy". Journal of Cell Biology 162, nr 7 (29.09.2003): 1233–44. http://dx.doi.org/10.1083/jcb.200303200.

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During retrovirus assembly, the polyprotein Gag directs protein multimerization, membrane binding, and RNA packaging. It is unknown whether assembly initiates through Gag–Gag interactions in the cytosol or at the plasma membrane. We used two fluorescence techniques—two-photon fluorescence resonance energy transfer and fluorescence correlation spectroscopy—to examine Rous sarcoma virus Gag–Gag and –membrane interactions in living cells. Both techniques provide strong evidence for interactions between Gag proteins in the cytoplasm. Fluorescence correlation spectroscopy measurements of mobility suggest that Gag is present in large cytosolic complexes, but these complexes are not entirely composed of Gag. Deletion of the nucleocapsid domain abolishes Gag interactions and membrane targeting. Deletion of the membrane-binding domain leads to enhanced cytosolic interactions. These results indicate that Gag–Gag interactions occur in the cytosol, are mediated by nucleocapsid domain, and are necessary for membrane targeting and budding. These methods also have general applicability to in vivo studies of protein–protein and –membrane interactions involved in the formation of complex macromolecular structures.

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Schwille, Petra, Ulrich Haupts, Sudipta Maiti i Watt W. Webb. "Molecular Dynamics in Living Cells Observed by Fluorescence Correlation Spectroscopy with One- and Two-Photon Excitation". Biophysical Journal 77, nr 4 (październik 1999): 2251–65. http://dx.doi.org/10.1016/s0006-3495(99)77065-7.

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Fukuda, Takafumi, Shigeko Kawai-Noma, Chan-Gi Pack i Hideki Taguchi. "Large-scale analysis of diffusional dynamics of proteins in living yeast cells using fluorescence correlation spectroscopy". Biochemical and Biophysical Research Communications 520, nr 2 (grudzień 2019): 237–42. http://dx.doi.org/10.1016/j.bbrc.2019.09.066.

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Tudor, Cicerone, Jérôme N. Feige, Harikishore Pingali, Vidya Bhushan Lohray, Walter Wahli, Béatrice Desvergne, Yves Engelborghs i Laurent Gelman. "Association with Coregulators Is the Major Determinant Governing Peroxisome Proliferator-activated Receptor Mobility in Living Cells". Journal of Biological Chemistry 282, nr 7 (12.12.2006): 4417–26. http://dx.doi.org/10.1074/jbc.m608172200.

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The nucleus is an extremely dynamic compartment, and protein mobility represents a key factor in transcriptional regulation. We showed in a previous study that the diffusion of peroxisome proliferator-activated receptors (PPARs), a family of nuclear receptors regulating major cellular and metabolic functions, is modulated by ligand binding. In this study, we combine fluorescence correlation spectroscopy, dual color fluorescence cross-correlation microscopy, and fluorescence resonance energy transfer to dissect the molecular mechanisms controlling PPAR mobility and transcriptional activity in living cells. First, we bring new evidence that in vivo a high percentage of PPARs and retinoid X receptors is associated even in the absence of ligand. Second, we demonstrate that coregulator recruitment (and not DNA binding) plays a crucial role in receptor mobility, suggesting that transcriptional complexes are formed prior to promoter binding. In addition, association with coactivators in the absence of a ligand in living cells, both through the N-terminal AB domain and the AF-2 function of the ligand binding domain, provides a molecular basis to explain PPAR constitutive activity.

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Bates, Ian R., Paul W. Wiseman i John W. Hanrahan. "Investigating membrane protein dynamics in living cellsThis paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Membrane Proteins in Health and Disease." Biochemistry and Cell Biology 84, nr 6 (grudzień 2006): 825–31. http://dx.doi.org/10.1139/o06-189.

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Live cell imaging is a powerful tool for understanding the function and regulation of membrane proteins. In this review, we briefly discuss 4 fluorescence-microscopy-based techniques for studying the transport dynamics of membrane proteins: fluorescence-correlation spectroscopy, image-correlation spectroscopy, fluorescence recovery after photobleaching, and single-particle and (or) molecule tracking. The advantages and limitations of each approach are illustrated using recent studies of an ion channel and cell adhesion molecules.

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Wiseman, P. W., J. C. Bouwer, S. Peltier i M. H. Ellisman. "High Speed Two Photon Excitation Microscopy in Live Cell Imaging using Image Correlation Spectroscopy (ICS)". Microscopy and Microanalysis 7, S2 (sierpień 2001): 22–23. http://dx.doi.org/10.1017/s1431927600026180.

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For live-cell imaging, two-photon excitation microscopy (TPEM) is proving to be a significant technological advancement. The unique features offered by TPEM are the ability to image thick sections, excellent optical sectioning capabilities, low damage to living cells, and less out of focus fluorescence and out of focus photobleaching. of these features, the most useful for the biological microscopist, is optical sectioning. Optical sectioning is an intrinsic property of the two-photon process, whereby, two infrared (IR) photons are absorbed quickly to excite a single UV/blue transition. The probability for exciting a two photon transition is proportional to the instantaneous excitation intensity squared. Therefore, for a focused laser beam, only light at the focal point of the excitation beam excites a fluorescent transition. Thus, the need for confocal apertures and time consuming deconvolution algorithms are, for the most part, eliminated.We have continued to develop and enhance our ability to perform high-speed, two-photon excitation fluorescence microscopy. in 1998, we successfully deployed a prototype, video-rate twophoton laser scanning system (30 frames/sec or faster at reduced scan width) developed with support from Nikon Corporation. That system was built upon a Nikon RCM 8000 confocal microscope.

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Smoyer, Christine J., Santharam S. Katta, Jennifer M. Gardner, Lynn Stoltz, Scott McCroskey, William D. Bradford, Melainia McClain i in. "Analysis of membrane proteins localizing to the inner nuclear envelope in living cells". Journal of Cell Biology 215, nr 4 (9.11.2016): 575–90. http://dx.doi.org/10.1083/jcb.201607043.

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Understanding the protein composition of the inner nuclear membrane (INM) is fundamental to elucidating its role in normal nuclear function and in disease; however, few tools exist to examine the INM in living cells, and the INM-specific proteome remains poorly characterized. Here, we adapted split green fluorescent protein (split-GFP) to systematically localize known and predicted integral membrane proteins in Saccharomyces cerevisiae to the INM as opposed to the outer nuclear membrane. Our data suggest that components of the endoplasmic reticulum (ER) as well as other organelles are able to access the INM, particularly if they contain a small extraluminal domain. By pairing split-GFP with fluorescence correlation spectroscopy, we compared the composition of complexes at the INM and ER, finding that at least one is unique: Sbh2, but not Sbh1, has access to the INM. Collectively, our work provides a comprehensive analysis of transmembrane protein localization to the INM and paves the way for further research into INM composition and function.

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Prasai, Avishek, Marketa Schmidt Cernohorska, Klara Ruppova, Veronika Niederlova, Monika Andelova, Peter Draber, Ondrej Stepanek i Martina Huranova. "The BBSome assembly is spatially controlled by BBS1 and BBS4 in human cells". Journal of Biological Chemistry 295, nr 42 (5.08.2020): 14279–90. http://dx.doi.org/10.1074/jbc.ra120.013905.

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Bardet–Biedl syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of primary cilia. More than half of BBS patients carry mutations in one of eight genes encoding for subunits of a protein complex, the BBSome, which mediates trafficking of ciliary cargoes. In this study, we elucidated the mechanisms of the BBSome assembly in living cells and how this process is spatially regulated. We generated a large library of human cell lines deficient in a particular BBSome subunit and expressing another subunit tagged with a fluorescent protein. We analyzed these cell lines utilizing biochemical assays, conventional and expansion microscopy, and quantitative fluorescence microscopy techniques: fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. Our data revealed that the BBSome formation is a sequential process. We show that the pre-BBSome is nucleated by BBS4 and assembled at pericentriolar satellites, followed by the translocation of the BBSome into the ciliary base mediated by BBS1. Our results provide a framework for elucidating how BBS-causative mutations interfere with the biogenesis of the BBSome.

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Ohsugi, Yu, Kenta Saito, Mamoru Tamura i Masataka Kinjo. "Lateral Mobility of Membrane-Binding Proteins in Living Cells Measured by Total Internal Reflection Fluorescence Correlation Spectroscopy". Biophysical Journal 91, nr 9 (listopad 2006): 3456–64. http://dx.doi.org/10.1529/biophysj.105.074625.

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Du, Zhixue, Chaoqing Dong i Jicun Ren. "A study of the dynamics of PTEN proteins in living cells using in vivo fluorescence correlation spectroscopy". Methods and Applications in Fluorescence 5, nr 2 (28.04.2017): 024008. http://dx.doi.org/10.1088/2050-6120/aa6b07.

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Winckler, Pascale, Aurélie Cailler, Régis Deturche, Pierre Jeannesson, Hamid Morjani i Rodolphe Jaffiol. "Microfluidity mapping using fluorescence correlation spectroscopy: A new way to investigate plasma membrane microorganization of living cells". Biochimica et Biophysica Acta (BBA) - Biomembranes 1818, nr 11 (listopad 2012): 2477–85. http://dx.doi.org/10.1016/j.bbamem.2012.05.018.

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Unsay, Joseph D., Fabronia Murad, Eduard Hermann, Jonas Ries i Ana J. García-Sáez. "Scanning Fluorescence Correlation Spectroscopy for Quantification of the Dynamics and Interactions in Tube Organelles of Living Cells". ChemPhysChem 19, nr 23 (18.10.2018): 3273–78. http://dx.doi.org/10.1002/cphc.201800705.

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Hayakawa, Eri H., Michiko Furutani, Rumiko Matsuoka i Yuichi Takakuwa. "Comparison of protein behavior between wild-type and G601S hERG in living cells by fluorescence correlation spectroscopy". Journal of Physiological Sciences 61, nr 4 (15.05.2011): 313–19. http://dx.doi.org/10.1007/s12576-011-0150-2.

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Kilpatrick, Laura E., i Stephen J. Hill. "The use of fluorescence correlation spectroscopy to characterize the molecular mobility of fluorescently labelled G protein-coupled receptors". Biochemical Society Transactions 44, nr 2 (11.04.2016): 624–29. http://dx.doi.org/10.1042/bst20150285.

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The membranes of living cells have been shown to be highly organized into distinct microdomains, which has spatial and temporal consequences for the interaction of membrane bound receptors and their signalling partners as complexes. Fluorescence correlation spectroscopy (FCS) is a technique with single cell sensitivity that sheds light on the molecular dynamics of fluorescently labelled receptors, ligands or signalling complexes within small plasma membrane regions of living cells. This review provides an overview of the use of FCS to probe the real time quantification of the diffusion and concentration of G protein-coupled receptors (GPCRs), primarily to gain insights into ligand–receptor interactions and the molecular composition of signalling complexes. In addition we document the use of photon counting histogram (PCH) analysis to investigate how changes in molecular brightness (ε) can be a sensitive indicator of changes in molecular mass of fluorescently labelled moieties.

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Dunsing, Valentin, Magnus Mayer, Filip Liebsch, Gerhard Multhaup i Salvatore Chiantia. "Direct evidence of amyloid precursor–like protein 1 trans interactions in cell–cell adhesion platforms investigated via fluorescence fluctuation spectroscopy". Molecular Biology of the Cell 28, nr 25 (grudzień 2017): 3609–20. http://dx.doi.org/10.1091/mbc.e17-07-0459.

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The amyloid precursor–like protein 1 (APLP1) is a type I transmembrane protein that plays a role in synaptic adhesion and synaptogenesis. Past investigations indicated that APLP1 is involved in the formation of protein–protein complexes that bridge the junctions between neighboring cells. Nevertheless, APLP1–APLP1 trans interactions have never been directly observed in higher eukaryotic cells. Here, we investigated APLP1 interactions and dynamics directly in living human embryonic kidney cells using fluorescence fluctuation spectroscopy techniques, namely cross-correlation scanning fluorescence correlation spectroscopy and number and brightness analysis. Our results show that APLP1 forms homotypic trans complexes at cell–cell contacts. In the presence of zinc ions, the protein forms macroscopic clusters, exhibiting an even higher degree of trans binding and strongly reduced dynamics. Further evidence from giant plasma membrane vesicles suggests that the presence of an intact cortical cytoskeleton is required for zinc-induced cis multimerization. Subsequently, large adhesion platforms bridging interacting cells are formed through APLP1–APLP1 trans interactions. Taken together, our results provide direct evidence that APLP1 functions as a neuronal zinc-dependent adhesion protein and allow a more detailed understanding of the molecular mechanisms driving the formation of APLP1 adhesion platforms.

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Di Bona, Melody, Simone Pelicci, Isotta Cainero, Giuseppe Vicidomini, Davide Mazza, Michael A. Mancini, Alberto Diaspro i Luca Lanzano'. "Intensity Sorted Fluorescence Correlation Spectroscopy: A Novel Method to Probe Nuclear Dynamics and Chromatin Organization in Living Cells". Biophysical Journal 116, nr 3 (luty 2019): 72a. http://dx.doi.org/10.1016/j.bpj.2018.11.429.

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Larson, Daniel R., Julie A. Gosse, David A. Holowka, Barbara A. Baird i Watt W. Webb. "Temporally resolved interactions between antigen-stimulated IgE receptors and Lyn kinase on living cells". Journal of Cell Biology 171, nr 3 (7.11.2005): 527–36. http://dx.doi.org/10.1083/jcb.200503110.

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Upon cross-linking by antigen, the high affinity receptor for immunoglobulin E (IgE), FcεRI, is phosphorylated by the Src family tyrosine kinase Lyn to initiate mast cell signaling, leading to degranulation. Using fluorescence correlation spectroscopy (FCS), we observe stimulation-dependent associations between fluorescently labeled IgE-FcεRI and Lyn-EGFP on individual cells. We also simultaneously measure temporal variations in the lateral diffusion of these proteins. Antigen-stimulated interactions between these proteins detected subsequent to the initiation of receptor phosphorylation exhibit time-dependent changes, suggesting multiple associations between FcεRI and Lyn-EGFP. During this period, we also observe a persistent decrease in Lyn-EGFP lateral diffusion that is dependent on Src family kinase activity. These stimulated interactions are not observed between FcεRI and a chimeric EGFP that contains only the membrane-targeting sequence from Lyn. Our results reveal real-time interactions between Lyn and cross-linked FcεRI implicated in downstream signaling events. They demonstrate the capacity of FCS cross-correlation analysis to investigate the mechanism of signaling-dependent protein–protein interactions in intact, living cells.

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Ohsugi, Yu, Mamoru Tamura i Masataka Kinjo. "2P523 Molecular dynamics of membrane-binding protein in living cells analyzed by total internal reflection fluorescence correlation spectroscopy(52. Bio-imaging,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)". Seibutsu Butsuri 46, supplement2 (2006): S426. http://dx.doi.org/10.2142/biophys.46.s426_3.

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Ohrt, Thomas, Wolfgang Staroske, Jörg Mütze, Karin Crell, Markus Landthaler i Petra Schwille. "Fluorescence Cross-Correlation Spectroscopy Reveals Mechanistic Insights into the Effect of 2′-O-Methyl Modified siRNAs in Living Cells". Biophysical Journal 100, nr 12 (czerwiec 2011): 2981–90. http://dx.doi.org/10.1016/j.bpj.2011.05.005.

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Dawes, Michael L., Christian Soeller i Steffen Scholpp. "Studying molecular interactions in the intact organism: fluorescence correlation spectroscopy in the living zebrafish embryo". Histochemistry and Cell Biology 154, nr 5 (16.10.2020): 507–19. http://dx.doi.org/10.1007/s00418-020-01930-5.

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AbstractCell behaviour and function is determined through the interactions of a multitude of molecules working in concert. To observe these molecular dynamics, biophysical studies have been developed that track single interactions. Fluorescence correlation spectroscopy (FCS) is an optical biophysical technique that non-invasively resolves single molecules through recording the signal intensity at the femtolitre scale. However, recording the behaviour of these biomolecules using in vitro-based assays often fails to recapitulate the full range of variables in vivo that directly confer dynamics. Therefore, there has been an increasing interest in observing the state of these biomolecules within living organisms such as the zebrafish Danio rerio. In this review, we explore the advancements of FCS within the zebrafish and compare and contrast these findings to those found in vitro.

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