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The hierarchical organization of the cell nucleus into specialized open reservoirs and the nucleoplasm overcrowding impose restrictions to the mobility of biomolecules and their interactions with nuclear targets. These properties determine that many nuclear functions such as transcription, replication, splicing or DNA repair are regulated by complex, dynamical processes that do not follow simple rules. Advanced fluorescence microscopy tools and, in particular, fluorescence correlation spectroscopy (FCS) provide complementary and exquisite information on the dynamics of fluorescent labeled molecules moving through the nuclear space and are helping us to comprehend the complexity of the nuclear structure. Here, we describe how FCS methods can be applied to reveal the dynamical organization of the nucleus in live cells. Specifically, we provide instructions for the preparation of cellular samples with fluorescent tagged proteins and detail how FCS can be easily instrumented in commercial confocal microscopes. In addition, we describe general rules to set the parameters for one and two-color experiments and the required controls for these experiments. Finally, we review the statistical analysis of the FCS data and summarize the use of numerical simulations as a complementary approach that helps us to understand the complex matrix of molecular interactions network within the nucleus. © 2017 Elsevier Inc.


Documento: Artículo
Título:Mapping the dynamical organization of the cell nucleus through fluorescence correlation spectroscopy
Autor:Stortz, M.; Angiolini, J.; Mocskos, E.; Wolosiuk, A.; Pecci, A.; Levi, V.
Filiación:CONICET – Universidad de Buenos Aires, IFIBYNE, Argentina
Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Argentina
CONICET – Universidad de Buenos Aires, IQUIBICEN, Argentina
Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Computación, Argentina
CONICET – Centro de Simulación Computacional para Aplicaciones Tecnológicas, Buenos Aires, Argentina
Gerencia Química, Centro Atómico Constituyentes, Comisión Nacional de Energía Atómica, CONICET, Argentina
Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Química Inorgánica, Analítica y Química Física, Argentina
Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Química Biológica, Argentina
Palabras clave:Fluorescence correlation spectroscopy; Fluorescence microscopy; Glucocorticoid receptor; Nucleus; Simulations; Transcription factor; glucocorticoid receptor; Article; cell nucleus; cytoplasm; fluorescence correlation spectroscopy; molecular dynamics; molecular interaction; nonhuman; statistical analysis; animal; cell culture technique; cell line; cell nucleus; devices; fluorescence microscopy; fluorescent antibody technique; intravital microscopy; laser; laser scanning cytometry; Mesocricetus; metabolism; procedures; spectrofluorometry; Animals; Cell Culture Techniques; Cell Line; Cell Nucleus; Fluorescent Antibody Technique; Intravital Microscopy; Laser Scanning Cytometry; Lasers; Mesocricetus; Microscopy, Fluorescence; Spectrometry, Fluorescence
Página de inicio:10
Página de fin:22
Título revista:Methods
Título revista abreviado:Methods


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---------- APA ----------
Stortz, M., Angiolini, J., Mocskos, E., Wolosiuk, A., Pecci, A. & Levi, V. (2018) . Mapping the dynamical organization of the cell nucleus through fluorescence correlation spectroscopy. Methods, 140-141, 10-22.
---------- CHICAGO ----------
Stortz, M., Angiolini, J., Mocskos, E., Wolosiuk, A., Pecci, A., Levi, V. "Mapping the dynamical organization of the cell nucleus through fluorescence correlation spectroscopy" . Methods 140-141 (2018) : 10-22.
---------- MLA ----------
Stortz, M., Angiolini, J., Mocskos, E., Wolosiuk, A., Pecci, A., Levi, V. "Mapping the dynamical organization of the cell nucleus through fluorescence correlation spectroscopy" . Methods, vol. 140-141, 2018, pp. 10-22.
---------- VANCOUVER ----------
Stortz, M., Angiolini, J., Mocskos, E., Wolosiuk, A., Pecci, A., Levi, V. Mapping the dynamical organization of the cell nucleus through fluorescence correlation spectroscopy. Methods. 2018;140-141:10-22.