Mapping the dynamical organization of the cell nucleus through fluorescence correlation spectroscopy.
Methods
; 140-141: 10-22, 2018 05 01.
Article
en En
| MEDLINE
| ID: mdl-29253641
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Espectrometría de Fluorescencia
/
Núcleo Celular
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Citometría de Barrido por Láser
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Microscopía Intravital
Límite:
Animals
Idioma:
En
Revista:
Methods
Asunto de la revista:
BIOQUIMICA
Año:
2018
Tipo del documento:
Article
País de afiliación:
Argentina
Pais de publicación:
Estados Unidos