IFIBYNE   05513
INSTITUTO DE FISIOLOGIA, BIOLOGIA MOLECULAR Y NEUROCIENCIAS
Unidad Ejecutora - UE
artículos
Título:
MONITORING CYTOSKELETAL DYNAMICS IN LIVING NEURONS USING
Autor/es:
ANNE GAUTHIER; CARINA WEISSMANN; HANS JÜRGEN REYHER; ROLAND BRANDT
Revista:
METHODS IN ENZYMOLOGY.
Editorial:
ELSEVIER ACADEMIC PRESS INC
Referencias:
Año: 2011
ISSN:
0076-6879
Resumen:
Neurons exhibit high temporal and spatial dynamics of their cytoskeletal organization,
which is critical for the development and maintenance of axons and dendrites. Live cell
imaging of fluorescence labeled proteins provides a powerful approach to scrutinize the
dynamics of cytoskeletal components in living neuronal cells. Here we describe a method
to monitor and quantitatively analyze the dissipation of populations of cytoskeletal
proteins in neurites of living cells using fluorescence photoactivation of fusion constructs
with photoactivatable GFP (PAGFP). We present considerations on the design of the
constructs, methods of gene transfer in neural cell lines and primary neurons, and
implementation of photoactivation experiments using standard confocal laser scanning
microscopy. In addition, we introduce general methods for data presentation and analysis
using paradigmatic experiments of imaging PAGFP-neurofilament, -tubulin and -tau in
neuronally differentiated PC12 cells and primary cortical cultures. Methods include the
generation of color-coded plots of 2D space-time intensity function, determination of
immobile fractions, intensity shift analyses, and modeling to determine effective
diffusion constants.