title: Monitoring cytoskeletal dynamics in living neurons using fluorescence photoactivation.
abstract: 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.
authors.affiliation: Department of Neurobiology, University of Osnabrück, Osnabrück, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Gauthier-Kemper
authors.forename: Carina
authors.initials: C
authors.lastname: Weissmann
authors.forename: Hans-Jürgen
authors.initials: HJ
authors.lastname: Reyher
authors.forename: Roland
authors.initials: R
authors.lastname: Brandt
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D058948:Cell Tracking / Q000379:methods*; D003599:Cytoskeleton / Q000378:metabolism*; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D018613:Microscopy, Confocal / Q000379:methods*; D008869:Microtubule-Associated Proteins / Q000032:analysis; D008870:Microtubules; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D016716:PC12 Cells; D051381:Rats; D011993:Recombinant Fusion Proteins; D014404:Tubulin / Q000032:analysis; D016875:tau Proteins / Q000032:analysis
chemical_list: D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D016875:tau Proteins; D049452:Green Fluorescent Proteins