Live cell imaging of cytoskeletal dynamics using fluorescence photoactivation

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Titel: Live cell imaging of cytoskeletal dynamics using fluorescence photoactivation
Autor(en): Gauthier-Kemper, Anne
Erstgutachter: Prof. Dr. Roland Brandt
Zweitgutachter: Prof. Dr. Jürgen J. Heinisch
Zusammenfassung: Neurodegeneration in selected brain areas, associated with abnormal behavior of cytoskeletal proteins or altered organization of the cytoskeletal filament network, exhibits a characteristic feature of many neurodegenerative diseases. Therefore, focusing on analyzing the dynamics of cytoskeletal proteins under disease-relevant conditions using live cell imaging approaches could provide a better understanding of the cellular mechanisms underlying neurodegeneration. Fluorescence photoactivation (FPA) provides a novel tool to label and track living cells, organelles, or even single molecules in living systems in a spatio-temporal manner with high sensitivity. Fusion of photoactivatable fluorescence proteins to cytoskeletal proteins allows analyzing cytoskeletal dynamics in neurons in real-time and provides the unique opportunity to determine the effect of disease-relevant conditions on cytoskeletal dynamics in living neurons. Aim of the thesis was to study the motion of different cytoskeletal proteins: the microtubule associated protein tau and the growth associated actin-binding protein GAP-43. Expression of both proteins is developmentally regulated and may play an important role in neuronal polarization. Furthermore, both proteins show enrichment at the distal part of the neurite, the growth cone. The mechanisms, how distal trapping of tau and trafficking and enrichment of GAP-43 at the tip are regulated, are unclear. To scrutinize the dissipation of both proteins in living neurons, we constructed a panel of PAGFP-tagged fusion constructs and expressed them in differentiated PC12 cells as a neuronal model system. Using FPA in combination with computer-assisted image processing, we could identify the dissipation and trapping mechanisms of both proteins. The data indicate that FPA provides a useful and versatile approach to determine protein distribution in living cells during development and disease-like conditions.
URL: https://repositorium.ub.uni-osnabrueck.de/handle/urn:nbn:de:gbv:700-201111048511
Schlagworte: photoactivation; cytoskeleton
Erscheinungsdatum: 4-Nov-2011
Enthalten in den Sammlungen:FB05 - E-Dissertationen

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