| Popis: |
In animal cells, the relationship between the Golgi apparatus and cytoskeleton has been well characterised but not much is known in plants. The functions of the Golgi apparatus are conserved amongst eukaryotes. It is one of the main stations in the secretory pathway and is involved in protein processing and sorting to different destinations. In plants, it is also involved in trafficking and positioning of cell wall components. In tobacco epidermal cells, fluorescent labelling with Golgi marker proteins has shown that the Golgi apparatus is made of hundreds of individual units scattered in the cortical cytoplasm and moving on the actin cytoskeleton. The contribution of actin filaments to Golgi body motility in plant has been extensively described, but this actin-centric view has recently been challenged. Emerging evidence suggests that microtubules may contribute to short distance movement and 'fine tuning' of Golgi body displacement. Moreover, proteomic studies linking the actin- cytoskeleton to microtubules have demonstrated that these two components of the cytoskeleton are closely related and a role of the microtubules in Golgi movement cannot be excluded. In this thesis, automated tracking of Golgi bodies was used to understand and quantify the contribution of actin filaments and microtubules to the organelle dynamics. The tracking technique is also used to assess how the labelling of the cytoskeleton, with a novel fluorescent nanoprobe, affects the dynamics and stability of the actin filaments and the movement of Golgi bodies; FRAP analysis (fluorescent recovery after photo-bleaching) was also used to investigate the binding properties of the fluorescent nanoprobe to the actin filaments. The nanoprobe was compared with another cytoskeletal marker, Lifeact-GFP, to evaluate their suitability for studying the organelle's motility in relation to the actin-cytoskeleton. Micromanipulation of Golgi bodies with optical tweezers was used to test if there are physical links between the organelles and the cytoskeleton. The widely accepted model is that organelles move on actin filaments and movement is powered by myosins. The hypothesis that actin filaments slide one of top of the other, and drag the organelles along, was tested using the FRAP technique. Kinesin-13a is the only microtubule motor protein localized on Golgi bodies by immunochemical studies. Its localization was investigated in vivo to evaluate if it is involved in linking Golgi bodies to microtubules. |
