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Cytoskeleton-associated protein complexes during plant cell division
Book - Dissertation
In recent years it has become apparent that major cell biological processes are not dependent on individual proteins, but are carried out by assemblies of protein molecules. Different proteins work together in a highly coordinated way, resembling U+201Cmolecular machinesU+201D. Several of these molecular machines can be found during division, when protein complexes precisely orchestrate profound changes in the structure and physiology of the cell. The plant microtubule (MT) cytoskeleton plays an essential role in cell division and undergoes fast rearrangements from cortical microtubules to a preprophase band, spindle and phragmoplast. This process requires the cooperation of several MT-associated proteins (MAPs). Although the knowledge on plant MAPs is gradually increasing, not much is known yet about the interactions between these MAPs, nor about the processes that regulate their activity, such as phosphorylation. In animal cells it is already described that the mitotic Aurora kinase is an important player in the cytoskeletal organization during division. Aurora kinase is therefore often compared to a conductor of a symphonic orchestra, interacting with several microtubule-binding partners, and coordinating the transitions through the different phases of the mitotic symphony. In plants however, not much is known yet about the function of the Aurora kinases or their interacting proteins. Consequently, in this research we aimed to identify MAP protein complexes that function together in the successful execution of mitosis and cytokinesis, specifically focusing on AURORA1 complexes. Yeast two-hybrid library screens and Tandem Affinity Purification experiments were performed to identify interaction partners (Chapter 2). To narrow down the resulting set of candidate interacting proteins, their GFP-localization was followed in dividing BY-2 cells. This strategy resulted in identification of candidate proteins that associate with the cytoskeleton or cell plate during cell division. In Chapter 3, the interaction between AURORA1 (AUR1) and its novel interacting partner, ARCTICA1 (ARC1) is analyzed in more detail. We provide evidence that ARC1 is an in vitro substrate of AUR1. Besides localizing to kinetochores and the cell plate during cell division, ARC1 associated with the plasma membrane in a polar manner. This membrane association was further characterized in Chapter 4. Finally, a similar strategy was followed to study the binding partners of the EB1 (End Binding 1) protein, that is known to form an interaction hub at the microtubule plus end in human cells (Chapter 5). Our interaction assays identified that the EB1 family of microtubule plus-end binding proteins dimerize in plants, and we further investigated the function of EB1 dimerization in the EB1 plus-end complex assembly.
Pages: 231 p.