Abstracts Category : Other

Fishing in the ocean : Use of mass spectrometry to study protein interactions involved in cytoskeletal organization and cell division

by Q Liu

Protein functions often depend on physical interactions with other proteins. Sketching a map of protein-protein interactions is an important step to understand cellular processes. Over past decades, mass spectrometry based proteomics has increasingly become the method of choice for the identification and characterization of protein interactions. In this thesis, we have combined various mass spectrometry-based methods, diverse biochemical and molecular genetics approaches and advanced imaging techniques to get a better insight in protein interactions involved in cytoskeletal organization and cell division. Chemical cross-linking mass spectrometry provides a novel way to analyse protein complexes. In this thesis, we examine an integrated workflow that combines chemical cross-linking mass spectrometry with genetic fragmentation technique to accurately locate the minimal binding domains responsible for protein interactions. We show that chemical cross-linking mass spectrometry can efficiently guide the design of truncation or deletion mutants, however, not all the cross-links we found are within the minimal binding domain. Furthermore, our data indicate that chemical cross-linking mass spectrometry-based prediction of protein interaction domains might work better for folded protein domains than for protein regions predicted to be intrinsically disordered. We apply chemical cross-linking mass spectrometry in studying the interaction between the flavoprotein monooxygenase MICAL3 and the centralspindlin component MKLP1 during cytokinesis. We show that MKLP1 interacts with and recruits MICAL3 to the midbody. We propose that during cytokinesis, MICAL3 acts as a protein-binding hub, which promotes maturation of the intercellular bridge and abscission. We also use mass spectrometry-based technique to identify new binding partners for the microtubule minus-end-interacting proteins. We further investigate the role of these proteins in the regulation of microtubule minus end organization in interphase and in mitosis.Advisors/Committee Members: Akhmanova, Anna, Heck, Albert, Altelaar, Maarten.