In vitro reconstitution and (photo)control of the microtubule plus-end tracking complex
Collaborators: Anna Akhmanova, Lucas Kapitein
Microtubules (MTs) are a dynamic part of the cytoskeleton and an essential part of the adaptive capabilities of cellular organization. In order to investigate the complex interaction network and obtain a detailed quantitative description of the system, it is necessary to reconstitute the system in a fully controlled environment. This project will focus on the reorganizational properties of MTs, termed dynamic instability, in response to regulatory signals in vitro. Growing MTs specifically accumulate a set of conserved factors at their end, known as MT plus end tracking proteins (+TIPs), that can regulate MT dynamics and serve as linkers between MTs and other cytoskeletal components. Furthermore, to dissect feedback mechanisms, a possibility must exist to control the parameters of the system in space and time.
To this end, we aim to achieve a molecular-level understanding of cooperative and competitive effects of microtubule regulators on microtubule dynamics by reconstituting a controllable cytoskeleton, in which we have the ability to control +TIP interactions with the use of light-controllable heterodimerization domains. At the core of the +TIP network are End Binding (EB) proteins, which can autonomously recognize growing plus ends. In order to control MT tip recruitment we will employ the interaction of this EB protein with a short peptide containing a linear SxIP motif. To achieve spatial and temporal control of this interaction, a light-switchable protein interaction system, based on the Arabidopsis cryptochrome CRY2 and its binding partner CIB1, will be employed. By activating CRY2, which will be able to bind to EB through an engineered SxIP peptide, it recruits CIB1 together with an attached +TIP to the microtubule end. This way, we are able to engage specific protein-protein interactions and study their effect on microtubule dynamics.