Reconstitution of the interface between microtubules and cortical actin – ReconstMT-Act

The interaction between microtubules and actin is indispensable for fundamental biological processes such as organ development or wound healing. Defects in proteins that link microtubules to actin are either lethal or cause diseases ranging from skin blistering to cancer. Thus, understanding the function of these proteins is prerequisite for development of treatments against the disorders associated with them. Unfortunately, microtubule-actin linking proteins are mostly gigantic in size and display an extremely intricate set of interactions. As a result, information on the structure and function of microtubule-actin linking complexes is extremely limited.
The yeast protein Kar9 forms a simple complex that links microtubules to actin filaments. Kar9 relates in function to the tumor suppressor Adenomatous Polyposis Coli (APC) and the spectraplakins, mammalian microtubule-actin linking proteins. The Kar9 complex utilizes the force of an unconventional myosin motor to pull the mitotic spindle to its correct position inside the cell and to achieve correct segregation of chromosomes. Subject of this proposal is the reconstitution of Kar9 interactions in vitro and the detailed structural, biophysical and functional analysis of Kar9 complex.
The project involves collaboration between two teams. Previous studies from the first team analyzed the function of the Kar9 complex in vivo and identified the posttranslational modifications that regulate its function. Kar9 is transported to microtubule ends, where it forms, together with myosin V, a protein complex that links microtubules to actin filaments. Team1 has developed the tools to study these processes in vivo and partly reconstitute the protein complexes required for Kar9 transport and actin interaction from purified proteins.
The second team is expert in reconstituting in vitro systems to study cytoskeleton dynamics. It has pioneered the use of micropatterning to geometrically control cytoskeleton assembly in vitro and has implemented this method for the quantitative analysis of the cytoskeleton. In addition, they will provide most of the laser nanosurgery, patterning platforms, evanescent wave (TIRF) microscopes and image analysis tools required in this proposal.
We will join forces to reconstitute and study the Kar9 microtubule-actin interface. Team1 will obtain information on the structure of the Kar9 protein by biochemical analysis and X-ray crystallography. It will map the interactions among the different proteins in the Kar9 complex and examine the role of posttranslational modifications in complex assembly. Moreover, it will identify the stoichiometry of the complex in solution and use electron and cryoelectron microscopy to identify the structure of the Kar9 complex bound to microtubules.
We will combine the tools generated by Team1 with the expertise of Team2 to achieve the next two objectives. The first is to reconstitute transport of purified Kar9 on microtubules in vitro and quantitatively analyze the process using TIRF microscopy. The second is to reconstitute the link between microtubules and actin. Team2 will develop micropatterning arrays consisting of actin and dynamically growing microtubules in defined geometrical arrangements. We will use these arrays to reconstitute interactions between microtubules and actin filaments using Kar9 complexes assembled from recombinant proteins or native Kar9 complexes purified from yeast. Using TIRF assays, we will quantitatively analyze the structure and the biophysical properties (i.e amount of generated forces or complex dynamics) of active protein assemblies that constitute the microtubule-actin interface. Whenever possible, the results obtained in vitro will be validated using quantitative fluorescence microscopy in living yeast cells. Ultimately, we could develop this novel technology to compare the Kar9 complex with more complex systems involving spectraplakins or the APC protein.