Reconstitution of the interface between microtubules and cortical actin
Final Report Abstract
The project proposed to study the biochemical properties, the stoichiometry and the molecular structure of the budding yeast Kar9 complex that links microtubules to actin with the aim to reconstitute the microtubule-actin interface and its regulation. The experiments proposed planned to uncover the molecular basis of force generation as astral microtubules interact with actin filaments during spindle positioning in asymmetric cell divisions. We have expressed and purified the components of the Kar9 complex and have reconstituted a stoichiometric Kar9 complex by purified components in vitro. In addition, we have mapped a number of interaction domains inside the Kar9 complex. Although we did not manage to obtain the three-dimensional structure of Kar9, we analyzed the structure of the protein Kar9 in silico, revealing that it includes a spectrin-like fold. Moreover, we have shown that Kar9 transport to the plus-ands of microtubules is regulated by phosphorylation of the Kip2 kinesin by cell cycle kinases. Lastly, we have performed the first steps towards the initial reconstitution of the microtubule-actin interface in vitro, regarding electron- and evanescent wave microscopy. These experiments were complicated by the incompatibility of the yeast proteins with the mammalian tubulin routinely utilized in these assays. We plan to overcome this difficulty by using assembly competent yeast tubulin and plan a continuation of the project.
Publications
- (2012). Spindle positioning: structures and new concepts. Curr. Opin. Cell Biol. 24, 816-24
Stevermann,L. and Liakopoulos,D.
- Yeast GSK-3 kinase regulates astral microtubule function via phosphorylation of the microtubule-stabilizing kinesin Kip2. J Cell Sci 2015, 128: 3910-3921
Drechsler,H., Tan,A.N., Liakopoulos,D.
(See online at https://doi.org/10.1242/jcs.166686)
