Identification and analysis of Dictyostelium discoideum microtubule associated proteins
The microtubule cytoskeleton and its dynamic ends are crucial for many cellular functions throughout the whole cell cycle. Microtubule associated proteins (MAPs) are known to interact with other proteins to fulfill these complex functions in balancing the dynamic instability of microtubules as well...
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|Summary:||The microtubule cytoskeleton and its dynamic ends are crucial for many cellular functions throughout the whole cell cycle. Microtubule associated proteins (MAPs) are known to interact with other proteins to fulfill these complex functions in balancing the dynamic instability of microtubules as well as anchoring microtubules at the cell cortex, guiding transport along them and controlling mitosis at the centrosome. Deficient function of these proteins leads to severe defects, including cancer. A major part of our understanding of these processes is knowing the proteins associated with the complexes at either end of microtubules.
To identify new members of these complexes, interactors of well characterized and conserved proteins of the EB1 and XMAP215 family of MAPs were searched for in the model organism Dictyostelium discoideum. DdEB1 and the Dictyostelium member of the XMAP215 protein family, DdCP224, are known to be part of complexes at the microtubule tips as well as at the centrosome. DdCP224 is involved in centrosome duplication and cytokinesis, whereas DdEB1 assists in spindle formation. At the microtubule tip these two proteins are part of a complex that is thought to link microtubules to the cell cortex.
In this study, two approaches were selected screen for novel interactors.
Employment of the yeast two hybrid system yielded five putative interactors of DdEB1 and DdCP224 that could not be verified by other means.
Tandem affinity purification (TAP) is a method originally established in yeast to isolate highly purified protein complexes in a very gentle and efficient way. In this study TAP was modified for Dictyostelium applications and proved to be a useful method to specifically isolate and identify microtubule-associated protein (MAP) complexes. Employing TAP and mass spectrometry the interaction between DdEB1 and DdCP224 was confirmed. Additionally, among several interactions that remain to be confirmed by other methods, an interaction between DdCP224 and a TACC-family protein could be shown for the first time in Dictyostelium and was confirmed by colocalization and co-immunoprecipitation analyses. Similar to findings in other species, the TACC domain is sufficient for the centrosomal localization of the protein and the interaction with the XMAP215 orthologue. Based on the results presented, DdTACC, the only member of this protein family present in Dictyostelium discoideum, was considered to be an essential gene product.|