Biology subjects

Rale, M. J.

Publications and source records attributed to Rale, M. J..

2 recordsLinked to original sources

bioRxiv2022

Integrated Model of the Vertebrate Augmin Complex

Accurate segregation of chromosomes is required to maintain genome integrity during cell division. This feat is accomplished by the microtubule-based spindle. To build a spindle rapidly and with high fidelity, cells take advantage of branching microtubule nucleation, which exponentially amplifies microtubules during cell division. Branching microtubule nucleation relies on the hetero-octameric augmin complex, but understanding how augmin promotes branching has been hindered by a lack of structural information about the complex. Here, we report an integrated model of vertebrate augmin, combining cryo-electron microscopy, advanced protein structural prediction, and the visualization of fused bulky tags via negative stain electron microscopy. This strategy allowed us to identify the location and orientation of each subunit within the structure. Evolutionary analysis of augmins structure reveals that it is highly conserved across diverse eukaryotes, and that augmin contains a previously-unidentified microtubule binding site. Moreover, we identify homology with the kinetochore-localized NDC80 complex. This new model of the augmin complex provides insight towards the mechanism and evolution of branching microtubule nucleation.

molecular biology↗

bioRxiv2022

The conserved centrosomal motif, γTuNA, forms a dimer that directly activates microtubule nucleation by the γ-tubulin ring complex (γTuRC).

1.To establish the microtubule cytoskeleton, the cell must tightly regulate when and where microtubules are nucleated. This regulation involves controlling the initial nucleation template, the {gamma}-tubulin ring complex ({gamma}TuRC). Although {gamma}TuRC is present throughout the cytoplasm, its activity is restricted to specific sites including the centrosome and Golgi. The well-conserved {gamma}-tubulin nucleation activator ({gamma}TuNA) domain has been reported to increase the number of microtubules generated by {gamma}TuRCs. Here we utilize Xenopus egg extract and in vitro single molecule imaging assays to show that {gamma}TuNA activates microtubule nucleation in extract and directly activates {gamma}TuRC in vitro. Via mutation analysis, we find that {gamma}TuNA is an obligate dimer. Moreover, efficient dimerization as well as {gamma}TuNAs L70, F75, and L77 residues are required for binding to and activation of {gamma}TuRC. Finally, we find that {gamma}TuNAs activating effect opposes inhibitory regulation by stathmin. In sum, our study illuminates how {gamma}TuRC is controlled in space and time in order to build specific cytoskeletal structures.

biochemistry↗