The distinct behavior of the spindle pole bodies (SPBs) during spindle orientation in yeast metaphase does not result from them being differently mature, but astral microtubule organization correlates with the subcellular position rather than the age of the SPBs.

γ-Tubulin has a well-established role in nucleating the assembly of microtubules, yet how phosphorylation regulates its activity remains unclear. Here, we use a time-resolved, fitness-based SGA approach to compare two γ-tubulin alleles, and find that the genetic interaction profile of γtub-Y362E is enriched in spindle positioning and cell polarity genes relative to that of γtub-Y445D, which is enriched in genes involved in spindle assembly and stability. In γtub-Y362E cells, we find a defect in spindle alignment and an increase in the number of astral microtubules at both spindle poles. Our results suggest that the γtub-Y362E allele is a separation-of-function mutation that reveals a role for γ-tubulin phospho-regulation in spindle alignment. We propose that phosphorylation of the evolutionarily conserved Y362 residue of budding yeast γ-tubulin contributes to regulating the number of astral microtubules associated with spindle poles, and promoting efficient pre-anaphase spindle alignment.

Protein complexes are not static, but rather highly dynamic with subunits that undergo 1-dimensional diffusion with respect to each other. Interactions within protein complexes are modulated through regulatory inputs that alter interactions and introduce new components and deplete existing components through exchange. While it is clear that the structure and function of any given protein complex is coupled to its dynamical properties, it remains a challenge to predict the possible conformations that complexes can adopt. Protein-fragment Complementation Assays detect physical interactions between protein pairs constrained to ≤8 nm from each other in living cells. This method has been used to build networks composed of 1000s of pair-wise interactions. Significantly, these networks contain a wealth of dynamic information, as the assay is fully reversible and the proteins are expressed in their natural context. In this study, we describe a method that extracts this valuable information in the form of predicted conformations, allowing the user to explore the conformational landscape, to search for structures that correlate with an activity state, and estimate the abundance of conformations in the living cell. The generator is based on a Markov Chain Monte Carlo simulation that uses the interaction dataset as input and is constrained by the physical resolution of the assay. We applied this method to an 18-member protein complex composed of the seven core proteins of the budding yeast Arp2/3 complex and 11 associated regulators and effector proteins. We generated 20,480 output structures and identified conformational states using principle component analysis. We interrogated the conformation landscape and found evidence of symmetry breaking, a mixture of likely active and inactive conformational states and dynamic exchange of the core protein Arc15 between core and regulatory components. Our method provides a novel tool for prediction and visualization of the hidden dynamics within protein interaction networks.

Kinesin-5 proteins are microtubule associated motors, which are highly conserved from yeast to human cells. They share high homology in their catalytic motor domain sequence, fulfill similar essential mitotic roles in spindle assembly and anaphase B spindle elongation and, until recently (Roostalu et al., Science, 2011), were all thought to move towards plus ends of microtubules. Mechanisms that regulate Kinesin-5 function, specifically during anaphase B, are not well understood. S. cerevisiae cells express two Kinesin-5 homologues, Cin8 and Kip1, which overlap in function. Here we have examined in vitro and in vivo functions and regulation of Cin8 during anaphase B. We followed Cin8 localization and carried out single molecule fluorescence motility assays to study Cin8 motile properties. We found that in vitro, Cin8 molecules are able to switch directionality along a single microtubule as a function of ionic strength conditions and that during anaphase B, Cin8 moves not only towards the plus, but also towards the minus ends of spindle microtubules. Compared to kinesin-5 homologues of higher eukaryotes, S. cerevisiae Cin8 carries a uniquely large insert in loop 8 in its motor domain. To probe the role of the large loop 8 in the directionality switch of Cin8, we studied a construct in which this segment was replaced with the seven amino acids of loop 8 in the related S. cerevisiae kinesin-5 Kip1 (Cin8D99) (Hoyt et al.,J Cell Biol, 1992). We examined the anaphase B localization and in vitro motile properties of the Cin8D99 variant. Using combined in vitro and in vivo approaches, we were able to characterize the role of loop 8 in controlling Cin8 motility and function during S. cerevisiae anaphase.

The Acknowledgements section in this Article is incomplete."The authors thank current and past members of the Vogel lab for stimulating discussions during this work, Brian Leung (McGill Biology) for input during the development of GAMER, and Elke Küster-Schöck and Guillaume Lesage for robotics support. " should read:"The authors thank current and past members of the Vogel lab for stimulating discussions during this work, Brian Leung (McGill Biology) for input during the development of GAMER, and Elke Küster-Schöck and Guillaume Lesage for robotics support.