Successive Kinesin-5 Microtubule Crosslinking and Sliding Promote Fast, Irreversible Formation of a Stereotyped Bipolar Spindle.

Separation of duplicated spindle poles is the first step in forming the mitotic spindle. Kinesin-5 crosslinks and slides anti-parallel microtubules (MTs), but it is unclear how these two activities contribute to the first steps in spindle formation. In this study, we report that in monopolar spindles, the duplicated spindle poles snap apart in a fast and irreversible step that produces a nascent bipolar spindle. Using mutations in Kinesin-5 that inhibit microtubule sliding, we show that the fast, irreversible pole separation is primarily driven by microtubule crosslinking. Electron tomography revealed microtubule pairs in monopolar spindles have short overlaps that intersect at high angles and are unsuited for ensemble Kinesin-5 sliding. However, maximal extension of a subset of anti-parallel microtubule pairs approaches the length of nascent bipolar spindles and is consistent with a Kinesin-5 crosslinking-driven transition. Nonetheless, microtubule sliding by Kinesin-5 contributes to stabilizing the nascent spindle and setting its stereotyped equilibrium length.

Author Correction: Interrogation of γ-tubulin alleles using high-resolution fitness measurements reveals a distinct cytoplasmic function in spindle alignment.

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Interrogation of γ-tubulin alleles using high-resolution fitness measurements reveals a distinct cytoplasmic function in spindle alignment.

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.

Combining the Optimized Yeast Cytosine Deaminase Protein Fragment Complementation Assay and an In Vitro Cdk1 Targeting Assay to Study the Regulation of the γ-Tubulin Complex.

Cdk1 is the essential cyclin-dependent kinase in the budding yeast Saccharomyces cerevisiae. Cdk1 orchestrates cell cycle control by phosphorylating target proteins with extraordinary temporal and spatial specificity by complexing with one of the nine cyclin regulatory subunits. The identification of the cyclin required for targeting Cdk1 to a substrate can help to place the regulation of that protein at a specific time point during the cell cycle and reveal information needed to elucidate the biological significance of the regulation. Here, we describe a combination of strategies to identify interaction partners of Cdk1, and associate these complexes to the appropriate cyclins using a cell-based protein-fragment complementation assay. Validation of the specific reliance of the OyCD interaction between Cdk1 and budding yeast γ-tubulin on the Clb3 cyclin, relative to the mitotic Clb2 cyclin, was performed by an in vitro kinase assay using the γ-tubulin complex as a substrate.

Functional genomics analysis of the Saccharomyces cerevisiae iron responsive transcription factor Aft1 reveals iron-independent functions.

The Saccharomyces cerevisiae transcription factor Aft1 is activated in iron-deficient cells to induce the expression of iron regulon genes, which coordinate the increase of iron uptake and remodel cellular metabolism to survive low-iron conditions. In addition, Aft1 has been implicated in numerous cellular processes including cell-cycle progression and chromosome stability; however, it is unclear if all cellular effects of Aft1 are mediated through iron homeostasis. To further investigate the cellular processes affected by Aft1, we identified >70 deletion mutants that are sensitive to perturbations in AFT1 levels using genome-wide synthetic lethal and synthetic dosage lethal screens. Our genetic network reveals that Aft1 affects a diverse range of cellular processes, including the RIM101 pH pathway, cell-wall stability, DNA damage, protein transport, chromosome stability, and mitochondrial function. Surprisingly, only a subset of mutants identified are sensitive to extracellular iron fluctuations or display genetic interactions with mutants of iron regulon genes AFT2 or FET3. We demonstrate that Aft1 works in parallel with the RIM101 pH pathway and the role of Aft1 in DNA damage repair is mediated by iron. In contrast, through both directed studies and microarray transcriptional profiling, we show that the role of Aft1 in chromosome maintenance and benomyl resistance is independent of its iron regulatory role, potentially through a nontranscriptional mechanism.