A mutation uncouples the tubulin conformational and GTPase cycles, revealing allosteric control of microtubule dynamics.

Microtubule dynamic instability depends on the GTPase activity of the polymerizing αß-tubulin subunits, which cycle through at least three distinct conformations as they move into and out of microtubules. How this conformational cycle contributes to microtubule growing, shrinking, and switching remains unknown. Here, we report that a buried mutation in αß-tubulin yields microtubules with dramatically reduced shrinking rate and catastrophe frequency. The mutation causes these effects by suppressing a conformational change that normally occurs in response to GTP hydrolysis in the lattice, without detectably changing the conformation of unpolymerized αß-tubulin. Thus, the mutation weakens the coupling between the conformational and GTPase cycles of αß-tubulin. By showing that the mutation predominantly affects post-GTPase conformational and dynamic properties of microtubules, our data reveal that the strength of the allosteric response to GDP in the lattice dictates the frequency of catastrophe and the severity of rapid shrinking.

Constitutive dynein activity in She1 mutants reveals differences in microtubule attachment at the yeast spindle pole body.

The organization of microtubules is determined in most cells by a microtubule-organizing center, which nucleates microtubule assembly and anchors their minus ends. In Saccharomyces cerevisiae cells lacking She1, cytoplasmic microtubules detach from the spindle pole body at high rates. Increased rates of detachment depend on dynein activity, supporting previous evidence that She1 inhibits dynein. Detachment rates are higher in G1 than in metaphase cells, and we show that this is primarily due to differences in the strengths of microtubule attachment to the spindle pole body during these stages of the cell cycle. The minus ends of detached microtubules are stabilized by the presence of γ-tubulin and Spc72, a protein that tethers the γ-tubulin complex to the spindle pole body. A Spc72-Kar1 fusion protein suppresses detachment in G1 cells, indicating that the interaction between these two proteins is critical to microtubule anchoring. Overexpression of She1 inhibits the loading of dynactin components, but not dynein, onto microtubule plus ends. In addition, She1 binds directly to microtubules in vitro, so it may compete with dynactin for access to microtubules. Overall, these results indicate that inhibition of dynein activity by She1 is important to prevent excessive detachment of cytoplasmic microtubules, particularly in G1 cells.

A quantitative, high-throughput reverse genetic screen reveals novel connections between Pre-mRNA splicing and 5' and 3' end transcript determinants.

Here we present the development and implementation of a genome-wide reverse genetic screen in the budding yeast, Saccharomyces cerevisiae, that couples high-throughput strain growth, robotic RNA isolation and cDNA synthesis, and quantitative PCR to allow for a robust determination of the level of nearly any cellular RNA in the background of ~5,500 different mutants. As an initial test of this approach, we sought to identify the full complement of factors that impact pre-mRNA splicing. Increasing lines of evidence suggest a relationship between pre-mRNA splicing and other cellular pathways including chromatin remodeling, transcription, and 3' end processing, yet in many cases the specific proteins responsible for functionally connecting these pathways remain unclear. Moreover, it is unclear whether all pathways that are coupled to splicing have been identified. As expected, our approach sensitively detects pre-mRNA accumulation in the vast majority of strains containing mutations in known splicing factors. Remarkably, however, several additional candidates were found to cause increases in pre-mRNA levels similar to that seen for canonical splicing mutants, none of which had previously been implicated in the splicing pathway. Instead, several of these factors have been previously implicated to play roles in chromatin remodeling, 3' end processing, and other novel categories. Further analysis of these factors using splicing-sensitive microarrays confirms that deletion of Bdf1, a factor that links transcription initiation and chromatin remodeling, leads to a global splicing defect, providing evidence for a novel connection between pre-mRNA splicing and this component of the SWR1 complex. By contrast, mutations in 3' end processing factors such as Cft2 and Yth1 also result in pre-mRNA splicing defects, although only for a subset of transcripts, suggesting that spliceosome assembly in S. cerevisiae may more closely resemble mammalian models of exon-definition. More broadly, our work demonstrates the capacity of this approach to identify novel regulators of various cellular RNAs.

Regulation of microtubule dynamics by Bim1 and Bik1, the budding yeast members of the EB1 and CLIP-170 families of plus-end tracking proteins.

Microtubule dynamics are regulated by plus-end tracking proteins (+TIPs), which bind microtubule ends and influence their polymerization properties. In addition to binding microtubules, most +TIPs physically associate with other +TIPs, creating a complex web of interactions. To fully understand how +TIPs regulate microtubule dynamics, it is essential to know the intrinsic biochemical activities of each +TIP and how +TIP interactions affect these activities. Here, we describe the activities of Bim1 and Bik1, two +TIP proteins from budding yeast and members of the EB1 and CLIP-170 families, respectively. We find that purified Bim1 and Bik1 form homodimers that interact with each other to form a tetramer. Bim1 binds along the microtubule lattice but with highest affinity for the microtubule end; however, Bik1 requires Bim1 for localization to the microtubule lattice and end. In vitro microtubule polymerization assays show that Bim1 promotes microtubule assembly, primarily by decreasing the frequency of catastrophes. In contrast, Bik1 inhibits microtubule assembly by slowing growth and, consequently, promoting catastrophes. Interestingly, the Bim1-Bik1 complex affects microtubule dynamics in much the same way as Bim1 alone. These studies reveal new activities for EB1 and CLIP-170 family members and demonstrate how interactions between two +TIP proteins influence their activities.

The Saccharomyces cerevisiae homolog of p24 is essential for maintaining the association of p150Glued with the dynactin complex.

Stu1 is the Saccharomyces cerevisiae member of the CLASP family of microtubule plus-end tracking proteins and is essential for spindle formation. A genomewide screen for gene deletions that are lethal in combination with the temperature-sensitive stu1-5 allele identified ldb18Delta. ldb18Delta cells exhibit defects in spindle orientation similar to those caused by a block in the dynein pathway. Consistent with this observation, ldb18Delta is synthetic lethal with mutations affecting the Kar9 spindle orientation pathway, but not with those affecting the dynein pathway. We show that Ldb18 is a component of dynactin, a complex required for dynein activity in yeast and mammalian cells. Ldb18 shares modest sequence and structural homology with the mammalian dynactin component p24. It interacts with dynactin proteins in two-hybrid and co-immunoprecipitation assays, and comigrates with them as a 20 S complex during sucrose gradient sedimentation. In ldb18Delta cells, the interaction between Nip100 (p150(Glued)) and Jnm1 (dynamitin) is disrupted, while the interaction between Jnm1 and Arp1 is not affected. These results indicate that p24 is required for attachment of the p150(Glued) arm to dynamitin and the remainder of the dynactin complex. The genetic interaction of ldb18Delta with stu1-5 also supports the notion that dynein/dynactin helps to generate a spindle pole separating force.

Requirement for the budding yeast polo kinase Cdc5 in proper microtubule growth and dynamics.

In many organisms, polo kinases appear to play multiple roles during M-phase progression. To provide new insights into the function of the budding yeast polo kinase Cdc5, we generated novel temperature-sensitive cdc5 mutants by mutagenizing the C-terminal noncatalytic polo box domain, a region that is critical for proper subcellular localization. One of these mutants, cdc5-11, exhibited a temperature-sensitive growth defect with an abnormal spindle morphology. Strikingly, provision of a moderate level of benomyl, a microtubule-depolymerizing drug, permitted cdc5-11 cells to grow significantly better than the isogenic CDC5 wild type in a FEAR (cdc Fourteen Early Anaphase Release)-independent manner. In addition, cdc5-11 required MAD2 for both cell growth and the benomyl-remedial phenotype. These results suggest that cdc5-11 is defective in proper spindle function. Consistent with this view, cdc5-11 exhibited abnormal spindle morphology, shorter spindle length, and delayed microtubule regrowth at the nonpermissive temperature. Overexpression of CDC5 moderately rescued the spc98-2 growth defect. Interestingly, both Cdc28 and Cdc5 were required for the proper modification of the spindle pole body components Nud1, Slk19, and Stu2 in vivo. They also phosphorylated these three proteins in vitro. Taken together, these observations suggest that concerted action of Cdc28 and Cdc5 on Nud1, Slk19, and Stu2 is important for proper spindle functions.

Dynamic microtubules are essential for efficient chromosome capture and biorientation in S. cerevisiae.

Attachment of chromosomes to the mitotic spindle has been proposed to require dynamic microtubules that randomly search three-dimensional space and become stabilized upon capture by kinetochores. In this study, we test this model by examining chromosome capture in Saccharomyces cerevisiae mutants with attenuated microtubule dynamics. Although viable, these cells are slow to progress through mitosis. Preanaphase cells contain a high proportion of chromosomes that are attached to only one spindle pole and missegregate in the absence of the spindle assembly checkpoint. Measurement of the rates of chromosome capture and biorientation demonstrate that both are severely decreased in the mutants. These results provide direct evidence that dynamic microtubules are critical for efficient chromosome capture and biorientation and support the hypothesis that microtubule search and capture plays a central role in assembly of the mitotic spindle.

The regulation of microtubule dynamics in Saccharomyces cerevisiae by three interacting plus-end tracking proteins.

Microtubule plus-end tracking proteins (+TIPs) are a diverse group of molecules that regulate microtubule dynamics and interactions of microtubules with other cellular structures. Many +TIPs have affinity for each other but the functional significance of these associations is unclear. Here we investigate the physical and functional interactions among three +TIPs in S. cerevisiae, Stu2, Bik1, and Bim1. Two-hybrid, coimmunoprecipitation, and in vitro binding assays demonstrate that they associate in all pairwise combinations, although the interaction between Stu2 and Bim1 may be indirect. Three-hybrid assays indicate that these proteins compete for binding to each other. Thus, Stu2, Bik1, and Bim1 interact physically but do not appear to be arranged in a single unique complex. We examined the functional interactions among pairs of proteins by comparing cytoplasmic and spindle microtubule dynamics in cells lacking either one or both proteins. On cytoplasmic microtubules, Stu2 and Bim1 act cooperatively to regulate dynamics in G1 but not in preanaphase, whereas Bik1 acts independently from Stu2 and Bim1. On kinetochore microtubules, Bik1 and Bim1 are redundant for regulating dynamics, whereas Stu2 acts independently from Bik1 and Bim1. These results indicate that interactions among +TIPS can play important roles in the regulation of microtubule dynamics.

Model for the yeast cofactor A-beta-tubulin complex based on computational docking and mutagensis.

Virtually every biological process involves protein-protein contact but relatively few protein-protein complexes have been solved by X-ray crystallography. As more individual protein structures become available, computational methods are likely to play increasingly important roles in defining these interactions. Tubulin folding and dimer formation are complex processes requiring a variety of protein cofactors. One of these is cofactor A, which interacts with beta-tubulin prior to assembly of the alpha-tubulin-beta-tubulin heterodimer. In the yeast Saccharomyces cerevisiae, beta-tubulin is encoded by TUB2 and cofactor A by RBL2. We have used computational docking and site-directed mutagenesis to generate a model of the Rbl2-Tub2 complex from the solved structures of these two proteins. Residues in the N termini and the loops of the Rbl2 homodimer appear to mediate binding to beta-tubulin. These interact with beta-tubulin residues in the region that contains helices H9 and H10. Rbl2 and alpha-tubulin share overlapping binding sites on the beta-tubulin molecule providing a structural explanation for the mutually exclusive binding of Rbl2 and alpha-tubulin to beta-tubulin.

Spindle orientation in Saccharomyces cerevisiae depends on the transport of microtubule ends along polarized actin cables.

Microtubules and actin filaments interact and cooperate in many processes in eukaryotic cells, but the functional implications of such interactions are not well understood. In the yeast Saccharomyces cerevisiae, both cytoplasmic microtubules and actin filaments are needed for spindle orientation. In addition, this process requires the type V myosin protein Myo2, the microtubule end-binding protein Bim1, and Kar9. Here, we show that fusing Bim1 to the tail of the Myo2 is sufficient to orient spindles in the absence of Kar9, suggesting that the role of Kar9 is to link Myo2 to Bim1. In addition, we show that Myo2 localizes to the plus ends of cytoplasmic microtubules, and that the rate of movement of these cytoplasmic microtubules to the bud neck depends on the intrinsic velocity of Myo2 along actin filaments. These results support a model for spindle orientation in which a Myo2-Kar9-Bim1 complex transports microtubule ends along polarized actin cables. We also present data suggesting that a similar process plays a role in orienting cytoplasmic microtubules in mating yeast cells.

The yeast ubiquitin protease, Ubp3p, promotes protein stability.

Stu1p is a microtubule-associated protein required for spindle assembly. In this article we show that the temperature-sensitive stu1-5 allele is synthetically lethal in combination with ubp3, gim1-gim5, and kem1 mutations. The primary focus of this article is on the stu1-5 ubp3 interaction. Ubp3 is a deubiquitination enzyme and a member of a large family of cysteine proteases that cleave ubiquitin moieties from protein substrates. UBP3 is the only one of 16 UBP genes in yeast whose loss is synthetically lethal with stu1-5. Stu1p levels in stu1-5 cells are several-fold lower than the levels in wild-type cells and the stu1-5 temperature sensitivity can be rescued by additional copies of stu1-5. These results indicate that the primary effect of the stu1-5 mutation is to make the protein less stable. The levels of Stu1p are even lower in ubp3Delta stu1-5 cells, suggesting that Ubp3p plays a role in promoting protein stability. We also found that ubp3Delta produces growth defects in combination with mutations in other genes that decrease protein stability. Overall, these data support the idea that Ubp3p has a general role in the reversal of protein ubiquitination.

Stu1p is physically associated with beta-tubulin and is required for structural integrity of the mitotic spindle.

Formation of the bipolar mitotic spindle relies on a balance of forces acting on the spindle poles. The primary outward force is generated by the kinesin-related proteins of the BimC family that cross-link antiparallel interpolar microtubules and slide them past each other. Here, we provide evidence that Stu1p is also required for the production of this outward force in the yeast Saccharomyces cerevisiae. In the temperature-sensitive stu1-5 mutant, spindle pole separation is inhibited, and preanaphase spindles collapse, with their previously separated poles being drawn together. The temperature sensitivity of stu1-5 can be suppressed by doubling the dosage of Cin8p, a yeast BimC kinesin-related protein. Stu1p was observed to be a component of the mitotic spindle localizing to the midregion of anaphase spindles. It also binds to microtubules in vitro, and we have examined the nature of this interaction. We show that Stu1p interacts specifically with beta-tubulin and identify the domains required for this interaction on both Stu1p and beta-tubulin. Taken together, these findings suggest that Stu1p binds to interpolar microtubules of the mitotic spindle and plays an essential role in their ability to provide an outward force on the spindle poles.