S Biggins, N Bhalla, A Chang, DL Smith, and AW Murray. 2001. “Genes involved in sister chromatid separation and segregation in the budding yeast Saccharomyces cerevisiae.” Genetics, 159, 2, Pp. 453-70. Publisher's VersionAbstract
Accurate chromosome segregation requires the precise coordination of events during the cell cycle. Replicated sister chromatids are held together while they are properly attached to and aligned by the mitotic spindle at metaphase. At anaphase, the links between sisters must be promptly dissolved to allow the mitotic spindle to rapidly separate them to opposite poles. To isolate genes involved in chromosome behavior during mitosis, we microscopically screened a temperature-sensitive collection of budding yeast mutants that contain a GFP-marked chromosome. Nine LOC (loss of cohesion) complementation groups that do not segregate sister chromatids at anaphase were identified. We cloned the corresponding genes and performed secondary tests to determine their function in chromosome behavior. We determined that three LOC genes, PDS1, ESP1, and YCS4, are required for sister chromatid separation and three other LOC genes, CSE4, IPL1, and SMT3, are required for chromosome segregation. We isolated alleles of two genes involved in splicing, PRP16 and PRP19, which impair alpha-tubulin synthesis thus preventing spindle assembly, as well as an allele of CDC7 that is defective in DNA replication. We also report an initial characterization of phenotypes associated with the SMT3/SUMO gene and the isolation of WSS1, a high-copy smt3 suppressor.
BM Stern and AW Murray. 2001. “Lack of tension at kinetochores activates the spindle checkpoint in budding yeast.” Curr Biol, 11, 18, Pp. 1462-7. Publisher's VersionAbstract
The spindle checkpoint delays the onset of anaphase until all pairs of sister chromatids are attached to the mitotic spindle. The checkpoint could monitor the attachment of microtubules to kinetochores, the tension that results from the two sister chromatids attaching to opposite spindle poles, or both. We tested the role of tension by allowing cells to enter mitosis without a prior round of DNA replication. The unreplicated chromatids are attached to spindle microtubules but are not under tension since they lack a sister chromatid that could attach to the opposite pole. Because the spindle checkpoint is activated in these cells, we conclude that the absence of tension at the yeast kinetochore is sufficient to activate the spindle checkpoint in mitosis.
AD Rudner, KG Hardwick, and AW Murray. 2000. “Cdc28 activates exit from mitosis in budding yeast.” J Cell Biol, 149, 7, Pp. 1361-76. Publisher's VersionAbstract
The activity of the cyclin-dependent kinase 1 (Cdk1), Cdc28, inhibits the transition from anaphase to G1 in budding yeast. CDC28-T18V, Y19F (CDC28-VF), a mutant that lacks inhibitory phosphorylation sites, delays the exit from mitosis and is hypersensitive to perturbations that arrest cells in mitosis. Surprisingly, this behavior is not due to a lack of inhibitory phosphorylation or increased kinase activity, but reflects reduced activity of the anaphase-promoting complex (APC), a defect shared with other mutants that lower Cdc28/Clb activity in mitosis. CDC28-VF has reduced Cdc20- dependent APC activity in mitosis, but normal Hct1- dependent APC activity in the G1 phase of the cell cycle. The defect in Cdc20-dependent APC activity in CDC28-VF correlates with reduced association of Cdc20 with the APC. The defects of CDC28-VF suggest that Cdc28 activity is required to induce the metaphase to anaphase transition and initiate the transition from anaphase to G1 in budding yeast.
AW Murray. 2000. “Journey to the centre of the cell.” Nat Cell Biol, 2, 7, Pp. E130-1. Publisher's VersionAbstract
Over the last 20 years, studies of the biochemical oscillator that drives cell reproduction have revolutionized our understanding of the cell cycle. A recent Jaques Monod Conference, at the Station Biologique in Roscoff (30 April - 3 May 2000), concentrated on dissecting the elaborate structural rearrangements that the oscillator induces in order to push cells from interphase to mitosis and then to divide them in two.
KG Hardwick, RC Johnston, DL Smith, and AW Murray. 2000. “MAD3 encodes a novel component of the spindle checkpoint which interacts with Bub3p, Cdc20p, and Mad2p.” J Cell Biol, 148, 5, Pp. 871-82. Publisher's VersionAbstract
We show that MAD3 encodes a novel 58-kD nuclear protein which is not essential for viability, but is an integral component of the spindle checkpoint in budding yeast. Sequence analysis reveals two regions of Mad3p that are 46 and 47% identical to sequences in the NH(2)-terminal region of the budding yeast Bub1 protein kinase. Bub1p is known to bind Bub3p (Roberts et al. 1994) and we use two-hybrid assays and coimmunoprecipitation experiments to show that Mad3p can also bind to Bub3p. In addition, we find that Mad3p interacts with Mad2p and the cell cycle regulator Cdc20p. We show that the two regions of homology between Mad3p and Bub1p are crucial for these interactions and identify loss of function mutations within each domain of Mad3p. We discuss roles for Mad3p and its interactions with other spindle checkpoint proteins and with Cdc20p, the target of the checkpoint.
AD Rudner and AW Murray. 2000. “Phosphorylation by Cdc28 activates the Cdc20-dependent activity of the anaphase-promoting complex.” J Cell Biol, 149, 7, Pp. 1377-90. Publisher's VersionAbstract
Budding yeast initiates anaphase by activating the Cdc20-dependent anaphase-promoting complex (APC). The mitotic activity of Cdc28 (Cdk1) is required to activate this form of the APC, and mutants that are impaired in mitotic Cdc28 function have difficulty leaving mitosis. This defect can be explained by a defect in APC phosphorylation, which depends on mitotic Cdc28 activity in vivo and can be catalyzed by purified Cdc28 in vitro. Mutating putative Cdc28 phosphorylation sites in three components of the APC, Cdc16, Cdc23, and Cdc27, makes the APC resistant to phosphorylation both in vivo and in vitro. The nonphosphorylatable APC has normal activity in G1, but its mitotic, Cdc20-dependent activity is compromised. These results show that Cdc28 activates the APC in budding yeast to trigger anaphase. Previous reports have shown that the budding yeast Cdc5 homologue, Plk, can also phosphorylate and activate the APC in vitro. We show that, like cdc28 mutants, cdc5 mutants affect APC phosphorylation in vivo. However, although Cdc5 can phosphorylate Cdc16 and Cdc27 in vitro, this in vitro phosphorylation does not occur on in vivo sites of phosphorylation.
MA Shonn, R McCarroll, and AW Murray. 2000. “Requirement of the spindle checkpoint for proper chromosome segregation in budding yeast meiosis.” Science, 289, 5477, Pp. 300-3. Publisher's VersionAbstract
The spindle checkpoint was characterized in meiosis of budding yeast. In the absence of the checkpoint, the frequency of meiosis I missegregation increased with increasing chromosome length, reaching 19% for the longest chromosome. Meiosis I nondisjunction in spindle checkpoint mutants could be prevented by delaying the onset of anaphase. In a recombination-defective mutant (spo11Delta), the checkpoint delays the biochemical events of anaphase I, suggesting that chromosomes that are attached to microtubules but are not under tension can activate the spindle checkpoint. Spindle checkpoint mutants reduce the accuracy of chromosome segregation in meiosis I much more than that in meiosis II, suggesting that checkpoint defects may contribute to Down syndrome.
BJ Howell, DB Hoffman, G Fang, AW Murray, and ED Salmon. 2000. “Visualization of Mad2 dynamics at kinetochores, along spindle fibers, and at spindle poles in living cells.” J Cell Biol, 150, 6, Pp. 1233-50. Publisher's VersionAbstract
The spindle checkpoint prevents errors in chromosome segregation by inhibiting anaphase onset until all chromosomes have aligned at the spindle equator through attachment of their sister kinetochores to microtubules from opposite spindle poles. A key checkpoint component is the mitotic arrest-deficient protein 2 (Mad2), which localizes to unattached kinetochores and inhibits activation of the anaphase-promoting complex (APC) through an interaction with Cdc20. Recent studies have suggested a catalytic model for kinetochore function where unattached kinetochores provide sites for assembling and releasing Mad2-Cdc20 complexes, which sequester Cdc20 and prevent it from activating the APC. To test this model, we examined Mad2 dynamics in living PtK1 cells that were either injected with fluorescently labeled Alexa 488-XMad2 or transfected with GFP-hMAD2. Real-time, digital imaging revealed fluorescent Mad2 localized to unattached kinetochores, spindle poles, and spindle fibers depending on the stage of mitosis. FRAP measurements showed that Mad2 is a transient component of unattached kinetochores, as predicted by the catalytic model, with a t(1/2) of approximately 24-28 s. Cells entered anaphase approximately 10 min after Mad2 was no longer detectable on the kinetochores of the last chromosome to congress to the metaphase plate. Several observations indicate that Mad2 binding sites are translocated from kinetochores to spindle poles along microtubules. First, Mad2 that bound to sites on a kinetochore was dynamically stretched in both directions upon microtubule interactions, and Mad2 particles moved from kinetochores toward the poles. Second, spindle fiber and pole fluorescence disappeared upon Mad2 disappearance at the kinetochores. Third, ATP depletion resulted in microtubule-dependent depletion of Mad2 fluorescence at kinetochores and increased fluorescence at spindle poles. Finally, in normal cells, the half-life of Mad2 turnover at poles, 23 s, was similar to kinetochores. Thus, kinetochore-derived sites along spindle fibers and at spindle poles may also catalyze Mad2 inhibitory complex formation.
AW Murray. 2000. “Whither genomics?” Genome Biol, 1, 1, Pp. COMMENT003. Publisher's VersionAbstract
The flood of data from genome-wide analysis is transforming biology. We need to develop new, interdisciplinary approaches to convert these data into information about the components and structures of individual biological pathways and to use the resulting information to yield knowledge about general principles that explain the functions and evolution of life.
H Funabiki and AW Murray. 2000. “The Xenopus chromokinesin Xkid is essential for metaphase chromosome alignment and must be degraded to allow anaphase chromosome movement.” Cell, 102, 4, Pp. 411-24. Publisher's VersionAbstract
At anaphase, the linkage betweeh sister chromatids is dissolved and the separated sisters move toward opposite poles of the spindle. We developed a method to purify metaphase and anaphase chromosomes from frog egg extracts and identified proteins that leave chromosomes at anaphase using a new form of expression screening. This approach identified Xkid, a Xenopus homolog of human Kid (kinesin-like DNA binding protein) as a protein that is degraded in anaphase by ubiquitin-mediated proteolysis. Immunodepleting Xkid from egg extracts prevented normal chromosome alignment on the metaphase spindle. Adding a mild excess of wild-type or nondegradable Xkid to egg extracts prevented the separated chromosomes from moving toward the poles. We propose that Xkid provides the metaphase force that pushes chromosome arms toward the equator of the spindle and that its destruction is needed for anaphase chromosome movement.
S Biggins, FF Severin, N Bhalla, I Sassoon, AA Hyman, and AW Murray. 1999. “The conserved protein kinase Ipl1 regulates microtubule binding to kinetochores in budding yeast.” Genes Dev, 13, 5, Pp. 532-44. Publisher's VersionAbstract
Chromosome segregation depends on kinetochores, the structures that mediate chromosome attachment to the mitotic spindle. We isolated mutants in IPL1, which encodes a protein kinase, in a screen for budding yeast mutants that have defects in sister chromatid separation and segregation. Cytological tests show that ipl1 mutants can separate sister chromatids but are defective in chromosome segregation. Kinetochores assembled in extracts from ipl1 mutants show altered binding to microtubules. Ipl1p phosphorylates the kinetochore component Ndc10p in vitro and we propose that Ipl1p regulates kinetochore function via Ndc10p phosphorylation. Ipl1p localizes to the mitotic spindle and its levels are regulated during the cell cycle. This pattern of localization and regulation is similar to that of Ipl1p homologs in higher eukaryotes, such as the human aurora2 protein. Because aurora2 has been implicated in oncogenesis, defects in kinetochore function may contribute to genetic instability in human tumors.
LH Hartwell, JJ Hopfield, S Leibler, and AW Murray. 1999. “From molecular to modular cell biology.” Nature, 402, 6761 Suppl, Pp. C47-52. Publisher's VersionAbstract
Cellular functions, such as signal transmission, are carried out by 'modules' made up of many species of interacting molecules. Understanding how modules work has depended on combining phenomenological analysis with molecular studies. General principles that govern the structure and behaviour of modules may be discovered with help from synthetic sciences such as engineering and computer science, from stronger interactions between experiment and theory in cell biology, and from an appreciation of evolutionary constraints.
TC Norman, DL Smith, PK Sorger, BL Drees, SM O'Rourke, TR Hughes, CJ Roberts, SH Friend, S Fields, and AW Murray. 1999. “Genetic selection of peptide inhibitors of biological pathways.” Science, 285, 5427, Pp. 591-5. Publisher's VersionAbstract
Genetic selections were used to find peptides that inhibit biological pathways in budding yeast. The peptides were presented inside cells as peptamers, surface loops on a highly expressed and biologically inert carrier protein, a catalytically inactive derivative of staphylococcal nuclease. Peptamers that inhibited the pheromone signaling pathway, transcriptional silencing, and the spindle checkpoint were isolated. Putative targets for the inhibitors were identified by a combination of two-hybrid analysis and genetic dissection of the target pathways. This analysis identified Ydr517w as a component of the spindle checkpoint and reinforced earlier indications that Ste50 has both positive and negative roles in pheromone signaling. Analysis of transcript arrays showed that the peptamers were highly specific in their effects, which suggests that they may be useful reagents in organisms that lack sophisticated genetics as well as for identifying components of existing biological pathways that are potential targets for drug discovery.
KG Hardwick, R Li, C Mistrot, RH Chen, P Dann, A Rudner, and AW Murray. 1999. “Lesions in many different spindle components activate the spindle checkpoint in the budding yeast Saccharomyces cerevisiae.” Genetics, 152, 2, Pp. 509-18. Publisher's VersionAbstract
The spindle checkpoint arrests cells in mitosis in response to defects in the assembly of the mitotic spindle or errors in chromosome alignment. We determined which spindle defects the checkpoint can detect by examining the interaction of mutations that compromise the checkpoint (mad1, mad2, and mad3) with those that damage various structural components of the spindle. Defects in microtubule polymerization, spindle pole body duplication, microtubule motors, and kinetochore components all activate the MAD-dependent checkpoint. In contrast, the cell cycle arrest caused by mutations that induce DNA damage (cdc13), inactivate the cyclin proteolysis machinery (cdc16 and cdc23), or arrest cells in anaphase (cdc15) is independent of the spindle checkpoint.
JC Waters, RH Chen, AW Murray, GJ Gorbsky, ED Salmon, and RB Nicklas. 1999. “Mad2 binding by phosphorylated kinetochores links error detection and checkpoint action in mitosis.” Curr Biol, 9, 12, Pp. 649-52. Publisher's VersionAbstract
The spindle checkpoint must detect the presence of unattached or improperly attached kinetochores and must then inhibit progression through the cell cycle until the offending condition is resolved. Detection probably involves attachment-sensitive kinetochore phosphorylation (reviewed in [1,2]). A key player in the checkpoint's response is the Mad2 protein, which prevents activation of the anaphase-promoting complex (APC) by the Cdc20 protein [3-8]. Microinjection of Mad2 antibodies results in premature anaphase onset [9,10], and excess Mad2 protein causes arrest in mitosis [5,11]. We have previously shown that Mad2 localizes to unattached kinetochores in vertebrate cells, and that this localization ceases as kinetochores accumulate microtubules [10,12,13]. But how is Mad2 binding limited to unattached kinetochores? Here, we used lysed PtK1 cells to study kinetochore phosphorylation and Mad2 binding. We found that Mad2 binds to phosphorylated kinetochores, but not to unphosphorylated ones. Our data suggest that it is kinetochore protein phosphorylation that promotes Mad2 binding to unattached kinetochores. Thus, we have identified a probable molecular link between attachment-sensitive kinetochore phosphorylation and the inhibition of anaphase. The complete pathway for error control in mitosis can now be outlined.
S Biggins and AW Murray. 1999. “Sister chromatid cohesion in mitosis.” Curr Opin Genet Dev, 9, 2, Pp. 230-6. Publisher's VersionAbstract
Sister chromatid cohesion is essential for accurate chromosome segregation during the cell cycle. Newly identified structural proteins are required for sister chromatid cohesion and there may be a link in some organisms between the processes of cohesion and condensation. Proteins that induce and regulate the separation of sister chromatids have also been recently identified. (This review is an updated version of one that was published in Current Opinion in Cell Biology 1998, 10:769-775.)
RH Chen, DM Brady, D Smith, AW Murray, and KG Hardwick. 1999. “The spindle checkpoint of budding yeast depends on a tight complex between the Mad1 and Mad2 proteins.” Mol Biol Cell, 10, 8, Pp. 2607-18. Publisher's VersionAbstract
The spindle checkpoint arrests the cell cycle at metaphase in the presence of defects in the mitotic spindle or in the attachment of chromosomes to the spindle. When spindle assembly is disrupted, the budding yeast mad and bub mutants fail to arrest and rapidly lose viability. We have cloned the MAD2 gene, which encodes a protein of 196 amino acids that remains at a constant level during the cell cycle. Gel filtration and co-immunoprecipitation analyses reveal that Mad2p tightly associates with another spindle checkpoint component, Mad1p. This association is independent of cell cycle stage and the presence or absence of other known checkpoint proteins. In addition, Mad2p binds to all of the different phosphorylated isoforms of Mad1p that can be resolved on SDS-PAGE. Deletion and mutational analysis of both proteins indicate that association of Mad2p with Mad1p is critical for checkpoint function and for hyperphosphorylation of Mad1p.
A Desai, A Murray, T. J. Mitchison, and CE Walczak. 1999. “The use of Xenopus egg extracts to study mitotic spindle assembly and function in vitro.” Methods Cell Biol, 61, Pp. 385-412. Publisher's Version
LH Hwang, LF Lau, DL Smith, CA Mistrot, KG Hardwick, ES Hwang, A Amon, and AW Murray. 1998. “Budding yeast Cdc20: a target of the spindle checkpoint.” Science, 279, 5353, Pp. 1041-4. Publisher's VersionAbstract
The spindle checkpoint regulates the cell division cycle by keeping cells with defective spindles from leaving mitosis. In the two-hybrid system, three proteins that are components of the checkpoint, Mad1, Mad2, and Mad3, were shown to interact with Cdc20, a protein required for exit from mitosis. Mad2 and Mad3 coprecipitated with Cdc20 at all stages of the cell cycle. The binding of Mad2 depended on Mad1 and that of Mad3 on Mad1 and Mad2. Overexpression of Cdc20 allowed cells with a depolymerized spindle or damaged DNA to leave mitosis but did not overcome the arrest caused by unreplicated DNA. Mutants in Cdc20 that were resistant to the spindle checkpoint no longer bound Mad proteins, suggesting that Cdc20 is the target of the spindle checkpoint.
K Nabeshima, T Nakagawa, AF Straight, A Murray, Y Chikashige, YM Yamashita, Y Hiraoka, and M Yanagida. 1998. “Dynamics of centromeres during metaphase-anaphase transition in fission yeast: Dis1 is implicated in force balance in metaphase bipolar spindle.” Mol Biol Cell, 9, 11, Pp. 3211-25. Publisher's VersionAbstract
In higher eukaryotic cells, the spindle forms along with chromosome condensation in mitotic prophase. In metaphase, chromosomes are aligned on the spindle with sister kinetochores facing toward the opposite poles. In anaphase A, sister chromatids separate from each other without spindle extension, whereas spindle elongation takes place during anaphase B. We have critically examined whether such mitotic stages also occur in a lower eukaryote, Schizosaccharomyces pombe. Using the green fluorescent protein tagging technique, early mitotic to late anaphase events were observed in living fission yeast cells. S. pombe has three phases in spindle dynamics, spindle formation (phase 1), constant spindle length (phase 2), and spindle extension (phase 3). Sister centromere separation (anaphase A) rapidly occurred at the end of phase 2. The centromere showed dynamic movements throughout phase 2 as it moved back and forth and was transiently split in two before its separation, suggesting that the centromere was positioned in a bioriented manner toward the poles at metaphase. Microtubule-associating Dis1 was required for the occurrence of constant spindle length and centromere movement in phase 2. Normal transition from phase 2 to 3 needed DNA topoisomerase II and Cut1 but not Cut14. The duration of each phase was highly dependent on temperature.