AW Murray, AB Desai, and ED Salmon. 1996. “Real time observation of anaphase in vitro.” Proc Natl Acad Sci U S A, 93, 22, Pp. 12327-32. Publisher's VersionAbstract
We used digital fluorescence microscopy to make real-time observations of anaphase chromosome movement and changes in microtubule organization in spindles assembled in Xenopus egg extracts. Anaphase chromosome movement in these extracts resembled that seen in living vertebrate cells. During anaphase chromosomes moved toward the spindle poles (anaphase A) and the majority reached positions very close to the spindle poles. The average rate of chromosome to pole movement (2.4 microns/min) was similar to earlier measurements of poleward microtubule flux during metaphase. An increase in pole-to-pole distance (anaphase B) occurred in some spindles. The polyploidy of the spindles we examined allowed us to observe two novel features of mitosis. First, during anaphase, multiple microtubule organizing centers migrated 40 microns or more away from the spindle poles. Second, in telophase, decondensing chromosomes often moved rapidly (7-23 microns/min) away from the spindle poles toward the centers of these asters. This telophase chromosome movement suggests that the surface of decondensing chromosomes, and by extension those of intact nuclei, bear minus-end-directed microtubule motors. Preventing the inactivation of Cdc2/cyclin B complexes by adding nondegradable cyclin B allowed anaphase A to occur at normal velocities, but reduced the ejection of asters from the spindles, blocked chromosome decondensation, and inhibited telophase chromosome movement. In the presence of nondegradable cyclin B, chromosome movement to the poles converted bipolar spindles into pairs of independent monopolar spindles, demonstrating the role of sister chromatid linkage in maintaining spindle bipolarity.
AD Rudner and AW Murray. 1996. “The spindle assembly checkpoint.” Curr Opin Cell Biol, 8, 6, Pp. 773-80. Publisher's VersionAbstract
The spindle assembly checkpoint monitors proper chromosome attachment to spindle microtubules and is conserved from yeast to humans. Checkpoint components reside on kinetochores of chromosomes and show changes in phosphorylation and localization as cells proceed through mitosis. Adaptation to prolonged checkpoint arrest can occur by inhibitory phosphorylation of Cdc2.
AW Murray. 1995. “Cell cycle. Tense spindles can relax.” Nature, 373, 6515, Pp. 560-1. Publisher's Version
A Murray. 1995. “Cyclin ubiquitination: the destructive end of mitosis.” Cell, 81, 2, Pp. 149-52. Publisher's Version
AW Murray. 1995. “The genetics of cell cycle checkpoints.” Curr Opin Genet Dev, 5, 1, Pp. 5-11. Publisher's VersionAbstract
Checkpoints help in the prevention of genetic damage by giving cells time to repair damaged structures before proceeding in the cell cycle. Genetic analyses in budding and fission yeast have identified a large number of cell cycle checkpoint genes. Several of these encode proteins related to components of other signal transduction pathways, including protein kinases, lipid kinases, and 14-3-3 proteins. In fission yeast, checkpoints play an important role in keeping cells from entering mitosis before they pass Start.
KG Hardwick and AW Murray. 1995. “Mad1p, a phosphoprotein component of the spindle assembly checkpoint in budding yeast.” J Cell Biol, 131, 3, Pp. 709-20. Publisher's VersionAbstract
The spindle assembly checkpoint prevents cells from initiating anaphase until the spindle has been fully assembled. We previously isolated mitotic arrest deficient (mad) mutants that inactivate this checkpoint and thus increase the sensitivity of cells to benomyl, a drug that interferes with mitotic spindle assembly by depolymerizing microtubules. We have cloned the MAD1 gene and show that when it is disrupted yeast cells have the same phenotype as the previously isolated mad1 mutants: they fail to delay the metaphase to anaphase transition in response to microtubule depolymerization. MAD1 is predicted to encode a 90-kD coiled-coil protein. Anti-Mad1p antibodies give a novel punctate nuclear staining pattern and cell fractionation reveals that the bulk of Mad1p is soluble. Mad1p becomes hyperphosphorylated when wild-type cells are arrested in mitosis by benomyl treatment, or by placing a cold sensitive tubulin mutant at the restrictive temperature. This modification does not occur in G1-arrested cells treated with benomyl or in cells arrested in mitosis by defects in the mitotic cyclin proteolysis machinery, suggesting that Mad1p hyperphosphorylation is a step in the activation of the spindle assembly checkpoint. Analysis of Mad1p phosphorylation in other spindle assembly checkpoint mutants reveals that this response to microtubule-disrupting agents is defective in some (mad2, bub1, and bub3) but not all (mad3, bub2) mutant strains. We discuss the possible functions of Mad1p at this cell cycle checkpoint.
DR Kellogg, A Kikuchi, T Fujii-Nakata, C. W. Turck, and AW Murray. 1995. “Members of the NAP/SET family of proteins interact specifically with B-type cyclins.” J Cell Biol, 130, 3, Pp. 661-73. Publisher's VersionAbstract
Cyclin-dependent kinase complexes that contain the same catalytic subunit are able to induce different events at different times during the cell cycle, but the mechanisms by which they do so remain largely unknown. To address this problem, we have used affinity chromatography to identify proteins that bind specifically to mitotic cyclins, with the goal of finding proteins that interact with mitotic cyclins to carry out the events of mitosis. This approach has led to the identification of a 60-kD protein called NAP1 that interacts specifically with members of the cyclin B family. This interaction has been highly conserved during evolution: NAP1 in the Xenopus embryo interacts with cyclins B1 and B2, but not with cyclin A, and the S. cerevisiae homolog of NAP1 interacts with Clb2 but not with Clb3. Genetic experiments in budding yeast indicate that NAP1 plays an important role in the function of Clb2, while biochemical experiments demonstrate that purified NAP1 can be phosphorylated by cyclin B/p34cdc2 kinase complexes, but not by cyclin A/p34cdc2 kinase complexes. These results suggest that NAP1 is a protein involved in the specific functions of cyclin B/p34cdc2 kinase complexes. In addition to NAP1, we found a 43-kD protein in Xenopus that is homologous to NAP1 and also interacts specifically with B-type cyclins. This protein is the Xenopus homolog of the human SET protein, which was previously identified as part of a putative oncogenic fusion protein (Von Lindern et al., 1992).
DR Kellogg and AW Murray. 1995. “NAP1 acts with Clb1 to perform mitotic functions and to suppress polar bud growth in budding yeast.” J Cell Biol, 130, 3, Pp. 675-85. Publisher's VersionAbstract
NAP1 is a 60-kD protein that interacts specifically with mitotic cyclins in budding yeast and frogs. We have examined the ability of the yeast mitotic cyclin Clb2 to function in cells that lack NAP1. Our results demonstrate that Clb2 is unable to carry out its full range of functions without NAP1, even though Clb2/p34CDC28-associated kinase activity rises to normal levels. In the absence of NAP1, Clb2 is unable to efficiently induce mitotic events, and cells undergo a prolonged delay at the short spindle stage with normal levels of Clb2/p34CDC28 kinase activity. NAP1 is also required for the ability of Clb2 to induce the switch from polar to isotropic bud growth. As a result, polar bud growth continues during mitosis, giving rise to highly elongated cells. Our experiments also suggest that NAP1 is required for the ability of the Clb2/p34CDC28 kinase complex to amplify its own production, and that NAP1 plays a role in regulation of microtubule dynamics during mitosis. Together, these results demonstrate that NAP1 is required for the normal function of the activated Clb2/p34CDC28 kinase complex, and provide a step towards understanding how cyclin-dependent kinase complexes induce specific events during the cell cycle.
A Murray. 1994. “Cell cycle checkpoints.” Curr Opin Cell Biol, 6, 6, Pp. 872-6. Publisher's VersionAbstract
Checkpoints help ensure that cell cycle events occur in the correct order. Studies on mammalian cells identified inhibitors of complexes of cyclins and cyclin-dependent kinases as components of cell cycle checkpoints and provide the first glimpse of the molecular pathways that prevent cells with damaged DNA from replicating their DNA. In embryos, the extent to which checkpoints arrest the cell cycle reflects the relative strength of inhibitory checkpoints and the machinery driving the cell cycle forward.
AW Murray. 1994. “Cell cycle. Rum tale of replication.” Nature, 367, 6460, Pp. 219-20. Publisher's Version
AW Murray. 1994. “Cyclin-dependent kinases: regulators of the cell cycle and more.” Chem Biol, 1, 4, Pp. 191-5. Publisher's VersionAbstract
Cyclin-dependent kinases determine the timing of key events in the cell cycle, and may also regulate other important cellular functions. Although some of the effects of activating these kinases are clear, the mechanisms by which the effects are produced are not; several types of chemical probes that might be enlightening can be imagined.
J Minshull, H Sun, N.K Tonks, and AW Murray. 1994. “A MAP kinase-dependent spindle assembly checkpoint in Xenopus egg extracts.” Cell, 79, 3, Pp. 475-86. Publisher's VersionAbstract
Like early Xenopus embryos, extracts made from Xenopus eggs lack the cell cycle checkpoint that keeps anaphase from occurring before spindle assembly is complete. At very high densities of sperm nuclei, however, microtubule depolymerization arrests the extracts in mitosis. The arrested extracts have high levels of maturation-promoting factor activity, fail to degrade cyclin B, and contain activated ERK2/mitogen-activated protein (MAP) kinase. The addition of the purified MAP kinase-specific phosphatase MKP-1 demonstrates that MAP kinase activity is required for both the establishment and maintenance of the mitotic arrest induced by spindle depolymerization. Increased calcium concentrations, which release unfertilized frog eggs from their natural arrest in metaphase of meiosis II, have no effect on the mitotic arrest.
AW Murray and T. J. Mitchison. 1994. “Mitosis. Kinetochores pass the IQ test.” Curr Biol, 4, 1, Pp. 38-41. Publisher's VersionAbstract
Kinetochores oscillate to and fro on the mitotic spindle. The oscillations seem to be biased by the forces acting on the kinetochore, explaining the variety of chromosome movements seen at different stages of mitosis.
A. Murray and T. Hunt. 1993. The cell cycle: an introduction. New York: Oxford University Press. Publisher's VersionAbstract
In the last decade there has been a revolution in our comprehension of how cells grow and divide. Results from experiments on yeast, embryos, and cultured mammalian cells have unified seemingly disparate viewpoints into a single set of principles for normal cellular reproduction in plants, animals and bacteria. Written by two leading participants in that revolution, The Cell Cycle provides the first thorough, authoritative account of the new philosophy of normal cellular reproduction and how it emerged. It is a vivid portrayal of the molecular logic of the cell: how the cell engine induces DNA replication and chromosome replication; how the integrity of genetic information is preserved; and how cell size and environmental signals regulate the cycle of growth and division. By describing important breakthroughs in their historical and experimental context, The Cell Cycle traces the development of the new vision of cell biology and shows its relevance to other areas of modern biology. It is the ideal introduction to the current understanding of cell growth and division for advanced undergraduate and graduate level cell biology courses.
SL Holloway, M Glotzer, R. W. King, and AW Murray. 1993. “Anaphase is initiated by proteolysis rather than by the inactivation of maturation-promoting factor.” Cell, 73, 7, Pp. 1393-402. Publisher's VersionAbstract
We have used frog egg extracts that assemble mitotic spindles to identify the event that triggers sister chromatid separation. Adding a nondegradable form of cyclin B prevents maturation-promoting factor (MPF) inactivation but does not block sister chromatid separation, showing that MPF inactivation is not needed to initiate anaphase. In contrast, adding an N-terminal fragment of cyclin, which acts as a specific competitor for cyclin degradation, produces a dose-dependent delay in MPF inactivation and sister chromatid separation. Methylated ubiquitin, which inhibits ubiquitin-mediated proteolysis, also delays sister chromatid separation, suggesting that ubiquitin-mediated proteolysis is necessary to initiate anaphase. The N-terminal cyclin fragment inhibits chromosome separation even in extracts that contain only nondegradable forms of cyclin, suggesting that proteins other than the known cyclins must be degraded to dissolve the linkage between sister chromatids.
AW Murray. 1993. “Cell cycle. Sunburnt fission yeast.” Nature, 363, 6427, Pp. 302. Publisher's Version
AW Murray. 1993. “Cell-cycle control: turning on mitosis.” Curr Biol, 3, 5, Pp. 291-3. Publisher's Version
R Li, C Havel, JA Watson, and AW Murray. 1993. “The mitotic feedback control gene MAD2 encodes the alpha-subunit of a prenyltransferase.” Nature, 366, 6450, Pp. 82-4. Publisher's VersionAbstract
The mad2-1 mutation inactivates the cell-cycle feedback control that prevents budding yeast cells from leaving mitosis until spindle assembly is complete. The gene product of MAD2 shows significant sequence similarity to the alpha-subunit of prenyltransferases. Here we isolate a new temperature-sensitive mad2 mutant, mad2-2ts, and find that Mad2p is required for the membrane association of Ypt1p and Sec4p, two prenylated small GTP-binding proteins involved in protein trafficking. Extracts from mad2-2ts mutant cells fail to geranylgeranylate a number of substrates at the non-permissive temperature. mad2-2ts is synthetically lethal with bet2-1, a mutation in the gene that encodes for the beta-subunit of the Ypt1p and Sec4p geranylgeranyl transferase. Therefore MAD2 and BET2 gene products may physically interact to form a geranylgeranyl transferase complex. In addition, the difference between the phenotypes of mad2-1 and mad2-2ts suggests that MAD2 has distinct roles in protein transport and the mitotic feedback control.
AW Murray. 1992. “Creative blocks: cell-cycle checkpoints and feedback controls.” Nature, 359, 6396, Pp. 599-604. Publisher's VersionAbstract
Before division, cells must ensure that they finish DNA replication, DNA repair and chromosome segregation. They do so by using feedback controls which can detect the failure to complete replication, repair or spindle assembly to arrest the progress of the cell cycle at one of three checkpoints. Failures in feedback controls can contribute to the generation of cancer.
MS Boguski, AW Murray, and S Powers. 1992. “Novel repetitive sequence motifs in the alpha and beta subunits of prenyl-protein transferases and homology of the alpha subunit to the MAD2 gene product of yeast.” New Biol, 4, 4, Pp. 408-11. Publisher's Version