AF Straight, AS Belmont, CC Robinett, and AW Murray. 1996. “GFP tagging of budding yeast chromosomes reveals that protein-protein interactions can mediate sister chromatid cohesion.” Curr Biol, 6, 12, Pp. 1599-608. Publisher's VersionAbstract
BACKGROUND: Precise control of sister chromatid separation is essential for the accurate transmission of genetic information. Sister chromatids must remain linked to each other from the time of DNA replication until the onset of chromosome segregation, when the linkage must be promptly dissolved. Recent studies suggest that the machinery that is responsible for the destruction of mitotic cyclins also degrades proteins that play a role in maintaining sister chromatid linkage, and that this machinery is regulated by the spindle-assembly checkpoint. Studies on these problems in budding yeast are hampered by the inability to resolve its chromosomes by light or electron microscopy. RESULTS: We have developed a novel method for visualizing specific DNA sequences in fixed and living budding yeast cells. A tandem array of 256 copies of the Lac operator is integrated at the desired site in the genome and detected by the binding of a green fluorescent protein (GFP)-Lac repressor fusion expressed from the HIS3 promoter. Using this method, we show that sister chromatid segregation precedes the destruction of cyclin B. In mad or bub cells, which lack the spindle-assembly checkpoint, sister chromatid separation can occur in the absence of microtubules. The expression of a tetramerizing form of the GFP-Lac repressor, which can bind Lac operators on two different DNA molecules, can hold sister chromatids together under conditions in which they would normally separate. CONCLUSIONS: We conclude that sister chromatid separation in budding yeast can occur in the absence of microtubule-dependent forces, and that protein complexes that can bind two different DNA molecules are capable of holding sister chromatids together.
CC Robinett, A Straight, G. Li, C Willhelm, G Sudlow, A Murray, and AS Belmont. 1996. “In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition.” J Cell Biol, 135, 6 Pt 2, Pp. 1685-700. Publisher's VersionAbstract
We report a new method for in situ localization of DNA sequences that allows excellent preservation of nuclear and chromosomal ultrastructure and direct, in vivo observations. 256 direct repeats of the lac operator were added to vector constructs used for transfection and served as a tag for labeling by lac repressor. This system was first characterized by visualization of chromosome homogeneously staining regions (HSRs) produced by gene amplification using a dihydrofolate reductase (DHFR) expression vector with methotrexate selection. Using electron microscopy, most HSRs showed approximately 100-nm fibers, as described previously for the bulk, large-scale chromatin organization in these cells, and by light microscopy, distinct, large-scale chromatin fibers could be traced in vivo up to 5 microns in length. Subsequent experiments demonstrated the potential for more general applications of this labeling technology. Single and multiple copies of the integrated vector could be detected in living CHO cells before gene amplification, and detection of a single 256 lac operator repeat and its stability during mitosis was demonstrated by its targeted insertion into budding yeast cells by homologous recombination. In both CHO cells and yeast, use of the green fluorescent protein-lac repressor protein allowed extended, in vivo observations of the operator-tagged chromosomal DNA. Future applications of this technology should facilitate structural, functional, and genetic analysis of chromatin organization, chromosome dynamics, and nuclear architecture.
J Minshull, A Straight, AD Rudner, AF Dernburg, A Belmont, and AW Murray. 1996. “Protein phosphatase 2A regulates MPF activity and sister chromatid cohesion in budding yeast.” Curr Biol, 6, 12, Pp. 1609-20. Publisher's VersionAbstract
BACKGROUND: Mitosis is regulated by MPF (maturation promoting factor), the active form of Cdc2/28-cyclin B complexes. Increasing levels of cyclin B abundance and the loss of inhibitory phosphates from Cdc2/28 drives cells into mitosis, whereas cyclin B destruction inactivates MPF and drives cells out of mitosis. Cells with defective spindles are arrested in mitosis by the spindle-assembly checkpoint, which prevents the destruction of mitotic cyclins and the inactivation of MPF. We have investigated the relationship between the spindle-assembly checkpoint, cyclin destruction, inhibitory phosphorylation of Cdc2/28, and exit from mitosis. RESULTS: The previously characterized budding yeast mad mutants lack the spindle-assembly checkpoint. Spindle depolymerization does not arrest them in mitosis because they cannot stabilize cyclin B. In contrast, a newly isolated mutant in the budding yeast CDC55 gene, which encodes a protein phosphatase 2A (PP2A) regulatory subunit, shows a different checkpoint defect. In the presence of a defective spindle, these cells separate their sister chromatids and leave mitosis without inducing cyclin B destruction. Despite the persistence of B-type cyclins, cdc55 mutant cells inactivate MPF. Two experiments show that this inactivation is due to inhibitory phosphorylation on Cdc28: phosphotyrosine accumulates on Cdc28 in cdc55 delta cells whose spindles have been depolymerized, and a cdc28 mutant that lacks inhibitory phosphorylation sites on Cdc28 allows spindle defects to arrest cdc55 mutants in mitosis with active MPF and unseparated sister chromatids. CONCLUSIONS: We conclude that perturbations of protein phosphatase activity allow MPF to be inactivated by inhibitory phosphorylation instead of by cyclin destruction. Under these conditions, sister chromatid separation appears to be regulated by MPF activity rather than by protein degradation. We discuss the role of PP2A and Cdc28 phosphorylation in cell-cycle control, and the possibility that the novel mitotic exit pathway plays a role in adaptation to prolonged activation of the spindle-assembly checkpoint.
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