GJ Gorbsky, RH Chen, and AW Murray. 1998. “Microinjection of antibody to Mad2 protein into mammalian cells in mitosis induces premature anaphase.” J Cell Biol, 141, 5, Pp. 1193-205. Publisher's VersionAbstract
In yeast, the Mad2 protein is required for the M phase arrest induced by microtubule inhibitors, but the protein is not essential under normal culture conditions. We tested whether the Mad2 protein participates in regulating the timing of anaphase onset in mammalian cells in the absence of microtubule drugs. When microinjected into living prophase or prometaphase PtK1 cells, anti-Mad2 antibody induced the onset of anaphase prematurely during prometaphase, before the chromosomes had assembled at the metaphase plate. Anti-Mad2 antibody-injected cells completed all aspects of anaphase including chromatid movement to the spindle poles and pole-pole separation. Identical results were obtained when primary human keratinocytes were injected with anti-Mad2 antibody. These studies suggest that Mad2 protein function is essential for the timing of anaphase onset in somatic cells at each mitosis. Thus, in mammalian somatic cells, the spindle checkpoint appears to be a component of the timing mechanism for normal mitosis, blocking anaphase onset until all chromosomes are aligned at the metaphase plate.
JF Charles, SL Jaspersen, RL Tinker-Kulberg, L.H. Wang, A Szidon, and DO Morgan. 1998. “The Polo-related kinase Cdc5 activates and is destroyed by the mitotic cyclin destruction machinery in S. cerevisiae.” Curr Biol, 8, 9, Pp. 497-507. Publisher's VersionAbstract
BACKGROUND: Following chromosome segregation in anaphase, ubiquitin-dependent degradation of mitotic cyclins contributes to the exit from mitosis. A key step in this process is catalyzed by a ubiquitin-protein ligase known as the anaphase-promoting complex (APC), the regulation of which is poorly understood. The Polo-related protein kinase Cdc5 in Saccharomyces cerevisiae might encode a regulator of the APC, because cdc5 mutant cells arrest with a late mitotic phenotype similar to that observed in cells with defective cyclin destruction. RESULTS: We investigated the role of Cdc5 in the regulation of mitotic cyclin degradation. In cdc5-1 mutant cells, we observed a defect in the destruction of cyclins and a reduction in the cyclin-ubiquitin ligase activity of the APC. Overexpression of CDC5 resulted in increased APC activity and mitotic cyclin destruction in asynchronous cells or in cells arrested in metaphase. CDC5 mutation or overexpression did not affect the degradation of the APC substrate Pds 1, which is normally degraded at the metaphase-to-anaphase transition. Cyclin-specific APC activity in cells overexpressing CDC5 was reduced in the absence of the APC regulatory proteins Hct 1 and Cdc20. In G1, Cdc5 itself was degraded by an APC-dependent and Hct1-dependent mechanism. CONCLUSIONS: We conclude that Cdc5 is a positive regulator of cyclin-specific APC activity in late mitosis. Degradation of Cdc5 in G1 might provide a feedback mechanism by which the APC destroys its activator at the onset of the next cell cycle.
S Biggins and AW Murray. 1998. “Sister chromatid cohesion in mitosis.” Curr Opin Cell Biol, 10, 6, Pp. 769-75. 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.
RH Chen, A Shevchenko, M Mann, and AW Murray. 1998. “Spindle checkpoint protein Xmad1 recruits Xmad2 to unattached kinetochores.” J Cell Biol, 143, 2, Pp. 283-95. Publisher's VersionAbstract
The spindle checkpoint prevents the metaphase to anaphase transition in cells containing defects in the mitotic spindle or in chromosome attachment to the spindle. When the checkpoint protein Xmad2 is depleted from Xenopus egg extracts, adding Xmad2 to its endogenous concentration fails to restore the checkpoint, suggesting that other checkpoint component(s) were depleted from the extract through their association with Xmad2. Mass spectrometry provided peptide sequences from an 85-kD protein that coimmunoprecipitates with Xmad2 from egg extracts. This information was used to clone XMAD1, which encodes a homologue of the budding yeast (Saccharomyces cerevisiae) checkpoint protein Mad1. Xmad1 is essential for establishing and maintaining the spindle checkpoint in egg extracts. Like Xmad2, Xmad1 localizes to the nuclear envelope and the nucleus during interphase, and to those kinetochores that are not bound to spindle microtubules during mitosis. Adding an anti-Xmad1 antibody to egg extracts inactivates the checkpoint and prevents Xmad2 from localizing to unbound kinetochores. In the presence of excess Xmad2, neither chromosomes nor Xmad1 are required to activate the spindle checkpoint, suggesting that the physiological role of Xmad1 is to recruit Xmad2 to kinetochores that have not bound microtubules.
AF Straight, JW Sedat, and AW Murray. 1998. “Time-lapse microscopy reveals unique roles for kinesins during anaphase in budding yeast.” J Cell Biol, 143, 3, Pp. 687-94. Publisher's VersionAbstract
The mitotic spindle is a complex and dynamic structure. Genetic analysis in budding yeast has identified two sets of kinesin-like motors, Cin8p and Kip1p, and Kar3p and Kip3p, that have overlapping functions in mitosis. We have studied the role of three of these motors by video microscopy of motor mutants whose microtubules and centromeres were marked with green fluorescent protein. Despite their functional overlap, each motor mutant has a specific defect in mitosis: cin8Delta mutants lack the rapid phase of anaphase B, kip1Delta mutants show defects in the slow phase of anaphase B, and kip3Delta mutants prolong the duration of anaphase to the point at which the spindle becomes longer than the cell. The kip3Delta and kip1Delta mutants affect the duration of anaphase, but cin8Delta does not.
CD Webb, A Teleman, S. Gordon, A Straight, A Belmont, DC Lin, AD Grossman, A Wright, and R. Losick. 1997. “Bipolar localization of the replication origin regions of chromosomes in vegetative and sporulating cells of B. subtilis.” Cell, 88, 5, Pp. 667-74. Publisher's VersionAbstract
To investigate chromosome segregation in B. subtilis, we introduced tandem copies of the lactose operon operator into the chromosome near the replication origin or terminus. We then visualized the position of the operator cassettes with green fluorescent protein fused to the Lac1 repressor. In sporulating bacteria, which undergo asymmetric cell division, origins localized near each pole of the cell whereas termini were restricted to the middle. In growing cells, which undergo binary fission, origins were observed at various positions but preferentially toward the poles early in the cell cycle. In contrast, termini showed little preference for the poles. These results indicate the existence of a mitotic-like apparatus that is responsible for moving the origin regions of newly formed chromosomes toward opposite ends of the cell.
RH Chen and A Murray. 1997. “Characterization of spindle assembly checkpoint in Xenopus egg extracts.” Methods Enzymol, 283, Pp. 572-84. Publisher's Version
GS Gordon, D Sitnikov, CD Webb, A Teleman, A Straight, R. Losick, AW Murray, and A Wright. 1997. “Chromosome and low copy plasmid segregation in E. coli: visual evidence for distinct mechanisms.” Cell, 90, 6, Pp. 1113-21. Publisher's VersionAbstract
We have investigated DNA segregation in E. coli by inserting multiple lac operator sequences into the chromosome near the origin of replication (oriC), in the hisC gene, a terminus marker, and into plasmids P1 and F. Expression of a GFP-LacI fusion protein allowed visualization of lac operator localization. oriC was shown to be specifically localized at or near the cell poles, and when duplicated, one copy moved to the site of new pole formation near the site of cell division. In contrast, P1 and F localized to the cell center and on duplication appeared to move rapidly to the quarter positions in the cell. Our analysis suggests that different active processes are involved in movement and localization of the chromosome and of the two plasmids during segregation.
LH Hartwell, P Szankasi, CJ Roberts, AW Murray, and SH Friend. 1997. “Integrating genetic approaches into the discovery of anticancer drugs.” Science, 278, 5340, Pp. 1064-8. Publisher's VersionAbstract
The discovery of anticancer drugs is now driven by the numerous molecular alterations identified in tumor cells over the past decade. To exploit these alterations, it is necessary to understand how they define a molecular context that allows increased sensitivity to particular compounds. Traditional genetic approaches together with the new wealth of genomic information for both human and model organisms open up strategies by which drugs can be profiled for their ability to selectively kill cells in a molecular context that matches those found in tumors. Similarly, it may be possible to identify and validate new targets for drugs that would selectively kill tumor cells with a particular molecular context. This article outlines some of the ways that yeast genetics can be used to streamline anticancer drug discovery.
WF Marshall, A Straight, JF Marko, J Swedlow, A Dernburg, A Belmont, AW Murray, DA Agard, and JW Sedat. 1997. “Interphase chromosomes undergo constrained diffusional motion in living cells.” Curr Biol, 7, 12, Pp. 930-9. Publisher's VersionAbstract
BACKGROUND: Structural studies of fixed cells have revealed that interphase chromosomes are highly organized into specific arrangements in the nucleus, and have led to a picture of the nucleus as a static structure with immobile chromosomes held in fixed positions, an impression apparently confirmed by recent photobleaching studies. Functional studies of chromosome behavior, however, suggest that many essential processes, such as recombination, require interphase chromosomes to move around within the nucleus. RESULTS: To reconcile these contradictory views, we exploited methods for tagging specific chromosome sites in living cells of Saccharomyces cerevisiae with green fluorescent protein and in Drosophila melanogaster with fluorescently labeled topoisomerase ll. Combining these techniques with submicrometer single-particle tracking, we directly measured the motion of interphase chromatin, at high resolution and in three dimensions. We found that chromatin does indeed undergo significant diffusive motion within the nucleus, but this motion is constrained such that a given chromatin segment is free to move within only a limited subregion of the nucleus. Chromatin diffusion was found to be insensitive to metabolic inhibitors, suggesting that it results from classical Brownian motion rather than from active motility. Nocodazole greatly reduced chromatin confinement, suggesting a role for the cytoskeleton in the maintenance of nuclear architecture. CONCLUSIONS: We conclude that chromatin is free to undergo substantial Brownian motion, but that a given chromatin segment is confined to a subregion of the nucleus. This constrained diffusion is consistent with a highly defined nuclear architecture, but also allows enough motion for processes requiring chromosome motility to take place. These results lead to a model for the regulation of chromosome interactions by nuclear architecture.
J Nunnari, WF Marshall, A Straight, A Murray, JW Sedat, and P Walter. 1997. “Mitochondrial transmission during mating in Saccharomyces cerevisiae is determined by mitochondrial fusion and fission and the intramitochondrial segregation of mitochondrial DNA.” Mol Biol Cell, 8, 7, Pp. 1233-42. Publisher's VersionAbstract
To gain insight into the process of mitochondrial transmission in yeast, we directly labeled mitochondrial proteins and mitochondrial DNA (mtDNA) and observed their fate after the fusion of two cells. To this end, mitochondrial proteins in haploid cells of opposite mating type were labeled with different fluorescent dyes and observed by fluorescence microscopy after mating of the cells. Parental mitochondrial protein markers rapidly redistributed and colocalized throughout zygotes, indicating that during mating, parental mitochondria fuse and their protein contents intermix, consistent with results previously obtained with a single parentally derived protein marker. Analysis of the three-dimensional structure and dynamics of mitochondria in living cells with wide-field fluorescence microscopy indicated that mitochondria form a single dynamic network, whose continuity is maintained by a balanced frequency of fission and fusion events. Thus, the complete mixing of mitochondrial proteins can be explained by the formation of one continuous mitochondrial compartment after mating. In marked contrast to the mixing of parental mitochondrial proteins after fusion, mtDNA (labeled with the thymidine analogue 5-bromodeoxyuridine) remained distinctly localized to one half of the zygotic cell. This observation provides a direct explanation for the genetically observed nonrandom patterns of mtDNA transmission. We propose that anchoring of mtDNA within the organelle is linked to an active segregation mechanism that ensures accurate inheritance of mtDNA along with the organelle.
AF Straight, WF Marshall, JW Sedat, and AW Murray. 1997. “Mitosis in living budding yeast: anaphase A but no metaphase plate.” Science, 277, 5325, Pp. 574-8. Publisher's VersionAbstract
Chromosome movements and spindle dynamics were visualized in living cells of the budding yeast Saccharomyces cerevisiae. Individual chromosomal loci were detected by expression of a protein fusion between green fluorescent protein (GFP) and the Lac repressor, which bound to an array of Lac operator binding sites integrated into the chromosome. Spindle microtubules were detected by expression of a protein fusion between GFP and Tub1, the major alpha tubulin. Spindle elongation and chromosome separation exhibited biphasic kinetics, and centromeres separated before telomeres. Budding yeast did not exhibit a conventional metaphase chromosome alignment but did show anaphase A, movement of the chromosomes to the poles.
LH Hwang and AW Murray. 1997. “A novel yeast screen for mitotic arrest mutants identifies DOC1, a new gene involved in cyclin proteolysis.” Mol Biol Cell, 8, 10, Pp. 1877-87. Publisher's VersionAbstract
B-type cyclins are rapidly degraded at the transition between metaphase and anaphase and their ubiquitin-mediated proteolysis is required for cells to exit mitosis. We used a novel enrichment to isolate new budding mutants that arrest the cell cycle in mitosis. Most of these mutants lie in the CDC16, CDC23, and CDC27 genes, which have already been shown to play a role in cyclin proteolysis and encode components of a 20S complex (called the cyclosome or anaphase promoting complex) that ubiquitinates mitotic cyclins. We show that mutations in CDC26 and a novel gene, DOC1, also prevent mitotic cyclin proteolysis. Mutants in either gene arrest as large budded cells with high levels of the major mitotic cyclin (Clb2) protein at 37 degrees C and cannot degrade Clb2 in G1-arrested cells. Cdc26 associates in vivo with Doc1, Cdc16, Cdc23, and Cdc27. In addition, the majority of Doc1 cosediments at 20S with Cdc27 in a sucrose gradient, indicating that Cdc26 and Doc1 are components of the anaphase promoting complex.
AF Straight and AW Murray. 1997. “The spindle assembly checkpoint in budding yeast.” Methods Enzymol, 283, Pp. 425-40. Publisher's Version
WA Wells and AW Murray. 1996. “Aberrantly segregating centromeres activate the spindle assembly checkpoint in budding yeast.” J Cell Biol, 133, 1, Pp. 75-84. Publisher's VersionAbstract
The spindle assembly checkpoint is the mechanism or set of mechanisms that prevents cells with defects in chromosome alignment or spindle assembly from passing through mitosis. We have investigated the effects of mini-chromosomes on this checkpoint in budding yeast by performing pedigree analysis. This method allowed us to observe the frequency and duration of cell cycle delays in individual cells. Short, centromeric linear mini-chromosomes, which have a low fidelity of segregation, cause frequent delays in mitosis. Their circular counterparts and longer linear mini-chromosomes, which segregate more efficiently, show a much lower frequency of mitotic delays, but these delays occur much more frequently in divisions where the mini-chromosome segregates to only one of the two daughter cells. Using a conditional centromere to increase the copy number of a circular mini-chromosome greatly increases the frequency of delayed divisions. In all cases the division delays are completely abolished by the mad mutants that inactivate the spindle assembly checkpoint, demonstrating that the Mad gene products are required to detect the subtle defects in chromosome behavior that have been observed to arrest higher eukaryotic cells in mitosis.
KG Hardwick, E Weiss, FC Luca, M Winey, and AW Murray. 1996. “Activation of the budding yeast spindle assembly checkpoint without mitotic spindle disruption.” Science, 273, 5277, Pp. 953-6. Publisher's VersionAbstract
The spindle assembly checkpoint keeps cells with defective spindles from initiating chromosome segregation. The protein kinase Mps1 phosphorylates the yeast protein Mad1p when this checkpoint is activated, and the overexpression of Mps1p induces modification of Mad1p and arrests wild-type yeast cells in mitosis with morphologically normal spindles. Spindle assembly checkpoint mutants overexpressing Mps1p pass through mitosis without delay and can produce viable progeny, which demonstrates that the arrest of wild-type cells results from inappropriate activation of the checkpoint in cells whose spindle is fully functional. Ectopic activation of cell-cycle checkpoints might be used to exploit the differences in checkpoint status between normal and tumor cells and thus improve the selectivity of chemotherapy.
RH Chen, JC Waters, ED Salmon, and AW Murray. 1996. “Association of spindle assembly checkpoint component XMAD2 with unattached kinetochores.” Science, 274, 5285, Pp. 242-6. Publisher's VersionAbstract
The spindle assembly checkpoint delays anaphase until all chromosomes are attached to a mitotic spindle. The mad (mitotic arrest-deficient) and bub (budding uninhibited by benzimidazole) mutants of budding yeast lack this checkpoint and fail to arrest the cell cycle when microtubules are depolymerized. A frog homolog of MAD2 (XMAD2) was isolated and found to play an essential role in the spindle assembly checkpoint in frog egg extracts. XMAD2 protein associated with unattached kinetochores in prometaphase and in nocodazole-treated cells and disappeared from kinetochores at metaphase in untreated cells, suggesting that XMAD2 plays a role in the activation of the checkpoint by unattached kinetochores. This study furthers understanding of the mechanism of cell cycle checkpoints in metazoa and provides a marker for studying the role of the spindle assembly checkpoint in the genetic instability of tumors.
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.