A Hec of a microtubule attachment.
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Animals↗
Biomedical subjects
Daniel J Burke
Publications and source records attributed to Daniel J Burke.
Pmr1, a Golgi Ca2+/Mn2+-ATPase, is a regulator of the target of rapamycin (TOR) signaling pathway in yeast.
The rapamycin.FKBP12 complex inhibits target of rapamycin (TOR) kinase in TORC1. We screened the yeast nonessential gene deletion collection to identify mutants that conferred rapamycin resistance, and we identified PMR1, encoding the Golgi Ca2+/Mn2+ -ATPase. Deleting PMR1 in two genetic backgrounds confers rapamycin resistance. Epistasis analyses show that Pmr1 functions upstream from Npr1 and Gln-3 in opposition to Lst8, a regulator of TOR. Npr1 kinase is largely cytoplasmic, and a portion localizes to the Golgi where amino acid permeases are modified and sorted. Nuclear translocation of Gln-3 and Gln-3 reporter activity in pmr1 cells are impaired, but expression of functional Gap1 in the plasma membrane of a pmr1 strain in response to nitrogen limitation is enhanced. These two phenotypes suggest up-regulation of Npr1 function in the absence of Pmr1. Together, our results establish that Pmr1-dependent Ca2+ and/or Mn2+ ion homeostasis is necessary for TOR signaling.
Amino Acid Transport Systems↗
The role of Cdc55 in the spindle checkpoint is through regulation of mitotic exit in Saccharomyces cerevisiae.
Cdc55, a B-type regulatory subunit of protein phosphatase 2A, has been implicated in mitotic spindle checkpoint activity and maintenance of sister chromatid cohesion during metaphase. The spindle checkpoint is composed of two independent pathways, one leading to inhibition of the metaphase-to-anaphase transition by checkpoint proteins, including Mad2, and the other to inhibition of mitotic exit by Bub2. We show that Cdc55 is a negative regulator of mitotic exit. A cdc55 mutant, like a bub2 mutant, prematurely releases Cdc14 phosphatase from the nucleolus during spindle checkpoint activation, and premature exit from mitosis indirectly leads to loss of sister chromatid cohesion and inviability in nocodazole. The role of Cdc55 is separable from Bub2 and inhibits release of Cdc14 through a mechanism independent of the known negative regulators of mitotic exit. Epistasis experiments indicate Cdc55 acts either downstream or independent of the mitotic exit network kinase Cdc15. Interestingly, the B-type cyclin Clb2 is partially stable during premature activation of mitotic exit in a cdc55 mutant, indicating mitotic exit is incomplete.
Cell Cycle Proteins↗
Diverse functions of spindle assembly checkpoint genes in Saccharomyces cerevisiae.
The spindle assembly checkpoint regulates the metaphase-to-anaphase transition from yeast to humans. We examined the genetic interactions with four spindle assembly checkpoint genes to identify nonessential genes involved in chromosome segregation, to identify the individual roles of the spindle assembly checkpoint genes within the checkpoint, and to reveal potential complexity that may exist. We used synthetic genetic array (SGA) analysis using spindle assembly checkpoint mutants mad1, mad2, mad3, and bub3. We found 228 synthetic interactions with the four spindle assembly checkpoint mutants with substantial overlap in the spectrum of interactions between mad1, mad2, and bub3. In contrast, there were many synthetic interactions that were common to mad1, mad2, and bub3 that were not shared by mad3. We found shared interactions between pairs of spindle assembly checkpoint mutants, suggesting additional complexity within the checkpoint and unique interactions for all of the spindle assembly checkpoint genes. We show that most genes in the interaction network, including ones with unique interactions, affect chromosome transmission or microtubule function, suggesting that the complexity of interactions reflects diverse roles for the checkpoint genes within the checkpoint. Our analysis expands our understanding of the spindle assembly checkpoint and identifies new candidate genes with possible roles in chromosome transmission and mitotic spindle function.
Cell Cycle Proteins↗
Eliminating gene conversion improves high-throughput genetics in Saccharomyces cerevisiae.
Synthetic genetic analysis was improved by eliminating leaky expression of the HIS3 reporter and gene conversion between the HIS3 reporter and his3Delta1. Leaky expression was eliminated using 3-aminotriazole and gene conversion was eliminated by using the Schizosaccharomyces pombe his5+ gene, resulting in a 5- to 10-fold improvement in the efficiency of SGA.
Amitrole↗
Two complexes of spindle checkpoint proteins containing Cdc20 and Mad2 assemble during mitosis independently of the kinetochore in Saccharomyces cerevisiae.
Favored models of spindle checkpoint signaling propose that two inhibitory complexes (Mad2-Cdc20 and Mad2-Mad3-Bub3-Cdc20) must be assembled at kinetochores in order to inhibit mitosis. We have directly tested this model in the budding yeast Saccharomyces cerevisiae. The proteins Mad2, Mad3, Bub3, Cdc20, and Cdc27 in yeast were quantified, and there are sufficient amounts to form stoichiometric inhibitors of Cdc20 and the anaphase-promoting complex. Mad2 is present in two separate complexes in cells arrested in mitosis with nocodazole. There is a small amount of Mad2-Mad3-Bub3-Cdc20 and a much larger amount of a complex that contains Mad2-Cdc20. We use conditional mutants to show that both Mad2 and Mad3 are essential for establishment and maintenance of the spindle checkpoint. Both spindle checkpoint complexes containing Mad2 form in mitosis, not in response to checkpoint activation. The kinetochore is not required to form either complex. We propose that the conversion of Mad1-Mad2 to Cdc20-Mad2, a key step in generating inhibitory checkpoint complexes, is limited to mitosis by the availability of Cdc20 and is kinetochore independent.
Cdc20 Proteins↗
Analyzing the spindle checkpoint in yeast and frogs.
The spindle checkpoint is an evolutionarily conserved regulatory mechanism that ensures correct segregation of chromosomes at mitosis and meiosis. The kinetochore plays an integral role in spindle checkpoint signaling by integrating chromosome attachment to the spindle with cell cycle progression. A single kinetochore can inhibit cell cycle progression in the absence of proper spindle attachment or tension from bipolar orientation. Recent advances have shed light on how the kinetochore measures these situations, transduces a signal, and inhibits the entry into anaphase.
Anaphase↗
Assaying the spindle checkpoint in the budding yeast Saccharomyces cerevisiae.
The spindle checkpoint is assayed in Saccharomyces cerevisiae using several criteria. Sensitivity to benzimidazole drugs is assayed in cells grown in liquid medium and cells grown on solid medium on petri plates. Cell cycle delays are measured using cells synchronized by treatment with mating pheromone alpha-factor, and the population is monitored by flow cytometry measuring DNA content in cells. There are two different transitions that are monitored, and cytological assays for individual cells and biochemical assays for populations of cells are presented. The metaphase to anaphase transition is assayed by monitoring sister chromatid separation using GFP-tagged chromosomes, Pds1 stability using immunofluorescence, and Mcd1/Scc1 association with chromatin using chromosome spreads. Pds1 and Mcd1/Scc1 stability is measured in populations by Western blots. The exit from mitosis is monitored by Cdc14 immunofluorescence and Clb2 Western blots.
Anaphase↗
Differential kinetochore requirements for establishment and maintenance of the spindle checkpoint are dependent on the mechanism of checkpoint activation in Saccharomyces cerevisiae.
The spindle checkpoint in the yeast Saccharomyces cerevisiae is an intracellular signal transduction pathway comprised of two branches that inhibit two different mitotic transitions in cells treated with benzimidazole drugs such as nocodazole. The kinetochore is an integral component of the MAD2 branch of the spindle checkpoint pathway. Current models propose that the kinetochore is required for both the establishment and maintenance of the spindle checkpoint but a role for the kinetochore in the maintenance of spindle checkpoint in yeast has never been directly tested. We used a temperature sensitive ndc10-1 mutant to inactivate kinetochores before and after arresting cells in mitosis to determine the role of kinetochores in the establishment and maintenance of the spindle checkpoint. We show that both establishment and maintenance requires kinetochore function in response to spindle damage induced by benzimidazole drugs. Excess expression of the Mps1 protein kinase causes wild type cells and ndc10-1 cells to arrest in mitosis. Unlike the spindle checkpoint arrest activated by benzimidazoles, this arrest can be maintained independently of kinetochores. The arrest induced by excess Mps1p is independent of BUB2. Therefore, mitotic arrest induced by excess Mps1p expression is due to the action of the MAD2 branch of the spindle checkpoint pathway and excess Mps1p acts downstream of the kinetochore.
Carrier Proteins↗
The highly conserved Ndc80 complex is required for kinetochore assembly, chromosome congression, and spindle checkpoint activity.
We show that the Xenopus homologs of Ndc80/Tid3/HEC1 (xNdc80) and Nuf2/MPP1/Him-10 (xNuf2) proteins physically interact in a 190-kD complex that associates with the outer kinetochore from prometaphase through anaphase. Injecting function-blocking antibodies to either xNdc80 or xNuf2 into XTC cells caused premature exit from mitosis without detectable chromosome congression or anaphase movements. Injected cells did not arrest in response to microtubule drugs, showing that the complex is required for the spindle checkpoint. Kinetochores assembled in Xenopus extracts after immunodepletion of the complex did not contain xRod, xZw10, xP150 glued (Dynactin), xMad1, xMad2, xBub1, and xBub3, demonstrating that the xNdc80 complex is required for functional kinetochore assembly. In contrast, function-blocking antibodies did not affect the localization of other kinetochore proteins when added to extracts containing previously assembled kinetochores. These extracts with intact kinetochores were deficient in checkpoint signaling, suggesting that the Ndc80 complex participates in the spindle checkpoint. We also demonstrate that the spindle checkpoint can arrest budding yeast cells lacking Ndc80 or Nuf2, whereas yeast lacking both proteins fail to arrest in mitosis. Systematic deletion of yeast kinetochore genes suggests that the Ndc80 complex has a unique role in spindle checkpoint signaling. We propose that the Ndc80 complex has conserved roles in kinetochore assembly, chromosome congression, and spindle checkpoint signaling.
Anaphase↗
The quest for the holy "G" of chromosomal passengers.
Twelve years after reporting a novel chromosomal passenger protein, Mollinari et al. in this issue of Developmental Cell finally report its molecular identification, and the results are stunning. TD-60 is a member of the RCC1 family of guanine nucleotide exchange factors implicating small GTPases in chromosome congression and cytokinesis.
Aurora Kinases↗
The spindle assembly and spindle position checkpoints.
The mitotic spindle segregates chromosomes to opposite ends of the cell in preparation for cell division. Chromosome attachment to the spindle is monitored by the spindle assembly checkpoint, and at least in yeast cells, penetration of one spindle pole into the bud is monitored by the spindle position checkpoint. We review the historical origins of these checkpoints and recent progress in understanding their surveillance pathways. We also highlight fascinating but as yet unresolved questions, and examine crosstalk between the checkpoints.
Cell Division↗
Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae.
Protein kinases are coded by more than 2,000 genes and thus constitute the largest single enzyme family in the human genome. Most cellular processes are in fact regulated by the reversible phosphorylation of proteins on serine, threonine, and tyrosine residues. At least 30% of all proteins are thought to contain covalently bound phosphate. Despite the importance and widespread occurrence of this modification, identification of sites of protein phosphorylation is still a challenge, even when performed on highly purified protein. Reported here is methodology that should make it possible to characterize most, if not all, phosphoproteins from a whole-cell lysate in a single experiment. Proteins are digested with trypsin and the resulting peptides are then converted to methyl esters, enriched for phosphopeptides by immobilized metal-affinity chromatography (IMAC), and analyzed by nanoflow HPLC/electrospray ionization mass spectrometry. More than 1,000 phosphopeptides were detected when the methodology was applied to the analysis of a whole-cell lysate from Saccharomyces cerevisiae. A total of 216 peptide sequences defining 383 sites of phosphorylation were determined. Of these, 60 were singly phosphorylated, 145 doubly phosphorylated, and 11 triply phosphorylated. Comparison with the literature revealed that 18 of these sites were previously identified, including the doubly phosphorylated motif pTXpY derived from the activation loop of two mitogen-activated protein (MAP) kinases. We note that the methodology can easily be extended to display and quantify differential expression of phosphoproteins in two different cell systems, and therefore demonstrates an approach for "phosphoprofiling" as a measure of cellular states.
Algorithms↗
The dephosphorylated form of the anaphase-promoting complex protein Cdc27/Apc3 concentrates on kinetochores and chromosome arms in mitosis.
Cell cycle regulated protein ubiquitination and degradation within subcellular domains may be essential for the normal progression of mitosis. Cdc27 is a conserved component of an essential M-phase ubiquitin-protein ligase called the anaphase-promoting complex/cyclosome. We examined the subcellular distribution of Cdc27 in greater detail in mammalian cells and found Cdc27 concentrated at spindle poles and on spindle microtubules as previously described, but also found Cdc27 at kinetochores and along chromosome arms. This localization was not dependent on intact microtubules. While the great majority of Cdc27 protein in M phase cells is highly phosphorylated, only the dephosphorylated form of Cdc27 was found associated with isolated chromosomes. Kinases that also associate with isolated chromosomes catalyzed the in vitro phosphorylation of the chromosome-associated Cdc27. Microinjection of anti-Cdc27 antibody into cells causes arrest at metaphase. Microinjection of cells with anti-Mad2 antibody normally induces premature anaphase onset resulting in catastrophic nondisjunction of the chromosomes. However, coinjection of anti-Cdc27 antibody with anti-Mad2 antibody resulted in metaphase arrest. The association of dephosphorylated APC/C components with mitotic chromosomes suggests mechanisms by which the spindle checkpoint may regulate APC/C activity at mitosis.
Animals↗