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T T Su

Publications and source records attributed to T T Su.

At least 19 recordsLinked to original sources
PubMed2001

mei-41 and bub1 block mitosis at two distinct steps in response to incomplete DNA replication in Drosophila embryos.

Drosophila double park encodes a homolog of Cdt1 that functions in initiation of DNA replication in fission yeast and Xenopus. dup mutants complete the first 15 embryonic cell cycles, presumably via maternal dup products, and show defects in the 16(th) S phase (S16). Cells carrying dup(a1) allele forgo S16 altogether but enter mitosis 16 (M16). We find that the timing of entry into M16 is similar in dup(a1) and heterozygous or wild-type (wt) controls. In contrast, we find that mutant cells carrying another allele, dup(a3), undergo a partial S16 and delay the entry into M16. Thus, initiation of S16 appears necessary for delaying M16. This delay is absent in double mutants of dup(a3) and mei-41 (Drosophila ATR), indicating that a mei-41-dependent checkpoint acts to delay the entry into mitosis in response to incomplete DNA replication. dup(a3) and dup(a1) mutant cells that enter M16 become arrested in M16. We find that mitotic cyclins are stabilized and that a spindle checkpoint protein, Bub1, localizes onto chromosomes during mitotic arrest in dup mutants. These features suggest an arrest prior to metaphase-anaphase transition. dup(a3) bub1 double mutant cells exit M16, indicating that a bub1-mediated checkpoint acts to block mitotic exit in dup mutants. To our knowledge, this is the first report of (1) incomplete DNA replication affecting both the entry into and the exit from mitosis in a single cell cycle via different mechanisms and (2) the role of bub1 in regulating mitotic exit in response to incomplete DNA replication.

Alleles↗
PubMed2001

DNA damage leads to a Cyclin A-dependent delay in metaphase-anaphase transition in the Drosophila gastrula.

BACKGROUND: In response to DNA damage, fission yeast, mammalian cells, and cells of the Drosophila gastrula inhibit Cdk1 to delay the entry into mitosis. In contrast, budding yeast delays metaphase-anaphase transition by stabilization of an anaphase inhibitor, Pds1p. A variation of the second response is seen in Drosophila cleavage embryos; when nuclei enter mitosis with damaged DNA, centrosomes lose gamma-tubulin, spindles lose astral microtubules, chromosomes fail to reach a metaphase configuration, and interphase resumes without an intervening anaphase. The resulting polyploid nuclei are eliminated. RESULTS: The cells of the Drosophila gastrula can also delay metaphase-anaphase transition in response to DNA damage. This delay accompanies the stabilization of Cyclin A, a known inhibitor of sister chromosome separation in Drosophila. Unlike in cleavage embryos, gamma-tubulin remains at the spindle poles, and anaphase always occurs after the delay. Cyclin A mutants fail to delay metaphase-anaphase transition after irradiation and show an increased frequency of chromosome breakage in the subsequent anaphase. CONCLUSIONS: DNA damage delays metaphase-anaphase transition in Drosophila by stabilizing Cyclin A. This delay may normally serve to preserve chromosomal integrity during segregation. To our knowledge this is the first report of a metazoan metaphase-anaphase transition being delayed in response to DNA damage. Though mitotic progression is modulated in response to DNA damage in both cleaving and gastruating embryos of Drosophila, different mechanisms operate. These differences are discussed in the context of differential cell cycle regulation in cleavage and gastrula stages.

Anaphase↗
PubMed2001

Cycling through development in Drosophila and other metazoa.

The cell-division cycle is an orchestrated sequence of events that results in the duplication of a cell. In metazoa, cell proliferation is regulated in response to differentiation signals and body-size parameters, which either induce cell duplication or arrest the cell cycle, to ensure that organs develop to the correct size. In addition, the cell cycle may be altered to meet specialized requirements. This can be seen in the rapid cleavage cycles of vertebrates and insects that lack gap phases, in the nested S phases of Drosophila, and in the endocycles of nematodes, insects, plants and mammals that lack mitosis. Here we present the various modes of cell-cycle regulation in metazoa and discuss their possible generation by a combination of universally conserved molecules and new regulatory circuits.

Animals↗
PubMed2001

Cell cycle roles for two 14-3-3 proteins during Drosophila development.

Drosophila 14-3-3 epsilon and 14-3-3 zeta proteins have been shown to function in RAS/MAP kinase pathways that influence the differentiation of the adult eye and the embryo. Because 14-3-3 proteins have a conserved involvement in cell cycle checkpoints in other systems, we asked (1) whether Drosophila 14-3-3 proteins also function in cell cycle regulation, and (2) whether cell proliferation during Drosophila development has different requirements for the two 14-3-3 proteins. We find that antibody staining for 14-3-3 family members is cytoplasmic in interphase and perichromosomal in mitosis. Using mutants of cyclins, Cdk1 and Cdc25(string) to manipulate Cdk1 activity, we found that the localization of 14-3-3 proteins is coupled to Cdk1 activity and cell cycle stage. Relocalization of 14-3-3 proteins with cell cycle progression suggested cell-cycle-specific roles. This notion is confirmed by the phenotypes of 14-3-3 epsilon and 14-3-3 zeta mutants: 14-3-3 epsilon is required to time mitosis in undisturbed post-blastoderm cell cycles and to delay mitosis following irradiation; 14-3-3 zeta is required for normal chromosome separation during syncytial mitoses. We suggest a model in which 14-3-3 proteins act in the undisturbed cell cycle to set a threshold for entry into mitosis by suppressing Cdk1 activity, to block mitosis following radiation damage and to facilitate proper exit from mitosis.

14-3-3 Proteins↗
PubMed2000

Activating the DNA damage checkpoint in a developmental context.

BACKGROUND: Studies in unicellular systems have established that DNA damage by irradiation invokes a checkpoint that acts to stall cell division. During metazoan development, the modulation of cell division by checkpoints must occur in the context of gastrulation, differential gene expression and changes in cell cycle regulation. To understand the effects of checkpoint activation in a developmental context, we examined the effect of X-rays on post-blastoderm embryos of Drosophila melanogaster. RESULTS: In Drosophila, DNA damage was previously found to delay anaphase chromosome separation during cleavage cycles that lack a G2 phase. In post-blastoderm cycles that included a G2 phase, we found that irradiation delayed the entry into mitosis. Gastrulation and the developmental program of string (Cdc25) gene expression, which normally regulates the timing of mitosis, occurred normally after irradiation. The radiation-induced delay of mitosis accompanied the exclusion of mitotic cyclins from the nucleus. Furthermore, a mutant form of the mitotic kinase Cdk1 that cannot be inhibited by phosphorylation drove a mitotic cyclin into the nucleus and overcame the delay of mitosis induced by irradiation. CONCLUSIONS: Developmental changes in the cell cycle, for example, the introduction of a G2 phase, dictate the response to checkpoint activation, for example, delaying mitosis instead of or in addition to delaying anaphase. This unprecedented finding suggests that different mechanisms are used at different points during metazoan development to stall cell division in response to checkpoint activation. The delay of mitosis in post-blastoderm embryos is due primarily to inhibitory phosphorylation of Cdk1, whereas nuclear exclusion of a cyclin-Cdk1 complex might play a secondary role. Delaying cell division has little effect on gastrulation and developmentally regulated string gene expression, supporting the view that development generally dictates cell proliferation and not vice versa.

Animals↗
PubMed2000

DNA defects target the centrosome.

When cells enter mitosis with DNA that is unfit to be segregated, the consequences appear to be loss of centrosome function, abnormal spindles and a failure to segregate chromosomes. These defects may result from the workings of a surveillance mechanism that acts to cull irreparable nuclei.

Animals↗
PubMed1999

Drosophila grapes/CHK1 mutants are defective in cyclin proteolysis and coordination of mitotic events.

The Drosophila grapes (grp) gene, which encodes a homolog of the Schizosaccharomyces pombe Chk1 kinase, provides a cell-cycle checkpoint that delays mitosis in response to inhibition of DNA replication [1]. Grp is also required in the undisturbed early embryonic cycles: in its absence, mitotic abnormalities appear in cycle 12 and chromosomes fail to fully separate in subsequent cycles [2] [3]. In other systems, Chk1 kinase phosphorylates and suppresses the activity of Cdc25 phosphatase: the resulting failure to remove inhibitory phosphate from cyclin-dependent kinase 1 (Cdk1) prevents entry into mitosis [4] [5]. Because in Drosophila embryos Cdk1 lacks inhibitory phosphate during cycles 11-13 [6], it is not clear that known actions of Grp/Chk1 suffice in these cycles. We found that the loss of grp compromised cyclin A proteolysis and delayed mitotic disjunction of sister chromosomes. These defects occurred before previously reported grp phenotypes. We conclude that Grp activates cyclin A degradation, and functions to time the disjunction of chromosomes in the early embryo. As cyclin A destruction is required for sister chromosome separation [7], a failure in Grp-promoted cyclin destruction can also explain the mitotic phenotype. The mitotic failure described previously for cycle 12 grp embryos might be a more severe form of the phenotypes that we describe in earlier embryos and we suggest that the underlying defect is reduced degradation of cyclin A.

Anaphase↗
PubMed1998

Size control: cell proliferation does not equal growth.

Division subdivides mass without increasing it. So one should not expect that an increase in cell division would make an organism bigger. Both classic and recent experiments confirm this simple rationale: altering proliferation produces normally sized body structures with either especially small or exceptionally large cells.

Animals↗
PubMed1998

Exit from mitosis in Drosophila syncytial embryos requires proteolysis and cyclin degradation, and is associated with localized dephosphorylation.

The cyclin proteolysis that accompanies the exit from mitosis in diverse systems appears to be essential for restoration of interphase. The early syncytial divisions of Drosophila embryos, however, occur without detectable oscillations in the total cyclin level or Cdk1 activity. Nonetheless, we found that injection of an established inhibitor of cyclin proteolysis, a cyclin B amino-terminal peptide, prevents exit from mitosis in syncytial embryos. Similarly, injection of a version of Drosophila cyclin B that is refractory to proteolysis results in mitotic arrest. We infer that proteolysis of cyclins is required for exit from syncytial mitoses. This inference can be reconciled with the failure to observe oscillations in total cyclin levels if only a small pool of cyclins is destroyed in each cycle. We find that antibody detection of histone H3 phosphorylation (PH3) acts as a reporter for Cdk1 activity. A gradient of PH3 along anaphase chromosomes suggests local Cdk1 inactivation near the spindle poles in syncytial embryos. This pattern of Cdk1 inactivation would be consistent with local cyclin destruction at centrosomes or kinetochores. The local loss of PH3 during anaphase is specific to the syncytial divisions and is not observed after cellularization. We suggest that exit from mitosis in syncytial cycles is modified to allow nuclear autonomy within a common cytoplasm.

Animals↗
PubMed1998

The cell cycle program in germ cells of the Drosophila embryo.

The germ cells of metazoans follow a program of proliferation that is distinct from proliferation programs of somatic cells. Despite their developmental importance, the cell proliferation program in the metazoan primordial germ cells is not well characterized and the regulatory controls are not understood. In Drosophila melanogaster, germ cell precursors (called pole cells) proliferate early in embryogenesis and then enter a prolonged quiescence. We found that polar nuclear divisions are asynchronous and lag behind somatic nuclear divisions during syncytial cycles 9 and 10. Thus, the polar division program deviates from the somatic division program when pole nuclei and somatic nuclei still share a common cytoplasm, earlier than previously thought. The lag in polar nuclear divisions is independent of grapes, which is required for lengthening somatic cell cycles 10-13. Mapping of the last S phase in pole cells and measurement of their DNA content indicate that pole cells become quiescent in G2 phase of the cell cycle. We were able to drive quiescent pole cells into mitosis by induction of either an activator of Cdc2 (Cdc25string phosphatase) or a mutant form of Cdc2 that cannot be inhibited by phosphorylation. In contrast, induction of wild-type Cdc2 with a mitotic cyclin did not induce mitosis in pole cells. We propose that inhibition of Cdc2 by phosphorylation contributes to G2 arrest in pole cells during embryogenesis. Furthermore, pole cells enter G1 following induced mitoses, indicating that entry into both mitosis and S phase is blocked in quiescent pole cells. These studies represent the first molecular characterization of proliferation in embryonic germ cells of Drosophila.

Animals↗
PubMed1998

Chromosome association of minichromosome maintenance proteins in Drosophila endoreplication cycles.

Minichromosome maintenance (MCM) proteins are essential eukaryotic DNA replication factors. The binding of MCMs to chromatin oscillates in conjunction with progress through the mitotic cell cycle. This oscillation is thought to play an important role in coupling DNA replication to mitosis and limiting chromosome duplication to once per cell cycle. The coupling of DNA replication to mitosis is absent in Drosophila endoreplication cycles (endocycles), during which discrete rounds of chromosome duplication occur without intervening mitoses. We examined the behavior of MCM proteins in endoreplicating larval salivary glands, to determine whether oscillation of MCM-chromosome localization occurs in conjunction with passage through an endocycle S phase. We found that MCMs in polytene nuclei exist in two states: associated with or dissociated from chromosomes. We demonstrate that cyclin E can drive chromosome association of DmMCM2 and that DNA synthesis erases this association. We conclude that mitosis is not required for oscillations in chromosome binding of MCMs and propose that cycles of MCM-chromosome association normally occur in endocycles. These results are discussed in a model in which the cycle of MCM-chromosome associations is uncoupled from mitosis because of the distinctive program of cyclin expression in endocycles.

Animals↗
PubMed1997

Chromosome association of minichromosome maintenance proteins in Drosophila mitotic cycles.

Minichromosome maintenance (MCM) proteins are essential DNA replication factors conserved among eukaryotes. MCMs cycle between chromatin bound and dissociated states during each cell cycle. Their absence on chromatin is thought to contribute to the inability of a G2 nucleus to replicate DNA. Passage through mitosis restores the ability of MCMs to bind chromatin and the ability to replicate DNA. In Drosophila early embryonic cell cycles, which lack a G1 phase, MCMs reassociate with condensed chromosomes toward the end of mitosis. To explore the coupling between mitosis and MCM-chromatin interaction, we tested whether this reassociation requires mitotic degradation of cyclins. Arrest of mitosis by induced expression of nondegradable forms of cyclins A and/or B showed that reassociation of MCMs to chromatin requires cyclin A destruction but not cyclin B destruction. In contrast to the earlier mitoses, mitosis 16 (M16) is followed by G1, and MCMs do not reassociate with chromatin at the end of M16. dacapo mutant embryos lack an inhibitor of cyclin E, do not enter G1 quiescence after M16, and show mitotic reassociation of MCM proteins. We propose that cyclin E, inhibited by Dacapo in M16, promotes chromosome binding of MCMs. We suggest that cyclins have both positive and negative roles in controlling MCM-chromatin association.

Animals↗
PubMed1997

Cloning of Drosophila MCM homologs and analysis of their requirement during embryogenesis.

MCM (minichromosome maintenance) gene family of Saccharomyces cerevisiae encodes essential DNA replication factors that participate in the initiation of DNA replication. In addition, their localization to the nucleus in a mitosis-dependent manner fueled the hypothesis that MCMs also act to couple DNA replication to mitosis. We report the identification of a Drosophila gene family with extensive sequence identity to the MCM genes. Results from antibody injection experiments suggest that MCMs play an essential role in DNA replication during embryogenesis. Evolutionary conservation of MCM sequences and function in Drosophila could potentially facilitate studies of how initiation of DNA replication is regulated and coupled to mitosis during metazoan development.

Amino Acid Sequence↗
PubMed1996

Magnetic resonance imaging of glomus tumour in the hand.

Glomus tumours of the hand are benign tumours that arise from the neuromyoarterial apparatus. The most common site for a glomus tumour is in the distal phalanx. They frequently display a classic triad of symptoms-pain, tenderness and temperature sensitivity. Ten treated cases examined with magnetic resonance imaging (MRI) preoperatively are described. All were female with a mean age of 43 years. All except 2 suffered the classic triad of symptoms. The mean duration from onset of symptoms to therapeutic intervention was 7.4 years. MRI depicted the tumours in excellent detail and aided diagnosis in the 4 patients without subungual blue discolouration. The lesions showed a low signal on the T1-weighted image and a high signal on the T2-weighted image. All except 3 were located in the subungual region. All patients underwent surgical excision with pathological confirmation. The postoperative course was smooth and there were no sequelae. High resolution MRI, providing a clear contrast between tumorous and normal tissues, has proved to be a useful non-invasive method for imaging glomus tumours in the hand.

Adolescent↗
PubMed1996

Drosophila MCM protein complexes.

MCM genes encode a family of evolutionarily conserved proteins required for DNA replication. In Saccharomyces cerevisiae, where they were first identified, MCM genes interact genetically with each other. Allele specificity in these interactions suggests that MCM proteins physically associate with one another and that this association is essential for function. We describe here an analysis of physical interactions among three Drosophila MCM proteins. Using specific antibodies we detect Drosophila MCMs almost exclusively in 600-kDa protein complexes. Co-immunoprecipitation data demonstrate the existence of at least two distinct types of 600-kDa complexes, one that contains DmCDC46 and one that appears to contain both DmMCM2 and Dpa (a CDC54 homologue). These complexes are stable throughout embryonic division cycles, are resistant to treatments with salt and detergent, and are present during development in tissues undergoing mitotic DNA replication as well as endoreplication. When extracts are prepared under low salt conditions all three MCM proteins co-immunoprecipitate. Consequently, we suggest that the 600-kDa complexes interact in a higher order complex.

Animals↗
PubMed1995

dpa, a member of the MCM family, is required for mitotic DNA replication but not endoreplication in Drosophila.

We have isolated the Drosophila disc proliferation abnormal (dpa) gene, a member of the MCM family of DNA replication factors. Members of this family of proteins are required for DNA replication in yeast. A dpa null mutant dies during pupal stages because imaginal tissues necessary for the formation of the adult fly fail to proliferate normally. Beginning in late embryogenesis BrdU labeling reveals DNA replication defects in mitotically proliferating cells. In contrast, dpa is dispensable for endoreplication, a specialized cell cycle consisting of consecutive rounds of S phases without intervening mitosis. Our studies suggest an essential role for dpa in mitotic DNA replication but not in endoreplication. Thus, dpa is not a general replication factor but may play a specialized regulatory role in DNA replication.

Amino Acid Sequence↗
PubMed1994

Selective binding of Escherichia coli RNA polymerase to topoisomers of minicircles carrying the TAC16 and TAC17 promoters.

We have studied the effect of DNA supercoiling on open complex formation by Escherichia coli RNA polymerase at the TAC16 and TAC17 promoters. A two-dimensional gel retardation assay was used to measure the relative rate of association between RNA polymerase and the TAC promoters on individual topoisomers. Plasmid DNAs usually have several promoters that complicate the analysis of RNA polymerase binding to only one of them. We avoided this problem by using minicircles of DNA carrying only a single promoter. These were generated in vivo by the site-specific recombination system of bacteriophage lambda. Both the TAC16 and TAC17 promoters exhibited a biphasic response to negative supercoiling. Between superhelical densities of 0 and -0.04, increases in the negative linking difference favored binding. Beyond -0.04, either no effect on binding (TAC16) or a slight inhibition of binding (TAC17) was observed for increases in the negative linking difference. The unwinding at the rate-limiting step was calculated for both TAC promoters at moderate superhelical densities. This unwinding was found to be a fraction (about 20-30%) of the total unwinding measured by topoisomerase I relaxation (about one turn). In addition, the 1-base pair difference in spacer length between the TAC16 and TAC17 promoters resulted in different extents of activation and inhibition by negative supercoiling.

DNA Topoisomerases, Type I↗