Given the essential role that Double parked plays in DNA replication, the expression and localization of Dup was examined. Antibodies were generated to the Dup protein. In stage 2-6 ovarian follicle cells that are proliferating mitotically, some nuclei stain brightly with anti-Dup antibodies, whereas others are faint. This pattern is similar to the pattern of staining observed with MCM and cyclin E antibodies. It is likely that the cells that stain brightly for Dup, MCM, and Cyclin E proteins are those in S phase. When follicle cells are undergoing endo cell cycles in stages 7-9, Dup appears to be mainly cytoplasmic, although there is some Dup staining in the nucleus. At stage 10A Dup is localized diffusely in the nucleus, but is mostly cytoplasmic. At early stage 10B, the stage when amplification is detectable by BrdU incorporation, two subnuclear foci are seen, but by late stage 10B primarily one prominent focus is seen in most nuclei. A similar localization pattern is seen at stage 11 (Whittaker, 2000).
Given the critical role Dup plays in replication it was postulated that, like other replication genes such as cyclin E and PCNA, dup is transcriptionally regulated by the E2F transcription factor. To examine the expression pattern of dup transcript, embryos of various developmental stages were hybridized in situ with dup riboprobes. The pattern of expression of dup transcript is very similar to that of other S phase genes. High levels of maternal transcript are present during cycles 1-13. At cycle 14, the transcript is downregulated. Ubiquitous expression of dup transcript is seen during the postblastoderm divisions. After cycle 16, concurrent with the addition of a G1 phase, dup transcript is downregulated in all tissues with the exception of the proliferating central nervous system (CNS) and peripheral nervous system (PNS). At cycle 17, dup transcript is expressed just prior to S phase and down regulated after S phase in the endoreplicating gut. Thus, like other S phase genes, expression of dup is regulated at the G1-S transition of the cell cycle (Whittaker, 2000).
The subnuclear localization pattern of Dup at stages 10B and 11 is strikingly similar to that seen for Orc2 and Orc1, which have been shown to localize to the sites of chorion gene amplification at these stages. To determine if Orc2 and Dup colocalize, ovaries were stained simultaneously with Orc2 and Dup antibodies. At stage 10B Dup and Orc2 colocalize in the follicle cells, although the Dup staining appears more diffuse than that of Orc2. By stage 11 only one focus of Orc2 is seen in follicle cell nuclei. Dup remains detectable at two foci in most nuclei, but one focus was clearly brighter than the other (Whittaker, 2000).
Two interesting differences are seen in the localization pattern of Orc2 and Dup. (1) At stage 10A, Orc2 has already localized to subnuclear foci. Dup, in contrast, is still diffusely localized in the nucleus. (2) Orc2 is not seen to localize to origins after stage 11, but BrdU incorporation continues for another seven hours, until stage 13. Prior results suggested that this BrdU incorporation is due to elongation. The levels of localized Orc1 protein also diminish after stage 10B. Interestingly, Dup is undiminished and remains detectable at subnuclear foci until stage 13, indicating that it may function during elongation. Alternatively, Dup could have no role in elongation but is not cleared from chromatin until after elongation has been completed (Whittaker, 2000).
Four alleles of the dup gene were isolated in a screen for mutations that alter a G1/S transcriptional program during embryogenesis (Royzman, 1997). A deficiency that uncovers dup, Df(2R)JP1 was identified and a previously existing mutation, l(2)51Ec was found to be an allele of dup. All five mutational alleles are embryonic lethal in trans to the deficiency. In addition to these strong dup mutations, the female-sterile mutation, fs(2)PA77, is an allele of dup. The dupa1-a4 mutations fail to complement fs(2)PA77; trans-heterozygotes have low viability and are female and male sterile. In contrast, l(2)51Ec and fs(2)PA77 complement and are both viable and fertile, thus they were previously thought to be separate genes. The ability of these alleles to complement may be because l(2)51Ec is a weaker allele than the other embryonic lethal mutations (Whittaker, 2000).
To determine whether overexpression of Geminin acts to inhibit DNA replication in vivo, transgenic flies were generated that contain geminin under control of the S. cerevisiae UAS(GAL4) promoter. Ectopic overexpression of Geminin during embryogenesis by heat shock induction of hsp70-GAL4 UAS-geminin flies results in a general decrease in BrdU-labeling cells in mitotic and endoreplicating tissues. To demonstrate this effect more clearly, the en-GAL4 driver was used to overexpress Geminin in a striped pattern during embryogenesis. Ectopic overexpression of Geminin results in a dramatic decrease in S-phase cells within the En stripe, relative to surrounding cells. Propidium iodide staining of En-Geminin-expressing cells reveals more condensed nuclei within the En stripes, suggesting that cells were attempting to enter mitosis. Staining with anti-PH3 to detect mitotic cells showsthat many cells (4× as many as normal) in the En-Geminin stripe are in mitosis. A similar phenotype is observed in Dup mutants despite the fact that they fail to replicate their DNA (Whittaker, 2000) and occurs presumably because the DNA replication checkpoint can only be triggered after the loading of DNA polymerase alpha onto the pre-RC. To determine the fate of these cells, TUNEL was carried out to detect apoptotic cells. Wild-type embryos at stage 11 normally show very little TUNEL staining, whereas the En-Geminin stage-11 embryos show numerous TUNEL-positive cells associated with the En stripes. These data show that ectopic overexpression of Geminin results in inhibition in DNA replication, increased numbers of metaphase cells, and increased apoptosis (Quinn, 2001).
Ectopic overexpression of Geminin using the eyeless(ey)-GAL4 driver, which is expressed during the early proliferative phase of the eye-antennal imaginal disc, also results in a dramatic decrease in S phases and in the size of third instar larvae eye discs and the size of the adult eye. Overexpression of Geminin using the GMR-GAL4 driver, which is expressed posterior to the MF in the eye imaginal discs of third instar larvae, leads to a 40%-50% decrease in S-phase cells within this region, but to severely rough adult eyes. Taken together, these data show that ectopic overexpression of Geminin leads to an inhibition of S phases in both mitotic and endoreplicative cycles and at different developmental stages (Quinn, 2001).
The GMR-geminin rough eye phenotype represents a good phenotype in which to examine genetic interactions. This phenotype is responsive to the dose of geminin because two copies of UAS-geminin results in a less severe phenotype compared with three copies of the transgene. In addition, reducing the dose of endogenous geminin by half using the strong P alleles results in a less severe phenotype. To determine whether geminin genetically interacts with dup(cdt1), the dosage of dup was reduced by half using a null allele (dupa1; Whittaker, 2000) in a GMR-GAL4 UAS-geminin (two copies) background. Halving the dosage of dup enhances the GMR-geminin eye phenotype, leading to a smaller, rougher eye. Moreover, the dupa1 mutant embryonic cycle 16 S-phase defect is suppressed by a geminin mutant. Therefore, consistent with the biochemical interaction observed between Geminin and Dup, geminin genetically interacts with dup (Quinn, 2001).
Chorion gene amplification in the ovaries of Drosophila is a powerful system for the study of metazoan DNA replication in vivo. Using a combination of high-resolution confocal and deconvolution microscopy and quantitative realtime PCR, it was found that initiation and elongation occur during separate developmental stages, thus permitting analysis of these two phases of replication in vivo. Bromodeoxyuridine, origin recognition complex, and the elongation factors minichromosome maintenance proteins (MCM)2-7 and proliferating cell nuclear antigen were precisely localized, and the DNA copy number along the third chromosome chorion amplicon was quantified during multiple developmental stages. These studies revealed that initiation takes place during stages 10B and 11 of egg chamber development, whereas only elongation of existing replication forks occurs during egg chamber stages 12 and 13. The ability to distinguish initiation from elongation makes this an outstanding model to decipher the roles of various replication factors during metazoan DNA replication. This system was used to demonstrate that the pre-replication complex component, Double-parked protein/Cdt1, is not only necessary for proper MCM2-7 localization, but, unexpectedly, is present during elongation (Claycomb, 2002).
The properties of the pre-RC component, Dup/Cdt1, were characterized. Dup/Cdt1 requires ORC2 to localize to chorion origins and DUP/Cdt1 homologues in yeast and Xenopus have been shown to interact with Cdc6/18 to load MCM2-7 onto origins. In Xenopus extracts, fission yeast, and budding yeast, Cdt1 is dispensable after initiation. Furthermore, Cdt1 appears to be lost from chromatin or the nucleus at the onset of S phase. These data suggest that Cdt1 is not necessary after performing its role in pre-RC formation. In contrast, the initial description of Dup/Cdt1 staining during amplification showed that Dup/Cdt1 localized to chorion loci throughout amplification, and was present during stage 13 in the double bar structure. Therefore, the localization pattern of DUP/Cdt1 during amplification was examined in relation to BrdU and ORC2, using confocal and deconvolution microscopy, to investigate whether DUP/Cdt1 could be traveling with replication forks (Claycomb, 2002).
Dup/Cdt1 colocalized with BrdU throughout amplification. In stage 10B, Dup/Cdt1 staining was detected as foci that overlapped completely with BrdU staining. By stage 13, Dup/Cdt1 staining resolved into the double bar structure and was coincident with BrdU. The fact that Dup/Cdt1 remained localized to chorion regions throughout the elongation phase suggests that Dup/Cdt1 travels with the replication forks (Claycomb, 2002).
Dup/Cdt1 was precisely localized with respect to ORC2 by deconvolution microscopy, and in contrast to the colocalization of Dup/Cdt1 and BrdU, the ORC2 and Dup/Cdt1 staining patterns diverged as amplification proceeded. In early stage 10B, ORC2 and Dup/Cdt1 staining overlapped, similar to the results with ORC2 and BrdU costaining. However, by late stage 10B and stage 11, Dup/Cdt1 staining became fainter at the origins and resolved into a coffee bean-like structure. This change in the Dup/Cdt1 localization pattern occurred while ORC2 remained bound to origins. By stage 13, Dup/Cdt1 was detected in the double bar structure, with no evidence of ORC2 staining at origins. Similar results were seen for Dup/Cdt1 and ORC1. The pattern of Dup/Cdt1 localization in relation to BrdU and the fact that Dup/Cdt1 clears from origin sequences while ORC2 remains bound strongly indicate that Dup/Cdt1 travels with elongating replication forks (Claycomb, 2002).
Given the unexpected presence of Dup/Cdt1 during elongation, it was of interest to know if Dup/Cdt1 functioned in this system to load MCM2-7 during initiation. To test this, the localization pattern of MCM2-7 was studied in the dupPA77 female-sterile mutant ovaries. These mutants have thin eggshells and decreased and delayed BrdU incorporation during amplification. In dupPA77 homozygous mutant ovaries, the localization of MCM2-7 to chorion loci was not detected at any stage of amplification . Furthermore, MCM2-7 appeared to cluster at the nuclear envelope, where it colocalized with nuclear lamins. These data indicate that Dup/Cdt1 is necessary to localize MCM2-7 to origins during chorion amplification, the same as the role of Dup/Cdt1 orthologues. The clustering of MCM2-7 at the nuclear periphery suggests that Dup/Cdt1 may be necessary for the nuclear transport of MCM2-7, consistent with the findings in S. cerevisiae that Cdt1 and MCM2-7 display an interdependence for nuclear trafficking (Claycomb, 2002).
In contrast to other systems, these results reveal that Dup/Cdt1 travels with replication forks during amplification. Although it could be argued that Dup/Cdt1 simply spreads along the chromatin as amplification proceeds, this is unlikely. DUP/Cdt1 and ORC2 colocalization studies show that although ORC2 remains at origins, the Dup/Cdt1 signal decreases at origins and subsequently flanks the ORC2 signal. Furthermore, during elongation Dup/Cdt1 does not spread across the entire chorion region. Rather, there is a gap between the double bars of Dup/Cdt1 staining which increases from 300 +/- 30 nm in stage 10B to 740 +/- 70 nm in stage 13 (Claycomb, 2002).
The presence of DUP/Cdt1 at forks during elongation strongly suggests it has a role in this phase of replication. Why might Dup/Cdt1 be required during elongation in this system? Chorion amplification is unique because replication forks chase forks, instead of converging as in normal eukaryotic replication. Given this peculiarity of amplification, and considering the steric constraints that arise and impede forks, Dup/Cdt1 may be necessary to maintain MCM2-7 at these lethargic forks. Dup/Cdt1 could function as a processivity factor for the MCM2-7 complex, holding it on the DNA, or it could continuously reload new MCM proteins as they fall off the progressing replication forks. It is formally possible that although Dup/Cdt1 travels with the forks it does not perform a function. Dup/Cdt1 could simply not be expelled from the replication machinery upon initiation and then be dragged along during elongation. Although this possibility is not favored, definitively proving that the Dup/Cdt1 at forks is necessary for elongation will require the use of a currently unavailable conditional allele. Such a mutation would permit inactivation of Dup/Cdt1 after initiation and allow a functional test for a role in elongation (Claycomb, 2002).
Chromosomes are dynamic structures that are reorganized during the cell cycle to optimize them for distinct functions. Structural maintenance of chromosomes (SMC) and non-SMC condensin proteins associate into complexes that have been implicated in the process of chromosome condensation. The roles of the individual non-SMC subunits of the complex are poorly understood, and mutations in the CAP-G subunit have not been described in metazoans. A role for Cap-G in chromosome condensation and cohesion has been demonstrated in Drosophila. The requirement of Cap-G for condensation during prophase and prometaphase is demonstrated; however, alternate mechanisms are demonstrated to ensure that replicated chromosomes are condensed prior to metaphase. In contrast, Cap-G is essential for chromosome condensation in metaphase of single, unreplicated sister chromatids, suggesting that there is an interplay between replicated chromatids and the condensin complex. In the Cap-G mutants, defects in sister-chromatid separation are also observed. Chromatid arms fail to resolve in prophase and are unable to separate at anaphase, whereas sister centromeres show aberrant separation in metaphase and successfully move to spindle poles at anaphase. A role for Cap-G during interphase in regulating heterochromatic gene expression is demonstrated (Dej, 2004).
Cap-G;double parked double mutants show that the condensin complex is dispensable for chromosome condensation by metaphase except in the absence of a replicated sister chromatid. What could replication provide to the condensation process? The cohesin complex could compensate for a faulty condensin complex, and replication is required to assemble the cohesin complex and establish cohesion. In this model the unreplicated sister chromatids in double parked mutants would contain inactive cohesin complex that would be unable to compensate for a faulty condensin complex in the process of chromosome condensation. Alternatively, experiments in Xenopus show that TopoII activity during replication is a prerequisite for setting up a structural axis required for the mitotic chromosome assembly (Dej, 2004).
Drosophila double park (dup) encodes a homolog of Cdt1 that functions in initiation of DNA replication in fission yeast and Xenopus. A study of double parked mutants demonstrate two ways in which mitosis is regulated in response to incomplete duplication of the genome: (1) entry into mitosis is delayed via mei-41 (Drosophila ATR); (2) exit from mitosis is blocked via a spindle checkpoint function, bub1. double park mutants complete the first 15 embryonic cell cycles, presumably via maternal dup products, and show defects in the 16th S phase (S16). Cells carrying dupa1 allele forgo S16 altogether but enter mitosis 16 (M16). The timing of entry into M16 is similar in dupa1 and heterozygous or wild-type (wt) controls. In contrast, mutant cells carrying another allele, dupa3, 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 dupa3 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. dupa3 and dupa1 mutant cells that enter M16 become arrested in M16. Mitotic cyclins are stabilized and a spindle checkpoint protein, Bub1 (Basu, 1999), localizes onto chromosomes during mitotic arrest in dup mutants. These features suggest an arrest prior to metaphase-anaphase transition. dupa3 bub1 double mutant cells exit M16, indicating that a bub1-mediated checkpoint acts to block mitotic exit in dup mutants. 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 (Garner, 2001).
Yeast mutants that cannot complete DNA replication arrest before mitosis, but yeast mutants that fail to initiate DNA replication fail to arrest; thus, initiation of DNA replication appears necessary to activate a checkpoint that couples mitosis to the completion of S phase. This can account for Drosophila dup mutants that fail to undergo S16 but enter M16. It was, however, surprising to find that cells of dupa1 and dupa3 mutant embryos, previously thought to behave similarly, enter M16 at different times. dupa1 mutant cells entered M16 approximately concurrent with heterozygous controls, whereas dupa3mutant cells entered M16 after heterozygotes. While the dupa1 allele results from a stop codon at 171 (out of a total of 743 amino acids), the dupa3allele results from a stop codon at position 592. Thus, dupa3 mutants may retain partial Dup activity that allows a partial S16 and consequently activates a checkpoint to delay M16. This idea is supported by two pieces of data: (1) a partial S16 is detected in dupa3 mutants, while confirming the observation that S16 is absent in dupa1 mutants; (2) the delay of M16 seen in dupa3 mutants is found to be absent in mei-41 dupa3double mutants. mei-41 encodes a homolog of the checkpoint kinase ATR and is required to delay mitosis upon inhibition of DNA synthesis in Drosophila syncytial embryos (cycles 11-13) (Sibon, 1999). It is concluded that partial DNA synthesis in dupa3mutants delays the entry into M16 via a Mei-41-dependent checkpoint (Garner, 2001).
The effect of dup mutations on entry into mitosis is in agreement with findings in yeast and Xenopus; the effect on exit from M16, however, is contrary to previous results. Budding yeast cells that lack CDC6 skip S phase and enter mitosis (similar to dupa1mutants) but are reported to subsequently exit mitosis with wild-type kinetics to undergo a 'reductional' anaphase. In contrast, epidermal cells of both dupa3and dupa1 mutants that enter M16, with and without a prior delay, become arrested. dupa1 cells are in M16 in stage 11 and mitotic cells are still seen in stage 13; dupa3cells are in M16 in stage 13 and mitotic cells are still visible at stage 15, if not later. Because the stages are at least 2 hr apart in each case, the arrest in M16 is likely to average at least 2 hr, significantly longer than normal embryonic mitosis of about 10 min (Garner, 2001 and references therein).
The apparent difference in the behavior of yeast and Drosophila cells that harbor unreplicated chromosomes led to an effort to determine the basis for mitotic arrest in dup mutants. To this end, mitotic spindles were examined by staining for alpha- and gamma-tubulin, and chromosomes were visualized by staining for a mitotic phosphoepitope on histone H3 (PH3). While the spindles are bipolar and appear to contain functional centrosomes, i.e., they contain gamma-tubulin and nucleate aster microtubules, chromosomes fail to align normally. Most chromosomes lie within the bipolar spindle but are scattered and not compacted into a metaphase plate. Severe alignment defects are readily visible in 84% (±11%) of mitotic cells in dupa1mutants and 80% (±7%) of mitotic cells in dupa3mutants; it is probable that higher resolution analyses may reveal higher incidences of defective alignment. Because chromosome configuration in dup mutants deviates from normal configurations, other markers of mitotic progression were examined to determine at which stage in mitosis dup mutant cells arrest (Garner, 2001).
In normal mitosis, Cyclin A degradation concludes in metaphase while Cyclin B degradation concludes in early anaphase. Spindle checkpoint proteins such as Bub1 that bind kinetochores upon spindle damage localize to kinetochores during unperturbed mitosis in metazoa, indicating that the spindle checkpoint is active through earlier parts of mitosis. Drosophila Bub1 localizes on kinetochores during prometaphase and dissociates during metaphase (Basu, 1999). In dup mutants, both cyclins persist during mitotic arrest, and Bub1 is present on discrete sites on chromosomes, presumably at kinetochores. These data suggest that dup mutant cells arrest prior to metaphase-anaphase transition (Garner, 2001).
The persistence of Bub1 on chromosomes and stabilization of Cyclin B occurs when the spindle checkpoint is active; therefore, mitotic arrest in dup mutants may be mediated by the spindle checkpoint. To test this directly, double mutants of dupa3and bub1 were examined. dupa3bub1 double mutants have fewer mitotic cells when compared to dupa3 single mutant embryos of similar stage. Two types of additional evidence indicate that this difference is due to dupa3bub1 double mutants exiting M16 (rather than reverting to previous interphase). (1) Approximately 10 times more cells are seen in the act of exiting mitosis (i.e., in telophase) in the double mutants. Most telophase cells show chromosome bridges, consistent with the failure to complete DNA replication in these mutants. (2) Nuclear density is higher in dupa3bub1 double mutants than in dupa3 single mutants, and it approaches that of heterozygotes or wt controls that complete M16. Collectively, these data indicate that a significant number of dupa3bub1 mutant cells exited M16 and suggest that bub1 is required for mitotic arrest in dup mutants (Garner, 2001).
During mitotic arrest by the spindle checkpoint, Cyclin A is degraded while Cyclin B remains stable. Therefore, persistence of Cyclin A during mitotic arrest in dup mutants suggests that additional control(s), in addition to the spindle checkpoint, operate to stabilize Cyclin A. DNA damage leads to stabilization of Cyclin A in Drosophila. Possibly, the presence of incompletely replicated DNA during mitosis also leads to stabilization of Cyclin A. bub1-mediated controls, however, appear more consequential because dupa3bub1 double mutants exited mitosis. This would be consistent with the finding that Cyclin A at endogenous levels cannot block mitotic exit in Drosophila (Garner, 2001).
CDC6-deficient cells are reported to exit mitosis with normal kinetics. An examination of these cells, however, reveals a mitotic arrest that requires MAD2. Thus, incomplete DNA replication in both yeast and Drosophila results in mitotic arrest mediated by members of the spindle checkpoint. Why might this be? A complete or partial absence of sister chromosomes would lead to a complete or partial absence of sister chromatid cohesion. The failure to duplicate centromeres, likely in dupa3mutants and certainly in dupa1mutants, would preclude the formation of kinetochore pairs. Either deficiency would preclude stable bipolar attachment of chromosomes to the spindle and thereby activate the spindle checkpoint (Garner, 2001).
In summary, dup mutants demonstrate two ways in which mitosis is regulated in response to incomplete duplication of the genome: (1) entry into mitosis is delayed via mei-41, Drosophila ATR; (2) exit from mitosis is blocked via a spindle checkpoint function, bub1. In Drosophila syncytial cycles, nuclei delay the entry into mitosis upon inhibition of DNA synthesis, but exit from mitosis is not blocked. Instead, chromosome separation fails during the exit from mitosis, resulting in polyploid nuclei that are subsequently eliminated. In other systems, either the entry into mitosis (in fission yeast and vertebrate cells) or the exit from mitosis (in budding yeast) is blocked in response to incomplete DNA synthesis. Therefore, this is the first report of mitosis in a single cell cycle being regulated at two points via two different mechanisms in response to incomplete DNA replication. Identification of these responses in Drosophila, a genetically tractable organism with superb cytology, should enable testing of candidate checkpoint genes and searching for new genes that function at each regulatory point (Garner, 2001).
Successful mitosis requires that anaphase chromosomes sustain a commitment to move to their assigned spindle poles. This requires stable spindle attachment of anaphase kinetochores. Prior to anaphase, stable spindle attachment depends on tension created by opposing forces on sister kinetochores. Because tension is lost when kinetochores disjoin, stable attachment in anaphase must have a different basis. After expression of nondegradable cyclin B (CYC-BS) in Drosophila embryos, sister chromosomes disjoined normally but their anaphase behavior is abnormal. Chromosomes exhibit cycles of reorientation from one pole to the other. Additionally, the unpaired kinetochores accumulate attachments to both poles (merotelic attachments), congress (again) to a pseudometaphase plate, and reacquire associations with checkpoint proteins more characteristic of prometaphase kinetochores. Unpaired prometaphase kinetochores, which occur in a mutant entering mitosis with unreplicated (unpaired) chromosomes, behave just like the anaphase kinetochores at the CYC-BS arrest. Finally, the normal anaphase release of AuroraB/INCENP from kinetochores is blocked by CYC-BS expression and, reciprocally, is advanced in a CycB mutant. Given its established role in destabilizing kinetochore-microtubule interactions, Aurora B dissociation is likely to be key to the change in kinetochore behavior. These findings show that, in addition to loss of sister chromosome cohesion, successful anaphase requires a kinetochore behavioral transition triggered by CYC-B destruction (Parry, 2003).
When Drosophila cells enter anaphase in the presence of CYC-BS, poleward movement of unpaired chromosomes is abortive and chromosome behavior is unusual. It has been suggested that this chromosome behavior might represent an extension of prometaphase/metaphase behavior, differing only in so far as the loss of kinetochore pairing at metaphase/anaphase alters the behavior. The behavior of unpaired prometaphase kinetochores has been examined in a mutant in maize, exhibiting premature loss of chromosome pairing and after microsurgical production of single kinetochore chromosomes in mammalian cells. In these experiments, single-kinetochore chromosomes behaved much as the chromosomes of Drosophila cells that progress to anaphase (to produce unpaired kinetochores) in the presence of CYC-BS (Parry, 2003 and references therein).
To further test this parallel, the Drosophila mutant, double parked was examined, in which unpaired chromosomes exist in prometaphase. Double Parked is an essential replication protein that is also required for a checkpoint function that ordinarily prevents cells from entering mitosis with unreplicated DNA, and like analogous mutants in S. cerevisiae (e.g., cdc6), Drosophila cells lacking Double Parked enter mitosis with unreplicated DNA. When a maternal supply of Double Parked is depleted, replication fails in double parked embryos and cells accumulate in mitosis. The mitotic arrest occurs because unpaired chromosomes are incapable of normal bipolar alignment and consequently induce the spindle checkpoint (Parry, 2003).
In fixed images of the double parked arrest, most chromosomes are scattered along the spindle, with some clustered in a central pseudometaphase plate, just as in CYC-BS-arrested cells. Real-time analysis shows that this is a dynamic situation, with chromosomes making oscillatory movements between the poles. This chromosome movement between the poles resembles that observed during the CYC-BS block and is consistent with reorientation of the kinetochore from one pole to the other, as occurs for prometaphase chromosomes (Parry, 2003).
Despite the absence of prior replication, INCENP and Aurora B localize to the unpaired kinetochores in the double parked arrest, as in the CYC-BS arrest. Furthermore, despite the presence of only a single kinetochore, many of the chromosomes congress to a pseudometaphase plate in double parked and CYC-BS arrests. It is concluded that, when CYC-B persists, unpaired chromosomes behave similarly before and after the metaphase/anaphase transition (Parry, 2003).
Although it was somewhat puzzling that some chromosomes congressed to a pseudometaphase plate in double parked embryos, a similar observation was made when single kinetochore chromosomes were present in prometaphase in mammals. These congressed single kinetochore chromosomes have attachments to both poles (merotelic attachment). Robust kinetochore fibers are observed in double parked spindles, and in cases that are not confounded by the clustering of chromosomes in the middle, it is apparent that kinetochore fibers from both poles impinge on single kinetochores. These observations are interpreted as an indication of frequent merotelic attachment in the double parked arrest; similar findings have been noted in the CYC-BS-arrested cells (Parry, 2003).
The finding that merotelic attachments accumulate in the double parked arrest suggests that kinetochore pairing normally helps to prevent merotelic attachments under prometaphase conditions. It is suggested that such an effect could be explained by an extension of the idea that trial and error processes contribute to bipolar attachment of paired kinetochores in prometaphase. Because kinetochore-spindle interactions are unstable in prometaphase, all modes of attachment can be sampled, at least transiently, but the most stable mode ultimately predominates. Consequently, the most stable (correct bipolar attachment) precludes less stable and incorrect attachments. Spindle tension stabilizes attachment, and it has been suggested that, upon bipolar arrangement, tension deforms the paired kinetochore, effectively 'pulling' the attachment sites away from a centrally localized destabilizing activity. Although tension also deforms a merotelically attached kinetochore, it is suggested that the distortion is not as orderly as in bipolar attachment and that the separation from the destabilizing activity is less effective. Consequently, when kinetochores are paired, bipolar attachments will accumulate as the most stable outcome and hence exclude merotelic attachments. When kinetochores are unpaired, the dynamics of formation and decay of merotelic attachments appears to favor their accumulation (Parry, 2003).
Mitotic cells assume a spherical shape by increasing their surface tension and osmotic pressure by extensively reorganizing their interphase actin cytoskeleton into a cortical meshwork and their microtubules into the mitotic spindle. Mitotic entry is known to interfere with tissue morphogenetic events that require cell-shape changes controlled by the interphase cytoskeleton, such as apical constriction. However, this study shows that mitosis plays an active role in the epithelial invagination of the Drosophila tracheal placode. Invagination begins with a slow phase under the control of epidermal growth factor receptor (EGFR) signalling; in this process, the central apically constricted cells, which are surrounded by intercalating cells, form a shallow pit. This slow phase is followed by a fast phase, in which the pit is rapidly depressed, accompanied by mitotic entry, which leads to the internalization of all the cells in the placode. It was found that mitotic cell rounding, but not cell division, of the central cells in the placode is required to accelerate invagination, in conjunction with EGFR-induced myosin II contractility in the surrounding cells. It is proposed that mitotic cell rounding causes the epithelium to buckle under pressure and acts as a switch for morphogenetic transition at the appropriate time (Kondo, 2013).
The invagination of epithelial placodes converts flat sheets into the three-dimensional structures that form complex organs, and it is a key morphogenetic process in animal development. A major mechanism of invagination is apical constriction, which is driven by actomyosin contraction. However, not all constricted cells invaginate, and some cell internalization occurs without apical constriction, suggesting that additional mechanisms of inward cell movement contribute to invagination (Kondo, 2013).
To obtain three-dimensional information about cell behaviour during invagination, live imaging was performed of the Drosophila tracheal placode. Ten pairs of tracheal placodes, each of which is composed of about 40 cells, are formed in the ectoderm at mid-embryogenesis, and each placode initiates invagination simultaneously. Using an adherens junction marker, DE-cadherin-green fluorescent protein (E-cad-GFP), it was found that the adherens junctions of the central placode cells slowly created a depression by apical constriction, which became the tracheal pit. After 30 to 60 min of slow movement (slow phase), the tracheal pit was suddenly enlarged, and the tracheal cells were rapidly internalized (fast phase) and eventually formed L-shaped tube structures (Kondo, 2013).
After the fast transition, all the tracheal cells and surrounding epidermal cells entered mitosis 16, the final round of embryonic mitosis. It was noticed that the fast invagination was always associated with the mitotic entry of central cells that were frequently the first to enter mitosis 16. Intriguingly, mitotic rounding of the central constricted cells occurred simultaneously with the rapid depression of their apices, followed by chromosome condensation 10 min later. In this study, this atypical mitotic rounding associated with apical depression in an internalized cell is called 'rounding', to distinguish it from canonical surface mitosis (surface cell rounding) (Kondo, 2013).
To determine whether cell rounding is required for invagination, zygotic mutants were examined of the cell-cycle gene Cyclin A (CycA), which fail to enter mitosis 16, and double parkeda3 (dupa3), which show a prolonged S phase 16 and delayed entry into mitosis 16. Tracheal invagination was initiated normally in the CycA and dupa3 mutants, but proceeded more slowly than in controls, indicating that entry into mitosis 16 is required for proper timing of the fast phase (Kondo, 2013).
Although delayed, the accelerated invagination in the CycA or dupa3 mutants eventually occurred, allowing the formation of tube structures and suggesting that additional mechanisms are involved. After invagination, fibroblast growth factor (FGF) signalling is activated in the tracheal cells to induce branching morphogenesis through chemotaxis. To examine the contribution of FGF signalling to invagination, mutants of the FGF ligand branchless (bnl) or the FGF receptor breathless (btl) were analyzed. These mutants invaginated normally, indicating that chemoattraction to FGF is dispensable for invagination (Kondo, 2013).
Next, to assess FGF's role in the mitosis-defective condition, double mutants were analyzed for CycA and bnl or CycA and btl, and it was found that they showed slower invagination than CycA single mutants. Furthermore, the invagination in these double mutants was incomplete, in that the cells failed to form L-shaped tubular structures. Therefore, FGF signalling is critical for invagination when mitosis is blocked, serving a back-up role. Tracheal-specific CycA expression rescued the defects in invagination speed and tube structure in the CycA btl mutants. In addition, mitosis of cells outside the pit was occasionally observed that occurred before the mitosis of the central apically constricted cells and was not correlated with the fast invagination phase. Thus, mitosis of the surrounding epidermal cells is dispensable for tracheal invagination. Taken together, it is concluded that mitotic entry of central cells is a major mechanism for accelerating tracheal invagination (Kondo, 2013).
To distinguish the role of cell rounding from that of cell division in the fast phase, the microtubule inhibitor colchicine was used to arrest the cell cycle after cell rounding. Colchicine treatment after mitosis 15 induced M-phase arrest at mitosis 16, but the fast invagination movement accompanied by cell rounding was not affected. This result indicates that cell rounding, but not cell division, is responsible for the acceleration phase of the tracheal invagination (Kondo, 2013).
Mitosis of cells in the columnar epithelium normally occurs at the apical surface after surface rounding. It was next asked how the apical surface of the central cells becomes depressed during internalized cell rounding. One possible model explains internalized cell rounding as cell-autonomously controlled by the association of the cells with the basement membrane or underlying mesodermal cells. However, genetic removal of basement-membrane adhesion by the maternal and zygotic mutation of βPS-integrin (also known as mys) did not compromise the speed of invagination, and snail-twist double-mutant embryos, which lack mesodermal cells, still showed tracheal invagination with internalized cell rounding. These results suggest that anchoring to the basal side is probably not required (Kondo, 2013).
A second model proposes that the apical depression of the rounding cells is driven by local planar interactions among the tracheal cells. Before and during tracheal invagination, myosin II is enriched at the cell boundaries tangential to the centre of the placode and regulates cell intercalation. It was noted that the myosin II level in the central cells was lower than in the surrounding, intercalating cells. Nevertheless, the apices of the central cells were constricted during the slow phase, strongly suggesting that the surrounding cells exerted centripetal pressure on the central cells through myosin II cables. Myosin II cables fail to form in EGFR signalling mutants (such as rho, the rhomboid endopeptidase required for EGF ligand maturation, and Egfr), and apical constriction is impaired in these mutants. The first few cells undergoing mitosis 16 in the tracheal placode of rho or Egfr mutants showed surface cell rounding with expanded apices, indicating that EGFR signalling is required to couple the mitotic cell rounding with fast apical depression. It is speculated that the columnar shape of the central cells resists centripetal movements, resulting in the accumulation of inward pressure during the slow phase. The existence of such resistance was supported by the results of a physical perturbation experiment using a pulsed ultraviolet lase. The cell rounding associated with mitotic entry would release the stored inward pressure by means of cytoskeletal remodelling that causes rapid depression of apical surface together with the active shortening of cell height, leading to rapid buckling of the apical surface and the fast phase of invagination (Kondo, 2013).
Even with the loss of both EGFR and FGF signalling, the tracheal placodes form moderately invaginated structures, compared to the flat tracheal placode observed in the rho-bnl-CycA triple mutant at the same stage, indicating that cells needed to undergo mitosis 16 to induce invagination, independent of EGFR and FGF signalling. In rho bnl double mutants, although the cells undergoing the earliest mitoses showed surface cell rounding, some of the subsequent mitotic events were coupled to apical depression and internalized cell rounding. Unlike the earlier mitotic events on the surface, the internalized rounding cells in the rho bnl embryos showed constricted apices and were surrounded by apically rounded cells before mitosis. Internalized rounding with a constricted apical surface were shared properties of cells in mitoses leading to invagination, in both control and rho bnl embryos. It is suggested that the first few cells undergoing surface cell rounding compress the adjacent interphase cells and restrict their apical area, so that they are forced to move internally after rounding, causing the epithelial layer to buckle and invaginate (Kondo, 2013).
Although invagination was largely blocked in the rho-bnl-CycA triple mutants, any double mutant combination permitted invagination to some degree, indicating that three qualitatively distinct mechanisms, mitotic cell rounding, myosin II contractility (EGFR) and active cell motility (FGFR), can independently trigger invagination. In the normal context of wild-type development the combination of cell rounding and EGFR signalling may optimize the timing and speed of invagination, and then invaginated tracheal sacs subsequently respond to FGF emanating from several target tissues guiding branching morphogenesis (Kondo, 2013).
These observations demonstrates a new role for mitosis in tissue morphogenesis to generate mechanical force through cell rounding, independent of cell division. This is distinct from previously described invagination mechanisms involving cell-autonomous constriction by the apical activation of actomyosin contractility, which is incompatible with mitosis. Mitosis 16 outside the tracheal placode occurs in clusters on the ectoderm surface, but does not lead to invagination, suggesting that the tracheal placode is sensitized to invaginate upon mitosis, independent of EGFR and FGFR signalling. Future research to uncover the properties of the tracheal placode that enables it to respond to clustered mitosis will explain not only this new mode of morphogenesis, but also the homeostasis mechanisms of epithelial architecture (Kondo, 2013).
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