dMyt1 localization was examined in cultured cells. S2 cells were transiently transfected with pdMyt1/V5 (dMyt containing a C-terminal epitope tag) for 18 h followed by immunostaining for dMyt1/V5. dMyt1/V5 expression appeared as a punctate cytoplasmic staining similar to the pattern of Golgi. To compare this pattern directly to a Golgi marker, the cells were co-stained with an antibody specific for a Drosophila integral Golgi protein. When these images were merged, green and red fluorescence overlap were observed as yellow. dMyt1/V5 expression correlated with Golgi, although not completely, and may have been a result of dMyt1/V5 expression in alternate Golgi subcompartments or in the endoplasmic reticulum. A DAPI counterstain was provided to indicate the nucleus. To control for the possibility that the V5 tag was inappropriately showing the Golgi localization, expression of Dif containing a C-terminal V5 tag was examined. Unlike dMyt1/V5, it was found that Dif/V5 was expressed evenly throughout the cytoplasm. These results suggest that dMyt1 subcellular localization is overlapping with Golgi, and similar to that of human Myt1 (Cornwell, 2002).
Because dMyt1 kinase appears to be cytoplasmically expressed in a pattern similar to the Golgi, the impact of the loss of dMyt1 on Golgi was examined. S2 cells were RNAi-treated for 3 and 7 days and immunostained for Golgi. S2 cells in interphase have an intact Golgi that appears with punctate staining for the Golgi marker. During normal mitosis, the Golgi apparatus fragments and can be observed as an elimination of punctate staining with a diffusion of fluorescent staining. However, in S2 cells that have been dMyt1 RNAi-treated, a modest reduction, but not a complete loss of punctate staining, was observed in mitotic cells. These results were consistent throughout mitosis. Furthermore, 3 days post-RNAi, a similar pattern of incompletely grouped Golgi vesicles was observed in S2 cells lacking both dMyt1 and dWee1 kinases. By day 7, the nuclei of dMyt1/dWee1 RNAi-treated cells appeared significantly smaller with fragmented Golgi and were also reduced significantly in viability. These results suggest that the absence of both dMyt1 and dWee1 kinases may be driving the cells into mitotic catastrophe (Cornwell, 2002).
Eukaryotic organisms use conserved checkpoint mechanisms that regulate Cdk1 by inhibitory phosphorylation to prevent mitosis from interfering with DNA replication or repair. In metazoans, this checkpoint mechanism is also used for coordinating mitosis with dynamic developmental processes. Inhibitory phosphorylation of Cdk1 is catalyzed by Wee1 kinases that phosphorylate tyrosine 15 (Y15) and dual-specificity Myt1 kinases found only in metazoans that phosphorylate Y15 and the adjacent threonine (T14) residue. Despite partially redundant roles in Cdk1 inhibitory phosphorylation, Wee1 and Myt1 serve specialized developmental functions that are not well understood. Wild type and phospho-acceptor mutant Cdk1 proteins were expresses in order to investigate how biochemical differences in Cdk1 inhibitory phosphorylation influence Drosophila imaginal development. Phosphorylation of Cdk1 on Y15 appeared to be crucial for developmental and DNA damage-induced G2 phase checkpoint arrest, consistent with other evidence that Myt1 is the major Y15-directed Cdk1 inhibitory kinase at this stage of development. Expression of non-inhibitable Cdk1 also caused chromosome defects in larval neuroblasts that were not observed with Cdk1(Y15F) mutant proteins that were phosphorylated on T14, implicating Myt1 in a novel mechanism promoting genome stability. Collectively, these results suggest that dual inhibitory phosphorylation of Cdk1 by Myt1 serves at least two functions during development. Phosphorylation of Y15 is essential for the pre-mitotic checkpoint mechanism, whereas T14 phosphorylation facilitates accumulation of dually inhibited Cdk1-Cyclin B complexes that can be rapidly activated once checkpoint-arrested G2 phase cells are ready for mitosis (Ayeni, 2013).
Aging influences stem cells, but the processes involved remain unclear. Insulin signaling, which controls cellular nutrient sensing and organismal aging, regulates the G2 phase of Drosophila female germ line stem cell (GSC) division cycle in response to diet; furthermore, this signaling pathway is attenuated with age. The role of insulin signaling in GSCs as organisms age, however, is also unclear. This study reports that aging results in the accumulation of tumorous GSCs, accompanied by a decline in GSC number and proliferation rate. Intriguingly, GSC loss with age is hastened by either accelerating (through eliminating expression of Myt1, a cell cycle inhibitory regulator) or delaying (through mutation of insulin receptor (dinR) GSC division, implying that disrupted cell cycle progression and insulin signaling contribute to age-dependent GSC loss. As flies age, DNA damage accumulates in GSCs, and the S phase of the GSC cell cycle is prolonged. In addition, GSC tumors (which escape the normal stem cell regulatory microenvironment, known as the niche) still respond to aging in a similar manner to normal GSCs, suggesting that niche signals are not required for GSCs to sense or respond to aging. Finally, GSCs from mated and unmated females behave similarly, indicating that female GSC-male communication does not affect GSCs with age. These results indicate the differential effects of aging and diet mediated by insulin signaling on the stem cell division cycle, highlight the complexity of the regulation of stem cell aging, and describe a link between ovarian cancer and aging (Kao, 2014).
Although aging results in a decline in stem cell proliferation, relatively few studies have addressed how stem cell cycle progression is altered by aging. DNA damage is mainly induced by by-products of cellular metabolism, such as reactive oxygen species (ROS) and environmentally induced lesions upon irradiation. Accumulation of irreversible genomic DNA damage has been implicated as a prominent cause of aging, both at the organismal and at the cellular levels. Cells respond to DNA damage by activating checkpoint pathways, which delay cell cycle progression and allow for repair of the defects. This study observed that aged GSCs exhibit accumulation of DNA damage and a prolonged S phase, suggesting that the former may be responsible for the latter in GSCs during aging. (Kao, 2015).
DNA breaks result in activation of ATM/ATR kinases (ataxia-telangiectasia mutated and Rad3 related), which phosphorylate a variant of histone H2A (H2AX); this histone variant is a critical factor in facilitating the assembly of specific DNA-repair complexes on damaged DNA. ATM/ATR kinase-mediated signaling is part of the intra-S phase checkpoint pathway, and its activation is often associated with a delay in S phase progression. However, ATR heterozygous mutant (mei-41D3/+) GSCs still exhibited a similar degree of S phase delay compared to wild-type, suggesting that ATR may be dispensable for age-induced S phase delay, although it is possible that disruption of one copy of ATR may not be sufficient to block the intra-S check point pathway (Kao, 2015).
Surprisingly, it was observed that there was a 65% increase of aged tufeatm-8/+ (atm heterozygous mutant) GSCs in S phase (1.98-fold increase relative to young tufeatm-8/+ GSCs), as compared to its sibling controls at the same age (1.33-fold increase relative to young control GSCs). Coincidently, a recent publication on Drosophila reported that ATM functions in DNA damage repair and exerts negative feedback control over the level of programmed double strand breaks (DSBs) during meiosis, and thus the number of H2AX foci (a marker of DNA damage) is dramatically increased in tufeatm-8 mutant germ cells. It was speculated that tufeatm-8/+ GSCs may induce more DNA damage via feedback regulation, thereby causing more severe S phase delay. However, in mice, Atm-/- undifferentiated spermatogonia are not maintained in the testis due to DNA damage-induced cell cycle G1 arrest, suggesting that ATM may function in the G1 phase in response to DNA damage. Nevertheless, it remains to be elucidated whether ATM mediates different cell cycle regulators in different cell contexts or in response to different types of stress-induced DNA damage (Kao, 2015).
With age, cells may accumulate DNA mutations that allow them to escape normal regulatory processes and become tumor cells. Although tumorigenesis is harmful to health in the long term, it may also serve as a survival and protective mechanism when the body is highly threatened. While the germarium normally houses differentiating 8- or 16-germ cell cysts interconnected with branched fusomes, this study found that the middle portion of the aged germarium was occupied by tumor-like GSCs, which express pMad (a Dpp signaling effector) and possess rounded fusomes. This result recalls an earlier report that forced stemness Dpp signaling causes differentiating germ cell cysts to revert into functional stem cells in Drosophila ovaries, through the induction of ring canal closure and fusome scission (Kao, 2015).
It has also been reported that aged human epidermal cells can dedifferentiate into stem cell-like cells via Wnt/β-catenin signaling, and injury can drive the dedifferentiation of epidermal cells via the β-integrin-mediated signaling pathway; these findings suggest that dedifferentiation is a process by which organisms address aging or tissue damage. Given that GSCs play a fundamental role in producing the next generation, this study suspects that these tumor-like GSCs may be derived from germ cell cysts through a dedifferentiation process triggered by aging; however, the possibility that these tumor-like GSCs are derived from the transformation of normal GSCs could not be ruled out (Kao, 2015).
In invertebrates, including C. elegans and Drosophila, mating is detrimental to the lifespan of females, to increase progeny production. In Drosophila, mating females die earlier than unmated females, and sex peptides, produced from the male accessory gland, may be responsible for this effect. In C. elegans, females shrink and die after mating, and this is partially due to the stimulation of GSC proliferation by sperm. This study, however, did not observe differences in GSC proliferation rates between mated and unmated females at any age, suggesting that the promotion of GSC proliferation by mating may be specific to C. elegans. In addition, the results also indicate that sex peptides do not affect GSCs, at least at the level of proliferation. Moreover, similar rates of aging-induced GSC loss were observed in mated and unmated females, suggesting that mating does not affect the physiological status of GSCs (Kao, 2015).
Entry into mitosis is regulated by inhibitory phosphorylation of cdc2/cyclin B, and these phosphorylations can be mediated by the Wee kinase family. This study presents the identification of Drosophila Myt1 kinase and examines the relationship of Myt1 and Wee activities in the context of Cdc2 phosphorylation. Myt1 kinase was found by BLAST-searching the complete Drosophila genome using the amino acid sequence of human Myt1 kinase. A single predicted polypeptide was identified that shared a 48% identity within the kinase domain with human and Xenopus Myt1. Consistent with its putative role as negative regulator of mitotic entry, overexpression of this protein in Drosophila S2 cells results in a reduced rate of cellular proliferation while the loss of expression via RNA interference (RNAi) results in an increased rate of proliferation. In addition, loss of Myt1 alone or in combination with Drosophila Wee1 (Wee1) results in a reduction of cells in G2/M phase and an increase in G1 phase cells. Finally, loss of Myt1 alone results in a significant reduction of phosphorylation of cdc2 on the threonine-14 (Thr-14) residue as expected. Surprisingly however, a reduction in the phosphorylation of Cdc2 on the tyrosine-15 (Tyr-15) residue is observed only when expression of both Myt1 and Wee expression is reduced via RNAi and not loss of expression of Wee alone. Most strikingly, in the absence of Myt1, Golgi fragmentation during mitosis is incomplete. These findings suggest that Myt1 and Wee have distinct roles in the regulation of Cdc2 phosphorylation and the regulation of mitotic events (Cornwell, 2002).
This study shows that loss of dMyt1 expression results in reduced phosphorylation of the Thr-14 residue of cdc2, an increased rate of cell proliferation, and a reduction of cells in G2/M with an increased number of cells in G1. The most biologically interesting observation was that loss of dMyt1 resulted in the incomplete fragmentation of the Golgi in mitotic cells. Taken together, these results indicate that dMyt1 kinase is involved in cdc2 regulation and necessary for proper Golgi fragmentation in mitosis (Cornwell, 2002).
RNAi in Drosophila S2 cells was used as a means to provide insight into the functional role of dMyt1 and dWee1 in the cell cycle beyond previously published biochemical studies. Consistent with Myt1 biochemical studies (i.e., negative regulation of cdc2/cyclin B, it was found that loss of dMyt1 expression but not of other genes (dWee1, Polo, or Dif) affects the phosphorylation state of cdc2 at the Thr-14 residue. The loss of dWee1 alone is insufficient to result in a reduced phosphorylation of the cdc2 Tyr-15 residue. These results are interesting in the context of previous biochemical analyses demonstrating that Myt1, although capable of phosphorylating both Thr-14 and Tyr-15, has a preference for phosphorylation of cdc2/Thr-14 and Wee1 is restricted to phosphorylation of cdc2/Tyr-15 only. However, the data suggest that in S2 cells the role of dMyt1 in Tyr-15 phosphorylation is greater than anticipated since loss of dWee1 did not significantly reduce Tyr-15 phosphorylation. Perhaps, this dual specific activity of dMyt1 is necessary to ensure that cdc2 localized to cytoplasmic compartments is fully inhibited until the onset of mitosis. Additional evidence that dMyt1 is a Myt1 kinase ortholog came from recombinant expression of tagged dMyt1 and localization to the Golgi (Cornwell, 2002).
Additional consequences were observed with the loss of dMyt1 expression, such as, alterations in the cell cycle. It was found that S2 cells RNAi-treated for dMyt1 contained fewer cells in G2/M by FACS and an increased number of mitotic cells via immunostaining. This suggests (as would be predicted) that the loss of dMyt1 shortens the G2 phase of the cell cycle by allowing premature activation of cdc2/cyclin B (Cornwell, 2002).
Because dMyt1 kinase is expressed in discrete cytoplasmic subcompartments that appear to overlap with the Golgi and active cdc2 has been shown to phosphorylate several Golgi-specific proteins at the onset of mitosis, it was of interest to exploring the impact on the Golgi following the loss of dMyt1 expression. At the onset of mitosis, the immunofluorescent vesicular (punctate-stained) Golgi is redistributed to a dispersed uniform staining pattern. Most interestingly, in S2 cells RNAi-treated for dMyt1, the Golgi staining pattern fails to redistribute and this phenotype persists throughout mitosis. This suggests that the Golgi is not devesiculating in the normal manner that occurs during mitosis. Several possible explanations to this observation are proposed. Although dMyt1 is a negative regulator of cdc2, it may also be required to sequester cdc2 to the Golgi. Previous studies have shown that Myt1 and cdc2/cyclin B can physically associate via domains other than the kinase domain. Myt1 may act as a sink to localize cdc2 to the Golgi or to certain Golgi compartments so that at mitosis cdc2/cyclin B is present and once activated can phosphorylate Golgi proteins before being transported to the nucleus. Alternatively, prematurely active cdc2 (due to the absence of dMyt1) drives events that block subsequent steps of the Golgi devesiculation process. Finally, these two models may overlap. The question of cdc2 transport and localization in the absence of dMyt1 are obvious and will be the focus of future studies. However, these data do not exclude the possibility that Myt1 has a novel function unrelated to cdc2 regulation that is required for Golgi devesiculation (Cornwell, 2002).
Wee1 kinases catalyze inhibitory phosphorylation of the mitotic regulator Cdk1, preventing mitosis during S phase and delaying it in response to DNA damage or developmental signals during G2. Unlike yeast, metazoans have two distinct Wee1-like kinases, a nuclear protein (Wee1) and a cytoplasmic protein (Myt1). The genes encoding Drosophila Wee1 and Myt1 have been isolated and genetic approaches are being used to dissect their functions during normal development. Overexpression of Dwee1 or Dmyt1 during eye development generates a rough adult eye phenotype. The phenotype can be modified by altering the gene dosage of known regulators of the G2/M transition, suggesting that these transgenic strains can be used in modifier screens to identify potential regulators of Wee1 and Myt1. To confirm this idea, a collection of deletions for loci that can modify the eye overexpression phenotypes was tested and several loci were identified as dominant modifiers. Mutations affecting the Delta/Notch signaling pathway strongly enhance a GMR-Dmyt1 eye phenotype but do not affect a GMR-Dwee1 eye phenotype, suggesting that Myt1 is potentially a downstream target for Notch activity during eye development. Interactions with p53 were observed, suggesting that Wee1 and Myt1 activity can block apoptosis (Price, 2002).
The G1/S and G2/M cell cycle transitions are temporally and spatially controlled during metazoan development, allowing growth and cell division to be coordinated with patterning and differentiation. Studies of G2/M checkpoint controls in metazoans have emphasized regulatory mechanisms affecting the Cdc25-like phosphatases, which activate the mitotic regulator Cdk1 by removing inhibitory phosphorylation. Regulatory mechanisms affecting the activity and protein stability of the Cdk1 inhibitory kinases are still poorly understood, but are probably just as important. There are ample precedents for these mechanisms from studies of Wee1 and Mik1 kinases in S. pombe and SWE1 in S. cerevisiae (Price, 2002).
During the third larval instar, the Drosophila eye disc undergoes progressive transformation from a relatively amorphous epithelial sac into the complex arrangement of ommatidial facets that comprises the adult compound eye. This transformation is marked by passage of a constriction called the morphogenetic furrow (MF) across the eye disc. Cells within the MF normally arrest in G1 and failure to synchronize cells at this stage disrupts ommatidial patterning. Following the MF, a population of cells called the second mitotic wave (SMW) undergoes a final cell cycle. If cells are blocked in G1 by overexpression of a p21 CKI homolog, insufficient cells are left to form all of the cell types required for normal ommatidia, resulting in a rough adult eye phenotype. In this report, GMR-driven misexpression of Dmyt1 immediately after the MF both delays the SMW divisions and reduces the numbers of mitotic cells, also resulting in a rough eye phenotype (Price, 2002).
Dwee1 and Dmyt1 overexpression eye phenotypes are sensitive to modification by mutations in known cell cycle regulatory genes, illustrating the feasibility of screening for mutations of genes that are potential regulators of either Wee1 or Myt1. Mutations in genes that promote mitosis, such as cdc2 and cdc25string, should dominantly enhance these overexpression phenotypes and this expectation was confirmed for both of these genes with Dmyt1. Although a GMR-Dwee1 eye phenotype is also enhanced by mutations in cdc2, it is not enhanced by mutations in cdc25string, providing evidence that Wee1 and Myt1 kinases have distinct Cdk1 regulatory effects in this developmental context. This result could be explained by a requirement for higher levels of cdc25string activity to overcome GMR-Dmyt1 inhibition of Cdk1 relative to GMR-Dwee1, perhaps because it is inherently more difficult to dephosphorylate Cdk1 inhibited on both T14 and Y15 by Myt1 activity, compared with Cdk1 inhibited on Y15 alone by Wee1 (Price, 2002).
The rux gene encodes a novel Cdk1 inhibitor that controls the onset of S phase during embryogenesis, eye development, and spermatogenesis. rux also plays a novel role in mitosis, by an unknown mechanism. Rux and Wee1 both negatively regulate Cdk1 activity; thus the observation that coexpression of these genes generates more extreme rough eye phenotypes than seen with either alone is consistent with known functions for these genes. Surprisingly, flies lacking both zygotic Dwee1 and rux functions show nearly complete synthetic lethality, with rare escapers exhibiting extensive adult bristle phenotypes. This interaction suggests that rux and Dwee1 may also cooperate in some other, as yet undefined regulatory mechanism. The extensive bristle phenotypes seen in rux;Dwee1 double mutant escapers could indicate disruption of cell cycle timing or abrogation of genome integrity checkpoints, similar to the phenotypes seen in mus304 mutants exposed to ionizing radiation, which are associated with increased genome instability. Another piece of evidence suggesting a role for Wee1 kinases in regulating genome stability is the interaction observed with Drosophila p53. In humans, the p53 tumor suppressor promotes apoptosis in cells that have suffered DNA damage. Overexpression of Drosophila p53 in the eye promotes extensive cell death by apoptosis, resulting in extremely defective eyes. There is significant suppression of the p53 overexpression eye phenotype by coexpression of either GMR-Dwee1 or GMR-Dmyt1, suggesting that these Cdk1 inhibitory kinases can negatively regulate p53-induced apoptosis. Since Cdk1 activity has been implicated in promoting apoptosis, this effect would be consistent with known functions of Wee1 and Myt1 in Cdk1 inhibition. Other reports relevant to this issue are somewhat contradictory, however. In human cell culture, Wee1 can inhibit granzyme B-induced apoptosis; furthermore, Wee1 appears to be downregulated through a p53-dependent mechanism, suggesting that p53 regulation of Wee1 might normally occur during this process. In contrast, Wee1 activity can actually promote apoptosis in a Xenopus oocyte extract system. Further studies are clearly needed to establish the physiological significance of any purported roles for Wee1 or Myt1 in regulating apoptosis, p53-dependent or otherwise (Price, 2002).
A screen for modulators of wee1 overexpression has been conducted in S. pombe, by isolating suppressors of wee1-induced lethality. These studies identified mutations in the gene encoding the Hsp90 chaperone as potent suppressors, suggesting a role for Hsp90 in promoting the assembly and/or disassembly of functional Wee1 protein complexes. In contrast, no hsp83 mutant alleles (encoding Drosophila Hsp90) were found to act as suppressors of a combined GMR-Dmyt1/GMR-Dwee1 transgene eye phenotype. Several other genetic loci have been identified as specific enhancers of eye phenotypes generated by GMR-Dwee1 or GMR-Dmyt1 alone, indicating that phenotypic effects mediated by Wee1 and Myt1 are responsive to lowered expression of different genes. These observations may reflect differences in threshold requirements for the relevant gene products in promoting mitosis (as suggested by the interactions with cdc25string) or they may signify differences in the regulation of Wee1 and Myt1 kinases that it will now be possible to dissect by identifying and characterizing the relevant modifier loci. Direct genetic screens to address this issue are being undertaken for mutations in genes that modify GMR-Dwee1 and GMR-Myt1 eye phenotypes. One of the loci identified as a specific enhancer of the GMR-Dmyt1 eye phenotype is Delta. This interaction could reflect defects in Dl-dependent neuronal specification that are enhanced by GMR-Dmyt1 activity, or it may indicate a novel role for Delta/Notch signaling in regulating Myt1 activity (Price, 2002).
In S. pombe, the DNA damage and DNA replication checkpoint pathways that regulate Cdk1 by inhibitory phosphorylation act by controlling the activity and stability of Wee1 and Mik1 kinases, as well as Cdc25 phosphatases. Although metazoan homologs of components of these checkpoint pathways show significant sequence conservation with their yeast homologs, the actual functions and interactions of individual components are not necessarily conserved. For example, Xenopus homologs of the checkpoint kinases Chk1 and Cds1, which respond to DNA damage and block DNA replication, respectively, in S. pombe, respond in the exact opposite manner to these stresses in Xenopus egg extracts. This example serves as a warning that simple predictions of metazoan gene function based on extrapolation from known functions of yeast genes can be misleading. Metazoan development requires that novel regulatory mechanisms exist to link specific developmental processes with the basic cell cycle machinery. Drosophila represents an ideal model for analyzing these developmental controls of the cell cycle, since the effects of specific mutations on complex processes like morphogenesis and differentiation can be established. The recent characterization of the tribbles gene in Drosophila illustrates this point. Trbl activity delays mitosis in invaginating G2 cells (mitotic domain 10) in a cycle 14 embryo. Although cdc25string transcription initiates in domain 10 before it is transcribed in other cells, these cells remain G2 arrested until they are completely internalized, well after cells in nine other mitotic domains have subsequently expressed cdc25string and entered mitosis. Trbl activity downregulates Cdc25string protein stability, providing an explanation for these observations. A similar purpose could be served by Trbl simultaneously upregulating Dwee1 or Dmyt1 activity. Intriguingly, Trbl contains motifs reminiscent of Nim1-type kinases, which negatively regulate Wee1 and Swe1 kinase activity and stability in S. pombe and S. cerevisiae. Despite these sequence similarities, the Trbl protein apparently lacks a functional catalytic domain, raising the possibility that Trbl could act in a 'dominant negative' manner to activate Wee1 (or Myt1) by interfering with the activities of Nim1-like inhibitors. Genetic interactions described in this study are consistent with this possibility (Price, 2002).
Two myt1 mutant alleles (originally designated as myt11 and myt12) were isolated in a genetic screen for hemizygous mutants with phenotypic defects that could be rescued by a P{myt1+} transgene. These alleles exhibit markedly different viability as hemizygotes; however, these differences were removed by out-crossing, indicating they were due to secondary lesions. Viable hemizygous myt1 mutants [myt/Df(3L)64D-F] exhibit bristle defects affecting the dorsal thorax, head and eye, and are male sterile. Although myt1 females are fertile, variable maternal effect lethal embryonic phenotypes were observed in their progeny. Genomic sequencing of the myt1 alleles identified identical mutations in each: a single nucleotide deletion at position 514 (amino acid 173). The fact that EMS mutagenesis usually causes CG-->TA transitions, combined with the unlikelihood that this mutation would have occurred twice independently, suggests that a spontaneous mutation occurred in the previously isogenized stock used for the screen. The myt1 mutation is predicted to cause a frame-shift alteration in the sequence of the protein, followed by a premature stop codon at nucleotide 689 (amino acid 232). This would truncate the protein within the kinase domain and also delete other conserved sequence motifs near the C terminus of the protein, suggesting that the mutants are likely functionally null. Moreover, myt1/Df(3L)64D-F hemizygotes display identical phenotypes as transheterozygous combinations of the original alleles, fulfilling classical genetic criteria that these myt1 alleles are functionally amorphic (Jin, 2005).
The male sterility of myt1 mutants led to a search for specific cell cycle defects during spermatogenesis, Male germline development begins with stem cell divisions that generate gonialblasts, which then undergo four synchronous mitotic divisions to produce cysts of 16 primary spermatocytes. These primary spermatocytes remain in G2 phase for ~90 hours before undergoing meiotic divisions to produce cysts containing 64 syncytial spermatids that differentiate into mature sperm. To analyze how loss of Myt1 function affects these cell divisions, an antibody was used that recognizes a phosphorylated form of histone H3 (PH3) as a marker for mitotic or meiotic cells. In control testes, small numbers of mitotic cells were usually seen near the tip of the testis. More distally along a control testis, one often observes a single PH3-positive meiotic cyst. A striking increase was observed in the numbers of PH3-positive cells in myt1 mutants. In addition to clearly demarcated germline cysts, isolated PH3-positive cells were observed along the length of myt1 mutant testes, as well as PH3-positive cells at the distal end of the testes that were never seen in controls. These cell proliferation defects were suppressed and male fertility was restored when a P{myt1+} transgene was introduced into the myt1 mutant background, confirming that these mutant phenotypes were due to a loss of Myt1 activity. The adult bristle phenotype observed in myt1 mutants was also rescued by this transgene (Jin, 2005).
To determine if there are other proliferation defects observable in the myt1 mutants, BrdU incorporation to assay for DNA replication was used in short-term (30 minute) cultures of dissected testes. BrdU incorporation was seen only in the cells near the tip of the testes in the controls, implying that pre-meiotic S phase is essentially complete by the time the primary spermatocyte cysts move away from the tip. A marked increase in BrdU-incorporating cells was observed near the apical tip of the testes in the myt1 mutants, relative to controls. These observations could be explained if cells in the myt1 mutants cycle faster than normal, or if they continue to cycle instead of undergoing developmental cell cycle arrest. Delays during S phase and mitosis could also contribute to these effects (Jin, 2005).
Male-sterile mutants with overproliferation defects can result from germline stem cells (GSC) or spermatogonia failing to differentiate properly, so that they continue to proliferate instead of entering meiosis. To determine if overproliferation in myt1 mutants is attributable to similar defects, established cell fate markers were used to examine germline stem cells, spermatogonia and spermatocytes. myt1 mutants have normal numbers of germline stem cells, assessed by immunostaining for the germline-specific marker Vasa and Fas3, which marks the somatic hub that GSCs associate with. There was, however, a significant increase in secondary spermatogonial cells marked by antibodies against BamC in the myt1 mutants, relative to controls. This could occur if the secondary spermatogonia undergo one or more extra rounds of cell division, in which case PH3-positive spermatogonial cysts with 16 or more cells would be expected (Jin, 2005).
To test this possibility, a BamC and PH3 colocalization experiment was performed. In controls, cysts were never observed containing more than eight cells that were both BamC and PH3 positive, consistent with spermatogonia only undergoing four mitotic divisions. By contrast, ~30% of the myt1 mutant testes examined contained at least one 16-cell cyst that was both BamC and PH3-positive, implying that these spermatogonia were undergoing an extra round of cell division. To further test this idea, germ cell cysts were examined by phase contrast microscopy, and the numbers of cells in each cyst were quantified. As expected, ~10% of the mutant cysts contain twice the expected numbers of primary spermatocytes or spermatids, a phenotype that is never seen in controls. In both 64-cell and 128-cell myt1 mutant spermatid cysts, there was consistent evidence of variable, aberrant-looking nuclei and nebenkern. These observations suggest that loss of Myt1 activity affects segregation of chromosomes and mitochondria during meiosis, in addition to the mitotic defects described earlier (Jin, 2005).
Next, whether additional defects in the cell cycle behavior of somatic cells might contribute to the myt1 over-proliferation phenotype was examined. Somatic stem cells located at the apical tip of the testes divide to generate cyst cells whose fate is intimately coupled with male germline development. Two cyst cells associate with each gonialblast and remain associated with the descendant cyst for the remainder of spermatogenesis. Normally, these somatic cyst cells do not undergo further cell division, suggesting that their differentiation is coupled with exit from the cell cycle. Antibodies against Eya were used to mark the cyst cells. There was a marked increase in the number of cyst cell nuclei in the myt1 mutants, relative to controls. Because the cyst cells are quiescent, they are PH3-negative in the controls. In myt1 mutants, however, cyst cell nuclei can be double-labeled with antibodies to PH3 and Eya. When Aly was used as a marker for spermatocytes, Aly-positive cysts with more than two Eya-positive cyst nuclei were never observed in controls, but were often seen in the mutants, implying that these extra nuclei remain associated with their germline cysts during meiosis. In addition to the ectopic division of cyst cells, it was also observed that terminal epithelial cells located at the distal end of the testes ectopically label with PH3 antibodies, unlike controls. These observations further distinguish myt1 mutants from previously described male-sterile over-proliferation mutants and implicate Myt1 in a molecular mechanism that promotes cell cycle exit during terminal differentiation. These defects may affect the ability of cyst and terminal cells to provide essential cell signaling or other support functions to their associated germline cells. Such effects could conceivably compromise sperm maturation or translocation and contribute to the observed sterility of male myt1 mutants. Mature sperm translocate into the seminal vesicle after spermatid differentiation and can be visualized by DNA staining. In myt1 mutants, the seminal vesicle appeared to be empty (Jin, 2005).
Previous studies in Drosophila have demonstrated that regulation of entry into mitosis and meiosis is controlled by inhibitory phosphorylation of Cdk1. Given that Myt1 is a Cdk1 inhibitory kinase, it was expected that phenotypic defects of myt1 mutants would be due to a defect in Cdk1 regulation. To test this idea, a heat shock-inducible, non-inhibitable allele of Cdk1 (hs-Cdk1AF) was expressed in testes, to see if it would phenocopy any of the defects observed in myt1 mutants. Expression of this transgene has previously been used to bypass a developmentally regulated G2 arrest in embryonic germline cells. As predicted, heat-shock induced expression of Cdk1AF caused germline over-proliferation defects similar to those seen in myt1 mutants. These included increased numbers of PH3-positive cells, relative to controls. It was confirmed, by BamC antibody co-localization, that some of these PH3-positive cells in the Cdk1AF-expressing testes were secondary spermatogonia undergoing an extra round of mitosis. Induction of hs-Cdk1AF also phenocopied the defects seen in germline-associated somatic cyst cells labeled with Eya. These results demonstrate that Myt1 inhibitory phosphorylation of Cdk1 is required for regulating multiple aspects of cell cycle behavior during spermatogenesis (Jin, 2005).
Female myt1 mutants are fertile; however, a high incidence of early lethality in maternally affected mutant embryos suggests that Myt1 might also function during oogenesis. Oogenesis initiates with stem cell divisions that produce cystoblasts that then undergo four synchronous mitotic divisions, to generate 16 cell cysts. A single cell in each cyst differentiates into an oocyte and progresses into prophase of meiosis I, where it remains arrested until ovulation. The 15 remaining cells in the cyst differentiate as nurse cells. Two or three germline stem cells (GSCs) are located at the tip of the germarium, each containing a ball-shaped fusome-related structure called a spectrosome. Fusomes are germline-specific membranous organelles that interconnect the cyst cells and are thought to coordinate their mitotic cell divisions. Using the position of GSCs and antibodies against Hts to label spectrosomes, it was determined that the number of GSCs is comparable in myt1 mutants and controls. The numbers of dividing GSCs, cystoblasts and cystocytes were counted, and a mitotic index was calculated for each cell type, using antibodies against Cnn and Hts to mark spindle poles and spectrosomes (or fusomes, in cystocytes), respectively, as well as the DNA-labeling dye Hoechst 33258 to mark condensed mitotic chromosomes. Female GSCs typically undergo one cell cycle per day, consequently mitotic GSCs are rarely observed and the mitotic index is very low. Only two mitotic GSCs were found in 130 germaria from control ovarioles. By contrast, 15 mitotic GSCs were found among 50 myt1 mutant germaria, a 20-fold increase in the mitotic index. The mutant cystoblasts and their cystocyte descendants also had a significantly higher mitotic index than normal, so it was not uncommon to find a metaphase stem cell and a metaphase cystoblast or cystocyte in a single myt1 mutant germarium. In the controls, at most a single dividing cyst was seen in each germarium. These results show that loss of myt1 activity causes germline overproliferation in females, as well as males. No egg chambers were observed with greater than 16 cells in myt1 mutants, however, indicating that ectopic germline cell divisions do not account for this defect, in females (Jin, 2005).
Oocyte and nurse cell differentiation appeared normal in the myt1 mutants, as assessed by antibody staining of Orb and Gurken in oocytes, and by nuclear morphology of the nurse cells. When myt1 mutant germaria were examined with antibodies against the Vasa germline marker, a significant increase was noted in the number of cysts (one- to two-fold), relative to controls. Although myt1 mutant cystoblasts and cystocytes are similar in size to controls, the GSCs appear slightly smaller. These data suggest that myt1 mutant GSCs are cycling more rapidly and therefore produce more germline cysts. As the differences are not as extreme as the GSC mitotic index measurements would predict, these results also imply that compensatory delays probably occur during these mitotic cell cycles that can account for this discrepancy (Jin, 2005).
To investigate whether there are effects on homologous chromosome segregation during female meiosis in myt1 mutant females, standard genetic tests were undertaken to identify non-disjunction (NDJ) events. These tests show that loss of Myt1 causes elevated non-disjunction, implicating Myt1 as a regulator of female meiosis. The NDJ frequency for myt1 mutants is much higher than controls for the X chromosome and the 4th chromosome. Further experiments indicated that exceptional progeny derive from NDJ during meiosis I in myt1 females. These data demonstrate that loss of Myt1 activity compromises female meiosis, specifically meiosis I (Jin, 2005).
Female germline cells are associated with somatic follicle cells derived from stem cell precursors. When each 16-cell germline cyst buds off from the germarium as an egg chamber, a layer of undifferentiated follicle cells surrounds it. These follicle cells then differentiate into functionally distinct subclasses. The stalk cells (located between each egg chamber) and the polar cells (located at each end of the chamber), cease dividing immediately after the egg chamber forms, whereas the remaining follicle cells proliferate asynchronously until stage 6 of oogenesis. Accordingly, early egg chambers have only small numbers of PH3-positive follicle cells. In myt1 mutant egg chambers, there was a marked increase in PH3-positive follicle cells before stage 6, as well as ectopic PH3-positive follicle cells after stage 6. Curiously, these ectopic PH3-positive follicle cells primarily appeared at the anterior and posterior ends of each egg chamber (Jin, 2005).
Each of the four major types of follicle cells can be distinguished by their cell shape and by expression of distinct molecular markers. Stalk cells have a unique disc-like shape and inter-egg chamber location; polar cells are located at the end of each egg chamber and express Fas3 before stage 9; border cells maintain Fas3 expression and migrate towards the posterior after stage 9, and stretched cells extend over the 15 nurse cells and express Eya. By these criteria, the different follicle cell types all appeared to be represented in myt1 mutants; however, unlike the controls, some of these cells were PH3 positive, suggesting that they were undergoing ectopic cell divisions. Consistent with this interpretation, there were more Eya-expressing cells in the mutants than in controls by stage 9, indicating that some of these cells are able to complete cell division. The typical 'stretched' morphology characteristic of this cell type was disrupted, presumably as a result of cytoskeleton reorganization accompanying mitosis. Also ectopic PH3-positive main body follicle cells were observed in mutant egg chambers after stage 9, long after these cells normally cease dividing. Thus, loss of Myt1 function causes germline-associated somatic cells to undergo ectopic cell division, in both males and females (Jin, 2005).
Mitosis is triggered by activation of Cdk1, a cyclin-dependent kinase. Conserved checkpoint mechanisms normally inhibit Cdk1 by inhibitory phosphorylation during interphase, ensuring that DNA replication and repair is completed before cells begin mitosis. In metazoans, this regulatory mechanism is also used to coordinate cell division with critical developmental processes, such as cell invagination. Two types of Cdk1 inhibitory kinases have been found in metazoans. They differ in subcellular localization and Cdk1 target-site specificity: one (Wee1) being nuclear and the other (Myt1), membrane-associated and cytoplasmic. Drosophila has one representative of each: dMyt1 and dWee1. Although dWee1 and dMyt1 are not essential for zygotic viability, loss of both resulted in synthetic lethality, indicating that they are partially functionally redundant. Bristle defects in myt1 mutant adult flies prompted a phenotypic analysis that revealed cell-cycle defects, ectopic apoptosis, and abnormal responses to ionizing radiation in the myt1 mutant imaginal wing discs that give rise to these mechanosensory organs. Cdk1 inhibitory phosphorylation was also aberrant in these myt1 mutant imaginal wing discs, indicating that dMyt1 serves Cdk1 regulatory functions that are important both for normal cell-cycle progression and for coordinating mitosis with critical developmental processes (Jin, 2008).
Multicellular organisms regulate Cdk1 by inhibitory phosphorylation to prevent mitosis when DNA is being replicated or repaired and to ensure that mitosis does not interfere with critical developmental processes that require remodeling of the cytoskeleton. Previous studies of Drosophila Wee1 and Myt1 revealed that these conserved Cdk1 inhibitory kinases were required during early embryogenesis and gametogenesis, respectively. This study has characterized imaginal and adult developmental defects caused by loss of dMyt1 activity (and to a much lesser extent, dWee1), that confirm the importance of Cdk1 inhibitory phosphorylation for coordinating cell-cycle events with critical developmental processes (Jin, 2008).
In Drosophila and other organisms, G2/M delays can be induced by overexpression of Myt1 kinases, suggesting a specific role for Myt1 in regulating this stage of the cell cycle. Further evidence of a role for Myt1 in G2/M regulation comes from studies of oocyte maturation in frogs, starfish, and nematodes. Not all data indicate that Myt1 is required for G2 phase arrest, however, and there is no evidence that dMyt1 regulates oocyte maturation in Drosophila. Nor is there evidence that dMyt1 activity is responsible for the timing of the G2/M meiotic transition that follows a prolonged 4-day-long G2 phase arrest, in Drosophila primary spermatocytes. Moreover, a recent study showed that functional depletion of human Myt1 by siRNA did not affect the proportion of cells in G2 phase, but instead affected membrane dynamics during mitotic exit (Nakajima, 2008). More needs to be learned about Myt1 mediated regulatory mechanisms before these apparent discrepancies in Myt1 functions are resolved (Jin, 2008).
Previous work showed that Cdk1 inhibitory phosphorylation is required for proper development of thoracic mechanosensory organs. This study has now identified dMyt1 as the primary Cdk1 inhibitory kinase for this developmental program. Several molecular mechanisms could explain the role of dMyt1 in mechanosensory bristle development. One obvious possibility is that myt1 mutant sensory organ precursor (SOP) cells and their descendants might divide prematurely due to a defect in G2/M regulation, resulting in aberrant segregation of cell fate determinants. If there was a relatively narrow window for coordinating specific developmental events with the G2/M transition, disrupting this regulatory mechanism could account for the observed loss and duplication of bristles and socket cells in myt1 mutants. Live analysis of mechanosensory organ development could test this possibility (Jin, 2008).
Alternatively, myt1 mutant phenotypes could reflect defects in Myt1-mediated regulatory mechanisms that are important for the control of intracellular membrane dynamics during mitosis, particularly the Golgi apparatus and endoplasmic reticulum. The Drosophila Golgi apparatus undergoes significant morphological changes that have been linked to specific developmental states and so the observed myt1 mutant developmental defects might reflect problems in the structure or function of this organelle. Further support for this idea comes from a recent study showing that asymmetrical segregation of mouse Numb (a conserved cell fate determinant) requires the Golgi apparatus, leading to the suggestion that Golgi fragmentation and reconstitution could represent a mechanism for coupling cell-fate specification and cell-cycle progression (Jin, 2008).
Another possible explanation for myt1 mutant defects concerns the large quantities of actin that are synthesized and packaged to form the large mechanosensory bristle shafts. This process involves extensive reorganization of the endoplasmic reticulum and Golgi apparatus to accommodate increased membrane trafficking. Defects in the structure or function of the Golgi apparatus and ER caused by loss of dMyt1 activity could therefore account for defects or diminution in these bristles. Resolving which of these potential mechanisms best explain the role of dMyt1 during mechanosensory organ development will be a major challenge of future research (Jin, 2008).
Intriguing cell-cycle defects (higher mitotic index, aberrant chromatin condensation, and ectopic apoptosis), as well as defects in responses to ionizing radiation in proliferating cells, were observed in myt1 mutant imaginal wing discs. These observations suggest an important role for dMyt1 in conserved cell-cycle checkpoint responses that target Cdk1 by inhibitory phosphorylation. It was not anticipated that dMyt1 would serve such functions, since Wee1 kinases are generally assumed to be responsible for checkpoint responses that protect the nucleus from premature Cdk1 activity. It was not clear that myt1 mutants were deficient in conventional premitotic checkpoint responses, however. Indeed, the partial decline in myt1 mutant PH3-labeled cells observed immediately after exposure to ionizing radiation could reflect activation of an otherwise dispensable Wee1-regulated premitotic checkpoint mechanism. The remaining PH3-positive cells that persisted long after irradiation in myt1 mutant discs could be arrested in mitosis by an alternative regulatory mechanism that was responsive to DNA damage. Further studies will be needed to clarify the respective roles of dMyt1 and dWee1 in cellular responses to DNA damage (Jin, 2008).
This study also observed profound defects in Cdk1 inhibitory phosphorylation in myt1 mutant imaginal discs. Phosphorylation of the T14 residue of Cdk1 was eliminated, demonstrating that dMyt1 is solely responsible for this regulatory modification, like Myt1 homologs described in other organisms. It was also observed that phosphorylation of the Y15 residue of Cdk1 was markedly reduced in myt1 mutant extracts, demonstrating for the first time that dMyt1 functions as a dual specificity Cdk1 inhibitory kinase, in vivo. Why dWee1 activity is insufficient for maintaining normal levels of phosphorylation of the Y15 residue is not clear, since Cdk1 complexes are thought to shuttle between the nucleus and cytoplasm. One possible explanation is that the doubly phosphorylated Cdk1 isoform may be more refractory to dephosphorylation by Cdc25 phosphatases, and hence more stably inhibited, than Cdk1 phosphorylated on a single residue. Another possibility is that the kinase-independent Myt1 mechanism proposed to tether phospho-inhibited Cdk1 complexes in the cytoplasm until cells are ready for mitosis might also protect them from dephosphorylation. Loss of either of these regulatory mechanisms could therefore underlie the cell-cycle defects observed in myt1 mutants. Testing these hypotheses promises to yield interesting new insights into cell-cycle regulation and the diverse developmental roles of dMyt1 and similar regulatory kinases in other organisms (Jin, 2008).
Ayeni, J. O., Varadarajan, R., Mukherjee, O., Stuart, D. T., Sprenger, F., Srayko, M. and Campbell, S. D. (2013). Dual phosphorylation of Cdk1 coordinates cell proliferation with key developmental processes in Drosophila. Genetics 196(1): 197-210. PubMed ID: 24214341
Booher, R. N., Holman, P. S. and Fattaey, A. (1997). Human Myt1 is a cell cycle-regulated kinase that inhibits Cdc2 but not Cdk2 activity. J. Biol. Chem. 272: 22300-22306. 9268380
Burrows, A. E., et al. (2006). The C. elegans Myt1 ortholog is required for the proper timing of oocyte maturation. Development 133(4): 697-709. 16421191
Cornwell, W. D., Kaminski, P. J. and Jackson, J. R. (2002). Identification of Drosophila Myt1 kinase and its role in Golgi during mitosis. Cell Signal 14(5): 467-76. 11882391
Furuno, N., Kawasaki, A. and Sagata, N. (2003). Expression of cell-cycle regulators during Xenopus oogenesis. Gene. Expr. Patterns 3: 165-168. 12711544
Gaffré, M., et al. (2011). A critical balance between Cyclin B synthesis and Myt1 activity controls meiosis entry in Xenopus oocytes. Development 138(17): 3735-44. PubMed Citation: 21795279
Inoue, D. and Sagata, N. (2005). The Polo-like kinase Plx1 interacts with and inhibits Myt1 after fertilization of Xenopus eggs. EMBO J. 24: 1057-1067. 15692562
Jackman, M., Lindon, C., Nigg, E. A. and Pines, J. (2003). Active cyclin B1-Cdk1 first appears on centrosomes in prophase. Nat. Cell Biol. 5: 143-148. 12524548
Jin, Z., Homola, E. M., Goldbach, P., Choi, Y., Brill, J. A. and Campbell, S. D. (2005). Drosophila Myt1 is a Cdk1 inhibitory kinase that regulates multiple aspects of cell cycle behavior during gametogenesis. Development 132(18): 4075-85. 16107480
Jin, Z., Homola, E., Tiong, S. and Campbell, S. D. (2008). Drosophila myt1 is the major cdk1 inhibitory kinase for wing imaginal disc development. Genetics 180(4): 2123-33. PubMed Citation: 18940789
Kao, S. H., Tseng, C. Y., Wan, C. L., Su, Y. H., Hsieh, C. C., Pi, H. and Hsu, H. J. (2014). Aging and insulin signaling differentially control normal and tumorous germline stem cells. Aging Cell 14(1): 25-34. PubMed ID: 25470527
Kalous, J., Solc, P., Baran, V., Kubelka, M., Schultz, R. M. and Motlik, J. (2005). PKB/AKT is involved in resumption of meiosis in mouse oocytes. Biol Cell. 98(2): 111-23. 15842198
Karaiskou, A., Lepretre, A. C., Pahlavan, G., Du Pasquier, D., Ozon, R. and Jessus, C. (2004). Polo-like kinase confers MPF autoamplification competence to growing Xenopus oocytes. Development 131: 1543-1552. 14985258
Kramer, A., Mailand, N., Lukas, C., Syljuasen, R. G., Wilkinson, C. J., Nigg, E. A., Bartek, J. and Lukas, J. (2004). Centrosome-associated Chk1 prevents premature activation of cyclin-B-Cdk1 kinase. Nat. Cell Biol. 6: 884-891. 15311285
Lamitina, S. T. and L'Hernault, S. W. (2002). Dominant mutations in the Caenorhabditis elegans Myt1 ortholog wee-1.3 reveal a novel domain that controls M-phase entry during spermatogenesis. Development 129: 5009-5018. 12397109
Leise, W. F. and Mueller, P. R. (2002). Multiple Cdk1 inhibitory kinases regulate the cell cycle during development. Dev. Bio. 249: 156-173. 12217326
Liu, F., Stanton, J. J., Wu, Z. and Piwnica-Worms, H. (1997). The human Myt1 kinase preferentially phosphorylates Cdc2 on threonine 14 and localizes to the endoplasmic reticulum and Golgi complex. Mol. Cell. Biol. 17: 571-583. 9001210
Liu, F., Rothblum-Oviatt, C., Ryan, C. E. and Piwnica-Worms, H. (1999). Overproduction of human Myt1 kinase induces a G2 cell cycle delay by interfering with the intracellular trafficking of Cdc2-cyclin B1 complexes. Mol. Cell. Biol. 19(7): 5113-23. 10373560
Mueller, P. R., Coleman, T. R., Kumagai, A. and Dunphy, W. G. (1995). Myt1: a membrane-associated inhibitory kinase that phosphorylates Cdc2 on both threonine-14 and tyrosine-15. Science 270: 86-90. 7569953
Murakami, M. S., Moody, S. A., Daar, I. O. and Morrison, D. K. (2004). Morphogenesis during Xenopus gastrulation requires Wee1-mediated inhibition of cell proliferation. Development 131: 571-580. 14711880
Nakajima, H., Toyoshima-Morimoto, F., Taniguchi, E. and Nishida, E. (2003). Identification of a consensus motif for Plk (Polo-like kinase) phosphorylation reveals Myt1 as a Plk1 substrate. J. Biol. Chem. 278(28): 25277-80. 12738781
Nakajima, H., et al. (2008). Myt1 protein kinase is essential for Golgi and ER assembly during mitotic exit. J. Cell Biol. 181: 89-103. PubMed Citation: 18378775
Nakajo, N., Yoshitome, S., Iwashita, J., Iida, M., Uto, K., Ueno, S., Okamoto, K. and Sagata, N. (2000). Absence of Wee1 ensures the meiotic cell cycle in Xenopus oocytes. Genes Dev. 14: 328-338. 10673504
Nakanishi, M., Ando, H., Watanabe, N., Kitamura, K., Ito, K., Okayama, H., Miyamoto, T., Agui, T. and Sasaki, M. (2000). Identification and characterization of human Wee1B, a new member of the Wee1 family of Cdk-inhibitory kinases. Genes Cells 5: 839-847. 11029659
Okamoto, K., Nakajo, N. and Sagata, N. (2002). The existence of two distinct Wee1 isoforms in Xenopus: implications for the developmental regulation of the cell cycle. EMBO J. 21: 2472-2484. 12006499
Okano-Uchida, T., Okumura, E., Iwashita, M., Yoshida, H., Tachibana, K. and Kishimoto, T. (2003). Distinct regulators for Plk1 activation in starfish meiotic and early embryonic cycles. EMBO J. 22: 5633-5642. 14532135
Okumura, E., Fukuhara, T., Yoshida, H., Hanada Si, S., Kozutsumi, R., Mori, M., Tachibana, K. and Kishimoto, T. (2002). Akt inhibits Myt1 in the signalling pathway that leads to meiotic G2/M-phase transition. Nat. Cell Biol. 4(2): 111-116. 11802161
Palmer, A., Gavin, A. C. and Nebreda, A. R. (1998). A link between MAP kinase and p34(cdc2)/cyclin B during oocyte maturation: p90(rsk) phosphorylates and inactivates the p34(cdc2) inhibitory kinase Myt1. EMBO J. 17(17): 5037-47. 9724639
Peter, M., et al. (2002). A new role for Mos in Xenopus oocyte maturation: targeting Myt1 independently of MAPK. Development 129: 2129-2139. 11959823
Price, D. M., Jin, Z., Rabinovitch, S. and Campbell, S. D. (2002). Ectopic expression of the Drosophila cdk1 inhibitory kinases, Wee1 and Myt1, interferes with the second mitotic wave and disrupts pattern formation during eye development. Genetics 161: 721-731. 12072468
Wells, N. J., Watanabe, N., Tokusumi, T., Jiang, W., Verdecia, M. A. and Hunter, T. (1999). The C-terminal domain of the Cdc2 inhibitory kinase Myt1 interacts with Cdc2 complexes and is required for inhibition of G(2)/M progression. J. Cell Sci. 112: 3361-3371. 10504341
Wilson, M. A., Hoch, R. V., Ashcroft, N. R., Kosinski, M. E. and Golden, A. (1999). A Caenorhabditis elegans wee1 homolog is expressed in a temporally and spatially restricted pattern during embryonic development. Biochim. Biophys. Acta 1445: 99-109. 10209262
date revised: 10 February 2015
Home page: The Interactive Fly © 2006 Thomas Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.