The Interactive Fly
Zygotically transcribed genes
How does the cell cycle function?
Molecular dissection of cytokinesis by RNA interference in Drosophila cultured cells
Identification of pathways regulating cell size and cell-cycle progression by RNAi
Cell Cycle in Drosophila
Drosophila development is characterized by the existence of four types of cycles of DNA replication, each appearing successively after fertilization. These four different cell cycles are: cleavage, post-cellularization mitosis, neurogenesis and endoreplication.
Immediately after fertilization, prior to cellularization, 13 cleavage cycles occur giving rise to the syncytial blastoderm. Nuclear division cycles occur rapidly and there is no G1 or G2 phase, that is DNA synthesis and mitosis alternate without pause. This type of cell division is driven entirely by maternally derived gene products.
The second type of replicative cycle, post-cellularization mitosis, occurs during cycles 14, 15 and 16. After cellularization cells no longer share a common pool of cell cycle regulatory proteins. The zygotically regulated cycles have a variable G2 phase (between DNA replication and cell division), but have no detectable G1 phase (following mitosis). Division during these three cycles follows a well defined spatio-temporal pattern that correlates with organ and tissue type. Progression through these cycles is regulated by String, a fly homolog of yeast phosphatase cdc25.
A third type of cell cycle occurs in neuroblasts and some other cells that continues after cycles 14, 15 and 16. After mitosis 16, most cells arrest in the G2 phase of cell cycle except for neuroblasts, cells in imaginal disc primordia, Malpighian tubules and gut. Cells in the nervous system continue to divide following the 16th mitosis, undergoing between five and eight additional divisions (Richardson, 1993 and references). Division of cells during neurogenesis is regulated by Cyclin E and is accompanied by a G1 phase, following mitosis.
The fourth type of replicative cycle, the endoreplication cycle,
refers to sequential rounds of replication that proceed in the absence
of cell division. This generates polytene chromosomes in which the DNA
may be duplicated many times over. Endoreplication occurs in larval salivary
gland cells and in nurse cells during oogenesis (see escargot). Larval salivary gland chromosomes undergo a endoreduplication. For information about polytene chromsomes, see Polytene chromosomes, endoreduplication and puffing.
The cell cycle is considered to start at the transition between G1, the ground state following mitosis, and S, the DNA synthesis phase of the cell cycle. This transition, a highly regulated event, is called Start, and commits the cell to the completion of a full mitotic cycle. A short tour of the cell cycle is given below. Seven processes are described: 1) Cyclin E promotion of transition from G1 to S; 2) E2F promotion of the S phase; 3) The promotion of G2-M transition by Cyclin A and Cyclin B; 4) the regulation of the Cyclin B/CDC2 heterodimer by String; 5) polo kinase regulation of progression through mitosis; 6) the termination of mitosis by fizzy, and 7) factors effecting G1 arrest in differentiating cells.
1) Cyclin E, a G1 specific Cyclin regulating entrance into S phase is present among maternal mRNAs, and translated immediately after fertilization. Sufficient Cyclin E is available to carry zygotic cells through cleavage, without pause for the first 13 mitotic cycles. If Cyclin E is caused to be limited during cleavage, these mitotic cycles stop. During cellularization, following cleavage, maternal Cyclin E degrades, bringing to an end the process of cleavage.
During cycles 14-16, zygotic Cyclin E mRNA shows no cell cycle associated variation and the level of Cyclin E expression is independent of cell cycle. In the peripheral nervous system, during mitosis following the 16th cycle, Cyclin E expression occurs during G1 in a brief pulse that initiates and overlaps with S phase (Richardson, 1993). Cyclin E is down-regulated during the final mitotic cycle of differentiating cells (Knoblich, 1994). Cyclin E and its associated cdc2c kinase are not normally inactivated during the G2 phase. Continued presence of Cyclin E and its partner cdc2c kinase could function to prohibit induction of a second S phase during the G2 phase, thus preventing endoreduplication of DNA (Sauer, 1995).
Mammalian cells are held in G1 by the action of RB, a product of retinoblastoma gene. RB binds to and inactivates the transcription factor E2F. Inactivation of RB at the G1-S transition promotes entry into S phase. A Drosophila protein, DP, is developmentally regulated in later embryonic stages and preferentially expressed in proliferating cells. The expression of Drosophila E2F in later stage embryos occurs in a segmentally restricted group of neural cells, whereas it is widely expressed in early embryos (Hao, 1995).
2) E2F and DP represent a protein complex that acts as a transcriptional activator responsible for the S phase properties of proliferating cells (Dynlacht, 1994). The E2F transcription factor is required for S phase in Drosophila. Cyclin E expression at G1-S requires E2F. Ectopic expression of Cyclin E can bypass the requirement for E2F for the induction of S phase. This implies that Cyclin E is a downstream gene of E2F. In rapidly proliferating cells of the CNS, the hierarchical relationship between Cyclin E and E2F is reversed and Cyclin E activates E2F in the CNS. These rapidly dividing cells show no obvious G1 phase (Duronio, 1995).
3)There are poorly defined mechanisms for detecting the end of the S phase, as well as mechanisms to assure that each chromosomal segment is replicated only once. After the end of S, both Cyclin A and Cyclin B, known as G2 Cyclins, are required for entry into and successful completion of mitosis. The dimerization partner of these two Cyclins is cdc2 kinase. When flies are generated that are mutant in both Cyclin A and B, cell cycle progression is blocked just before the exhaustion of the maternally contributed Cyclin A and B stores (Knoblich, 1993). The partner of Cyclins A and B, cdc2 kinase, is held inactive during S phase by phosphorylation. In mammals, this inhibitory phosphorylation is carried out by Wee1 and Mlk kinases. The heterodimer between Cyclin A and Cdc2 (the cyclin dependent kinase) is kept at high levels in abdominal histoblasts by the action of escargot, preventing endoreduplication and consequent polyploidy (Hayashi, 1996).
4) In Drosophila, the Cyclin B/cdc2 kinase complex is activated just before mitosis. string gene encodes a phosphatase activity which is rate limiting for entry into postblastodermal mitosis. The G2-M transition is the only differentially regulated transition in cell cycle progression during these cycles, and String activity is rate limiting for the transition. Conversely, String cannot induce mitosis during S phase, indicating that a mechanism exists for preventing this from happening. By removing phosphate residues from cdc2 kinase, String activates Cyclin/cdc2 kinase and promotes S phase in dividing Drosophila cells (Edgar, 1990). During interphase 14, programmed degradation of maternal String protein leads to inhibitory phosphorylation of cdc2 and cell cycle arrest. Subsequently, mitotic phases 14-16 are triggered by pulses of zygotic string transcription (Edgar, 1994). 5)
The polo gene product accumulates in mitotic cells late in mitosis during anaphase and telophase. Mutations in polo cause abnormal mitotic and meiotic divisions. The polo mutation results in a high mitotic index in larval neuroblasts with a wide range of defects including monopolar spindles and spindles with broad poles. This suggests that polo mutants may regulate crucial-to-cell-division microtubule behavior. Polo-like kinases have been isolated from organisms as diverse as yeast and humans (Fenton, 1993).
6) Exit from mitosis requires inactivation of cdc2 kinase. Inactivation results from proleotylic degradation of the regulatory Cyclins A and B during mitosis. Mutations in the Drosophila gene fizzy block the mitotic degradation of these Cyclins. In an appealing model, Sigrist (1995) suggests that successive degradation of Cyclins A and B, each having a different role in promoting sequential mitotic steps (chromosome separation, regulation and telophase), allows for exit from mitosis.
7) This overview of mitosis in Drosophila concludes with G1, the post-mitotic state of dividing cells. In mammals, cessation of mitosis and initiation of differentiation is regulated by Cyclin-dependent kinase inhibitors. One example is P21 (C1P1/WAF1). P21 homologs have not yet been isolated in non-mammalian species, so it is perhaps too early to estimate the importance of kinase inhibitors in promotion of differentiation. Human P21 has been expressed in the Drosophila eye to discover what the effect would be on eye differentiation. When mitosis is blocked in mitotic eye cells by human P21, cell differentiation takes place according to each cellís predetermined fate. P21 expression does not alter cell fate, but it is able to block cell cycle progression. This suggests that the mechanism of inhibition of cell cycle progression by P21 is general and conserved across species, and that similar mechanisms act to promote differentiation in the fly (de Nooij, 1995).
Double-stranded RNA-mediated interference (RNAi) was used to study Drosophila cytokinesis. Double-stranded RNAs for anillin, RacGAP50C, pavarotti, rho1, pebble, spaghetti squash, syntaxin1A, and twinstar all disrupt cytokinesis in S2 tissue culture cells, causing gene-specific phenotypes. The phenotypic analyses identify genes required for different aspects of cytokinesis, such as central spindle formation, actin accumulation at the cell equator, contractile ring assembly or disassembly, and membrane behavior. Moreover, the cytological phenotypes elicited by RNAi reveal simultaneous disruption of multiple aspects of cytokinesis. These phenotypes suggest interactions between central spindle microtubules, the actin-based contractile ring, and the plasma membrane, and led to a proposal that the central spindle and the contractile ring are interdependent structures. Finally, these results indicate that RNAi in S2 cells is a highly efficient method to detect cytokinetic genes, and predict that genome-wide studies using this method will permit identification of the majority of genes involved in Drosophila mitotic cytokinesis (Somma, 2002).
The finding that chicadee, four wheel drive (fwd), and Kinesin-like protein at 3A (klp3A) are not required for cytokinesis in S2 cells is not surprising, because previous studies pointed toward a specific involvement of these genes in meiotic cytokinesis of males. Null mutations in klp3A, a gene encoding a kinesin-like protein expressed both in testes and somatic tissues, disrupt meiotic cytokinesis but have no effect on larval neuroblast division. Similarly, flies homozygous for null mutations in fwd, which encodes a phosphatidyl-inositol kinase, are viable but male sterile, and are specifically defective in male meiotic cytokinesis. In contrast with fwd and klp3A that are not required for viability, chic is an essential gene that specifies a Drosophila homolog of profilin. However, both male sterile chic mutants and heteroallelic chic combinations resulting in lethality, display severe disruptions in meiotic cytokinesis but have no defects in neuroblast cytokinesis (Somma, 2002).
It was initially surprising to find that RNAi depletion of the Pnut protein, which shares homology with the yeast septins, does not markedly affect cytokinesis in S2 cells. This protein concentrates in the cleavage furrow of several Drosophila cell types; null pnut mutants die at the larval/pupal boundary and exhibit polyploid cells in their brains, consistent with a defect in cytokinesis. It is possible that the lack of an effect in pnut (RNAi) cells reflects a small amount of residual Pnut protein in these cells. However, it is instead believed that Pnut's role in cytokinesis is not fundamental to the process. The larval brains of null pnut mutants were reexamined and the presence of polyploid cells was confirmed. However, polyploid cells represent only 10.5% of the mitotic figures, indicating that most neuroblasts can undergo cytokinesis even in the absence of Pnut. In addition, Pnut is not required for cytokinesis during either male meiosis or the cystoblast divisions in the female germline. Taken together, these findings indicate that the Pnut function is either partially or totally dispensable for cytokinesis in Drosophila (Somma, 2002).
The phenotypical analyses of RNAi-induced mutants in the RacGAP50C, rho1, and sqh genes provide the first description of the cytological defects that lead to cytokinesis failures when the function of these genes is ablated. Previous studies have shown that mutations in rho1 and sqh disrupt mitotic cytokinesis but have not defined the cytological phenotypes elicited by these mutations. In addition, pav and pbl (RNAi) cells have been characterized; the phenotypes of these (RNAi) cells are consistent with those previously observed in animals homozygous for mutations in these genes (Somma, 2002 and references therein).
Cells in which the RacGAP50C, pav, pbl, rho1, and sqh genes are ablated by RNAi normally undergo anaphase A, but they then fail to elongate and to undergo anaphase B. After anaphase A, mutant cells proceed toward telophase and decondense their chromosomes, forming typical telophase nuclei. However, these cells fail to develop a central spindle, to assemble an actomyosin contractile ring and to concentrate anillin in the cleavage furrow. This results in the formation of short, aberrant telophases that are unable to undergo cytokinesis and will thus give rise to binucleated cells (Somma, 2002).
The functional ablation of genes influencing either the actin or the microtubule cytoskeleton have similar effects on cytokinesis. The genes pbl, rho1, and sqh likely play primary roles in controlling the actin cytoskeleton. The sqh gene encodes a regulatory light chain of myosin II. Rho1 is a member of the Rho family GTPases that cycle from an inactive GDP-bound state to an active GTP-bound state under the regulation of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). GEFs enhance the exchange of bound GDP for GTP, whereas GAPs increase the GTPase activity of Rho. Rho proteins and Rho GEFs, such as Drosophila Pbl and human ECT2, localize to the cleavage furrow and are required for contractile ring assembly. In contrast, the activities of RacGAP50C and pav are likely to primarily influence the function of the central spindle. The Pav kinesin-like protein, a homolog of the C. elegans ZEN-4, is localized in the central spindle, and is thought to mediate microtubule cross-linking at the central spindle midzone. The RacGAP50C gene encodes a Rho GAP, and it is orthologous to the cyk-4 gene of C. elegans. CYK-4 interacts with ZEN-4, and the two proteins are mutually dependent for their localization to the central spindle. The complete absence of Pav immunostaining in RacGAP50C (RNAi) telophases suggests a similar interaction between RacGAP50C and Pav, pointing to a role of RacGAP50C in central spindle assembly. In summary, the cytological phenotypes of pbl, rho1, and sqh (RNAi) cells indicate that a primary defect in acto-myosin ring formation results in a secondary defect in central spindle assembly. The phenotypes of RacGAP50C- and Pav-depleted cells suggest the converse: that a primary defect in the central spindle can secondarily disrupt contractile ring formation. Thus, taken together, these data indicate that the central spindle and the actomyosin ring are interrelated structures. Although the molecular mechanisms underlying the cross talk between these structures is not conpletely understood, two possibilities can be envisioned. The formation and maintenance of both the central spindle and the actomyosin ring could be mediated by physical interactions between interzonal microtubules and components of the contractile ring. Alternatively, the central spindle and the contractile ring could be coupled by a checkpoint-like regulatory mechanism, which would inhibit the formation of either of these structures when the other is not properly assembled (Somma, 2002).
Although RacGAP50C, pav, pbl, rho1, and sqh (RNAi) cells display similar terminal phenotypes, the aberrant telophases observed in these cultures differ in both actin and anillin distribution. In rho1 telophases these proteins are excluded from the cell equator, in pbl they are uniformly distributed, and in RacGAP50C, pav, and sqh they concentrate in a wide equatorial band. This suggests that rho1 and pbl are required for actin and anillin accumulation in the equatorial region of the dividing cell. In contrast RacGAP50C, pav, and sqh seem to be required for the assembly of the contractile machinery from proteins already concentrated at the cell equator. In sqh (RNAi) cells the failure to assemble an actomyosin ring is likely to be a direct consequence of the depletion of an essential component of the ring. In RacGAP50C and pav cells this failure is instead likely to be a secondary effect of problems in central spindle assembly (Somma, 2002).
An interplay between the central spindle and the contractile ring has been suggested by studies on Drosophila male meiosis. Mutant spermatocytes in the chic, and dia loci, which encode products thought to be involved in contractile ring formation, and mutants in the kinesin-encoding gene klp3A, all display severe defects in both structures. Although all the extant results on Drosophila cells strongly suggest an interdependence of the central spindle and the contractile ring, it is currently unclear whether this is true in all animal cells. Studies on mammalian cells have shown that central spindle plays an essential role during cytokinesis. However, these experiments have provided limited information on whether perturbations in the actomyosin ring assembly disrupt the central spindle. The best evidence of an interplay between the central spindle and the contractile ring has been in rat kidney cells. By puncturing these cells with a blunt needle a physical barrier is created between the central spindle and the equatorial cortex. This barrier not only abrogates actomyosin ring assembly on the side of perforation facing the cortex, but also disrupts the organization of central spindle microtubules on the opposite side (Somma, 2002 and references therein).
In contrast, studies on C. elegans embryos indicate that, at least in the early stages of cytokinesis, the actomyosin ring and the central spindle can assemble independently. Why do Drosophila, and possibly mammalian cells, differ from C. elegans in the interactions between the central spindle and the contractile ring? It is believed that the answer to this question reflects differences in the distance between the central spindle and the equatorial cortex. In Drosophila and mammalian cells during central spindle assembly the equatorial cortex is very close to the interzonal microtubules. In contrast, in C. elegans embryos the central spindle assembles in the center of the cell when the cleavage furrow has just began to ingress, so that during their assembly the actomyosin ring and the central spindle lie a considerable distance apart. Only later in cell division, after substantial furrow ingression, can the actomyosin ring and the central spindle come into contact. It is thus hypothesized that in embryonic cells of C. elegans the cytokinetic process consists of two steps: an early step, where the central spindle and the contractile ring assemble independently in distant cellular regions, and a late step that begins when the central spindle and the contractile ring have come into contact. The early stage might be mediated by interactions between astral (rather than central spindle) microtubules and the contractile ring. The late step of C. elegans cytokinesis may then require that the contractile ring and the central spindle interact cooperatively to complete cytokinesis successfully. This two-step hypothesis also applies to other large cells, such as echinoderm eggs, where the central spindle and the cortex are separated by large masses of cytoplasmic material and seem to assemble independently (Somma, 2002 and references therein).
The syx1A gene, which encodes a t-SNARE, plays an essential role in embryonic cellularization, but its direct role in cytokinesis has not been demonstrated. In syx1A (RNAi) cells approximately half of the telophases are shorter that those of control cells and display severe defects in both the central spindle and the contractile ring. These findings are rather surprising, because there is abundant evidence that syntaxins are specifically involved in membrane fusion processes. Thus, the observations on syx1A (RNAi) cells raise the question of how a defect in membrane formation can affect both the central spindle and contractile ring assembly. Studies of C. elegans embryos depleted of the cytokinesis-specific Syntaxin-4 protein by RNAi have shown that in some of these embryos there is a complete failure of cleavage furrow ingression, suggesting an underlying defect in the contractile ring machinery. It has been thus proposed that formation of new membrane may positively regulate contractile ring assembly. In agreement with this hypothesis, it is suggested that RNAi-induced Syx1A depletion in S2 cells disrupts membrane formation at the site of cleavage furrow, causing a secondary defect in contractile ring formation and thus also in central spindle assembly (Somma, 2002).
Many high-throughput loss-of-function analyses of the eukaryotic cell cycle have relied on the unicellular yeast species Saccharomyces cerevisiae and Schizosaccharomyces pombe. In multicellular organisms, however, additional control mechanisms regulate the cell cycle to specify the size of the organism and its constituent organs. To identify such genes, this study analysed the effect of the loss of function of 70% of Drosophila genes (including 90% of genes conserved in human) on cell-cycle progression of S2 cells using flow cytometry. To address redundancy, genes involved in protein phosphorylation were also targeted simultaneously with their homologues. Genes that control cell size, cytokinesis, cell death and/or apoptosis, and the G1 and G2/M phases of the cell cycle were identified. Classification of the genes into pathways by unsupervised hierarchical clustering on the basis of these phenotypes shows that, in addition to classical regulatory mechanisms such as Myc/Max, Cyclin/Cdk and E2F, cell-cycle progression in S2 cells is controlled by vesicular and nuclear transport proteins, COP9 signalosome activity and four extracellular-signal-regulated pathways (Wnt, p38βMAPK, FRAP/TOR and JAK/STAT). In addition, by simultaneously analysing several phenotypes, a translational regulator, eIF-3p66, was identified that specifically affects the Cyclin/Cdk pathway activity (Bjorklund, 2006).
The cell cycle can be divided into distinct phases including a synthesis (S) phase, where DNA is replicated, and a mitosis (M) phase, where cell division occurs. In animal cells, growth and the synthesis of components required for these phases are regulated by extracellular growth factors and occur mainly in two gap phases, G1 (between M and S) and G2 (between S and M). The ordered progression of the cell-cycle phases is orchestrated by cyclin-dependent kinases (Cdks), whose activity is controlled by phosphorylation and by association with specific regulatory subunits, the cyclins (Bjorklund, 2006).
The cell cycle is a robust system in which compensatory mechanisms control its overall length. Therefore the effect of RNA-mediated interference (RNAi)-induced loss of function of Drosophila genes was analyzed in S2 cell-cycle distribution using flow cytometry, which allows direct determination of the fraction of cells in different phases of the cell cycle (Bjorklund, 2006).
Consistent with the high efficiency of RNAi in Drosophila, clear phenotypes including arrest in G1, G2/M and S, cytokinesis and DNA replication defects, and apoptosis and/or cell death were observed when targeting known regulators of these processes. A pilot screen was carried out targeting Drosophila kinases and phosphatases individually, and in pools of homologues to address redundancy. Unexpectedly, the analysis of redundancy did not reveal any additional cell-cycle regulators. These results suggest that a considerably lower fraction of Drosophila kinases regulate the cell cycle (19%) than has been reported previously (35%). Despite this, the screen identified also kinases for which a role in the S2 cell cycle has not been appreciated (for example, AKT1, CG7177 and MEKK1/MEKK4) (Bjorklund, 2006).
It was reasoned that, by screening most Drosophila genes, it should be possible to identify the pathways regulating the S2 cell cycle. Screening of 11,971 double-stranded RNAs (dsRNAs) generated from the Drosophila Gene Collection (DGC) releases 1 and 2 showed that loss of 270 and 169 genes resulted in significant changes in G1 and G2 populations, respectively. A strong correlation between cell size at the G1 and G2 phases was observed in individual samples, suggesting that Drosophila cells do not have a 'strong' cell-size checkpoint that forces cells to a particular size at a defined cell-cycle phase. Components acting on the same direction in a particular process, such as ribosomal proteins or Dp, E2f and Cyclin E, appeared in discrete areas of a plot describing G1 cell size as a function of fraction of cells in G1. The strongest phenotypes, characterized by specifically decreased cell size, were observed with dsRNAs targeting Rbf, a negative regulator of E2f pathway, and cropped (crp), the Drosophila AP4 transcription factor (Bjorklund, 2006).
To identify genes involved in cell death and cytokinesis, changes were analyzed in the populations of cells with less than 2N DNA or more than 4N DNA, respectively. The increase in the <2N population in all cases consisted of apoptotic or dead cells; the genes whose loss caused this phenotype included previously unknown effectors, the known inhibitor of apoptosis Thread and several mitosis regulators, consistent with induction of apoptosis by mitotic catastrophe. The increase in the >4N population was due to cells that had undergone two rounds of DNA replication without cytokinesis (8N DNA); dsRNAs causing this phenotype targeted several previously undescribed genes and known cytokinesis regulators (Bjorklund, 2006).
There have been several large-scale RNAi screens, and in many there is very little overlap between the gene sets identified even when the same phenotypes are analysed. This is apparently due to a very large number of false positives. To reduce the number of false positives, all phenotypes were analysed by using criteria that excluded all 564 control samples. Roughly 4% of genes had a cell-size, cell-cycle or cell-death phenotype. From an analysis of 19 different pathways and/or protein complexes, it was estimated that the screen identified ~80% of strong non-redundant cell-cycle regulators. Despite the general effectiveness of the RNAi, however, some genes with clearly defined functions in the cell cycle were not identified. In different cases, the weak RNAi phenotype could be explained by high protein levels or stability, redundant function and/or the fact that defects in mitotic or DNA replication fidelity often do not appreciably alter overall cell-cycle phase distribution (Bjorklund, 2006).
Of the 78 known interactions between the genes identified, 70 were consistent with the observed phenotypes. A high proportion of the diseases linked to the human orthologues of the identified genes are related to cancer, consistent with the known relationship between cell-cycle regulation and cancer. As expected, the Drosophila protein–protein interaction map indicated a much larger number of interactions between proteins than were identified than between a random set of proteins, and most interactions were consistent with the observed phenotypes (Bjorklund, 2006).
Of the 488 genes that identified, 319 have not been identified in previous Drosophila and mammalian RNAi screens. A principal difference between this screen and previous studies is that, by using flow cytometry, six distinct phenotypes can be simultaneously identified, allowing unbiased classification of the identified genes into pathways using hierarchical clustering. Positive components of a given pathway should segregate into the same cluster, because their loss is expected to cause similar phenotypes. This was indeed observed, for example 43 of 56 dsRNAs in one 'translation' cluster targeted ribosomal subunits. Another 'G1 cell-cycle' cluster contained the classical positive G1 regulators of the Cdk/E2f pathway, including E2f, Dp, Cyclin E, Cdk2 and Cdk4. Because of an opposite phenotype, Rbf, a known negative regulator of this pathway, was not included in this cluster (Bjorklund, 2006).
The other genes in the G1 cell-cycle cluster included two cullins (Cul-1/lin19 and Cul-4), involved in ubiquitin-mediated protein degradation, and five components of the COP9 signalosome, a protein complex linked to regulation of cullin activity. In mammals Cul-4 has been linked to DNA damage response, but the results indicate that Cul-4 is also involved in normal cell-cycle regulation in animals. Consistent with the similar phenotypes of the cullins, COP9 subunits (CSNs) and several Cdk/E2f pathway components, the increase in G1 induced by dsRNAs targeting CSNs could be reversed by simultaneously targeting the Cdk inhibitor Dacapo. The results are thus consistent with a loss of COP9 leading to G1 arrest owing to failure of cullin-mediated degradation of Dacapo. The positive role of COP9 in the cell cycle is in agreement with mouse knockout studies. In Drosophila ovaries, however, COP9 negatively regulates the cell cycle, suggesting that its effects may be tissue-specific (Bjorklund, 2006).
Notably, a translational initiation factor, eIF-3p66, clustered with the G1 cell-cycle regulators. Expression of eIF-3p66 also correlates well with that of Rbf, E2f and Cyclin E during Drosophila development. dsRNAs targeting eIF-3p66 resulted in an increased cell size, indicating that, unlike loss of other translational regulators, loss of eIF-3p66 does not generally inhibit translation. Furthermore, the phenotype caused by dsRNA targeting eIF-3p66 was reversed by dsRNA targeting dacapo, indicating that eIF-3p66 specifically affects the Cyclin/Cdk pathway (Bjorklund, 2006).
The cluster of genes whose loss resulted in an increase in G1 cell size with no G1 DNA content phenotype contained several genes involved in transcription and messenger RNA processing. The lack of G1 phenotype is probably due to a uniform effect of transcriptional inhibition on all cell-cycle phases, and continued growth of the cells is possible owing to translation of stable mRNAs. The distinct phenotype of transcriptional and translational regulators suggests that cell size may be controlled at the RNA level, either directly by regulatory RNAs or through differential stability of mRNAs of proteins controlling cell division and growth (Bjorklund, 2006).
A number of genes whose loss resulted in increased G1 content were classified into a defined 'signalling' cluster, which contained two regulators of translation (eIF-4G and Ef1α48D), a ubiquitin E3-ligase (URE-B1/CG8184), and components of several extracellular ligand-regulated signalling pathways. These genes differed from those in the G1 cell-cycle cluster in that the dsRNAs targeting them resulted in decreased cell size. Thus, the signalling pathways either affect cell division through regulation of growth, or affect both cell growth and cell division in parallel (Bjorklund, 2006).
By manually comparing the genes identified with literature, four signalling pathways were identified that regulate the cell cycle: the FRAP/TOR, JAK/STAT, Wnt and p38βMAPK pathways. The FRAP/TOR pathway is known to regulate cell proliferation and, consistently, eight known components of this pathway were identifed as G1 regulators. In S2 cells, another known cell-cycle regulatory pathway, the JAK/STAT pathway, had a relatively weak effect, as indicated by the mild phenotypes observed by dsRNAs targeting several of its components. The identified genes known to regulate the Wnt pathway indicated that this pathway is not active in S2 cells, but that treatments that activate it inhibit S2 cell proliferation (Bjorklund, 2006).
Several genes linked to the p38βMAPK pathway were also identified, and the function of MEKK1/MEKK4 and MEK3 in this pathway was varified by analysing p38βMAPK phosphorylation in response to dsRNA treatments. Although regulation of the cell cycle by the p38βMAPK pathway in the absence of stress or DNA damage has not previously been appreciated, the data are consistent with the identification of MAPk-Ak2 (Manke, 2005) as a checkpoint kinase (Bjorklund, 2006).
Loss of several kinases that negatively regulate Cdk1 activity led to an increase in G1 content, probably owing to an accelerated G2/M phase. It was also observed that Chk1 and MAPk-Ak2, two kinases that have been linked to DNA damage checkpoints, operate in normal cell-cycle regulation of cultured S2 cells. Together with the finding that in human cells Chk1 shields Cdk1 from premature activation, the results suggest that during normal cell cycle these kinases have basal activity that slows down G2 progression. The basal activity could represent partial activation that occurs locally in response to damage during normal replication, possibly effecting localized changes in DNA replication and repair (Bjorklund, 2006).
In summary, several pathways and processes are involved in cell-cycle and cell-size regulation, including basal transcription, vesicular trafficking, nuclear export, ubiquitin-mediated protein degradation, and the Cdk/E2f, JAK/STAT, FRAP/TOR, p38βMAPK and Wnt signalling pathways. The genes identified are likely to include previously unknown components of these pathways, ubiquitin ligase and kinase substrates, and targets of transcription factors such as E2f and Myc/Max that are relevant for cell-cycle progression. Thus, these results will also serve as a solid foundation for a systems biology analysis of the metazoan cell cycle (Bjorklund, 2006).
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date revised: 15 April 2007
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