The Interactive Fly
Zygotically transcribed genes
High abundance of CDC45 inhibits cell proliferation through elevation of HSPA6
Meiosis
Oogenesis, Spermatogenesis and Meiosis
Meiosis in Females
Meiosis in Males
Genetic background impacts the timing of synaptonemal complex breakdown in Drosophila melanogaster
Superresolution expansion microscopy reveals the three-dimensional organization of the Drosophila synaptonemal complex
Unconventional conservation reveals structure-function relationships in the synaptonemal complex
The meiotic recombination checkpoint suppresses NHK-1 kinase to prevent reorganisation of the oocyte nucleus in Drosophila
A meiotic switch in lysosome activity supports spermatocyte development in young flies but collapses with age
Meiotic, genomic and evolutionary properties of crossover distribution in Drosophila yakuba
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 cells 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).
In newly hatched Drosophila larvae, quiescent cells reenter the cell cycle in response to dietary amino acids. To understand this process, larval nutrition was varied and effects on cell cycle initiation and maintenance were monitored in the mitotic neuroblasts and imaginal disc cells, as well as the endoreplicating cells in other larval tissues. After cell cycle activation, mitotic and endoreplicating cells respond differently to the withdrawal of nutrition: mitotic cells continue to proliferate in a nutrition-independent manner, while most endoreplicating cells reenter a quiescent state. Ectopic expression of Drosophila Cyclin E or the E2F transcription factor can drive quiescent endoreplicating cells, but not quiescent imaginal neuroblasts, into S-phase. Conversely, quiescent imaginal neuroblasts, but not quiescent endoreplicating cells, can be induced to enter the cell cycle when co-cultured with larval fat body in vitro. These results demonstrate a fundamental difference in the control of cell cycle activation and maintenance in these two cell types, and imply the existence of a novel mitogen generated by the larval fat body in response to nutrition (Britton, 1998).
These results suggest that multiple pathways are involved in regulating the onset of cell proliferation in different tissue types in response to the global nutritional cue. Mitotic and endoreplicating cell cycles are regulated differently in response to the nutritional state: the endoreplicating tissues (ERTs) require continuous nutrition to cycle, whereas the mitotic cells cycle in a nutrition-independent manner once activated. In addition, the mechanism of cell cycle arrest in the two types of quiescent cells is different: quiescent ERTs can be driven into S-phase by ectopic expression of either of the G1/S regulators E2F or Cyclin E, while neither of these regulators can induce quiescent neuroblasts to enter S-phase.Conversely, quiescent neuroblasts but not quiescent ERTs are induced to reenter the cell cycle in response to a mitogen produced by the larval fat body (Britton, 1998).
The differential responses of the mitotic and endoreplicative cell cycles to nutrient withdrawal may provide an important mechanism for survival of the organism and reproduction in the face of food shortages in the wild. When nutrients become limiting, available resources can be dedicated to maintaining growth and proliferation in the mitotic tissues which are required to form the reproductive adult. Indeed, larvae are capable of pupating at a much smaller size than they normally do. A 'critical size' has been defined at which larvae are able to pupariate without further feeding. The small pupae which are formed by these larvae produce normal, fertile, but small adult flies (Britton, 1998).
Embryonic neuroblasts have an intrinsic program of cell proliferation. Each type of neuroblast has a specific identity, expresses unique and dynamic combinations of sublineage genes, and will give rise to a precise number and type of progeny before exiting the cell cycle. Interestingly, temporal control of sublineage gene expression in embryonic neuroblasts can be independent of cell cycle progression. Thus arresting a proliferating neuroblast in mid-lineage could lead to the desynchronization of sublineage gene expression and the loss of certain types of progeny, a result which could have disastrous consequences for the developing CNS (Britton, 1998).
In a food withdrawal experiment it was observed that many activated neuroblasts continued to proliferate for up to 7 days after food withdrawal, however a subset of them did not. This observation was most striking in the abdominal region of the VNC. The abdominal neuroblast lineages are much shorter than those of the majority of brain and thoracic neuroblasts, with a single abdominal neuroblast producing as few as four neurons during its postembryonic period of proliferation. Since the abdominal neuroblasts generally complete their entire larval program of proliferation in less than 2 days, it is not surprising that after 7 days of culture on sucrose the majority of these neuroblasts have exited the cell cycle. It is suspected that the reduction in labeled neuroblasts observed in all regions of the CNS over the course of this experiment is due to a subset of neuroblasts completing their intrinsic program of proliferation and exiting the cell cycle (Britton, 1998).
The insect fat body is the source of the majority of hemolymph proteins, including lipid binding proteins, juvenile hormone binding proteins and esterases, peptides which mediate the insect immune response, and vitellogenins involved in oocyte maturation in the adult female. The fat body is also responsible for synthesizing the stores of protein, lipid and glycogen which sustain the animal throughout metamorphosis. Ultrastructurally, the fat body shows a dramatic response to starvation. In Calpodes larvae, starvation leads to a rapid reorganization of the fat body including loss of mitochondria and rough endoplasmic reticulum (RER) by autophagy and depletion of stored metabolites. Refeeding induces mitochondrial divisions andincreases in RER content as well as the eventual replenishment of depleted stores. This study observed dramatic changes in the larval fat body in the course of starvation experiments, including a loss of tissue cohesion and changes in opacity. These changes probably reflect the alteration in composition the fat body cells undergo as stores of metabolites are mobilized to support proliferating mitotic tissues during starvation (Britton, 1998).
Previous studies have demonstrated that the adult female fat body is able to regulate yolk gene transcription in response to the nutritional environment. Interestingly, there is evidence that a component of the adult female abdomen is also capable of supporting the proliferation of larval tissues in a nutrition dependent manner. It has been demonstrated that the proliferation of imaginal disc fragments transplanted into the abdominal cavity of adult female hosts is dependent onnutrition. This study has found that when quiescent central nervous systems from starved larvae are transplanted into the abdomens of fed adult female hosts, larval neuroblasts reenter the cell cycle in what appears to be a normal spatiotemporal pattern. An appealing hypothesis is that production of the neuroblast mitogen in the fat body is regulated at the transcriptional level under the control of nutritional enhancers similar to those identified in the regions upstream of yolk protein genes. The ability of something in the adult female abdomen to activate proliferation in quiescent neuroblasts suggests that similar fat body-derived mitogens are produced in the larval and adult female fat bodies. This adult mitogen could have a role in controlling proliferation in the adult, perhaps functioning to regulate some oogenic process in response to the nutritional state. Indeed, oogenesis is inhibited in adult females fed on sucrose (Britton, 1998).
The dramatic response of the fat body to starvation, the demonstration that there is a mechanism for nutritional controlof transcription in adult female fat body, and the similar abilities of the adult female abdomen and the larval fat body to support nutrition-dependent cell cycle activation lend support to the proposal that the fat body is responsible for mediating the nutritional response in larval neuroblasts. The results of co-culture experiment demonstrate that the fat body supplies a diffusible factor which stimulates larval neuroblasts to enter the cell cycle (Britton, 1998).
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).
Fast, early embryonic cell cycles have correspondingly fast S phases. In early Drosophila embryos, forks starting from closely spaced origins replicate the whole genome in 3.4 min, ten times faster than in embryonic cycle 14 and a hundred times faster than in a wing disc. It is not known how S phase duration is regulated. This study examined prolongation of embryonic S phases, its coupling to development, and its relationship to the appearance of heterochromatin. Imaging of fluorescent nucleotide incorporation and GFP-PCNA gave exquisite time resolution of S phase events. In the early S phases, satellite sequences replicated rapidly despite a compact chromatin structure. In S phases 11-13, a delay in satellite replication emerged in sync with modest and progressive prolongation of S phase. In S phase 14, major and distinct delays ordered the replication of satellites into a sequence that occupied much of S phase. This onset of late replication required transcription. Satellites only accumulated abundant heterochromatin protein 1 (HP1) after replicating in S phase 14. By cycle 15, satellites clustered in a compact HP1-positive mass, but replication occurred at decondensed foci at the surface of this mass. It is concluded that the slowing of S phase is an active process, not a titration of maternal replication machinery. Most sequences continue to replicate rapidly in successive cycles, but increasing delays in the replication of satellite sequences extend S phase. Although called constitutively heterochromatic, satellites acquire the distinctive features of heterochromatin, compaction, late replication, HP1 binding, and aggregation at the chromocenter, in successive steps coordinated with developmental progress (Shermoen, 2010).
This work shows that heterochromatin, long recognized as a key factor in the developmental programming of gene expression, also plays an integral role in the timing of the early embryonic cell cycles. Satellite sequences successively acquire features of heterochromatin, becoming late replicating by cycle 14, which prolongs S phase. This prolongation of S phase slows the early cell cycles and allows the progression to MBT (Shermoen, 2010).
This description of the successive introduction of the features of heterochromatin reveals a lack of interdependency of these features. For example, because it occurs earlier, the compaction of the satellite sequences is independent of late replication and of HP1 binding. It was also possible to visualize the events of late replication with unprecedented spatial and temporal resolution that gives insights into the replication of compacted HP1-bound chromatin (Shermoen, 2010).
Origin spacing could contribute to S phase length. However, electron microscopy studies show that origin spacing changes only slightly, from 7.9 to 10.6 kb, from preblastoderm embryos to cycle 14. Because forks are thought to converge at a rate of 3 kb/min, the additional separation would extend S phase by about 1 min, a minor contribution to the change from a 3.4 to a 50 min S phase (Shermoen, 2010).
S phase duration would also increase if all of the replicons did not replicate at the same time. However, asynchrony in replicon firing can occur in two ways, organized and unorganized. Unorganized asynchrony means that origins fire at different times without regard to their position in the genome. In this case, early- and late-firing origins can be juxtaposed. When replication from an early-firing origin reaches an adjacent later-firing origin just before it fires, one fork, rather than two, replicates the interorigin distance, doubling replication time. Greater unorganized asynchrony will result in passive replication of later origins and reduce the number of origins. Thus, given the known origin spacing, unorganized asynchrony is unlikely to make a very major contribution to the more than 10-fold increase in S phase length between preblastoderm cycles and cycle 14 (Shermoen, 2010).
If replication asynchrony is organized so that large regions of the genome (replication units) have many similarly behaving replicons, early-initiated forks invading a late region from the outside will not have time to replicate a significant portion of the large domain. The insulation resulting from distance can greatly magnify the impact of asynchrony on S phase duration. Organization of genomes into large replication units is widespread but poorly understood. This study shows that the satellite sequences are replication units and that embryonic changes in S phase duration result from change in the schedules of their replication. In preblastoderm cycles, satellite sequences replicate early, finishing in synch with general replication. Subsequently, satellite replication is increasingly delayed in parallel to S phase prolongation. Importantly, when satellite replication is late, it is deferred, not slow. For example, a 3.4 Mb Y-chromosomal repeat of the simple sequence AATAC begins to replicate 18 min into S phase 14. Each type of satellite sequence exhibits distinct replication delays. The 11 Mb X-chromosomal repeat of 359 bases, termed the 359 sequence has almost no delay in S phase 14, whereas AATAT and AATAACATAG finish replicating after 359 but before AATAC. The stereotyped schedules suggest that each replication unit has a characteristic 'lateness' parameter. This lateness parameter appears to be continuously variable in that there are many replication units, each with its own schedule of replication (Shermoen, 2010).
Though its replication is delayed, a unit such as AATAC replicates quickly once initiated (10 min). Although the 359 satellite is more slowly replicating (~15 min), it is suggested that it may be composed of separately and asynchronously replicating subdomains that were sometimes resolved. It is concluded that the dynamics of replication within a replication unit change only modestly during the early cycles (from 3.4 to roughly 10 min) (Shermoen, 2010).
In summary, the results argue that by S phase 14, the genome is replicated as a series of units, each of which replicates relatively quickly, but that a temporal program of sequential replication of these units creates a long S phase. This replication program resembles a consensus view of replication in slowly replicating cells of mammals and plants. It is concluded that progression from coincident replication of all of the replication units in a rapid S phase to sequential replication in a prolonged S phase 14 underlies prolongation of early embryonic S phases in Drosophila (Shermoen, 2010).
In widely divergent species and biological settings, heterochromatin has common characteristics including compaction, transcriptional quiescence, late replication, 'repressive' histone modifications, and association of specific heterochromatin proteins. This intimate association suggests mechanistic coupling of these features. If this were so, the various heterochromatin characteristics would emerge coordinately at the same moment during development. Instead, the current observations show temporal uncoupling during early Drosophila embryogenesis (Shermoen, 2010).
Although it was suggested that heterochromatin appears at cycle 14, both the cytological and biochemical manifestations of heterochromatin develop progressively. Foci of compacted chromatin that align with satellite sequences appeared in preblastoderm embryos prior to, and hence independently of, late replication and HP1 recruitment. Furthermore, HP1 binding to satellite sequences occurred late in cycle 14, after the onset of late replication. Because HP1 would have to decorate the satellite sequences at the onset of cycle 14 if it were required to suppress early replication and promote late replication, it is concluded that the late replication of satellite sequences is specified independently of the HP1 binding. Finally, satellite sequences reorganize; the previously independent foci of satellites aggregate into a large coherent HP1-positive region at the very beginning of interphase 15. This intimate association of satellites, which makes the chromocenter more coherent, is downstream of cycle 14 events and the MBT (Shermoen, 2010).
Together, these findings show that satellite sequences acquire the features of heterochromatin progressively. Compaction is present early, late replication is introduced subsequently, and recruitment of HP1 and then chromocenter maturation follow. Onset of position-effect variegation suggests that heterochromatic suppression of transcription begins in G2 of cycle 14 and mounts subsequently. Thus, heterochromatin does not form in a single step, and it acquires increasing influence during critical developmental events surrounding the MBT and gastrulation (Shermoen, 2010).
If compaction of chromatin prevents replication, decompaction might accompany or provoke replication. Real-time observations of PCNA and HP1 in cycle 15 show replication adjacent to, but not overlapping, HP1-bright foci of compacted chromatin. A more diffuse HP1 region appears adjacent to bright HP1 foci; PCNA overlies these fainter partner foci. Each partner focus appears and disappears as the PCNA signal rises and declines. It is concluded from this that replication does not occur in the compacted domain and that the sequences in the compacted HP1-bright focus unfurl during replication (Shermoen, 2010).
The persistence of in situ foci for 359 and AATAC shows that the satellites are not fully decondensed during replication. The size of partner HP1 foci also argues for limited decompaction. If an entire focus of compacted HP1-bright chromatin were to disperse, it would expand in volume, but the partner focus is about the same size as the brighter parent focus. Thus, it is suggested that a partner focus represents decompaction of a portion of the sequences harbored in the adjacent HP1-bright focus (Shermoen, 2010).
Following replication, heterochromatic sequences rapidly recompact. After an initial expansion, the partner HP1 focus does not grow throughout replication, and it shrinks and disappears as replication declines. When pulsed with fluorescent nucleotides for less than the replication time of the satellite, fluorescence overlies the compacted satellite sequence. Thus, it is suggested that DNA is “spooled” out of compacted foci, replicated, and returned to compacted foci shortly after replication. It the duration of replication-associated decompaction in embryonic cycle 15 is roughly estimated as 1 min. The dynamics, which are not easily consistent with decompaction of large topological domains, suggest that active replication forks drive local unfolding of chromatin structure, but the possibility cannot be excluded that transient decompaction might promote replication (Shermoen, 2010).
Mechanisms that couple the changing cell-cycle behavior with development are of great interest. Previous work suggested that the gradual prolongation of early cycles is secondary to gradual prolongation of S phase. A model in which the exponentially increasing amounts of DNA titrate replication components to prolong S phase is attractive but not presently supported (Shermoen, 2010).
The current results show that if a titration mechanism governs S phase duration, it is indirect. Injection of α-amanitin in cycle 13 prevented onset of late replication, accelerated S phase 14, and caused an early synchronous mitosis. Thus, activity of at least one of the DNA-dependent RNA polymerases is required to slow S phase, and the replication “hardware” needed for a rapid S phase is not limiting. Accordingly, if a titration mechanism were involved, the titrated component would regulate an upstream process. For example, transcription is restricted prior to cycle 14, and titration of a repressor might derepress transcription in late cycle 13, indirectly triggering onset of late replication (Shermoen, 2010).
Three findings suggest an abrupt switch to late replication at the beginning of cycle 14: the dramatic increase in S phase length, the accompanying switch of satellite sequences to delayed replication, and the requirement for transcription in cycle 13 for this transition. However, the late replication program of cycle 14 is anticipated by slight delays in replication of satellite sequences in cycles 12 and 13. These early changes suggest a more progressive process. It is proposed that early slight changes in replication timing and transcription initiate a positive feedback process that precipitates an abrupt change at the MBT. Rapid cell cycles suppress transcription and limit the time available to modify newly replicated chromatin, but, once the cycle begins to slow, transcription and heterochromatin modifications would accelerate to create conditions permissive for late replication, which would further slow the cycle (Shermoen, 2010).
Transmission of active transcriptional states from mother to daughter cells has the potential to foster precision in the gene expression programs underlying development. Such transcriptional memory has been specifically proposed to promote rapid reactivation of complex gene expression profiles after successive mitoses in Drosophila development. By monitoring transcription in living Drosophila embryos, this study provides the first evidence for transcriptional memory in animal development. The activities of stochastically expressed transgenes were measured in order to distinguish active and inactive mother cells and the behaviors of their daughter nuclei after mitosis. Quantitative analyses reveal that there is a 4-fold higher probability for rapid reactivation after mitosis when the mother experiences transcription. Moreover, memory nuclei activate transcription twice as fast as neighboring inactive mothers, thus leading to augmented levels of gene expression. The study proposes that transcriptional memory is a mechanism of precision, which helps coordinate gene activity during embryogenesis (Ferraro, 2015).
Cell based RNAi is a powerful approach to screen for modulators of many cellular processes. However, resulting candidate gene lists from cell-based assays comprise diverse effectors, both direct and indirect, and further dissecting their functions can be challenging. This study screened a genome-wide RNAi library for modulators of mitosis and cytokinesis in Drosophila S2 cells. The screen identified many previously known genes as well as modulators that have previously not been connected to cell cycle control. Approximately 300 candidate modifiers where characterized further by genetic interaction analysis using double RNAi and a multiparametric, imaging-based assay. Analyzing cell-cycle relevant phenotypes increased the sensitivity for associating novel gene function. Genetic interaction maps based on mitotic index and nuclear size grouped candidates into known regulatory complexes of mitosis or cytokinesis, respectively, and predicted previously uncharacterized components of known processes. For example, a role was confirmed for the Drosophila CCR4 mRNA processing complex component l(2)NC136 during the mitotic exit. These results show that the combination of genome-scale RNAi screening and genetic interaction analysis using process-directed phenotypes provides a powerful two-step approach to assign components to specific pathways and complexes (Billmann, 2016).
The beautiful mitotic waves that characterize nuclear divisions in the early Drosophila embryo have been the subject of intense research to identify the elements that control mitosis. Calcium waves in phase with mitotic waves suggest that calcium signals control this synchronized pattern of nuclear divisions. However, protein targets that would translate these signals into mitotic control have not been described. This study investigated the role of the calcium-dependent protease Calpain A in mitosis. Impaired Calpain A function was shown to result in loss of mitotic synchrony and ultimately halted embryonic development. The presence of defective microtubules and chromosomal architecture at the mitotic spindle during metaphase and anaphase and perturbed levels of Cyclin B indicate that Calpain A is required for the metaphase-to-anaphase transition. The results suggest that Calpain A functions as part of a timing module in mitosis, at the interface between calcium signals and mitotic cycles of the Drosophila embryo (Vieira, 2016).
The egg contains maternal RNAs and proteins, which have instrumental functions in patterning and morphogenesis. Besides these, the egg also contains metabolites, whose developmental functions have been little investigated. For example, the rapid increase of DNA content during the fast embryonic cell cycles poses high demands on the supply of deoxyribonucleotides (dNTPs), which may be synthesized in the embryo or maternally provided. This study analyzed the role of dNTP in early Drosophila embryos. dNTP levels initially decreased about 2-fold before reaching stable levels at the transition from syncytial to cellular blastoderm. Employing a mutant of the metabolic enzyme serine hydroxymethyl transferase (SHMT), which is impaired in the embryonic synthesis of deoxythymidine triphosphate (dTTP), it was found that the maternal supply of dTTP was specifically depleted by interphase 13. SHMT mutants showed persistent S phase, replication stress, and a checkpoint-dependent cell-cycle arrest in NC13, depending on the loss of dTTP. The cell-cycle arrest in SHMT mutants was suppressed by reduced zygotic transcription. Consistent with the requirement of dTTP for cell-cycle progression, increased dNTP levels accelerated the cell cycle in embryos lacking zygotic transcription. A model is proposed that both a limiting dNTP supply and interference of zygotic transcription with DNA replication elicit DNA replication stress and checkpoint activation. This study reveals a specific mechanism of how dNTP metabolites contribute to the embryonic cell-cycle control (Liu, 2019).
During development cell proliferation and differentiation must be tightly
coordinated to ensure proper tissue morphogenesis. Because steroid
hormones are central regulators of developmental timing, understanding the
links between steroid hormone signaling and cell proliferation is crucial
to understanding the molecular basis of morphogenesis. This study examined
the mechanism by which the steroid hormone ecdysone regulates the cell cycle in Drosophila. A cell cycle arrest induced by ecdysone in Drosophila cell culture is analogous to a G2 cell cycle arrest observed in the early pupa.
In the wing, ecdysone signaling at the
larva to puparium transition induces Broad
which in turn represses the cdc25c phosphatase String. The repression of String generates a temporary G2 arrest that synchronizes the cell cycle in the wing epithelium during early pupa wing elongation
and flattening. As ecdysone levels decline after the larva to puparium
pulse during early metamorphosis, Broad expression plummets allowing
String to become re-activated, which promotes rapid G2/M progression and a
subsequent synchronized final cell cycle in the wing. In this manner,
pulses of ecdysone can both synchronize the final cell cycle and promote the coordinated acquisition of terminal differentiation characteristics in the wing (Guo, 2016).
This study presents a model for how the pulse of ecdysone at the larval to pupal transition
impacts the cell cycle dynamics in the wing during metamorphosis.
Ecdysone signaling at the larva to puparium transition induces Broad, which in turn
represses Stg to generate a temporary G2 arrest, which synchronizes the cell cycle
in the wing epithelium. As ecdysone levels decline, Broad expression plummets,
allowing Stg to be re-activated resulting in a pulse of cdc2 activity that promotes a
rapid G2/M progression during the final cell cycle in the wing. This ultimately
culminates in the relatively synchronized cell cycle exit at 24h APF, coinciding with the second large pulse of
ecdysone. This second pulse in the pupa activates a different set of transcription
factors (not Broad), promoting the acquisition of terminal differentiation
characteristics in the wing. In this way, two pulses of ecdysone signaling can both
synchronize the final cell cycle by a temporary G2 arrest and coordinate permanent
cell cycle exit with the acquisition of terminal differentiation characteristics in the
wing (Guo, 2016).
Over 30 years ago it was shown that 20-HE exposure in
Drosophila tissue culture cells induces a cell cycle arrest in G2-phase. This response appears to be shared among 3 different cell lines, Cl-8, Kc and S2. This study shows that in Kc cells, pulsed 20-HE
exposure also leads to a G2 arrest followed by rapid cell cycle re-entry after 20-HE
removal and a subsequent prolonged G1. This cell cycle response to a pulse
of 20-HE is reminiscent of the cell cycle changes that occur during early
metamorphosis in the pupal wings and legs (Guo, 2016).
It is worth considering why Kc and S2 cells, which are thought to be derived
from embryonic hemocytes would exhibit a similar cell cycle response to 20-HE to
the imaginal discs. Relatively little is known about how ecdysone signaling impacts
embryonic hemocytes, although recent work suggests that ecdysone signaling
induces embryonic hemocyte cell death under sensitized conditions. More is known about larval hemocytes, which differentiate into phagocytic macrophages and disperse into the hemolymph during the first 8h of
metamorphosis. Ecdysone is involved in this maturation process, as lymph glands of
ecdysoneless (ecd) mutants fail to disperse mature
hemocytes and become hypertrophic in the developmentally arrested mutants. This suggests that the high levels of systemic ecdysone signaling at the larval-puparium transition mediates a switch from proliferation to
cell cycle arrest and terminal differentiation for lymph gland hemocytes during
metamorphosis. Without ecdysone signaling, hemocytes may continue to proliferate
and fail to undergo terminal differentiation leading to the hypertrophic lymph gland
phenotype observed. Interestingly, while the loss of
broad also prevents proper differentiation of hemocytes similar to loss of
ecd, loss of broad does not lead to the
hypertrophy observed in ecd mutants. Further studies will
be needed to examine whether the ecdysone induced cell cycle arrest in larval
hemocytes occurs in the G2 phase, or whether their cell cycle arrest proceeds via a
similar pathway to that shown in this study for the wing (Guo, 2016).
Multiple lines of evidence suggest that the ecdysone receptor complex in the larval
wing acts as a repressor for certain early pupa targets and that the binding of
ecdysone to the receptor relieves this repression. For example loss of EcR by
RNAi or loss of the EcR dimerization partner USP, de-represses ecdysone target
genes that are high in the early pupal wing such as Broad-Z1 and βFtz-F1. The EcR/USP heterodimer also cooperates with the SMRTR co-repressor in the wing to prevent precocious
expression of ecdysone target genes such as Broad-Z1. Consistent
with the hypothesis that a repressive EcR/USP complex prevents precocious
expression of Broad-Z1 and thereby a precocious G2 arrest, inhibition of SMRTR can
also cause a G2 arrest. Thus, in the context of the early pupal wing,
it is proposed that the significant pulse of ecdysone at the larval to puparium
transition relieves the inhibition of a repressive receptor complex, leading to Broad-Z1 activation. Consistent with this model, high levels of Broad-Z1 in the larval wing lead to precocious neural differentiation at the margin and precocious inhibition of stg
expression in the wing pouch. Interestingly, a switch in Broad isoform expression also occurs during the
final cell cycle in the larval eye, such that Broad-Z1 becomes high in cells undergoing
their final cell cycle and entering into terminal differentiation.
However in this case, Broad-Z1 expression is not associated with a G2 arrest and
occurs in an area of high Stg expression, suggesting the downstream Broad-Z1
targets in the eye may be distinct or regulated differently from those in the wing (Guo, 2016).
The ecdysone receptor has also been shown to down regulate Wingless
expression via the transcription factor Crol at the wing margin, to indirectly
promote CycB expression. While a loss of EcR at the margin
decreased CycB protein levels, the effects of EcR loss on CycB levels in the wing
blade outside of the margin area were not obvious. It is suggested
that in the wing, the role for EcR outside of the margin acts on the cell cycle via a
different mechanism through stg. Consistent with a distinct mechanism acting in the
wing blade, over-expression of Cyclin B in the early prepupal wing could not
promote increased G2 progression or bypass the prepupal G2 arrest. Instead
the results on the prepupal G2 arrest are consistent with previous findings that Stg
is the rate-limiting component for G2-M cell cycle progression in the fly wing pouch
and blade (Guo, 2016).
In order to identify the gene expression changes in the wing that occur in response
to the major peaks of ecdysone during metamorphosis, RNAseq was performed on a
timecourse of pupal wings. Major changes were observed in gene expression in this
tissue during metamorphosis. In addition, known ecdysone targets were identified that are affected differently in the wing during the first larval-to-pupal ecdysone pulse and the second, larger pulse at 24h APF.
Ecdysone signaling induces different direct targets with distinct kinetics. Furthermore specific targets, for example Ftz-F1 can modulate the expression of other ecdysone targets, to shape the response to the
hormone. Thus, it is expected that a pulse of ecdysone
signaling leads to sustained effects on gene expression and the cell cycle, even after
the ecdysone titer returns to its initial state. These factors together with the
differences in the magnitude of the ecdysone pulse may contribute to the differences
in the response to the early vs. later pulses in the wing (Guo, 2016).
Ecdysone signaling can also affect the cell cycle and cell cycle exit via indirect
mechanisms such as altering cellular metabolism. This is used to promote cell cycle
exit and terminal differentiation in neuroblasts, where a switch toward oxidative
phosphorylation leads to progressive reductive divisions, (divisions in the absence
of growth) leading to reduced neuroblast cell size and eventually terminal
differentiation. Although reductive divisions do occur in the final cell cycle of the pupa wing, this
type of mechanism does not provide a temporary arrest to synchronize the final cell
cycle in neuroblasts as is see in wings. Importantly, a striking reduction is seen
in the expression of genes involved in protein synthesis and ribosome biogenesis in
the wing during metamorphosis, consistent with the lack of cellular growth. Instead the increased surface area of the pupal wing comes from a flattening, elongation and apical expansion of the cells due to interactions
with the extracellular matrix creating tension and influencing cell shape changes. This is also consistent with the findings that a significant number of genes associated with protein targeting to the membrane are increased as the wing begins elongation in the early pupa. Further studies will be
needed to determine whether the changes in expression of genes involved in
ribosome biogenesis and protein targeting to the membrane are controlled by
ecdysone signaling, or some other downstream event during early wing metamorphosis (Guo, 2016).
Perhaps the most interesting and least understood aspect of steroid hormone
signaling is how a diversity of cell-type and tissue-specific responses are generated
to an individual hormone. Cell cycle responses to ecdysone signaling are highly cell
type specific. For example abdominal histoblasts, the progenitors of the adult
abdominal epidermis, become specified during embryogenesis and remain
quiescent in G2 phase during larval stages. During pupal development, the
abdominal histoblasts must be triggered to proliferate rapidly by a pulse of
ecdysone to quickly replace the dying larval abdominal epidermis. This is in contrast
to the behavior of the wing imaginal disc, where epithelial cells undergo
asynchronous rapid proliferation during larval stages, but during metamorphosis
the cell cycle dynamics become restructured to include a G2 arrest followed by a
final cell cycle and entry into a permanently postmitotic state, in a manner
coordinated with tissue morphogenesis and terminal differentiation (Guo, 2016).
How does the same system-wide pulse of ecdysone at the larval to puparium
transition lead to such divergent effects on the cell cycle in adult progenitors?
Surprisingly it seems to be through divergent effects on tissue specific pathways
that act on the same cell cycle targets. In the abdominal histoblasts the larval to
puparium pulse of ecdysone triggers cell cycle re-entry and proliferation via indirect
activation of Stg, by modulating the expression of a microRNA
miR-965 that targets Stg. This addition of the microRNA essentially allows ecdysone signaling to act oppositely on the same cell cycle regulatory target as Broad-Z1 does in the wing. Thus, tissue specific programs of gene regulatory networks can create divergent outcomes from the same system-
wide hormonal signal, even when they ultimately act on the same target (Guo, 2016).
Time-lapse microscopy is a powerful tool to investigate cellular and developmental dynamics. In Drosophila melanogaster, it can be used to study division cycles in embryogenesis. Image analysis of maternal-haploid (mh) embryos revealed that a fraction of haploid syncytial nuclei fused to give rise to nuclei of higher ploidy (2n, 3n, 4n). Moreover, nuclear densities in mh embryos at the mid-blastula transition varied over threefold. By tracking synchronized nuclei of different karyotypes side-by-side, DNA content was shown to determine nuclear growth rate and size in early, while the nuclear to cytoplasmic ratio constrains nuclear growth during late interphase. mh encodes the Drosophila ortholog of human Spartan, a protein involved in DNA damage tolerance. To explore the link between mh and chromosome instability, Mh protein was fluorescently tagged to study its subcellular localization. Mh-mKO2 was shown to localize to nuclear speckles that increase in numbers as nuclei expand in interphase. In summary, quantitative microscopy can provide new insights into well-studied genes and biological processes (Puah, 2017).
Chromatin condenses several folds to form mitotic chromosomes during cell division and decondenses post-mitotically to re-occupy their nuclear territory and re-gain their specific transcriptional profile in a precisely lineage specific manner. This necessitates that the features of nuclear architecture and DNA topology persist through mitosis. This study compared the proteome of nuclease and high salt resistant fraction of interphase nucleus known as nuclear matrix (NuMat) and an equivalent biochemical fraction in the mitotic chromosome known as mitotic chromosome scaffold (MiCS). This study elucidates that as much as 67% of the NuMat proteins are retained in the MiCS indicating that the features of nuclear architecture in interphase nucleus are retained on the mitotic chromosomes. Proteins of the NuMat/MiCS have large dynamic range of MS signal and were detected in sub-femtomolar amounts. Chromatin/RNA binding proteins with hydrolase and helicase activity are highly enriched in NuMat as well as MiCS. While several transcription factors involved in functioning of interphase nucleus are present exclusively in NuMat, protein components responsible for assembly of membrane-less nuclear bodies are uniquely retained in MiCS. This study clearly indicates that the features of nuclear architecture, in the structural context of NuMat, are retained in MiCS and possibly play an important role in maintenance of cell lineage specific transcriptional status during cell division and thereby, serve as components of cellular memory (Sureka, 2018).
Sister chromatid cohesion is crucial to ensure chromosome bi-orientation and equal chromosome segregation. Cohesin removal via mitotic kinases and Wapl has to be prevented in pericentromeric regions in order to protect cohesion until metaphase, but the mechanisms of mitotic cohesion protection remain elusive in Drosophila This study shows that dalmatian (Dmt), an ortholog of the vertebrate cohesin-associated protein sororin, is required for protection of mitotic cohesion in flies. Dmt is essential for cohesion establishment during interphase and is enriched on pericentromeric heterochromatin. Dmt is recruited through direct association with heterochromatin protein-1 (HP1), and this interaction is required for cohesion. During mitosis, Dmt interdependently recruits protein phosphatase 2A (PP2A) to pericentromeric regions, and PP2A binding is required for Dmt to protect cohesion. Intriguingly, Dmt is sufficient to protect cohesion upon heterologous expression in human cells. These findings of a hybrid system, in which Dmt exerts both sororin-like establishment functions and shugoshin-like (see mei-S332) heterochromatin-based protection roles, provide clues to the evolutionary modulation of eukaryotic cohesion regulation systems (Yamada, 2017).
The synaptonemal complex (SC) assembles between homologous chromosomes and is essential for accurate chromosome segregation at the first meiotic division. In Drosophila, many SC components within the complex have been dissected through a combination of genetic analyses and superresolution and electron microscopy. The inability to optically resolve the minute distances between proteins in the complex has precluded its 3D characterization. A recently described technology termed expansion microscopy (ExM) uniformly increases the size of a biological sample, thereby circumventing the limits of optical resolution. By adapting the ExM protocol to render it compatible with structured illumination microscopy, it is possible to examine the 3D organization of several known Drosophila SC components. These data provide evidence that two layers of SC are assembled. It is further speculated that each SC layer may connect two nonsister chromatids, and a 3D model of the Drosophila SC is presented based on these findings (Cahoon, 2017).
Experiments performed in different genetic backgrounds occasionally exhibit failure in experimental reproducibility. This is a serious issue in Drosophila where there are no standard control stocks. This study illustrates the importance of controlling genetic background by showing that the timing of a major meiotic event, the breakdown of the synaptonemal complex (SC), varies in different genetic backgrounds. SC breakdown was assessed in three different control stocks and found that in one control stock, y w; sv(spa-pol), the SC broke down earlier than in Oregon-R and w(1118) stocks. SC breakdown was further examined in these three control backgrounds with flies heterozygous for a null mutation in c(3)G, which encodes a key structural component of the SC. Flies heterozygous for c(3)G displayed differences in the timing of SC breakdown in different control backgrounds, providing evidence of a sensitizing effect of this mutation. These observations suggest that SC maintenance is associated with the dosage of c(3)G in some backgrounds. Lastly, chromosome segregation was not affected by premature SC breakdown in mid-prophase, consistent with previous findings that chromosome segregation is not dependent on full-length SC in mid-prophase. Thus, genetic background is an important variable to consider with respect to SC behavior during Drosophila meiosis (Wesley, 2020).
Functional requirements constrain protein evolution, commonly manifesting in a conserved amino acid sequence. This idea is extended to secondary structural features by tracking their conservation in essential meiotic proteins with highly diverged sequences. The synaptonemal complex (SC) is a ~100-nm-wide ladder-like meiotic structure present in all eukaryotic clades, where it aligns parental chromosomes and regulates exchanges between them. Despite the conserved ultrastructure and functions of the SC, SC proteins are highly divergent within Caenorhabditis. However, SC proteins have highly conserved length and coiled-coil domain structure. The same unconventional conservation signature was found in Drosophila and mammals, and it was used to identify a novel SC protein in Pristionchus pacificus, Ppa-SYP-1. This work suggests that coiled-coils play wide-ranging roles in the structure and function of the SC, and more broadly, that expanding sequence analysis beyond measures of per-site similarity can enhance understanding of protein evolution and function (Kursel, 2021).
The number and location of crossovers across genomes are highly regulated during meiosis, yet the key components controlling them are fast evolving, hindering understanding of the mechanistic causes and evolutionary consequences of changes in crossover rates. This study applied a novel and highly efficient approach to generate whole-genome high-resolution crossover maps in D. yakuba to tackle multiple questions that benefit from being addressed collectively within an appropriate phylogenetic framework, in this case the D. melanogaster species subgroup. The genotyping of more than 1,600 individual meiotic events allowed identification of several key distinct properties relative to D. melanogaster. D. yakuba, in addition to higher crossover rates than D. melanogaster, has a stronger centromere effect and crossover assurance than any Drosophila species analyzed to date. The presence of an active crossover-associated meiotic drive mechanism is reported for the X chromosome that results in the preferential inclusion in oocytes of chromatids with crossovers. This evolutionary and genomic analyses suggest that the genome-wide landscape of crossover rates in D. yakuba has been fairly stable and captures a significant signal of the ancestral crossover landscape for the whole D. melanogaster subgroup, even informative for the D. melanogaster lineage. Contemporary crossover rates in D. melanogaster, on the other hand, do not recapitulate ancestral crossovers landscapes. As a result, the temporal stability of crossover landscapes observed in D. yakuba makes this species an ideal system for applying population genetic models of selection and linkage, given that these models assume temporal constancy in linkage effects (Pettie, 2022).
Quiescent stem cells in adult tissues can be activated for homeostasis or repair. Neural stem cells (NSCs) in Drosophila are reactivated from quiescence in response to nutrition by the insulin signaling pathway. It is widely accepted that quiescent stem cells are arrested in G0. This study, however, demonstrates that quiescent NSCs (qNSCs) are arrested in either G2 or G0. G2-G0 heterogeneity directs NSC behavior: G2 qNSCs reactivate before G0 qNSCs. In addition, this study shows that the evolutionarily conserved pseudokinase Tribbles (Trbl) induces G2 NSCs to enter quiescence by promoting degradation of Cdc25String and that it subsequently maintains quiescence by inhibiting Akt activation. Insulin signaling overrides repression of Akt and silences trbl transcription, allowing NSCs to exit quiescence. These results have implications for identifying and manipulating quiescent stem cells for regenerative purposes (Otsuki, 2018).
Neural stem cells (NSCs) in Drosophila, like those in mammals, proliferate during embryogenesis, become quiescent in the late embryo, and then proliferate again (reactivate) postembryonically to produce neurons and glia. A nutritional stimulus induces reactivation; specifically, dietary amino acids induce glial cells in the blood- brain barrier to secrete Drosophila insulin-like peptides (dILPs). dILPs activate the insulin signaling pathway in neighboring quiescent NSCs (qNSCs), prompting the NSCs to exit quiescence (Otsuki, 2018).
Quiescent stem cells are widely accepted to be arrested in G0, a poorly understood state characterized by a 2n DNA content and a lack of expression of cell cycle progression factors. This study assessed whether Drosophila qNSCs are arrested in G0. As expected, the M phase marker phospho-histone H3 (pH3) was not detected in qNSCs. Previous studies demonstrated that qNSCs do not express the G1 marker cyclin E or incorporate the S phase marker 5-bromo-2'-deoxyuridine (BrdU) or 5-ethynyl-2'-deoxyuridine (EdU). However, it was found that 73% of qNSCs expressed the G2 markers cyclin A (CycA) and cyclin B (CycB). This finding suggests that most qNSCs are arrested in G2 and that qNSCs are arrested heterogeneously in the cell cycle (Otsuki, 2018).
It was verified that ~75% of qNSCs were arrested in G2 by comparing the fluorescent ubiquitination- based cell cycle indicator (FUCCI)-pH3 profiles of qNSCs and proliferating NSCs. CycA-positive (CycA+) qNSCs had twice the DNA content of CycA-negative (CycA-) qNSCs and larger nuclei than CycA- qNSCs. Thus, qNSCs exhibited two types of stem cell quiescence: The majority were arrested in G2, and a minority were arrested in G0. G2 quiescence has not been reported previously for stem cells in mammals or Drosophila (Otsuki, 2018).
The choice of G2 or G0 arrest could be stochastic or preprogrammed. Seven G0 qNSCs were found in the first thoracic hemisegment, T1, and eight G0 qNSCs each in T2 and T3. A consistent subset of qNSCs were always arrested in G0, namely, NB2-2, NB2-4, NB2-5, NB3-4, NB5-3, and NB7-4 (cells are named according to their spatial origin in the neuroectoderm). Of these qNSCs, NB2-4 disappears from T1 during embryogenesis, explaining why fewer qNSCs are arrested in G0 in T1 than in T2 and T3. NB5-4 and NB5-7 were arrested in G2 in 50% of hemisegments but were not always arrested in the same cell cycle phase on either side of the midline. It is concluded that, with the exception of NB5-4 and NB5-7, the choice of G2 or G0 quiescence is entirely invariant (Otsuki, 2018).
Is G2-G0 heterogeneity in qNSCs significant? The reactivation of G2 and G0 qNSCs
by tracking the expression of the reactivation
marker worniu (wor). More than 86% of
G qNSCs reactivated by 20 hours after larval 2
hatching (ALH), compared with 20% of G0 qNSCs. For example, NB3-4, a G0 qNSC, reactivated in fewer than 7% of hemisegments (n = 10 tVNCs, six hemisegments each) . All NSCs reactivated by 48 hours ALH. Thus, G2 qNSCs are faster-reactivating stem cells than G0 qNSCs (Otsuki, 2018).
Next, gene expression was profiled in qNSCs using targeted DamID (TaDa), identifying 1656 genes. Corresponding Gene Ontology (GO) terms included 'nervous system development' (35 genes) and 'neuroblast [NSC] development' (10 genes). To identify quiescence-specific genes, genes common to quiescent and proliferating NSCs, such as deadpan (dpn), were eliminated. tribbles (trbl) is one of the most significantly expressed protein-encoding genes specific to qNSCs. trbl encodes an evolutionarily conserved pseudokinase with three human homologs that have been implicated in insulin and mitogen-
activated protein kinase signaling. It was confirmed that trbl labels quiescent but not proliferating NSCs in vivo. To date, no other gene that labels qNSCs specifically has been identified (Otsuki, 2018).
trbl is necessary for quiescence entry, as NSCs continued to divide during late embryogenesis in trbl hypomorphic mutants or when trbl was knocked down specifically in NSCs. trbl regulates quiescence entry specifically, without affecting division mode or cell viability. The ectopically dividing NSCs in the trblEP3519 mutant were G2,
not G0, qNSCs. G2 but not G0 qNSCs also became significantly smaller in trblEP3519 mutants. As embryonic NSCs do not regrow between cell divisions, the size reduction is consistent with excessive divisions. Consistent with a function in G2 quiescence, Trbl was expressed primarily in G2 qNSCs (Otsuki, 2018).
Trbl is also required to maintain quiescence. RNA interference-mediated knockdown of trbl in qNSCs caused NSCs to leave quiescence and divide. Transgenic flies were generated carrying upstream activation sequence-green fluorescent protein (GFP)-Trbl and drove expression with grainyhead (grh)-GAL4 (4) to assess whether Trbl is sufficient to maintain G2 quiescence. grh-GAL4 expression is initiated at quiescence entry and occurs in ~67% of NSCs, allowing comparison between neighboring GFP-Trbl-expressing and nonexpressing NSCs. Almost all GFP-Trbl-expressing NSCs remained in G2 quiescence and expressed CycA. GFP-Trbl-expressing NSCs retained the primary process that is extended specifically by quiescent NSCs, unlike control NSCs, which had begun to divide. Thus, Trbl is sufficient to maintain G2 quiescence (Otsuki, 2018).
In the embryonic mesoderm, trbl induces G2 arrest by promoting Cdc25String protein degradation. This study found that Cdc25String protein was reduced in NSCs at quiescence entry but that Cdc25String mRNA was maintained (Fig. 4A). Therefore, Cdc25String is regulated posttranscriptionally at quiescence entry. Significantly more NSCs were positive for Cdc25String protein in trblEP3519 mutants than in controls. This increase in Cdc25String is sufficient to explain the excessive NSC proliferation in trbl mutants. Thus, Trbl initiates quiescence entry by promoting Cdc25String protein degradation during late embryogenesis (Otsuki, 2018).
Trbl also maintains NSC quiescence postembryonically; however, it must act through another mechanism, as Cdc25String is no longer expressed in postembryonic qNSCs. Trbl is known to inhibit insulin signaling by binding Akt and preventing its phosphorylation. Consistent with this, Trbl-expressing NSCs had less phosphorylated translation initiation factor 4E-binding protein (p4E-BP) than control NSCs. If Trbl inhibits Akt to maintain quiescence, constitutively active Akt (myr-Akt; here-
after AktACT) should counteract Trbl-induced quiescence. AktACT fully rescued NSC reactivation. In contrast, as Trbl is thought to act downstream of phosphatidylinositol 3-kinase (PI3K), constitutively active PI3K (dp110CAAX; hereafter PI3KACT)should not rescue reactivation, which it did not. Thus, Trbl maintains quiescence by
blocking activation of Akt. This role is specific to postembryonic NSCs, as embryonic NSCs do not depend on insulin signaling to proliferate (Otsuki, 2018).
trbl expression must be repressed to allow NSC reactivation. It was found that insulin signaling is necessary and sufficient to repress trbl transcription. NSCs misexpressing phosphatase and tensin homolog (PTEN), an insulin pathway inhibitor, failed to down-regulate trbl transcription . In contrast, activating the insulin pathway by expressing AktACT in NSCs was sufficient to switch off trbl transcription (Otsuki, 2018).
This study has discovered the mechanisms by which Drosophila NSCs enter, remain in, and exit quiescence in response to nutrition. First, Trbl pseudokinase promotes degradation of Cdc25String protein to induce quiescence; second, it blocks insulin signaling by inhibiting Akt in the same NSCs to maintain quiescence; and third, it is overridden by nutrition-dependent secretion of dILPs from blood-brain barrier glia, which activate insulin signaling in qNSCs, repress trbl expression, and enable reactivation (Otsuki, 2018).
qNSCs were found to be preprogrammed for arrest in G2 or G0, contrary to accepted doctrine. G2 qNSCs are the first to reactivate and generate neurons; this is followed by reactivation of G0 qNSCs. This pattern may ensure that neurons form the correct circuits in the appropriate order during brain development. G2 arrest also enables high-fidelity homologous recombination- mediated repair in response to DNA damage, preserving genomic integrity during quiescence. Quiescent stem cells in mammals may also arrest in G2, with implications for isolating and manipulating quiescent stem cells for therapeutic purposes (Otsuki, 2018).
Cytokinesis is a highly ordered cellular process driven by interactions between central spindle microtubules and the actomyosin contractile ring linked to the dynamic remodelling of the plasma membrane. The mechanisms responsible for reorganizing the plasma membrane at the cell equator and its coupling to the contractile ring in cytokinesis are poorly understood. This study reports that Syndapin, a protein containing an F-BAR domain required for membrane curvature, contributes to the remodelling of the plasma membrane around the contractile ring for cytokinesis. Syndapin colocalizes with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) at the cleavage furrow, where it directly interacts with a contractile ring component, Anillin. Accordingly, Anillin is mislocalized during cytokinesis in Syndapin mutants. Elevated or diminished expression of Syndapin leads to cytokinesis defects with abnormal cortical dynamics. The minimal segment of Syndapin, which is able to localize to the cleavage furrow and induce cytokinesis defects, is the F-BAR domain and its immediate C-terminal sequences. Phosphorylation of this region prevents this functional interaction, resulting in reduced ability of Syndapin to bind to and deform membranes. Thus, the dephosphorylated form of Syndapin mediates both remodelling of the plasma membrane and its proper coupling to the cytokinetic machinery (Takeda, 2013).
This study provides the first compelling description of the requirements for an F-BAR protein in cytokinesis in animal cells. This work does not exclude other F-BAR proteins from participating in cytokinesis, but it does show a positive role for Syndapin in cortical membrane dynamics at the cleavage furrow. Syndapin's localization to the cleavage furrow and its in vitro membrane binding and tubulation are regulated by phosphorylation. The defects in cytokinesis ensuing from phosphomimetic mutants imply that phosphorylation of Syndapin regulates cytokinesis by affecting its membrane association. However, this does not exclude a possible indirect effect whereby phosphorylation may influence the association between Syndapin's SH3 and F-BAR domains, as has been proposed to auto-inhibit its membrane association. These findings would suggest that auto-inhibition results in reduced membrane binding, and yet no increased membrane binding and tubulation is seen with the full-length 12ST>A mutant. This implies that the major effect of phosphorylation is directly upon its membrane association. The phosphorylation of Syndapin could be a mechanism to prevent its premature association with the membrane at the cleavage furrow, as with phosphoregulation of Cdc15p during cytokinesis. Thus, Syndapin joins the F-BAR proteins of S. pombe and S. cerevisiae (Cdc15p and Hof1p, respectively) as proteins that are also phosphoregulated during cytokinesis (Takeda, 2013).
Syndapin's localization to the cleavage furrow requires its association with anionic lipids via its F-BAR domain. Syndapin also colocalizes with and directly binds to Anillin, but this interaction is dispensable for Syndapin localization. By contrast, Anillin is mislocalized during cytokinesis at least in primary spermatocytes of Syndapin mutants. Together these results led to a hypothesis that Syndapin may be a component of the coupling between the plasma membrane and the Anillin ring and hence the contractile ring. Alternatively, as Anillin itself has been proposed to have a role in linking the plasma membrane and the contractile ring, it is possible that Syndapin and Anillin share redundant function, and they may function cooperatively at the interface of plasma membrane and the contractile ring. Other candidate proteins for linking the contractile ring to the plasma membrane in cytokinesis are the C1 domain-containing MgcRacGAP of human cells and the C2 domain-containing protein Inn1 of budding yeast. Interestingly, Inn1 interacts with the F-BAR protein, Hof1p, and together they may cooperatively regulate membrane dynamics during cytokinesis in this organism. The Drosophila genome encodes several uncharacterized C2 domain-containing proteins, and it will be interesting to examine whether any of these proteins function cooperatively with Syndapin in cytokinesis. It is also possible that some of the other Drosophila F-BAR proteins (Cip4, Nwk, FCHo/CG8176, Fps85D and NOSTRIN/CG42388) function in cytokinesis. Such proteins could provide some functional redundancy to the molecular mechanism. Indeed, it cannot be excluded that other molecular components can participate in safeguarding the linkage of the membrane to the contractile ring. The importance of such molecules might vary between tissues, thus accounting for the differences in severity of Syndapin phenotypes between different cell types (Takeda, 2013).
Structure-function analyses demonstrated that expression of Syndapin fragments comprising the minimum segments required for the cleavage furrow localization (i.e. the F-BAR domain plus its C-terminal 65 amino acids) could induce dominant, strong cytokinesis defects. Expression of exogenous Syndapin also induced similar abnormal cortical behaviour with furrow ingression failure and severe generalized blebbing. Surprisingly, despite the robustness of these cytokinesis defects, the localization of the major components of the central spindle (Pavarotti) was not affected, although contractile ring components were misplaced around the central part of the cell. By contrast, expression of Syndapin segments that fail to bind to anionic lipids and localize to the cleavage furrow (i.e. ΔFBAR and K5E) did not affect cytokinesis. These results suggest that Syndapin affects cortical dynamics during cytokinesis by directly associating with anionic lipids on the plasma membrane. The S. pombe F-BAR protein, Cdc15p, has roles in organizing membrane domains into lipid rafts as well as in the contractile ring formation. Thus, it will be of future interest to determine whether Syndapin has an equivalent role in organizing membrane during cytokinesis in animal cells as a means of regulating cortical stiffness and dynamics (Takeda, 2013).
Syndapin is required for synaptic vesicle recycling both in mice and in flies, and an involvement in neuronal morphogenesis is regulated by developmentally controlled phosphorylation. This raises the question of whether the functions of Syndapin in synaptic vesicle trafficking and other developmental processes might follow similar regulatory processes. The shape of a membrane can be described by the radius of curvature in two perpendicular arcs. At the cleavage furrow, the radius of curvature along the axis of cell division will be positive, and perpendicular to this it will be negative. Similar curvatures will arise during vesicle recycling at the interface between the cap of a nascent vesicle and its parent membrane. The banana-shaped structure of F-BAR domains may make them ideal for associating with membrane in the context of such curvature, provided that all molecules orient in the one direction. An involvement of Syndapin in both cytokinesis and synaptic vesicle recycling would suggest that it can generate or stabilize varying degrees of positive curvature. When overexpressed in D.Mel-2 cells, narrow tubules decorated by Syndapin were sometime seen, and in vitro tubules were observed with approximate diameter of 55 nm. However, the diameter of positive curvature of a cleavage furrow will be at least one order of magnitude greater than this. Either Syndapin participates in forming smaller buds that become incorporated into the cleavage furrow or it indeed associates with membranes having larger diameters of curvature than may be suggested by the diameter of the concave face of its F-BAR domain. This latter possibility has some credibility because the extent of curvature will depend on the local membrane concentration of the F-BAR domain, and it would not be expected for the membrane to be saturated with the protein in vivo (otherwise, other membrane interacting proteins would be outcompeted). Thus, only narrow tubules are expected to be formed either in vitro or, as a result of overexpression, in vivo when membrane sites could be saturated (Takeda, 2013).
Several future challenges lay ahead beforethe regulation and roles of Syndapin in cytokinesis can be fully understood. Although the OA sensitivity of the protein phosphatase that dephosphorylates Syndapin suggests it is in the PP1 family, further studies are required to identify precisely the protein phosphatase(s) involved. Similarly, future studies will be necessary to identify the kinase(s) required for Syndapin's phosphoregulation. An understanding of Syndapin's precise cytokinetic role will be aided by more detailed description of its interacting partners. Although Syndapin interacts with Anillin, a full description of its functions in cytokinetic network is still needed. Only with this knowledge will it be understood how it might contribute to the coupling between the contractile ring and central spindle MTs underlying the cleavage furrow and the invaginating membrane (Takeda, 2013).
During cytokinesis, an actomyosin contractile ring drives the separation of the two daughter cells. A key molecule in this process is the inositol lipid PtdIns(4,5)P2, which recruits numerous factors to the equatorial region for contractile ring assembly. Despite the importance of PtdIns(4,5)P2 in cytokinesis, the regulation of this lipid in cell division remains poorly understood. This study identified a role for IPIP27 in mediating cellular PtdIns(4,5)P2 homeostasis. IPIP27 scaffolds the inositol phosphatase oculocerebrorenal syndrome of Lowe (OCRL) by coupling it to endocytic BAR domain proteins. Loss of IPIP27 causes accumulation of PtdIns(4,5)P2 on aberrant endomembrane vacuoles, mislocalization of the cytokinetic machinery, and extensive cortical membrane blebbing. This phenotype is observed in Drosophila and human cells and can result in cytokinesis failure. This study has therefore identified IPIP27 as a key modulator of cellular PtdIns(4,5)P2 homeostasis required for normal cytokinesis. The results indicate that scaffolding of inositol phosphatase activity is critical for maintaining PtdIns(4,5)P2 homeostasis and highlight a critical role for this process in cell division (Carim, 2019).
This study provides a detailed characterization of all phases of S2 cell mitosis visualized by transmission electron microscopy (TEM). A random sample of 144 cells undergoing mitosis was analyzed by TEM, focusing on intracellular membrane and microtubule (MT) behaviors. S2 cells were found to exhibit a behavior of intracellular membranes, involving the formation of a quadruple nuclear membrane in early prometaphase and its disassembly during late prometaphase. After nuclear envelope disassembly, the mitotic apparatus becomes encased by a discontinuous network of endoplasmic reticulum membranes, which associate with mitochondria, presumably to prevent their diffusion into the spindle area. A peculiar metaphase spindle organization was observed. Kinetochores with attached k-fibers are almost invariably associated with lateral MT bundles that can be either interpolar bundles or k-fibers connected to a different kinetochore. This spindle organization is likely to favor chromosome alignment at metaphase and subsequent segregation during anaphase. This study has discovered several previously unknown features of membrane and MT organization during S2 cell mitosis. The genetic determinants of these mitotic features can now be investigated, for instance by using an RNAi-based approach, which is particularly easy and efficient in S2 cells (Strunov, 2018).
Absence of the spindle assembly checkpoint restores mitotic fidelity upon loss of sister chromatid cohesion
The fidelity of mitosis depends on cohesive forces that keep sister chromatids together. This is mediated by cohesin. Cleavage of cohesin marks anaphase onset. Unscheduled loss of sister chromatid cohesion is prevented by a safeguard mechanism known as the spindle assembly checkpoint (SAC). To identify specific conditions capable of restoring defects associated with cohesion loss, a screen was carried out for genes whose depletion modulates Drosophila wing development when sister chromatid cohesion is impaired. Cohesion deficiency was induced by knockdown of the acetyltransferase separation anxiety (San)/Naa50, a cohesin complex stabilizer. Several genes whose function impacts wing development upon cohesion loss were identified. Surprisingly, knockdown of key SAC proteins, Mad2 and Mps1, suppressed developmental defects associated with San depletion. SAC impairment upon cohesin removal, triggered by San depletion or artificial removal of the cohesin complex, prevented extensive genome shuffling, reduced segregation defects, and restored cell survival. This counterintuitive phenotypic suppression was caused by an intrinsic bias for efficient chromosome biorientation at mitotic entry, coupled with slow engagement of error-correction reactions. Thus, in contrast to SAC's role as a safeguard mechanism for mitotic fidelity, removal of this checkpoint alleviates mitotic errors when sister chromatid cohesion is compromised (Silva, 2018).
In agreement with its 'safeguard' function, mitotic errors are often exacerbated by impairment of the SAC. These include defects associated with multiple centrosomes, defective microtubule assembly, or kinetochore structure. This study demonstrates that the opposite happens with cohesion defects. Absence of the SAC alleviated mitotic errors and improved mitotic fidelity after cohesion loss. Cells with a functional SAC undergo extensive chromosome shuffling and consequent genome randomization, whereas virtually no shuffling is observed in absence of the SAC. The detrimental nature of the SAC in the absence of cohesin is likely related to irreversibility of cohesion loss. Most mitotic defects can be corrected over time, but premature cohesin loss is an irreversible error and prolonging mitosis further enhances genome randomization (Silva, 2018).
Improved mitotic fidelity after cohesion loss in absence of the SAC is likely a consequence of slow kinetics of error-correction engagement coupled with a bias toward correct chromosome alignment. Several mechanisms are known to bias chromosome segregation toward the right orientation, including chromosome positioning, centromere geometry, bias on microtubule growth toward the kinetochores, and/or kinetochore-mediated microtubule nucleation. Among these, chromosome geometry is believed to facilitate bipolar attachment by facing one kinetochore to the opposite pole upon attachment. If so, what ensures geometric arrangement during the initial mitotic stages, even in the absence of cohesin? A possible mechanism enabling transient orientation of chromatids toward opposing poles is the incomplete resolution of sister chromatid intertwines. Yet residual catenation in metaphase chromosomes is unable to confer functional cohesion, as removal of cohesin is sufficient to induce immediate chromatid separation. Additional mechanisms may thus impair resolution of sister chromatids during early mitosis, in contrast to metaphase chromosomes. Spindle forces enhance decatenation, and thus resolution of DNA intertwines may only be achieved upon chromosome capture. Recent findings propose that efficient decatenation requires constant 'guiding action' from condensin I, whose maximal levels are observed on chromosomes in late metaphase or anaphase. This gradual condensin loading could limit full decatenation to later stages of mitosis, making residual catenation sufficient to allow transient pseudo-metaphase alignment that biases initial chromosome attachment toward biorientation. Although error prone, this process would be more accurate than total genome randomization resulting from chromosome shuffling (Silva, 2018).
Why cohesion loss is insufficient at triggering error correction during early mitosis remains to be addressed. This could be related to a partial tension state facilitated by pseudo-metaphase chromosomal configuration. Additionally, an intrinsic delayed action of error-correction machinery may further account for observed late shuffling onset. Indeed, slow kinetics or a lag time of Aurora-B-mediated chromosome detachment has been hypothesized in several theoretical studies with little experimental validation. Such intrinsic delay would solve the 'problem of initiation of biorientation' whereby initial low-tension interactions could survive such a tension-sensitive error correction (Silva, 2018).
Interestingly, the interplay between mitotic timing and sister chromatid cohesion has been previously reported in mammalian cells whereby extension of mitosis predisposes to sister chromatid cohesion defects. Cells arrested in mitosis for long periods were shown to display sister chromatid separation (referred as 'cohesion fatigue'). Moreover, defective sister chromatid cohesion was described to be synthetically lethal with impaired APC/C function in Warsaw breakage syndrome (WABS) patient-derived cells as well as several cancer cell lines with cohesion defects. The current observations demonstrate how reduction of mitotic timing is sufficient to rescue segregation defects associated with premature cohesin loss. Importantly, these experiments highlight the detrimental effect of the SAC upon cohesion defects. When sister chromatid cohesion is compromised and mitotic fidelity irreversibly affected, the SAC exacerbates mitotic errors in contrast to its canonical protective function (Silva, 2018).
Cell division is critical for development, organ growth, and tissue repair. The later stages of cell division include the formation of the microtubule (MT)-rich central spindle in anaphase, which is required to properly define the cell equator, guide the assembly of the acto-myosin contractile ring, and ultimately ensure complete separation and isolation of the two daughter cells via abscission. Much is known about the molecular machinery that forms the central spindle, including proteins needed to generate the antiparallel overlapping interzonal MTs. One critical protein that has garnered great attention is Protein Regulator of Cytokinesis 1 (PRC1), or Fascetto (in Drosophila, which forms a homodimer to crosslink interzonal MTs, ensuring proper central spindle formation and cytokinesis. This study reports on a new direct protein interactor and regulator of Feo named Fascetto Interacting Protein (FIP; Sip2). Loss of FIP results in a reduction in Feo localization, rapid disassembly of interzonal MTs, and several defects related to cytokinesis failure, include polyploidization of neural stem cells. Simultaneous reduction in Feo and FIP results in very large, tumor-like, DNA-filled masses in the brain that contain hundreds of centrosomes. In aggregate, these data show that FIP acts directly on Feo to ensure fully accurate cell division (Swider, 2019).
The abnormal wing discs (awd) gene encodes the Drosophila homolog of NME1/NME2 metastasis suppressor genes. Awd acts in multiple tissues where its function is critical in establishing and maintaining epithelial integrity. This study analysed awd gene function in Drosophila epithelial cells using transgene-mediated RNA interference and genetic mosaic analysis. awd knockdown in larval wing disc epithelium leads to chromosomal instability (CIN) and induces apoptosis mediated by activation of c-Jun N-terminal kinase. Forced maintenance of Awd depleted cells, by expressing the cell death inhibitor p35, downregulates atypical protein kinase C and DE-Cadherin. Consistent with their loss of cell polarity and enhanced level of matrix metalloproteinase 1, cells delaminate from wing disc epithelium. Furthermore, the DNA content profile of these cells indicates that they are aneuploid. Overall, these data demonstrate a novel function for awd in maintenance of genomic stability. These results are consistent with other studies reporting that NME1 down-regulation induces CIN in human cell lines and suggest that Drosophila model could be successfully used to study in vivo the impact of NME/Awd induced genomic instability on tumour development and metastasis formation (Romani, 2017).
Genomic stability is critical for cell survival and development and several cellular mechanisms act to maintain genomic integrity. Failure of these mechanisms underlies aging and can lead to malignancies such as cancer and age-related neurodegenerative diseases. Chromosomal instability (CIN) is a form of genomic instability that often leads to aneuploidy, a deleterious condition characterised by copy number changes affecting part or whole chromosomes. Several dysfunctions could lead to CIN. Defective activity of the spindle assembly checkpoint (SAC), a signalling pathway that blocks anaphase onset in response to mis-attachment of chromosomes to the mitotic spindle, leads to CIN and aneuploidy. Work in Drosophila showed that loss of function of SAC genes as well as loss of function of genes involved in spindle assembly, chromatin condensation and cytokinesis induce CIN. More recent work in larval disc epithelia has shown that down-regulation of these genes causes apoptotic cell death trough activation of the c-Jun N-terminal kinase (JNK) pathway. Interestingly, blocking CIN-induced apoptotic cell death induces tumourigenic behaviour including basement membrane degradation, cell delamination, tissue overgrowth and aneuploidy (Romani, 2017).
The abnormal wing discs (awd) gene encodes the Drosophila homolog of NME1/2 metastasis suppressor genes. Awd is a well-known endocytic mediator whose function is required in multiple tissues during development. Genetic studies showed that Awd endocytic function ensures appropriate internalisation of chemotactic signalling receptors such as platelet-derived growth factor/VEGF receptor (PVR) and fibroblast growth factor receptor (FGFR) and thus it regulates invasion and cellular motility. Furthermore, this endocytic function regulates Notch receptor trafficking and is required for maintenance of epithelial integrity as it controls the turnover of adherens junction components in ovarian somatic follicle cells. Consistent with the high degree of functional conservation between Awd and its mammalian counterparts, recent studies have shown a role for the NME1/2 proteins in vesicular transport (Romani, 2017).
This work has extended an analysis of the functional conservation between Awd and NME1/2 proteins. Since loss of NME1 gene function in human cell culture leads to polyploidy, this study have explored the role of Awd in maintenance of genomic stability. The data show that knockdown of awd in wing disc cells leads to CIN and to the CIN-induced biological responses mediated through JNK activation. Furthermore, when combined with block of apoptosis, down-regulation of awd leads to cell delamination and aneuploidy. Thus, the results of this in vivo analysis show a novel function for awd in maintenance of genomic stability (Romani, 2017).
Depletion of Awd triggers JNK-mediated cell death of wing disc cells and blocking the cell death machinery results in aneuploidy and cell delamination without overt hyperproliferative effect. Overgrowth of wing disc hosting aneuploid cells is due to activation of the JNK pathway that promotes expression of Wingless (Wg) upon blocking of apoptotic cell death. Wg is a mitogenic molecule required in the imaginal discs for growth and patterning and its expression in the aneuploid, delaminating CIN cells triggers growth of neighbouring non-delaminating cells. However, awd J2A4 mutant wing disc cells do not express Wg as a consequence of faulty Notch signalling; therefore, these cells cannot promote hyperplasia of the surrounding tissue. Furthermore, lack of hyperproliferation is also observed when an aneuploid condition arises from impaired activity of genes controlling karyokinesis. The diaphanous gene (dia) codes for an actin-regulatory molecule that is required during acto-myosin driven contraction of metaphase furrows. Simultaneous depletion of dia gene expression and blocking of apoptosis do not lead to hyperplastic growth probably due to defective karyokinesis. Intriguingly, Awd is a microtubule-associated nucleoside diphosphate kinase that converts GDP to GTP and the analysis of awd mutant larval brain showed mitotic defects correlated with defective microtubule polymerisation. This raises the possibility that the Awd kinase function plays a role in GTP supply to proteins such as Orbit that are required for stabilisation of spindle microtubules (Romani, 2017).
Two lines of evidence further support the hypothesis that Awd could be involved in karyokinesis. The first comes from studies showing that endosome trafficking and transport to the intercellular bridges of dividing cells plays a critical role during abscission, the last step of karyokinesis. In addition, remodelling the of plasma membrane that underlies nuclear divisions in the syncytial embryo and cellularisation also requires endocytosis. Embryonic cellularisation requires the dynamin encoded by the shibire (shi) locus and Rab5 GTPase function, since loss of function of either genes arrests ingression of metaphase furrows. Awd functionally interacts with shi locus and Awd is also required for Rab5 function in early endosomes. Thus, a possible role for Awd in cytokinesis should be considered (Romani, 2017).
The second line of evidence comes from studies on NME1, the human homolog of awd gene. This metastasis suppressor gene shares about 78% of amino acid identity with the awd gene. Down-regulation of NME1 gene expression in diploid cells results in cytokinesis failure and leads to tetraploidy. The in vivo results show that Awd plays a role in maintenance of genomic stability, confirming the high degree of conservation between NME1 and Awd proteins. Drosophila studies have already been crucial in identification of NME1 function in epithelial morphogenesis, and the present work shows that this function can be a useful model for impact on tumour development and progression (Romani, 2017).
Endoreplication is a cell cycle variant that entails cell growth and periodic genome duplication without cell division, and results in large, polyploid cells. Cells switch from mitotic cycles to endoreplication cycles during development, and also in response to conditional stimuli during wound healing, regeneration, aging, and cancer. This study used integrated approaches in Drosophila to determine how mitotic cycles are remodeled into endoreplication cycles, and how similar this remodeling is between induced and developmental endoreplicating cells (iECs and devECs). The evidence suggests that Cyclin A / CDK directly activates the Myb-MuvB (MMB) complex to induce transcription of a battery of genes required for mitosis, and that repression of CDK activity dampens this MMB mitotic transcriptome to promote endoreplication in both iECs and devECs. iECs and devECs differed, however, in that devECs had reduced expression of E2f1-dependent genes that function in S phase, whereas repression of the MMB transcriptome in iECs was sufficient to induce endoreplication without a reduction in S phase gene expression. Among the MMB regulated genes, knockdown of AurB protein and other subunits of the chromosomal passenger complex (CPC) induced endoreplication, as did knockdown of CPC-regulated cytokinetic, but not kinetochore, proteins. Together, these results indicate that the status of a CycA-Myb-MuvB-AurB network determines the decision to commit to mitosis or switch to endoreplication in both iECs and devECs, and suggest that regulation of different steps of this network may explain the known diversity of polyploid cycle types in development and disease (Rotelli, 2019).
Endoreplication is a common cell cycle variant that entails periodic genome duplication without cell division and results in large polyploid cells. Two variations on endoreplication are the endocycle, a repeated G/S cycle that completely skips mitosis, and endomitosis, wherein cells enter but do not complete mitosis and / or cytokinesis before duplicating their genome again. In a wide array of organisms, specific cell types switch from mitotic cycles to endoreplication cycles as part of normal tissue growth during development. Cells also can switch to endoreplication in response to conditional inputs, for example during wound healing, tissue regeneration, aging, and cancer. It is still not fully understood, however, how the cell cycle is remodeled when cells switch from mitotic cycles to endoreplication (Rotelli, 2019).
There are common themes across plants and animals for how cells switch to endoreplication during development. One common theme is that developmental signaling pathways induce endoreplication by inhibiting the mitotic cyclin dependent kinase 1 (CDK1). After CDK1 activity is repressed, repeated G / S cell cycle phases are controlled by alternating activity of the ubiquitin ligase APC/CCDH1 and Cyclin E / CDK2. Work in Drosophila has defined mechanisms by which APC/CCDH1 and CycE / Cdk2 regulate G / S progression, and ensure that the genome is duplicated only once per cycle. Despite these conserved themes, how endoreplication is regulated can vary among organisms, as well as tissues within an organism. These variations include the identity of the signaling pathways that induce endoreplication, the mechanism of CDK1 inhibition, and the downstream effects on cell cycle remodeling into either an endomitotic cycle (partial mitosis) or endocycle (skip mitosis). In many cases, however, the identity of the developmental signals and the molecular mechanisms of cell cycle remodeling are not known (Rotelli, 2019).
To gain insight into the regulation of variant polyploid cell cycles, two-color microarrays have been used to compare the transcriptomes of endocycling and mitotic cycling cells in Drosophila tissues (Maqbool, 2010). Endocycling cells of larval fat body and salivary gland have been shown to have dampened expression of genes that are normally induced by E2F1, a surprising result for these highly polyploid cells given that many of these genes are required for DNA synthesis. Nonetheless, subsequent studies showed that the expression of the E2F-regulated mouse orthologs of these genes is reduced in endoreplicating cells of mouse liver, megakaryocytes, and trophoblast giant cells. Drosophila endocycling cells also had dampened expression of genes regulated by the Myb transcription factor, the ortholog of the human B-Myb oncogene (MYBL2). Myb is part of a larger complex called Myb-MuvB (MMB), whose subunit composition and functions are mostly conserved from flies to humans. One conserved function of the MMB is the induction of periodic transcription of genes that are required for mitosis and cytokinesis. It was these mitotic and cytokinetic genes whose expression was dampened in Drosophila endocycles, suggesting that this repressed MMB transcriptome may promote the switch to endocycles that skip mitosis. It is not known, however, how E2F1 and Myb activity are repressed during endocycles, nor which of the downregulated genes are key for the remodeling of mitotic cycles into endocycles (Rotelli, 2019).
In addition to endoreplication during development, there are a growing number of examples of cells switching to endoreplication cycles in response to conditional stresses and environmental inputs. These cells will be called induced endoreplicating cells (iECs) to distinguish them from developmental endoreplicating cells (devECs). For example, iECs contribute to tissue regeneration after injury in flies, mice, humans, and the zebrafish heart, and evidence suggests that a transient switch to endoreplication contributes to genome instability in cancer. Cardiovascular hypertension stress also promotes an endoreplication that increases the size and ploidy of heart muscle cells, and this hypertrophy contributes to cardiac disease. It remains little understood how similar the cell cycles of iECs are to devECs (Rotelli, 2019).
Similar to the developmental repression of CDK1 activity to promote endocycles, it has been shown that experimental inhibition of CDK1 activity is sufficient to induce endoreplication in flies, mouse, and human cells. These experimental iECs in Drosophila are similar to devECs in that they skip mitosis, have oscillating CycE / Cdk2 activity, periodically duplicate their genome during G / S cycles, and repress the apoptotic response to genotoxic stress. This study uses these experimental iECs to determine how the cell cycle is remodeled when cells switch from mitotic cycles to endoreplication cycles, and how similar this remodeling is between iECs and devECs. The findings indicate that the status of a CycA-Myb-AurB network determines the choice between mitotic cycles and endoreplication cycles in both iECs and devECs (Rotelli, 2019).
This study has investigated how the cell cycle is remodeled when mitotic cycling cells switch into endoreplication cycles, and how similar this remodeling is between devECs and experimental iECs. Repression of a CycA-Myb-AurB mitotic network promotes a switch to endoreplication in both devECs and iECs. Although a dampened E2F1 transcriptome of S phase genes is a common property of devECs in flies and mice, this study found that repression of the Myb transcriptome is sufficient to induce endoreplication in the absence of reduced expression of the E2F1 transcriptome. Knockdown of different components of the CycA-Myb-AurB network resulted in endoreplication cycles that repressed mitosis to different extents, which suggests that regulation of different steps of this pathway may explain the known diversity of endoreplication cycles in vivo. Overall, these findings define how cells either commit to mitosis or switch to different types of endoreplication cycles, with broader relevance to understanding the regulation of these variant cell cycles and their contribution to development, tissue regeneration, and cancer (Rotelli, 2019).
The findings indicate that the status of the CycA-Myb-AurB network determines the choice between mitotic or endoreplication cycles (The CycA-Myb-AurB network regulates the choice between cell cycle programs). These proteins are essential for the function of their respective protein complexes: CycA activates CDK1 to regulate mitotic entry, Myb is required for transcriptional activation of mitotic genes by the MMB transcription factor complex, and AurB is the kinase subunit of the four-subunit CPC. While each of these complexes were previously known to have important mitotic functions, the data indicate that they are key nodes of a network whose activity level determines whether cells switch to the alternative growth program of endoreplication. The results are consistent with previous evidence in several organisms that lower activity of the Myb transcription factor results in polyploidization, and further shows that repressing the function of the CPC and cytokinetic proteins downstream of Myb also promotes endoreplication. Importantly, genetic evidence indicates that not all types of mitotic inhibition result in a switch to endoreplication. For example, knockdown of the Spc25 and Spc105R kinetochore proteins or the Polo kinase resulted in a mitotic arrest, not a switch to repeated endoreplication cycles. These observations are consistent with CycA / CDK, MMB, and the CPC playing principal roles in the mitotic network hierarchy and the decision to either commit to mitosis or switch to endoreplication cycles (Rotelli, 2019).
While knockdown of different proteins in the CycA-Myb-AurB network were each sufficient to induce endoreplication cycles, these iEC populations had different fractions of cells with multiple nuclei diagnostic of an endomitotic cycle. Knockdown of cytokinetic genes pav and tum resulted in the highest fraction of endomitotic cells, followed by the CPC subunits, then Myb, and finally CycA, with knockdown of this cyclin resulting in the fewest endomitotic cells. These results suggest that knocking down genes higher in this branching mitotic network (e.g. CycA) inhibits more mitotic functions and preferentially promotes G / S endocycles that skip mitosis, whereas inhibition of functions further downstream in the network promote endomitosis. Moreover, different levels of CPC function also resulted in different subtypes of endoreplication. Strong knockdown of AurB inhibited chromosome segregation and cytokinesis resulting in cells with a single polyploid nucleus, whereas a mild knockdown resulted in successful chromosome segregation but failed cytokinesis, suggesting that cytokinesis requires more CPC function than chromosome segregation. It thus appears that different thresholds of mitotic function result in different types of endoreplication cycles. This idea that endomitosis and endocycles are points on an endoreplication continuum is consistent with evidence that treatment of human cells with low concentrations of CDK1 or AurB inhibitors induces endomitosis, whereas higher concentrations induce endocycles. The results raise the possibility that in tissues of flies and mammals both conditional and developmental inputs may repress different steps of the CycA-Myb-AurB network to induce slightly different types of endoreplication cycles that partially or completely skip mitosis. Together, these findings show that there are different paths to polyploidy depending on both the types and degree to which different mitotic functions are repressed (Rotelli, 2019).
The findings are relevant to the regulation of periodic MMB transcription factor activity during the canonical mitotic cycle. Knockdown of CycA compromised MMB transcriptional activation of mitotic gene expression, and their physical association suggests that the activation of the MMB by CycA may be direct. The MMB-regulated mitotic genes were expressed at lower levels in CycA iECs, even though Myb protein levels were not reduced. This result is consistent with the hypothesis that CycA / CDK phosphorylation of the MMB is required for its induction of mitotic gene expression. Moreover, misexpression of Myb in CycA knockdown follicle cells did not prevent the switch to endoreplication, further evidence that CycA / CDK is required for MMB activity and mitotic cycles. While the dependency of the MMB on CycA was not previously known in Drosophila, it was previously reported that in human cells CycA / CDK2 phosphorylates and activates human B-Myb in late S phase, and also triggers its degradation. While further experiments are needed to prove that CycA / CDK regulation of the MMB is direct, interrogation of the results of multiple phosphoproteome studies using iProteinDB indicated that Drosophila Myb protein is phosphorylated at three CDK consensus sites including one, S381 that is of a similar sequence and position to a CDK phosphorylated site on human B-Myb (T447). The hypothesis is favored that it is CycA complexed to CDK1 that regulates the MMB because, unlike human cells, in Drosophila CycA / CDK2 is not required for S phase, and Myb is degraded later in the cell cycle during mitosis. Moreover, it is known that mutations in CDK1, but not CDK2, induce endocycles in Drosophila, mouse, and other organisms. A cogent hypothesis is that CycA / CDK1 phosphorylates Myb, and perhaps other MMB subunits, to stimulate MMB activity as a transcriptional activator of mitotic genes, explaining how pulses of mitotic gene expression are integrated with the master cell cycle control machinery. It remains formally possible, however, that both CycA / CDK2 and CycA / CDK1 activate the MMB in Drosophila. The early reports that CycA / CDK2 activates B-Myb in human cells were before the discovery that it functions as part of the MMB and the identification of many MMB target genes, and further experiments are needed to fully define how MMB activity is coordinated with the central cell cycle oscillator in fly and human cells (Rotelli, 2019).
Endocycles were experimentally induced by knockdown of CycA to mimic the repression of CDK1 that occurs in devECs. The data revealed both similarities and differences between these experimental iECs and devECs. Both iECs and SG devECs had a repressed CycA-Myb-AurB network of mitotic genes. In contrast, only devECs had reduced expression of large numbers of E2F1-dependent S phase genes, a conserved property of devECs in fly and mouse. In CycA iECs, many of these key S phase genes were not downregulated, including Cyclin E, PCNA, and subunits of the pre-Replicative complex, among others. This difference between CycA dsRNA iECs and SG devECs indicates that repression of these S phase genes is not essential for endoreplication. In fact, none of the E2F1 -dependent S phase genes were downregulated in Myb dsRNA iEC. Instead, the 12 E2F1-dependent genes that were commonly downregulated in Myb dsRNA iEC, CycA dsRNA iEC, and SG devEC all have functions in mitosis. These 12 mitotic genes are, therefore, dependent on both Myb and E2F1 for their expression, including the cytokinetic gene tum whose knockdown induced endomitotic cycles. This observation leads to the hypothesis that downregulation of the E2F transcriptome in fly and mouse devECs may serve to repress the expression of these mitotic genes, and that the repression of S phase genes is a secondary consequence of this regulation. These genomic data, together with the genetic evidence in S2 cells and tissues, indicates that in Drosophila the repression of the Myb transcriptome is sufficient to induce endoreplication without repression of the E2F1 transcriptome. The observation that both CycAdsRNA iECs and devECs both have lower CycA / CDK activity, but differ in expression of E2F1 regulated S phase genes, also implies that there are CDK-independent mechanisms by which developmental signals repress the E2F1 transcriptome in devECs (Rotelli, 2019).
The results have broader relevance to the growing number of biological contexts that induce endoreplication. Endoreplicating cells are induced and contribute to wound healing and regeneration in a number of tissues in fly and mouse, and, depending on cell type, can either inhibit or promote regeneration of the zebrafish heart. An important remaining question is whether these iECs, like experimental iECs and devECs, have a repressed CycA-Myb-AurB network. If so, manipulation of this network may improve regenerative therapies. In the cancer cell, evidence suggests that DNA damage and mitotic stress, including that induced by cancer therapies, can switch cells into an endoreplication cycle. These therapies include CDK and AurB inhibitors, which induce human cells to polyploidize, consistent with the fly data that CycA / CDK and the CPC are key network nodes whose repression promotes the switch to endoreplication. Upon withdrawal of these inhibitors, transient cancer iECs return to an error-prone mitosis that generates aneuploid cells, which have the potential to contribute to therapy resistance and more aggressive cancer progression. The finding that the Myb transcriptome is repressed in iECs opens the possibility that these mitotic errors may be due in part to a failure to properly orchestrate a return of mitotic gene expression. Understanding how this and other networks are remodeled in polyploid cancer cells will empower development of new approaches to prevent cancer progression (Rotelli, 2019).
Polyploidy frequently arises in response to injury, aging, and disease. Despite its prevalence, major gaps exist in understanding of how polyploid cells alter tissue function. In the adult Drosophila epithelium, wound healing is dependent on the generation of multinucleated polyploid cells resulting in a permanent change in the epithelial architecture. This study shows how the wound-induced polyploid cells affect tissue function by altering epithelial mechanics. The mechanosensor nonmuscle myosin II is activated and upregulated in wound-induced polyploid cells and persists after healing completes. Polyploidy enhances relative epithelial tension, which is dependent on the endocycle and not cell fusion post injury. Remarkably, the enhanced epithelial tension mimics the relative tension of the lateral muscle fibers, which are permanently severed by the injury. As a result, this study found that the wound-induced polyploid cells remodel the epithelium to maintain fly abdominal movements, which may help compensate for lost tissue tension (Losick, 2021).
According to the prevailing 'clock' model, chromosome decondensation and nuclear envelope reformation when cells exit mitosis are byproducts of Cdk1 inactivation at the metaphase-anaphase transition, controlled by the spindle assembly checkpoint. However, mitotic exit was recently shown to be a function of chromosome separation during anaphase, assisted by a midzone Aurora B phosphorylation gradient - the 'ruler' model. This study found that Cdk1 remains active during anaphase due to ongoing APC/C(Cdc20)- and APC/C(Cdh1)-mediated degradation of B-type Cyclins in Drosophila and human cells. Failure to degrade B-type Cyclins during anaphase prevented mitotic exit in a Cdk1-dependent manner. Cyclin B1-Cdk1 localized at the spindle midzone in an Aurora B-dependent manner, with incompletely separated chromosomes showing the highest Cdk1 activity. Slowing down anaphase chromosome motion delayed Cyclin B1 degradation and mitotic exit in an Aurora B-dependent manner. Thus, a crosstalk between molecular 'rulers' and 'clocks' licenses mitotic exit only after proper chromosome separation (Afonso, 2019).
Taken together, this work reveals that degradation of B-type Cyclins specifically during anaphase is rate-limiting for mitotic exit among animals that diverged more than 900 million years ago. Most importantly, Cdk1 activity during anaphase was shown to be a function of chromosome separation and is spatially regulated by Aurora B localization and activity at the spindle midzone. In concert with previous work, the findings unveil an unexpected crosstalk between molecular 'rulers' (Aurora B) and 'clocks' (B-type Cyclins-Cdk1) that ensures that cells only exit mitosis after proper chromosome separation during anaphase, consistent with the previously proposed chromosome separation checkpoint hypothesis. An Aurora B-dependent spatial control mechanism regulating normal NER in human cells has been recently confirmed. However, nuclear envelope defects associated with incomplete chromosome separation during anaphase (namely, anaphase lagging chromosomes due to mitotic errors) were proposed as an inevitable pathological condition. The present work provides yet additional evidence for a molecular network operating during anaphase that promotes chromosome segregation fidelity by controlling mitotic exit in space and time. According to this model, APC/CCdc20 mediates the initial degradation of Cyclin B1 during metaphase under SAC control. The consequent decrease in Cdk1 activity as cells enter anaphase targets Aurora B to the spindle midzone (via Subito/Mklp2/kinesin-6); Aurora B at the spindle midzone (counteracted by PP1/PP2A phosphatases on chromatin establishes a phosphorylation gradient that locally delays APC/CCdc20- and APC/CCdh1-mediated degradation of residual Cyclin B1 (and possibly B3) at the spindle midzone, at least in Drosophila cells. Localization experiments in human cells suggest that Cdk1 itself might be enriched at the spindle midzone. Consequently, as chromosomes separate and move away from the spindle midzone, Cdk1 activity decreases, allowing the PP1/PP2A-mediated dephosphorylation of Cdk1 and Aurora B substrates (e.g., Lamin B and Condensin I) necessary for mitotic exit. This model is consistent with the recent demonstration that Cdk1 inactivation promotes the recruitment of PP1 phosphatase to chromosomes to locally oppose Aurora B phosphorylation and recent findings in budding yeast demonstrating equivalent phosphorylation and dephosphorylation events during mitotic exit. It is also consistent with a premature Greatwall inactivation and PP2A:B55 reactivation that would be predicted after acute Cdk1 inactivation during anaphase. Most important, this model provides an explanation for the coordinated action of two unrelated protein kinases that likely regulate multiple substrates required for mitotic exit (Afonso, 2019).
Previous landmark work has carefully monitored the kinetics of Cyclin B1 degradation in living human HeLa and rat kangaroo Ptk1 cells during mitosis, and concluded that Cyclin B1 was degraded by the end of metaphase, becoming essentially undetectable as cells entered anaphase. However, it was noticed that, consistent with the current findings, a small pool of Cyclin B1 continued to be degraded during anaphase in Ptk1 cells. Subsequent work investigating cellular response to anti-mitotic drugs has also shown that human DLD-1 cells undergoing normal mitosis entered anaphase with as much as 32% of Cyclin B1 compared to metaphase levels, suggesting that human cells enter anaphase with significant Cdk1 activity. Indeed, quantitative analysis with a FRET biosensor in human HeLa cells also revealed residual Cdk1 activity during anaphase. However, the significance of persistent Cdk1 activity for the control of anaphase duration and mitotic exit was not investigated in these original studies. Previous works also clearly demonstrated that forcing Cdk1 activity during anaphase through expression of non-degradable Cyclin B1 (and Cyclin B3 in Drosophila) prevents chromosome decondensation and NER. However, while these works suggested the existence of different Cyclin B1 thresholds that regulate distinct mitotic transitions, expression of non-degradable Cyclin B1 could be interpreted as an artificial gain of function that preserves Cdk1 activity during anaphase. For example, it was shown that expression of non-degradable Cyclin B1 during anaphase 'reactivates' the SAC, inhibiting APC/CCdc20. The current work demonstrates in five different experimental systems, from flies to humans, including primary tissues, that Cdk1 activity persists during anaphase and is rate-limiting for the control of mitotic exit. Failure to degrade B-type Cyclins during anaphase blocked cells in an anaphase-like state with separated sister chromatids that remained condensed for several hours, whereas complete Cdk1 inactivation in anaphase triggered chromosome decondensation and NER. Importantly, if a positive feedback loop imposed by phosphatases was sufficient to drive mitotic exit simply by reverting the effect of Cdk1 phosphorylation prior to anaphase, cells would exit mitosis regardless of the remaining pool of B-type Cyclins that sustains Cdk1 activity during anaphase. The main conceptual implication of these findings is that, contrary to what was previously assumed, mitotic exit is determined during anaphase and not at the metaphase-anaphase transition under SAC control (Afonso, 2019).
This model also implies that persistent Cyclin B1-Cdk1 in anaphase is spatially regulated by a midzone Aurora B gradient. Indeed, a residual pool of Cyclin B1-Cdk1 was identified enriched at the spindle midzone and midbody, this localization was dependent on Aurora B activity and localization at the spindle midzone. Interestingly, human Cyclin B2 (which contains a recognizable KEN box), as well as Cdk1, were identified at the midbody and Cdk1 inactivation during late mitosis was required for the timely completion of cytokinesis in human cells. Thus, it is possible that in human cells, Cdk1 activity during anaphase is regulated not only by Cyclin B1, but also by Cyclin B2. Importantly, this model predicted the existence of a midzone-centered Cdk1 activity gradient during anaphase, which was confirmed experimentally by targeting a FRET reporter of Cdk1 activity to chromosomes (Afonso, 2019).
Finally, the experiments indicate that Aurora B activity regulates Cyclin B1 homeostasis and consequently anaphase duration in the presence of incompletely separated chromosomes. One possibility is that direct Cyclin B1 phosphorylation by Aurora B spatially regulates Cyclin B1 degradation during anaphase, mediated by both APC/CCdc20 and APC/CCdh1. Another non-mutually exclusive possibility is that Aurora B indirectly controls Cyclin B1 during anaphase by regulating APC/CCdc20 and/or APC/CCdh1 activity, as recently shown for Cdk1. Future work will be necessary to test these hypotheses (Afonso, 2019).
In conclusion, this study has uncovered an unexpected level of regulation at the end of mitosis in metazoans and reconciled what were thought to be antagonistic models of mitotic exit relying either on molecular 'clocks' or on 'rulers'. These findings have profound implications to fundamental understanding of how tissue homeostasis is regulated, perturbation of which is a hallmark of human cancers (Afonso, 2019).
Meiosis and oocyte maturation are tightly regulated processes. The meiosis arrest female 1 (MARF1) gene is essential for meiotic progression in animals; however, its detailed function remains unclear. This study examined the molecular mechanism of dMarf1, a Drosophila homolog of MARF1 encoding an OST and RNA Recognition Motif (RRM) -containing protein for meiotic progression and oocyte maturation. Although oogenesis progressed in females carrying a dMarf1 loss-of-function allele, the dMarf1 mutant oocytes were found to contain arrested meiotic spindles or disrupted microtubule structures, indicating that the transition from meiosis I to II was compromised in these oocytes. The expression of the full-length dMarf1 transgene, but none of the variants lacking the OST and RRM motifs or the 47 conserved C-terminal residues among insect groups, rescued the meiotic defect in dMarf1 mutant oocytes. These results indicate that these conserved residues are important for dMarf1 function. Immunoprecipitation of Myc-dMarf1 revealed that several mRNAs are bound to dMarf1. Of those, the protein expression of nanos (nos), but not its mRNA, was affected in the absence of dMarf1. In the control, the expression of Nos protein became downregulated during the late stages of oogenesis, while it remained high in dMarf1 mutant oocytes. It is proposed that dMarf1 translationally represses nos by binding to its mRNA. Furthermore, the downregulation of Nos induces cycB expression, which in turn activates the CycB/Cdk1 complex at the onset of oocyte maturation (Kawaguchi, 2020).
Nos is an evolutionary conserved protein that play important roles in early embryogenesis, formation of primordial germ cells and maintenance of germline stem cells (GSCs). However, the function of Nos during late oogenesis has not been described yet. Nos protein expression reaches the maximum around stage 10 of oogenesis and immediately reduces thereafter. This suggests that Nos expression is tightly regulated during late oogenesis. The current results indicate that dMarf1 regulates Nos expression by inhibiting its translation in late oogenesis. In the absence of dMarf1 expression during early-to-mid oogenesis, Nos might repress cycB to prevent the premature release of meiotic arrest until stage 10 of oogenesis. These results suggest that dMarf1 may coordinate oocyte maturation during late oogenesis in Drosophila; moreover, dMarf1 is dominantly expressed after stage 10, and binds to nos mRNA to repress its translation and reduce its expression. Consequently, Nos expression almost disappears at stages 12-14. Reduced expression of Nos induces the release of cycB mRNA from repression and promotes CycB translation. Subsequently, CycB binds to Cdk1 to form the active MPF complex to promote meiosis. Therefore, dMarf1 plays an important role in the release of the second meiotic arrest to drive embryogenesis (Kawaguchi, 2020).
The MARF1 gene is evolutionarily conserved in animals; most proteins of the MARF1 family contain three major domains: NYN, OST (also known as LOTUS), and RRM. Although the ribonuclease activity of NYN is essential for MARF1 function in mouse, it is not required in Drosophila dMarf1. The OST domain is present in the proteins of several species ranging from bacteria to humans. Drosophila melanogaster has has four members of OST domain-containing proteins: dMarf1, Oskar (Osk), Tejas (Tej), and Tapas (Tap). All of these proteins except dMarf1 contain a single OST domain, and are predominantly expressed in germline cells. Structural and biochemical studies of Osk, Tej, and Tap OST domains have revealed the ability of this domain to bind to Vasa, an RNA helicase expressed exclusively in germline cells. Interestingly, the OST domain(s) of MARF1 family members is smaller than those of other proteins and does not bind to Vasa. Instead, a recent study reported that dMarf1 could bind to CCR4-Not deadenylase complex via the OST domain(s); however, the importance and cooperation between multiple OST domains remains elusive. In addition to these two domains, the MARF1 family members contain one or two RRM domains. This study showed that dMarf1 translationally repressed nos mRNA. nos may not be the only target of dMarf1; other mRNAs such as tra2 and abo can also be targeted by dMarf1, although the biological significance of these interactions remains unclear. Zhu has recently reported that dMarf1 can bind to cyclin A mRNA via its RRM domain (Zhu, 2018). However, RNA-IP analysis did not detect cyclin A in the dMarf1-bound mRNA fraction. Further studies of the molecular mechanism underlying the specificity of dMarf1 RNA binding will reveal the range of mRNA regulation during oocyte maturation (Kawaguchi, 2020).
The C-terminal region of the MARF1 family members is highly conserved among higher animals, except insects, despite not forming any secondary structure. The C-terminal region of human MARF1 has been shown to directly interact with the decapping complex component Ge-1; however, this interaction was not observed in Drosophila. Moreover, the C-terminal region of the MARF1 family members is conserved among different insect species, but different from that of higher animals, indicating that it may bind to a unique partner in insects. This hypothesis was supported by results showing that transgenic dMarf1 mutant lacking 47 C-terminal residues (ΔC47) could not rescue dMarf1KO321 mutant phenotype (Kawaguchi, 2020).
In addition to binding to the RNA decapping complex subunit human MARF1 can localize to processing body (P-body), which is often related to translational repression and mRNA decay. Mouse MARF1 has also been shown to degrade target mRNA via its NYN domain, which is absent in Drosophila dMarf1. Transcriptome analysis of mouse MARF1 mutant oocytes revealed that 1,470 transcripts were upregulated in the steady state, whereas 103 transcripts were downregulated, indicating a global impact on RNA homeostasis in the mutant oocytes. By contrast, the expression of a few RNAs was downregulated and dp1 mRNA expression was upregulated in Drosophila dMarf1 mutant ovaries. These results suggest that mammalian MARF1 may regulate the global transcriptome predominantly by degradation, while dMarf1 represses translation of target proteins such as CycB and CycA by modulating Nos/Pumilio and the CCR4-NOT deadenylase complex, respectively (Kawaguchi, 2020).
In addition to nos mRNA, dMarf1 can bind to other mRNAs, including tra2 and abo. The mRNA expression of tra2 was not significantly affected in stage 14 dMarf1KO312 egg chambers, suggesting that dMarf1 post-transcriptionally regulates tra2 expression. abo is a negative regulator of histone gene expression and its expression is downregulated in mature oocytes to produce more histones. The expression of abo mRNA was upregulated by approximately three-fold in stage 14 dMarf1KO312 egg chambers. This may result in the overexpression of Abo protein in dMarf1 mutant oocytes, which in turn causes the downregulation of histone proteins that are required for embryogenesis (Kawaguchi, 2020).
The Ppp2cb gene encodes a protein phosphatase that is involved in cell cycle regulation. Ppp2cb has been previously reported as a major downstream effector of mouse MARF1. The high expression of Ppp2CB phosphatase in the MARF1 mutant ovaries of mouse can disrupt meiosis. However, the expression of mts, a Drosophila homolog of Ppp2cb, was not affected in dMarf1 mutant ovaries, suggesting that the signaling pathway for the activation of the M phase promoting factor, CycB/Cdk1, is not conserved between mouse and Drosophila. Although the direct activation of MPF in mouse MARF1 mutant oocytes by the inhibition of Ppp2cb rescued meiotic defect, embryogenesis of mutant oocytes was affected, suggesting that Ppp2cb may have additional functions in addition to MPF activation. Similarly, dMarf1KO312 ovaries exhibited not only meiotic defects, but also translationally downregulated some proteins required for embryogenesis, such as Dhd and Gnu. In conclusion, MARF1 may trigger oocyte maturation and coordinate multiple events during late oogenesis and fertilization (Kawaguchi, 2020).
The early stages of development involve complex sequences of morphological changes that are both reproducible from embryo to embryo and often robust to environmental variability. To investigate the relationship between reproducibility and robustness this study examined cell cycle progression in early Drosophila embryos at different temperatures. The experiments show that while the subdivision of cell cycle steps is conserved across a wide range of temperatures (5-35 °C), the relative duration of individual steps varies with temperature. The transition into prometaphase is delayed at lower temperatures relative to other cell cycle events, arguing that it has a different mechanism of regulation. Using an in vivo biosensor, the ratio of activities were quantified of the major mitotic kinase, Cdk1 and one of the major mitotic phosphatases PP1. Comparing activation profile with cell cycle transition times at different temperatures indicates that in early fly embryos activation of Cdk1 drives entry into prometaphase but is not required for earlier cell cycle events. In fact, chromosome condensation can still occur when Cdk1 activity is inhibited pharmacologically. These results demonstrate that different kinases are rate-limiting for different steps of mitosis, arguing that robust inter-regulation may be needed for rapid and ordered mitosis (Falahati, 2021).
Using a gain-of-function screen in Drosophila, the Kruppel-like factor Cabut (Cbt) as a positive regulator of cell cycle gene expression and cell proliferation. Enforced cbt expression is sufficient to induce an extra cell division in the differentiating fly wing or eye, and also promotes intestinal stem cell divisions in the adult gut. Although inappropriate cell proliferation also results from forced expression of the E2f1 transcription factor or its target, Cyclin E, Cbt does not increase E2F1 or Cyclin E activity. Instead, Cbt regulates a large set of E2F1 target genes independently of E2F1, and the data suggest that Cbt acts via distinct binding sites in target gene promoters. Although Cbt was not required for cell proliferation during wing or eye development, Cbt is required for normal intestinal stem cell divisions in the midgut, which expresses E2F1 at relatively low levels. The E2F1-like functions of Cbt identify a distinct mechanism for cell cycle regulation that may be important in certain normal cell cycles, or in cells that cycle inappropriately, such as cancer cells (Zhang, 2021).
The timing mechanism for mitotic progression is still poorly understood. The ">spindle assembly checkpoint (SAC), whose reversal upon chromosome alignment is thought to time anaphase, is functional during the rapid mitotic cycles of the Drosophila embryo; but its genetic inactivation had no consequence on the timing of the early mitoses. Mitotic cyclins-Cyclin A, Cyclin B, and Cyclin B3-influence mitotic progression and are degraded in a stereotyped sequence. RNAi knockdown of Cyclins A and B resulted in a Cyclin B3-only mitosis in which anaphase initiated prior to chromosome alignment. Furthermore, in such a Cyclin B3-only mitosis, colchicine-induced SAC activation failed to block Cyclin B3 destruction, chromosome decondensation, or nuclear membrane re-assembly. Injection of Cyclin B proteins restored the ability of SAC to prevent Cyclin B3 destruction. Thus, SAC function depends on particular cyclin types. Changing Cyclin B3 levels showed that it accelerated progress to anaphase, even in the absence of SAC function. The impact of Cyclin B3 on anaphase initiation appeared to decline with developmental progress. These results show that different cyclin types affect anaphase timing differently in the early embryonic divisions. The early-destroyed cyclins-Cyclins A and B-restrain anaphase-promoting complex/cyclosome (APC/C) function, whereas the late-destroyed cyclin, Cyclin B3, stimulates function. It is proposed that the destruction schedule of cyclin types guides mitotic exit by affecting both Cdk1 and APC/C, whose activities change as each cyclin type is lost (Yuan, 2015).
Work in tissue culture cells suggested a 'wait-until-ready' model for the control of anaphase onset, wherein unattached chromosomes activate the spindle assembly checkpoint (SAC) to prevent anaphase-promoting complex/cyclosome (APC/C) activation until all the chromosomes are attached (i.e., ready for anaphase). A shortcoming of this mechanism appears in a multinucleate cell where the first spindle to satisfy the SAC activates anaphase in the entire cell. Since this activation by the lead nucleus short-circuits regulation in all slower nuclei, this mode of timing control appears inappropriate for the syncytial Drosophila embryo (Yuan, 2015).
The mitotic cyclins are degraded in an orderly sequence, with Cyclin A disappearing in metaphase, Cyclin B near the time of onset of anaphase, and Cyclin B3 during anaphase. Moreover, destruction of each cyclin is required for progress to the next stage of mitosis. Hence, the schedule of destruction ought to pace the progress of mitotic exit. To test the basis of timing control of anaphase initiation during the syncytial mitotic cycles of early Drosophila embryos, SAC was inactivated or levels of the different mitotic cyclins were manipulted, and the duration of metaphase was analyzed (Yuan, 2015).
The function of the SAC is dispensable for Drosophila, as mad2 null and bubR1ΔKEN mad2 double mutants were viable and fertile. Although loss of the SAC slightly accelerated progress to anaphase in larval neuroblasts, no difference was detected in metaphase length in early embryos. Thus, the SAC did not contribute to the timing of metaphase-anaphase transition at this developmental stage.
As reported previously, RNAi knockdown of the three mitotic cyclins in embryos blocks the cell cycle rapidly and effectively (McCleland, 2008), and knockdown of individual or pairs of cyclins gives distinct phenotypes (McCleland, 2009; Yuan, 2012). Knocking down the early-degraded cyclins, Cyclin A and Cyclin B, accelerated progress to anaphase and led to mitoses without metaphase. Embryos entered anaphase prematurely and chromosomes were randomly segregated. Reciprocally, injection of mRNA encoding these early-degraded cyclins delayed chromosome segregation. Surprisingly, Cyclin B3 knockdown moderately extended metaphase, and injection of Cyclin B3 mRNA slightly advanced anaphase. It is concluded that the early-degraded cyclins delay anaphase, whereas Cyclin B3 advances it (Yuan, 2015).
How do the early cyclins inhibit anaphase entry? The APC/C system has a poorly understood capacity to degrade different substrates in an orderly progression, and Cyclin A and Cyclin B are among the most preferred substrates. These cyclins enjoy several cyclin-type-specific interactions that promote their early recruitment to the APC/C. These include a binding interaction between cyclin-dependent kinase regulatory subunit 1 (Cks1) and phosphorylated APC/C and complex interactions with a category of APC/C inhibitory proteins, Rca1/Emi1/Emi2. The strong interactions may commit APC/C to these preferred substrates to enforce ordered destruction. As long as the APC/C is preoccupied with destruction of these early cyclins, its action on other substrates will be inhibited, deferring their destruction. If the early cyclins suppress APC/C-mediated destruction of later substrates, perhaps they also contribute to checkpoint suppression of APC/C activity (Yuan, 2015).
To investigate cyclin influence on SAC function, embryos with were treated with colchicine after RNAi knockdown of pairs of cyclins. Control embryos treated with colchicine stably arrested with condensed chromosomes and had no detectable nuclear membrane. Embryos with only Cyclin B also exhibited persistent chromosome condensation. The chromosomes in embryos with only Cyclin A started to decondense after a moderate arrest, consistent with the previously reported continued degradation of Cyclin A at a checkpoint arrest. The chromosomes of embryos with only Cyclin B3 began to decondense after a short arrest, and nuclear membrane staining appeared. This study concluded that spindle disruption does not stably arrest a Cyclin B3-only mitosis despite the expectation that SAC should stabilize this late-degrading cyclin (Yuan, 2015)
The failure of SAC in the Cyclin B3-only mitosis might reflect an inability to activate the spindle checkpoint. However, Mad2 recruitment to the prometaphase kinetochores, a hallmark of SAC activation, still occurs in the Cyclin B3-only mitosis. This focused the attention of this study on the function of the SAC (Yuan, 2015).
Since a stable form of Cyclin B3 arrests cells in late mitosis with condensed chromosomes, escape from a mitotic arrest ought to be associated with Cyclin B3 destruction. To characterize cyclin degradation, mRNAs encoding mitotic cyclins were made with an EGFP tag fused to their C termini. Cyclin A-GFP and Cyclin B-GFP were enriched on centrosomes and kinetochores in mitosis. Interestingly, Cyclin B3-GFP was enriched on nuclear envelope/ER-like membranous structures that bracket the spindle in early Drosophila mitoses. Unlike Cyclin A, Cyclin B3 was stabilized in colchicine-injected wild-type embryos. However, when Cyclin A and Cyclin B were knocked down, Cyclin B3 was degraded in the presence of colchicine. Injection of recombinant Cyclin B proteins into these embryos blocked Cyclin B3 degradation. Degradation of Cyclin B3 during a Cyclin B3-only mitosis was mediated by APC/C, as inhibition of APC/C activity by injecting UbcH10C114S prevented its destruction. It is concluded that SAC needs the early cyclins in order to stabilize the late-degrading Cyclin B3 (Yuan, 2015).
The prevailing model for SAC is not sufficient to explain why the SAC requires early-degrading cyclins. Substrate is recruited to APC/C via the interaction between the destruction box of substrate and the WD40 domains of Cdc20. The SAC, once activated, is thought to function by blocking substrate binding to Cdc20. SAC inhibition of Cdc20 should inhibit Cyclin B3 destruction without reliance on an early cyclin. Apparently, the inhibition of APC/C by the SAC is more complex. It is suggested that Cyclin B continues to engage the APC/C during an SAC arrest, perhaps via Cdc20-independent interactions, and that SAC suppresses the ability of APC/C to degrade Cyclin B, effectively freezing ordered degradation and holding the APC/C in abeyance (Yuan, 2015).
Cyclin B3 knockdown extended metaphase. Perhaps, this is the result of activation of SAC upon Cyclin B3 knockdown. The Cyclin B3 RNAi experiment was repeated in the mad2 null embryos. Cyclin B3 knockdown still caused a metaphase delay in the SAC-deficient embryos (Yuan, 2015).
Although independent of SAC, it was thought that Cyclin B3 might alter early mitotic events with secondary effects on the APC/C and transition to anaphase. The mad2 mutant offered a context in which it was possible disrupt the spindle and normal events of mitotic progress with colchicine and still assess mitotic exit. To test whether Cyclin B3 influences mitotic progression in these embryos, Cyclin B3 was knocked down and the duration of the 'mitotic phase' was followed based on the degree of DNA condensation. Cyclin B3 knockdown, but not knockdown of the other cyclins, extended this mitotic phase. It is concluded that Cyclin B3 normally promotes the mitotic exit program and that this action is independent of both SAC and the spindle.
If Cyclin B3 directly regulates APC/C function, it might influence its cellular dynamics. Cdc27 is an APC/C subunit whose phosphorylation by Cdk1-cyclin appears to activate APC/C function and direct it to anaphase chromosomes. The anaphase localization of Cdc27 was studied in both wild-type and single cyclin RNAi-treated embryos. Cdc27 was recruited to anaphase chromosomes in wild-type, and Cyclin A or Cyclin B knockdown embryos; however, Cyclin B3 knockdown greatly reduced the anaphase chromosomal localization of Cdc27 (Yuan, 2015).
Cyclins generally inhibit mitotic exit. At least their destruction underlies downregulation of the mitotic Cdk, and their stabilization blocks mitotic exit. This makes acceleration of anaphase onset by Cyclin B3 appear incongruous. However, cyclins are thought to promote their own demise by activating the APC/C, and the best APC/C activator among the mitotic cyclins will promote mitotic exit. Suppression of APC/C by early-degraded cyclins and activation by a late-degraded cyclin would first stabilize the metaphase state, but initial cyclin destruction would ramp up APC/C activity in a decisive transition. As a late-degrading cyclin that does not block anaphase, Cyclin B3 is particularly well suited as an activator of the APC/C (Yuan, 2015).
It is not known how Cyclin B3 stimulates anaphase. However, cyclin:Cdk kinase activity phosphorylates certain APC/C subunits, such as Cdc27, as well as phosphorylating and inactivating an inhibitor of the APC/C, Emi2. It is suggest that Cyclin B3:Cdk1 has unique substrate specificity and spatial-temporal distribution that make it an effective activator of APC/C. Regardless of the detailed mechanism, this study shows that cyclin types influence APC/C function, and hence regulate their own destruction schedule (Yuan, 2015).
Flies without zygotic Cyclin B3 can develop into healthy adults, but the females are infertile. Cyclin B3 null females had normal ovaries and laid fertilized eggs, but these exhibited early cell-cycle defects. It is hypothesized that the female sterility, or more properly maternal-effect lethality, in the Cyclin B3 null flies resulted from a deficiency in exit from mitosis or meiosis (Yuan, 2015).
To test this, eggs were imaged from homozygous Cyclin B3 mutant mothers and controls. Eggs from Cyclin B3 null mothers were reported to be defective in exiting from meiosis. However, this study observed some eggs with centrosomes and polar bodies, indicators of fertilization and completion of meiosis, respectively. A Cyclin B3-deficient egg with a seemingly normal polar body and a metaphase spindle with four centrosomes adjacent to it is illustrated. These centrosomes were detached from the spindle, and the metaphase spindle was acentrosomal. In the older eggs, numerous microtubule-organizing centers were formed and DNA became highly fragmented. The data suggested that some of the mutant eggs finished meiosis but then arrested in metaphase of the subsequent mitotic division. These observations are consistent with a role of Cyclin B3 in stimulation of anaphase as is seen in later-staged embryos. It is suggested that Cyclin B3 is so important in meiosis and in the earliest mitoses that its absence disrupts restoration of interphase at these stages (Yuan, 2015).
Next, Cyclin B3 function was examined in larval neuroblasts, a more differentiated cell type that gives rise to neurons. Surprisingly, the metaphase duration in the neuroblasts of Cyclin B3 null larvae was indistinguishable from that in the control heterozygous siblings. Thus it is concluded that the impact of Cyclin B3 on anaphase onset is developmentally regulated. Meiosis and early embryonic mitoses need Cyclin B3, but later neuroblast divisions do not. Interestingly, the constitution of the APC/C holoenzyme is also under developmental regulation. During female meiosis and early embryonic mitoses, a distinct Cdc20-like protein, CORT, is expressed. It would be of great interest to explore the interaction between Cyclin B3 and this novel APC/C activator (Yuan, 2015).
The lack of an effect of Cyclin B3 in later cell cycles is not due to lack of Cyclin B3. It is expressed widely in Drosophila and was shown to regulate onset of cytokinesis in later cycles. This suggests that Cyclin B3 is not always important for timing and that timing duty shifts from a program of cyclin destruction at early stages to a SAC-dependent wait-until-ready mechanism. This raises the issue of how these two mechanisms are controlled to guarantee accurate mitotic progress. While it might seem dangerous to have different mechanisms timing mitosis come and go, it should be recalled that the checkpoint mechanism is beautifully appropriate as a backup mechanism. It is suggested that whenever spindle establishment slows to the point that misaligned chromosomes are still present after the cyclin-based timer has run its course, the SAC takes over. By removing the danger of misregulation, the SAC may have freed evolution and development to alter timing inputs governing mitotic progress (Yuan, 2015).
In mitosis and meiosis, chromosome segregation is triggered by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit ubiquitin ligase that targets proteins for degradation, leading to the separation of chromatids. APC/C activation requires phosphorylation of its APC3 and APC1 subunits, which allows the APC/C to bind its co-activator Cdc20. The identity of the kinase(s) responsible for APC/C activation in vivo is unclear. Cyclin B3 (CycB3) is an activator of the Cyclin-Dependent Kinase 1 (Cdk1) that is required for meiotic anaphase in flies, worms and vertebrates. It has been hypothesized that CycB3-Cdk1 may be responsible for APC/C activation in meiosis but this remains to be determined. Using Drosophila, this study found that mutations in CycB3 genetically enhance mutations in tws, which encodes the B55 regulatory subunit of Protein Phosphatase 2A (PP2A) known to promote mitotic exit. Females heterozygous for CycB3 and tws loss-of-function alleles lay embryos that arrest in mitotic metaphase in a maternal effect, indicating that CycB3 promotes anaphase in mitosis in addition to meiosis. This metaphase arrest is not due to the Spindle Assembly Checkpoint (SAC) because mutation of mad2 that inactivates the SAC does not rescue the development of embryos from CycB3-/+, tws-/+ females. Moreover, CycB3 was found to promote APC/C activity and anaphase in cells in culture. CycB3 physically associates with the APC/C, is required for phosphorylation of APC3, and promotes APC/C association with its Cdc20 co-activators Fizzy and Cortex. These results strongly suggest that CycB3-Cdk1 directly activates the APC/C to promote anaphase in both meiosis and mitosis (Garrido, 2020).
Mitosis and meiosis (collectively referred to as M-phase) are distinct modes of nuclear division resulting in diploid or haploid products, respectively. In animals, both require the breakdown of the nuclear envelope, the condensation of chromosomes and their correct attachment on a microtubule-based spindle, where chromosomes are under tension and chromatids are held together by cohesins. Progression through these initial phases requires multiple phosphorylation events of various protein substrates by mitotic kinases including Cyclin-Dependent Kinases (CDKs) activated by their mitotic cyclin partners. M-phase completion from this point (mitotic exit) requires the degradation of mitotic cyclins, and the dephosphorylation of several mitotic phosphoproteins by phosphatases including Protein Phosphatase 2A (PP2A). Mitotic exit begins with the segregation of chromosomes in anaphase. In mitosis, sister chromatids segregate. In meiosis I, replicated homologous chromosomes segregate, and in the subsequent meiosis II, sister chromatids segregate. Nuclear divisions are completed with the reassembly of a nuclear envelope concomitant with the decondensation of chromosomes. How mitosis and meiosis are alike and differ in the molecular mechanisms of their exit programs is not completely understood (Garrido, 2020).
Chromosome segregation is triggered by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase. By catalysing the addition of ubiquitin chains on the separase inhibitor securin, the APC/C targets it for degradation by the proteasome. As a result, separase cleaves cohesins, allowing separated chromosomes to migrate towards opposing poles of the spindle. Activation of the APC/C in mitosis requires its recruitment of its co-factor Cdc20. This recruitment can be prevented by the Spindle-Assembly Checkpoint (SAC), a complex mechanism that allows the sequestration of Cdc20 until all chromosomes are correctly attached on the spindle. Cdc20 binding to the APC/C is also inhibited by its phosphorylation at CDK sites. Phosphatase activity is then required to dephosphorylate Cdc20 and allow its binding of the APC/C for its activation of anaphase. In addition, phosphorylation of the APC/C itself is required to allow Cdc20 binding. Phosphorylation of APC3/Cdc27 and APC1 is key to this process. Phosphorylation of APC3 at CDK sites promotes the subsequent phosphorylation of APC1, inducing a conformational change in APC1 that opens the Cdc20 binding site. However, the precise identity of the kinase(s) involved in this process in vivo is unknown (Garrido, 2020).
At least 3 types of cyclins contribute to M-phase in animals: Cyclins A, B and B3. The Cyclin A type (A1 and A2 in mammals) can activate Cdk1 or Cdk2 and is required for mitotic entry, at least in part by allowing the phosphorylation of Cdc20 to prevent its binding and activation of the APC/C. This allows mitotic cyclins to accumulate without being ubiquitinated prematurely by the APC/C and degraded. The Cyclin B type (B1 and B2 in mammals) also promotes mitotic entry and is required for mitotic progression by allowing the phosphorylation of several substrates by Cdk1. Mammalian Cyclin B3, which can associate with both Cdk1 and Cdk2, is required for meiosis but its contribution to mitosis is less clear in view of its low expression in somatic cells. Drosophila possesses a single gene for each M-phase cyclin: CycA (Cyclin A), CycB (Cyclin B) and CycB3 (Cyclin B3) that collaborate to ensure mitotic progression by activating Cdk1. Genetic and RNAi results suggest that they act sequentially, CycA being required before prometaphase, CycB before metaphase and CycB3 at the metaphase-anaphase transition. CycA is the only essential cyclin, as it is required for mitotic entry. CycB and CycB3 mutants are viable, but mutations of CycB and CycB3 are synthetic-lethal, suggesting redundant roles in mitosis. However, mutation of CycB renders females sterile due to defects in ovary development, and mutant males are also sterile (Garrido, 2020).
Drosophila CycB3 associates with Cdk1 and is required for female meiosis (Jacobs, 1998). In Drosophila, eggs normally stay arrested in metaphase I of meiosis until egg laying triggers entry into anaphase I and the subsequent meiosis II. However, CycB3 mutant eggs predominantly stay arrested in meiosis I (Bourouh, 2016). In addition, silencing CycB3 expression in early embryos delays anaphase onset during the syncytial mitotic divisions (Yuan, 2015). Cyclin B3 is also required for anaphase in female meiosis of vertebrates and worms. In mice, RNAi Knock-down of Cyclin B3 in oocytes inhibits the metaphase-anaphase transition in meiosis I. Recently, two groups independently knocked out the Cyclin B3-coding Ccnb3 gene in mice and found that they were viable but female-sterile due to a highly penetrant arrest in meiotic metaphase I. In C. elegans, the closest Cyclin B3 homolog, CYB-3 is required for anaphase in meiosis and mitosis (Garrido, 2020).
How Cyclin B3 promotes anaphase in any system is unknown. One possibility is that it is required for Cdk1 to phosphorylate the APC/C on at least one of its activating subunits, APC3 or APC1. This has not been investigated. Another possibility is that inactivation of Cyclin B3 leads to an early mitotic defect that activates the SAC. This appears to be the case in C. elegans, because inactivation of the SAC rescues normal anaphase onset in the absence of CYB-3. However, in Drosophila, inactivation of the SAC by the mutation of mad2 did not eliminate the delay in anaphase onset observed when CycB3 is silenced in syncytial embryos. Similarly, in mouse oocytes, silencing Mad2 does not rescue the meiotic metaphase arrest upon Cyclin B3 depletion. In other studies, SAC markers on kinetochores did not persist in metaphase-arrested Ccnb3 KO oocytes, and SAC inactivation by chemical inhibition of Mps1 did not restore anaphase. Finally, it is also possible that Cyclin B3 is required upstream of another event required for APC/C activation, for example the activation of a phosphatase required for Cdc20 dephosphorylation and subsequent recruitment to the APC/C (Garrido, 2020).
This study has investigated how CycB3 promotes anaphase in Drosophila. Several lines of evidence are reported indicating that CycB3 directly activates the APC/C in both meiosis and mitosis (Garrido, 2020).
Altogether, the results strongly suggest that CycB3-Cdk1 directly activates the APC/C by phosphorylation, promoting its function at the metaphase-anaphase transition in meiosis and in both maternally driven early embryonic mitoses and somatic cell divisions. This regulation is likely mediated by the phosphorylation in the activation loop of APC3 by CycB3-Cdk1 that ultimately promotes the recruitment of the Cdc20-type co-activators Fizzy and Cortex. Previous work has shown that APC3 phosphorylation and APC/C activation by cyclin-CDK complexes require their CKS subunit (see Cks30A). CKS subunits can act as processivity factors that bind phosphorylated sites to promote additional phosphorylation by the CDK. Thus, phosphorylation of APC3 would prime the binding of a cyclin-CDK-CKS complex to promote the additional phosphorylation of APC1, allowing for Cdc20 binding. It has been shown that mutation of phosphorylation sites into Asp or Glu residues cannot substitute for the presence of phosphate in the CKS binding site. Therefore, it was not possible to generate a mutation in APC3 that would have mimicked phosphorylation at S316 to enhance cyclin-CDK-CKS binding. Such a mutation in APC3, if it were possible, would have potentially rescued APC/C activity in the absence of CycB3 according to this model. However, it is likely that this analysis did not detect all phosphorylation sites in the APC/C. Thus, the possibility cannot be exclustion that other phosphorylation events, mediated by CycB3-Cdk1 or another kinase, may be required for complete APC/C activation. For example, other phosphorylation events have been proposed to regulate APC/C localization. It is even formally possible that CycB3-Cdk1 is required to activate another proline-directed kinase that phosphorylates APC3 at S316. The interdependence between CycB3 and Tws that this study uncovered may reflect a role of PP2A-Tws in the recruitment of Cdc20 co-activators to the APC/C. Cdc20 must be dephosphorylated at CDK sites before binding the APC/C, and in human cells both PP2A-B55 and PP2A-B56 promote this event (Garrido, 2020).
CycB3 is strongly required for APC/C activation in meiosis and in the early syncytial mitoses, and to a lesser extent in other mitotic divisions, despite the presence of two additional mitotic cyclins, CycA and CycB, capable of activating Cdk1. There are many possible reasons for this requirement. Overexpression of stabilized forms of CycA or CycB can block or slow down anaphase, suggesting that they may interfere with APC/C function in this transition. However, under normal expression levels, CycA or CycB or both may contribute to activate the APC/C like CycB3. CycB3 mutant flies develop until adulthood, which implies that the APC/C can be activated to induce anaphase in at least a vast proportion of mitotic cells, and this activation could be mediated by CycA and/or CycB. CycA is essential for viability and CycB mutants show strong female germline development defects, complicating the examination of potential roles for these cyclins at the metaphase-anaphase transition. Thus, in principle, the requirements for CycB3 in female meiosis, in embryos and in mitotic cells in culture could merely reflect the need for a minimal threshold of total mitotic cyclins. This possibility is considered unlikely because CycB3 is expressed at much lower levels than CycB in early embryos. Moreover, while maternal heterozygosity for mutations in CycB3 and tws causes a metaphase arrest in embryos, heterozygosity for mutations in CycB and tws does not cause embryonic defects. In fact, genetic results suggest that the function of CycB is antagonized by PP2A-Tws in embryos, while CycB3 and PP2A-Tws collaborate for APC/C activation in embryos. Thus, although it is possible that CycA and CycB can participate in APC/C activation, CycB3 probably has some unique feature that makes it particularly capable of promoting APC/C activation (Garrido, 2020).
By what mechanism could CycB3 be particularly suited for APC/C activation? Cyclins can play specific roles by contributing to CDK substrate recognition or by directing CDK activity in space and time. This study did not investigate the precise nature of the molecular recognition of the APC/C by CycB3. It may be that CycB3 possesses a specific binding site for the APC/C that is lacking in CycA and CycB. Another possibility is that differences in localization between cyclins dictate their requirements. In particular, while CycA and CycB are cytoplasmic in interphase, CycB3 is nuclear. It is surmised that the nuclear localization of CycB3 may help concentrate CycB3 in the spindle area upon germinal vesicle breakdown, when the very large oocyte enters meiosis. In future studies, it will be interesting to compare the ability of different mitotic cyclins to activate the APC/C and to determine the molecular basis of potential differences (Garrido, 2020).
In any case, the results show that CycB3 activates the APC/C and that this regulation is essential in Drosophila. Cyclin B3 has been shown to be required for anaphase in female meiosis of insects (Drosophila), worms (C. elegans) and vertebrates (mice). It is tempting to conclude that the activation of the APC/C is a function of Cyclin B3 conserved in all these species. However, in C. elegans embryos, the metaphase arrest upon CYB-3 (Cyclin B3) inactivation requires SAC activity. The underlying mechanism and whether it also occurs in other systems remain to be determined. However, CYB-3 plays roles in C. elegans that have not been detected for Cyclin B3 in flies or vertebrates, including a major role in mitotic entry, where CYB-3 mediates the inhibitory phosphorylation of Cdc20. In this regard, C. elegans CYB-3 may be more orthologous to Cyclin A. Yet, given that Cyclin B3 is required for anaphase in a SAC-independent manner in flies and mice, it seems reasonable to suggest that the direct activation of the APC/C by Cyclin B3 is conserved in vertebrates (Garrido, 2020).
As organs and tissues approach their normal size during development or regeneration, growth slows down, and cell proliferation progressively comes to a halt. Among the various processes suggested to contribute to growth termination, mechanical feedback, perhaps via adherens junctions, has been suggested to play a role. However, since adherens junctions are only present in a narrow plane of the subapical region, other structures are likely needed to sense mechanical stresses along the apical-basal (A-B) axis, especially in a thick pseudostratified epithelium. This could be achieved by nuclei, which have been implicated in mechanotransduction in tissue culture. In addition, mechanical constraints imposed by nuclear crowding and spatial confinement could affect interkinetic nuclear migration (IKNM), which allows G2 nuclei to reach the apical surface, where they normally undergo mitosis. To explore how mechanical constraints affect IKNM, an individual-based model was devised that treats nuclei as deformable objects constrained by the cell cortex and the presence of other nuclei. The model predicts changes in the proportion of cell-cycle phases during growth, which were validated with the cell-cycle phase reporter FUCCI (Fluorescent Ubiquitination-based Cell Cycle Indicator). However, this model does not preclude indefinite growth, leading to a postulate that nuclei must migrate basally to access a putative basal signal required for S phase entry. With this refinement, the updated model accounts for the observed progressive slowing down of growth and explains how pseudostratified epithelia reach a stereotypical thickness upon completion of growth (Hecht, 2022).
Embryogenesis depends on a tightly regulated balance between mitosis, differentiation, and morphogenesis. Understanding how the embryo uses a relatively small number of proteins to transition between growth and morphogenesis is a central question of developmental biology, but the mechanisms controlling mitosis and differentiation are considered to be fundamentally distinct. This study shows the mitotic kinase Polo, which regulates all steps of mitosis in Drosophila, also directs cellular morphogenesis after cell cycle exit. In mitotic cells, the Aurora kinases activate Polo to control a cytoskeletal regulatory module that directs cytokinesis. In the post-mitotic mesoderm, the control of Polo activity transitions from the Aurora kinases to the uncharacterized kinase Back Seat Driver (Bsd), where Bsd and Polo cooperate to regulate muscle morphogenesis. Polo and its effectors therefore direct mitosis and cellular morphogenesis, but the transition from growth to morphogenesis is determined by the spatiotemporal expression of upstream activating kinases (Yang, 2022).
High abundance of CDC45 inhibits cell proliferation through elevation of HSPA6<
CDC45 is the core component of CMG (CDC45-MCMs-GINS) complex that plays important role in the initial step of DNA replication in eukaryotic cells. The expression level of cdc45 is under the critical control for the accurate cell cycle progression. A systematic analysis of the effect of high dose of CDC45 on cell physiology and behaviors is unclear. The present study aimed to investigate the effects and mechanisms of high dose of CDC45 on cell behaviors. cdc45 was overexpressed in cultured cell lines, Ciona, and Drosophila embryos, respectively. The cell cycle progression. High levels of cdc45 from different species (human, mammal, ascidian, and Drosophila) were found to inhibit cell cycle in vitro and in vivo. High dose of CDC45 blocks cells entering into S phase. However, DNA damage and cell apoptosis were not detected. hspa6 was the most upregulated gene in HeLa cells overexpressing cdc45 as detected using RNA-seq analysis and qRT-PCR validation. Overexpression of Hs-hspa6 inhibited proliferation rate and DNA replication in HeLa cells, mimicking the phenotype of cdc45 overexpression. RNAi against hspa6 partially rescued the cell proliferation defect caused by high dose of CDC45. This study suggests that high abundance of CDC45 stops cell cycle. Instead of inducing apoptosis, excessive CDC45 prevents cell entering S phase probably due to promoting hspa6 expression (Fu, 2022).
Polyploidy arises from the gain of complete chromosome sets, and it is known to promote cancer genome evolution. Recent evidence suggests that a large proportion of human tumors experience whole-genome duplications (WGDs), which might favor the generation of highly abnormal karyotypes within a short time frame, rather than in a stepwise manner. However, the molecular mechanisms linking whole-genome duplication to genetic instability remain poorly understood. Using repeated cytokinesis failure to induce polyploidization of Drosophila neural stem cells (NSCs) (also called neuroblasts [NBs]), this study investigated the consequences of polyploidy in vivo. Surprisingly, DNA damage was found generated in a subset of nuclei of polyploid NBs during mitosis. Importantly, the observations in flies were confirmed in mouse NSCs (mNSCs) and human cancer cells after acute cytokinesis inhibition. Interestingly, DNA damage occurs in nuclei that were not ready to enter mitosis but were forced to do so when exposed to the mitotic environment of neighboring nuclei within the same cell. Additionally, it was found that polyploid cells are cell-cycle asynchronous and forcing cell-cycle synchronization was sufficient to lower the levels of DNA damage generated during mitosis. Overall, this work supports a model in which DNA damage at mitotic entry can generate DNA structural abnormalities that might contribute to the onset of genetic instability (Nano, 2020).
To maintain cellular identities during development, gene expression profiles must be faithfully propagated through cell generations. The reestablishment of gene expression patterns upon mitotic exit is mediated, in part, by transcription factors (TF) mitotic bookmarking. However, the mechanisms and functions of TF mitotic bookmarking during early embryogenesis remain poorly understood. This study took advantage of the naturally synchronized mitoses of Drosophila early embryos, providing evidence that GAGA pioneer factor (GAF) acts as a stable mitotic bookmarker during zygotic genome activation. During mitosis, GAF remains associated to a large fraction of its interphase targets, including at cis-regulatory sequences of key developmental genes with both active and repressive chromatin signatures. GAF mitotic targets are globally accessible during mitosis and are bookmarked via histone acetylation (H4K8ac). By monitoring the kinetics of transcriptional activation in living embryos, this study reports that GAF binding establishes competence for rapid activation upon mitotic exit (Bellec, 2022).
This study set out to determine how gene regulation by a transcription factor might be propagated through mitosis in a developing embryo. By using a combination of quantitative live imaging and genomics, evidence is provided that the pioneer-like factor GAF acts as a stable mitotic bookmarker during zygotic genome activation in Drosophila embryos (Bellec, 2022).
The results indicate that during mitosis, GAF binds to an important fraction of its interphase targets, largely representing cis-regulatory sequences of key developmental genes. It was noticed that GAF mitotically retained targets contain a larger number of GAGA repeats than GAF interphase-only targets and that this number of GAGA repeats correlates with the broadness of accessibility. Multiple experiments, with model genes in vitro (e.g., hsp70, hsp26) or from genome-wide approaches clearly demonstrated that GAF contributes to the generation of nucleosome-free regions. The general view is that this capacity is permitted through the interaction of GAF with nucleosome remodeling factors as PBAP (SWI/SNIF), NURF (ISWI), or FACT. Although not yet confirmed with live imaging, immunostaining data suggest that NURF is removed during metaphase but re-engages chromatin by anaphase. If the other partners of GAF implicated in chromatin remodeling are evicted during early mitosis, chromatin accessibility at GAF mitotic targets could be established prior to mitosis onset and then maintained through mitosis owing to the remarkable stability of GAF binding. However, GAF interactions with other chromatin remodelers (e.g., PBAP) during mitosis and a scenario whereby mitotic accessibility at GAF targets would be dynamically established during mitosis thanks to the coordinated action of GAF and its partners cannot be excluded (Bellec, 2022).
It is proposed that the function of GAF as a mitotic bookmarker is possible because GAF has the intrinsic property to remain bound to chromatin for long periods (residence time in the order of minutes). This long engagement of GAF to DNA is in sharp contrast with the binding kinetics of many other TF, such as Zelda or Bicoid in Drosophila embryos or pluripotency TF in mouse ES cells. Another particularity of GAF binding, contrasting with other TF, resides in the multimerization of its DNA-binding sites as GAGAG repeats in a subset of its targets (76% of mitotically retained peaks display four or more repetitions of GAGAG motifs). Given the known oligomerization of GAF70 and as GAF is able to regulate transcription in a cooperative manner, it is tempting to speculate that GAF cooperative binding on long stretches of GAGAG motifs may contribute to a long residence time (Bellec, 2022).
Collectively, it is proposed that the combination of long residence time and the organization of GAF-binding sites in the genome may allow the stable bookmarking of a subset of GAF targets during mitosis (Bellec, 2022).
In this study, it was also discovered that a combination of GAF and histone modification could be at play to maintain the chromatin state during mitosis. Indeed, mitotic bookmarking may also be supported by the propagation of histone tail modifications from mother to daughter cells. Work from mammalian cultured cells revealed widespread mitotic bookmarking by epigenetic modifications, such as H3K27ac and H4K16ac. Moreover, H4K16ac transmission from maternal germline to embryos has recently been established. In the case of GAF, it is proposed that the combinatorial action of GAF and epigenetic marks, possibly selected via GAF interacting partners, will contribute to the propagation of various epigenetic programs. It would be therefore interesting to employ the established mitotic ChIP method to survey the extent to which cis-regulatory regions exhibit different mitotic histone mark modifications during embryogenesis (Bellec, 2022).
A key aspect of mitotic bookmarking is to relate mitotic binding to the rapid transcriptional activation after mitosis. This study has shown that GAF plays a role in the timing of reactivation after mitosis. However, it is noted that GAF binding during mitosis is not the only means to accelerate gene activation. Indeed, it has been shown shown that mechanisms such as enhancer priming by Zelda, paused polymerase or redundant enhancers contribute to fast gene activation. Moreover, a transcriptional memory bias can occur for a transgene not regulated by GAF. By modeling the transcriptional activation of the gene scylla, it was revealed that GAF accelerates the epigenetic steps prior to activation, selectively in the descendants of active nuclei. A model is proposed where GAF binding helps in the decision-making of the postmitotic epigenetic path. In this model, mitotic bookmarking by GAF would favor an epigenetic path with fast transitions after mitosis. In the context of embryogenesis, bookmarking would lead to the fast transmission of select epigenetic states and may contribute to gene expression precision (Bellec, 2022).
Interestingly, GAF vertebrate homolog (vGAF/Th-POK) has recently been implicated in the maintenance of chromatin domains during zebrafish development. It is therefore suspected that GAF action as a stable bookmarking factor controlling transcriptional memory during Drosophila ZGA might be conserved in vertebrates (Bellec, 2022).
list of genes active in
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