The Interactive Fly

Zygotically transcribed genes

Cell Cycle Genes

How does the cell cycle function?
Centrosomal/Centriolar Proteins
Centomere and Kinetochore Proteins
Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms
Molecular dissection of cytokinesis by RNA interference in Drosophila cultured cells
Identification of pathways regulating cell size and cell-cycle progression by RNAi
Developmental control of late replication and S phase length
Transcriptional memory in the Drosophila embryo
A genetic interaction map of cell cycle regulators
Calpain A controls mitotic synchrony in the Drosophila blastoderm embryo
A link between deoxyribonucleotide metabolites and embryonic cell-cycle control
Ecdysone signaling induces two phases of cell cycle exit in Drosophila cells
Quantitative microscopy uncovers ploidy changes during mitosis in live Drosophila embryos and their effect on nuclear size
Comparison of Nuclear Matrix and Mitotic Chromosome Scaffold proteins in Drosophila S2 cells - Transmission of hallmarks of nuclear organization through mitosis
Drosophila Dalmatian combines sororin and shugoshin roles in establishment and protection of cohesion
Superresolution expansion microscopy reveals the three-dimensional organization of the Drosophila synaptonemal complex
Chromatin organization changes during the establishment and maintenance of the postmitotic state
Cell cycle heterogeneity directs the timing of neural stem cell activation from quiescence
Drosophila F-BAR protein Syndapin contributes to coupling the plasma membrane and contractile ring in cytokinesis
IPIP27 coordinates PtdIns(4,5)P2 homeostasis for successful cytokinesis
Ultrastructural analysis of mitotic Drosophila S2 cells identifies distinctive microtubule and intracellular membrane behaviors
Absence of the spindle assembly checkpoint restores mitotic fidelity upon loss of sister chromatid cohesion
Fascetto (PRC1) interacting protein (FIP) ensures proper cytokinesis and ploidy

Female meiosis

Genes involved in cell cycle

The Cyclins

Cyclin dependent kinases

Cohesin and Condensin

Meiotic and Mitotic Checkpoint Proteins

Anaphase promoting complex and its regulators

Origin Recognition Complex

Other genes

How does the cell cycle function?

Cell Cycle in Drosophila

Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms

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).

Molecular dissection of cytokinesis by RNA interference in Drosophila cultured cells

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).

Identification of pathways regulating cell size and cell-cycle progression by RNAi

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).

Developmental control of late replication and S phase length

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).

Transcriptional memory in the Drosophila embryo

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).

A genetic interaction map of cell cycle regulators

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).

Calpain A controls mitotic synchrony in the Drosophila blastoderm embryo

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).

A link between deoxyribonucleotide metabolites and embryonic cell-cycle control

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).

Ecdysone signaling induces two phases of cell cycle exit in Drosophila cells

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).

Quantitative microscopy uncovers ploidy changes during mitosis in live Drosophila embryos and their effect on nuclear size

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).

Comparison of Nuclear Matrix and Mitotic Chromosome Scaffold proteins in Drosophila S2 cells - Transmission of hallmarks of nuclear organization through mitosis

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).

Drosophila Dalmatian combines sororin and shugoshin roles in establishment and protection of cohesion

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).

Superresolution expansion microscopy reveals the three-dimensional organization of the Drosophila synaptonemal complex

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).

Chromatin organization changes during the establishment and maintenance of the postmitotic state
Genome organization changes during development as cells differentiate. Chromatin motion becomes increasingly constrained and heterochromatin clusters as cells become restricted in their developmental potential. These changes coincide with slowing of the cell cycle, which can also influence chromatin organization and dynamics. Terminal differentiation is often coupled with permanent exit from the cell cycle, and existing data suggest a close relationship between a repressive chromatin structure and silencing of the cell cycle in postmitotic cells. This study examined the relationship between chromatin organization, terminal differentiation and cell cycle exit. These studies focused on the Drosophila wing, where epithelial cells transition from active proliferation to a postmitotic state in a temporally controlled manner. Two stages of G0 were found in this tissue, a flexible G0 period where cells can be induced to reenter the cell cycle under specific genetic manipulations and a state that is called "robust," where cells become strongly refractory to cell cycle reentry. Compromising the flexible G0 by driving ectopic expression of cell cycle activators causes a global disruption of the clustering of heterochromatin-associated histone modifications such as H3K27 trimethylation and H3K9 trimethylation, as well as their associated repressors, Polycomb and Heterochromatin protein 1 (HP1). However, this disruption is reversible. When cells enter a robust G0 state, even in the presence of ectopic cell cycle activity, clustering of heterochromatin-associated modifications is restored. If cell cycle exit is bypassed, cells in the wing continue to terminally differentiate, but heterochromatin clustering is severely disrupted. Heterochromatin-dependent gene silencing does not appear to be required for cell cycle exit, as compromising the H3K27 methyltransferase Enhancer of zeste, and/or HP1 cannot prevent the robust cell cycle exit, even in the face of normally oncogenic cell cycle activities. It is concluded that heterochromatin clustering during terminal differentiation is a consequence of cell cycle exit, rather than differentiation. Compromising heterochromatin-dependent gene silencing does not disrupt cell cycle exit (Ma, 2017).

Cell cycle heterogeneity directs the timing of neural stem cell activation from quiescence

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).

Drosophila F-BAR protein Syndapin contributes to coupling the plasma membrane and contractile ring in cytokinesis

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).

IPIP27 coordinates PtdIns(4,5)P2 homeostasis for successful cytokinesis

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).

Ultrastructural analysis of mitotic Drosophila S2 cells identifies distinctive microtubule and intracellular membrane behaviors

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).

Fascetto (PRC1) interacting protein (FIP) ensures proper cytokinesis and ploidy

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).

list of genes active in cell cycle


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Carim, S. C., Ben El Kadhi, K., Yan, G., Sweeney, S. T., Hickson, G. R., Carreno, S. and Lowe, M. (2019). IPIP27 coordinates PtdIns(4,5)P2 homeostasis for successful cytokinesis. Curr Biol 29(5): 775-789. PubMed ID: 30799246

de Nooij, J.C. and Hariharan, I.K. (1995). Uncoupling cell fate determination from patterned cell division in the Drosophila eye. Science 270: 983-985. 7481802

Duronio, R.J. and O'Farrell, P.H. (1995). Developmental control of the G1 to S transition in Drosophila: Cyclin E is a limiting downstream target of E2F. Genes Dev 9: 1456-68. 7601350

Dynlacht, B.D., Brook, A., Dembski, M., Yenush, L. and Dyson, N. (1994). DNA-binding and trans-activation properties of Drosophila E2F and DP proteins. Proc Natl Acad Sci 91: 6359-6363. 8022789

Edgar, B.A. and O'Farrell, P.H. (1990). The three postblastoderm cell cycles of Drosophila embryogenesis are regulated in G2 by string. Cell 62: 469-480. 2199063

Edgar, B.A., Sprenger, F., Duronio, R.J., Leopold, P. and O'Farrell, P.H. (1994). Distinct molecular mechanisms regulate cell cycle timing at successive stages of Drosophila embryogenesis. Genes Dev 8: 440-452. 7510257

Fenton, B. and Glover, D.M. (1993). A conserved mitotic kinase active in late anaphase-telophase in syncytial Drosophila embryos. Nature 363: 637-640. 8510757

Ferraro, T., Esposito, E., Mancini, L., Ng, S., Lucas, T., Coppey, M., Dostatni, N., Walczak, A.M., Levine, M. and Lagha, M. (2015). Transcriptional memory in the Drosophila embryo. Curr Biol [Epub ahead of print]. PubMed ID: 26748851

Guo, Y., Flegel, K., Kumar, J., McKay, D.J. and Buttitta, L.A. (2016). Ecdysone signaling induces two phases of cell cycle exit in Drosophila cells. Biol Open 5(11):1648-1661. PubMed ID: 27737823

Hao, X.F., Alphey, L., Bandara, L.R., Lam, E.W., Glover, D. and LaThangue, N.B. (1995). Functional onservation of the cell cycle-regulating transcription factor DRTF1/E2F and its pathway of control in Drosophila melanogaster. J Cell Sci 108: 2945-2954. 8537434

Hayashi, S. (1996). A Cdc2 dependent checkpoint maintains diploidy in Drosophila. Development 122: 1051-1058. 8620832

Knoblich, J.A. and Lehner, C.F. (1993). Synergistic action of Drosophila Cyclins A and B during the G2-M transition. EMBO J. 12: 65-74. 8428595

Knoblich, J. A., et al. (1994). Cyclin E controls S phase progression and its down-regulation during Drosophila embryogenesis is required for the arrest of cell proliferation. Cell 77: 107-120. 8156587

Liu, B., Winkler, F., Herde, M., Witte, C. P. and Grosshans, J. (2019). A link between deoxyribonucleotide metabolites and embryonic cell-cycle control. Curr Biol. PubMed ID: 30880011

Ma, Y. and Buttitta, L. (2017). Chromatin organization changes during the establishment and maintenance of the postmitotic state. Epigenetics Chromatin 10(1): 53. PubMed ID: 29126440

Manke, I. A. et al. (2005). MAPKAP kinase-2 is a cell cycle checkpoint kinase that regulates the G2/M transition and S phase progression in response to UV irradiation. Mol. Cell 17: 37-48. Medline abstract: 15629715

Otsuki, L. and Brand, A. H. (2018). Cell cycle heterogeneity directs the timing of neural stem cell activation from quiescence. Science 360(6384): 99-102. PubMed ID: 29622651

Puah, W. C., Chinta, R. and Wasser, M. (2017). Quantitative microscopy uncovers ploidy changes during mitosis in live Drosophila embryos and their effect on nuclear size. Biol Open [Epub ahead of print]. PubMed ID: 28108477

Richardson, H.E., O'Keefe, L.V., Reed, S.I. and Saint, R. (1993). A Drosophila G1-specific Cyclin E homolog exhibitis different modes of expression during embryogenesis. Development 119: 673-690. 8187637

Sauer, K., Knoblich, J.A., Richardson, H. and Lehner, C.F. (1995). Distinct modes of Cyclin E/cdc2c kinase regulation and S-phase control in mitotic and endoreduplication cycles of Drosophila embryogenesis. Genes Dev 9: 1327-1339. 7797073

Sigrist, S., Jacobs, H., Stratmann, R. and Lehner, C.F. (1995). Exit from mitosis is regulated by Drosophila fizzy and the sequential destruction of Cyclins A,B and B3. EMBO J 14: 4827-4838. 7588612

Shermoen, A. W., McCleland, M. L. and O'Farrell, P. H. (2010). Developmental control of late replication and S phase length. Curr. Biol. 20(23): 2067-77. PubMed Citation: 21074439

Silva, R. D., Mirkovic, M., Guilgur, L. G., Rathore, O. S., Martinho, R. G. and Oliveira, R. A. (2018). Absence of the spindle assembly checkpoint restores mitotic fidelity upon loss of sister chromatid cohesion. Curr Biol 28(17):2837-2844. PubMed ID: 30122528

Somma, M. P., et al. (2002). Molecular dissection of cytokinesis by RNA interference in Drosophila cultured cells. Mol. Biol. Cell 13: 2448-2460. 12134082

Strunov, A., Boldyreva, L. V., Andreyeva, E. N., Pavlova, G. A., Popova, J. V., Razuvaeva, A. V., Anders, A. F., Renda, F., Pindyurin, A. V., Gatti, M. and Kiseleva, E. (2018). Ultrastructural analysis of mitotic Drosophila S2 cells identifies distinctive microtubule and intracellular membrane behaviors. BMC Biol 16(1): 68. PubMed ID: 29907103

Sureka, R., Wadhwa, R., Thakur, S. S., Pathak, R. U. and Mishra, R. K. (2018). Comparison of Nuclear Matrix and Mitotic Chromosome Scaffold proteins in Drosophila S2 cells - Transmission of hallmarks of nuclear organization through mitosis. Mol Cell Proteomics. PubMed ID: 29991507

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Takeda, T., Robinson, I. M., Savoian, M. M., Griffiths, J. R., Whetton, A. D., McMahon, H. T. and Glover, D. M. (2013). Drosophila F-BAR protein Syndapin contributes to coupling the plasma membrane and contractile ring in cytokinesis. Open Biol 3(8): 130081. PubMed ID: 23926047

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date revised: 10 August 2017

Zygotically transcribed genes

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