Phenotypic characterization of diamond (dind), a Drosophila gene required for multiple aspects of cell division.

Many genes are required for the assembly of the mitotic apparatus and for proper chromosome behavior during mitosis and meiosis. A fruitful approach to elucidate the mechanisms underlying cell division is the accurate phenotypic characterization of mutations in these genes. Here, we report the identification and characterization of diamond (dind), an essential Drosophila gene required both for mitosis of larval brain cells and for male meiosis. Larvae homozygous for any of the five EMS-induced mutations die in the third-instar stage and exhibit multiple mitotic defects. Mutant brain cells exhibit poorly condensed chromosomes and frequent chromosome breaks and rearrangements; they also show centriole fragmentation, disorganized mitotic spindles, defective chromosome segregation, endoreduplicated metaphases, and hyperploid and polyploid cells. Comparable phenotypes occur in mutant spermatogonia and spermatocytes. The dind gene encodes a non-conserved protein with no known functional motifs. Although the Dind protein exhibits a rather diffuse localization in both interphase and mitotic cells, fractionation experiments indicate that some Dind is tightly associated with the chromatin. Collectively, these results suggest that loss of Dind affects chromatin organization leading to defects in chromosome condensation and integrity, which in turn affect centriole stability and spindle assembly. However, our results do not exclude the possibility that Dind directly affects some behaviors of the spindle and centrosomes.

Cell cycle-dependent regulation of Greatwall kinase by protein phosphatase 1 and regulatory subunit 3B.

Greatwall (Gwl) kinase plays an essential role in the regulation of mitotic entry and progression. Mitotic activation of Gwl requires both cyclin-dependent kinase 1 (CDK1)-dependent phosphorylation and its autophosphorylation at an evolutionarily conserved serine residue near the carboxyl terminus (Ser-883 in Xenopus). In this study we show that Gwl associates with protein phosphatase 1 (PP1), particularly PP1γ, which mediates the dephosphorylation of Gwl Ser-883. Consistent with the mitotic activation of Gwl, its association with PP1 is disrupted in mitotic cells and egg extracts. During mitotic exit, PP1-dependent dephosphorylation of Gwl Ser-883 occurs prior to dephosphorylation of other mitotic substrates; replacing endogenous Gwl with a phosphomimetic S883E mutant blocks mitotic exit. Moreover, we identified PP1 regulatory subunit 3B (PPP1R3B) as a targeting subunit that can direct PP1 activity toward Gwl. PPP1R3B bridges PP1 and Gwl association and promotes Gwl Ser-883 dephosphorylation. Consistent with the cell cycle-dependent association of Gwl and PP1, Gwl and PPP1R3B dissociate in M phase. Interestingly, up-regulation of PPP1R3B facilitates mitotic exit and blocks mitotic entry. Thus, our study suggests PPP1R3B as a new cell cycle regulator that functions by governing Gwl dephosphorylation.

A convenient synthesis of difficult medium-sized cyclic peptides by Staudinger mediated ring-closure.

Novel, efficient and mild preparation of 7- and 8-membered cyclic di- and 10-membered cyclic tripeptides containing α-, ß- or γ-amino acid residues is effected by a Staudinger-mediated ring closure. Medium-sized cyclic di- and tripeptides--recognized as difficult targets--were obtained in moderate to good yields according to a straightforward sequence. Empirical force-field calculations were undertaken to determine their conformational behaviors and showed high levels of similarity with X-ray results. A computational study at the B3LYP/6-31+G** level of theory afforded information regarding the impact of the sequence, ring-size and substitution on the activation barriers for the cyclization of azido peptide thioesters.

The Drosophila calcipressin sarah is required for several aspects of egg activation.

Activation of mature oocytes initiates development by releasing the prior arrest of female meiosis, degrading certain maternal mRNAs while initiating the translation of others, and modifying egg coverings. In vertebrates and marine invertebrates, the fertilizing sperm triggers activation events through a rise in free calcium within the egg. In insects, egg activation occurs independently of sperm and is instead triggered by passage of the egg through the female reproductive tract ; it is unknown whether calcium signaling is involved. We report here that mutations in sarah, which encodes an inhibitor of the calcium-dependent phosphatase calcineurin, disrupt several aspects of egg activation in Drosophila. Eggs laid by sarah mutant females arrest in anaphase of meiosis I and fail to fully polyadenylate and translate bicoid mRNA. Furthermore, sarah mutant eggs show elevated cyclin B levels, indicating a failure to inactivate M-phase promoting factor (MPF). Taken together, these results demonstrate that calcium signaling is involved in Drosophila egg activation and suggest a molecular mechanism for the sarah phenotype. We also find the conversion of the sperm nucleus into a functional male pronucleus is compromised in sarah mutant eggs, indicating that the Drosophila egg's competence to support male pronuclear maturation is acquired during activation.

Protein polyglutamylation catalyzed by the bacterial calmodulin-dependent pseudokinase SidJ.

Pseudokinases are considered to be the inactive counterparts of conventional protein kinases and comprise approximately 10% of the human and mouse kinomes. Here, we report the crystal structure of the Legionella pneumophila effector protein, SidJ, in complex with the eukaryotic Ca2+-binding regulator, calmodulin (CaM). The structure reveals that SidJ contains a protein kinase-like fold domain, which retains a majority of the characteristic kinase catalytic motifs. However, SidJ fails to demonstrate kinase activity. Instead, mass spectrometry and in vitro biochemical analyses demonstrate that SidJ modifies another Legionella effector SdeA, an unconventional phosphoribosyl ubiquitin ligase, by adding glutamate molecules to a specific residue of SdeA in a CaM-dependent manner. Furthermore, we show that SidJ-mediated polyglutamylation suppresses the ADP-ribosylation activity. Our work further implies that some pseudokinases may possess ATP-dependent activities other than conventional phosphorylation.

Two putative acetyltransferases, san and deco, are required for establishing sister chromatid cohesion in Drosophila.

BACKGROUND: Sister chromatid cohesion is needed for proper alignment and segregation of chromosomes during cell division. Chromatids are linked by the multiprotein cohesin complex, which binds to DNA during G(1) and then establishes cohesion during S phase DNA replication. However, many aspects of the mechanisms that establish and maintain cohesion during mitosis remain unclear. RESULTS: We found that mutations in two evolutionarily conserved Drosophila genes, san (separation anxiety) and deco (Drosophila eco1), disrupt centromeric sister chromatid cohesion very early in division. This failure of sister chromatid cohesion does not require separase and is correlated with a failure of the cohesin component Scc1 to accumulate in centromeric regions. It thus appears that these mutations interfere with the establishment of centromeric sister chromatid cohesion. Secondary consequences of these mutations include activation of the spindle checkpoint, causing metaphase delay or arrest. Some cells eventually escape the block but incur many errors in anaphase chromosome segregation. Both san and deco are predicted to encode acetyltransferases, which transfer acetyl groups either to internal lysine residues or to the N terminus of other proteins. The San protein is itself acetylated, and it associates with the Nat1 and Ard1 subunits of the NatA acetyltransferase. CONCLUSIONS: At least two diverse acetyltransferases play vital roles in regulating sister chromatid cohesion during Drosophila mitosis.

Zwilch, a new component of the ZW10/ROD complex required for kinetochore functions.

The Zeste-White 10 (ZW10) and Rough Deal (ROD) proteins are part of a complex necessary for accurate chromosome segregation. This complex recruits cytoplasmic dynein to the kinetochore and participates in the spindle checkpoint. We used immunoaffinity chromatography and mass spectroscopy to identify the Drosophila proteins in this complex. We found that the complex contains an additional protein we name Zwilch. Zwilch localizes to kinetochores and kinetochore microtubules in a manner identical to ZW10 and ROD. We have also isolated a zwilch mutant, which exhibits the same mitotic phenotypes associated with zw10 and rod mutations: lagging chromosomes at anaphase and precocious sister chromatid separation upon activation of the spindle checkpoint. Zwilch's role within the context of this complex is evolutionarily conserved. The human Zwilch protein (hZwilch) coimmunoprecipitates with hZW10 and hROD from HeLa cell extracts and localizes to the kinetochores at prometaphase. Finally, we discuss immunoaffinity chromatography results that suggest the existence of a weak interaction between the ZW10/ROD/Zwilch complex and the kinesin-like kinetochore component CENP-meta.

Unfair competition governs the interaction of pCPI-17 with myosin phosphatase (PP1-MYPT1).

The small phosphoprotein pCPI-17 inhibits myosin light-chain phosphatase (MLCP). Current models postulate that during muscle relaxation, phosphatases other than MLCP dephosphorylate and inactivate pCPI-17 to restore MLCP activity. We show here that such hypotheses are insufficient to account for the observed rapidity of pCPI-17 inactivation in mammalian smooth muscles. Instead, MLCP itself is the critical enzyme for pCPI-17 dephosphorylation. We call the mutual sequestration mechanism through which pCPI-17 and MLCP interact inhibition by unfair competition: MLCP protects pCPI-17 from other phosphatases, while pCPI-17 blocks other substrates from MLCP's active site. MLCP dephosphorylates pCPI-17 at a slow rate that is, nonetheless, both sufficient and necessary to explain the speed of pCPI-17 dephosphorylation and the consequent MLCP activation during muscle relaxation.

Greatwall-phosphorylated Endosulfine is both an inhibitor and a substrate of PP2A-B55 heterotrimers.

During M phase, Endosulfine (Endos) family proteins are phosphorylated by Greatwall kinase (Gwl), and the resultant pEndos inhibits the phosphatase PP2A-B55, which would otherwise prematurely reverse many CDK-driven phosphorylations. We show here that PP2A-B55 is the enzyme responsible for dephosphorylating pEndos during M phase exit. The kinetic parameters for PP2A-B55's action on pEndos are orders of magnitude lower than those for CDK-phosphorylated substrates, suggesting a simple model for PP2A-B55 regulation that we call inhibition by unfair competition. As the name suggests, during M phase PP2A-B55's attention is diverted to pEndos, which binds much more avidly and is dephosphorylated more slowly than other substrates. When Gwl is inactivated during the M phase-to-interphase transition, the dynamic balance changes: pEndos dephosphorylated by PP2A-B55 cannot be replaced, so the phosphatase can refocus its attention on CDK-phosphorylated substrates. This mechanism explains simultaneously how PP2A-B55 and Gwl together regulate pEndos, and how pEndos controls PP2A-B55. DOI: http://dx.doi.org/10.7554/eLife.01695.001.

Determinants for activation of the atypical AGC kinase Greatwall during M phase entry.

The atypical AGC kinase Greatwall (Gwl) mediates a pathway that prevents the precocious removal of phosphorylations added to target proteins by M phase-promoting factor (MPF); Gwl is thus essential for M phase entry and maintenance. Gwl itself is activated by M phase-specific phosphorylations that are investigated here. Many phosphorylations are nonessential, being located within a long nonconserved region, any part of which can be deleted without effect. Using mass spectrometry and mutagenesis, we have identified 3 phosphorylation sites (phosphosites) critical to Gwl activation (pT193, pT206, and pS883 in Xenopus laevis) located in evolutionarily conserved domains that differentiate Gwl from related kinases. We propose a model in which the initiating event for Gwl activation is phosphorylation by MPF of the proline-directed sites T193 and T206 in the presumptive activation loop. After this priming step, Gwl can intramolecularly phosphorylate its C-terminal tail at pS883; this site probably plays a role similar to that of the tail/Z motif of other AGC kinases. These events largely (but not completely) explain the full activation of Gwl at M phase.

Bypassing the Greatwall-Endosulfine pathway: plasticity of a pivotal cell-cycle regulatory module in Drosophila melanogaster and Caenorhabditis elegans.

In vertebrates, mitotic and meiotic M phase is facilitated by the kinase Greatwall (Gwl), which phosphorylates a conserved sequence in the effector Endosulfine (Endos). Phosphorylated Endos inactivates the phosphatase PP2A/B55 to stabilize M-phase-specific phosphorylations added to many proteins by cyclin-dependent kinases (CDKs). We show here that this module functions essentially identically in Drosophila melanogaster and is necessary for proper mitotic and meiotic cell division in a wide variety of tissues. Despite the importance and evolutionary conservation of this pathway between insects and vertebrates, it can be bypassed in at least two situations. First, heterozygosity for loss-of-function mutations of twins, which encodes the Drosophila B55 protein, suppresses the effects of endos or gwl mutations. Several types of cell division occur normally in twins heterozygotes in the complete absence of Endos or the near absence of Gwl. Second, this module is nonessential in the nematode Caenorhaditis elegans. The worm genome does not contain an obvious ortholog of gwl, although it encodes a single Endos protein with a surprisingly well-conserved Gwl target site. Deletion of this site from worm Endos has no obvious effects on cell divisions involved in viability or reproduction under normal laboratory conditions. In contrast to these situations, removal of one copy of twins does not completely bypass the requirement for endos or gwl for Drosophila female fertility, although reducing twins dosage reverses the meiotic maturation defects of hypomorphic gwl mutants. These results have interesting implications for the function and evolution of the mechanisms modulating removal of CDK-directed phosphorylations.

Regulation of Greatwall kinase during Xenopus oocyte maturation.

Greatwall kinase has been identified as a key element in M phase initiation and maintenance in Drosophila, Xenopus oocytes/eggs, and mammalian cells. In M phase, Greatwall phosphorylates endosulfine and related proteins that bind to and inhibit protein phosphatase 2A/B55, the principal phosphatase for Cdk-phosphorylated substrates. We show that Greatwall binds active PP2A/B55 in G2 phase oocytes but dissociates from it when progesterone-treated oocytes reach M phase. This dissociation does not require Greatwall kinase activity or phosphorylation at T748 in the presumptive T loop of the kinase. A mutant K71M Greatwall, also known as Scant in Drosophila, induces M phase in the absence of progesterone when expressed in oocytes, despite its reduced stability and elevated degradation by the proteasome. M phase induction by Scant Greatwall requires protein synthesis but is not associated with altered binding or release of PP2A/B55 as compared to wild-type Greatwall. However, in vitro studies with Greatwall proteins purified from interphase cells indicate that Scant, but not wild-type Greatwall, has low but detectable activity against endosulfine. These results demonstrate progesterone-dependent regulation of the PP2A/B55-Greatwall interaction during oocyte maturation and suggest that the cognate Scant Greatwall mutation has sufficient constitutive kinase activity to promote M phase in Xenopus oocytes.

The M phase kinase Greatwall (Gwl) promotes inactivation of PP2A/B55delta, a phosphatase directed against CDK phosphosites.

We have previously shown that Greatwall kinase (Gwl) is required for M phase entry and maintenance in Xenopus egg extracts. Here, we demonstrate that Gwl plays a crucial role in a novel biochemical pathway that inactivates, specifically during M phase, "antimitotic" phosphatases directed against phosphorylations catalyzed by cyclin-dependent kinases (CDKs). A major component of this phosphatase activity is heterotrimeric PP2A containing the B55delta regulatory subunit. Gwl is activated during M phase by Cdk1/cyclin B (MPF), but once activated, Gwl promotes PP2A/B55delta inhibition with no further requirement for MPF. In the absence of Gwl, PP2A/B55delta remains active even when MPF levels are high. The removal of PP2A/B55delta corrects the inability of Gwl-depleted extracts to enter M phase. These findings support the hypothesis that M phase requires not only high levels of MPF function, but also the suppression, through a Gwl-dependent mechanism, of phosphatase(s) that would otherwise remove MPF-driven phosphorylations.

Phenotypic characterization of Drosophila ida mutants: defining the role of APC5 in cell cycle progression.

We have cloned and characterized the ida gene that is required for proliferation of imaginal disc cells during Drosophila development. IDA is homologous to APC5, a subunit of the anaphase-promoting complex (APC/cyclosome). ida mRNA is detected in most cell types throughout development, but it accumulates to its highest levels during early embryogenesis. A maternal component of IDA is required for the production of eggs and viable embryos. Homozygous ida mutants display mitotic defects: they die during prepupal development, lack all mature imaginal disc structures, and have abnormally small optic lobes. Cytological observations show that ida mutant brains have a high mitotic index and many imaginal cells contain an aneuploid number of aberrant overcondensed chromosomes. However, cells are not stalled in metaphase, as mitotic stages in which chromosomes are orientated at the equatorial plate are never observed. Interestingly, some APC/C-target substrates such as cyclin B are not degraded in ida mutants, whereas others controlling sister-chromatid separation appear to be turned over. Taken together, these results suggest a model in which IDA/APC5 controls regulatory subfunctions of the anaphase-promoting complex.

intersex, a gene required for female sexual development in Drosophila, is expressed in both sexes and functions together with doublesex to regulate terminal differentiation.

Previous genetic studies indicated intersex (ix) functions only in females and that it acts near the end of the sex determination hierarchy to control somatic sexual differentiation in Drosophila melanogaster. We have cloned ix and characterized its function genetically, molecularly and biochemically. The ix pre-mRNA is not spliced, and ix mRNA is produced in both sexes. The ix gene encodes a 188 amino acid protein, which has a sequence similar to mammalian proteins thought to function as transcriptional activators, and a Caenorhabditis elegans protein that is thought to function as a transcription factor. Bringing together the facts that (1) the ix phenotype is female-specific and (2) functions at the end of the sex determination hierarchy, yet (3) is expressed sex non-specifically and appears likely to encode a transcription factor with no known DNA-binding domain, leads to the inference that ix may require the female-specific protein product of the doublesex (dsx) gene in order to function. Consistent with this inference, we find that for all sexually dimorphic cuticular structures examined, ix and dsx are dependent on each other to promote female differentiation. This dependent relationship also holds for the only known direct target of dsx, the Yolk protein (Yp) genes. Using yeast 2-hybrid assay, immunoprecipitation of recombinant tagged IX and DSX proteins from Drosophila S2 cell extracts, and gel shifts with the tagged IX and DSX(F) proteins, we demonstrate that IX interacts with DSX(F), but not DSX(M). Taken together, the above findings strongly suggest that IX and DSX(F) function in a complex, in which IX acts as a transcriptional co-factor for the DNA-binding DSX(F).