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.

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 ). In this study we show that Gwl associates with protein phosphatase 1 (PP1), particularly PP1γ, which mediates the dephosphorylation of Gwl Ser-883.

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.

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.

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-, β- 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.