Publications by authors named "Michael L Goldberg"

33 Publications

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

Elife 2019 11 4;8. Epub 2019 Nov 4.

Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, United States.

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 effector protein, SidJ, in complex with the eukaryotic Ca-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 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.
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http://dx.doi.org/10.7554/eLife.51162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858067PMC
November 2019

PLEKHA4/kramer Attenuates Dishevelled Ubiquitination to Modulate Wnt and Planar Cell Polarity Signaling.

Cell Rep 2019 05;27(7):2157-2170.e8

Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA; Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA. Electronic address:

Wnt signaling pathways direct key physiological decisions in development. Here, we establish a role for a pleckstrin homology domain-containing protein, PLEKHA4, as a modulator of signaling strength in Wnt-receiving cells. PLEKHA4 oligomerizes into clusters at PI(4,5)P-rich regions of the plasma membrane and recruits the Cullin-3 (CUL3) E3 ubiquitin ligase substrate adaptor Kelch-like protein 12 (KLHL12) to these assemblies. This recruitment decreases CUL3-KLHL12-mediated polyubiquitination of Dishevelled, a central intermediate in canonical and non-canonical Wnt signaling. Knockdown of PLEKHA4 in mammalian cells demonstrates that PLEKHA4 positively regulates canonical and non-canonical Wnt signaling via these effects on the Dishevelled polyubiquitination machinery. In vivo knockout of the Drosophila melanogaster PLEKHA4 homolog, kramer, selectively affects the non-canonical, planar cell polarity (PCP) signaling pathway. We propose that PLEKHA4 tunes the sensitivities of cells toward the stimulation of Wnt or PCP signaling by sequestering a key E3 ligase adaptor controlling Dishevelled polyubiquitination within PI(4,5)P-rich plasma membrane clusters.
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http://dx.doi.org/10.1016/j.celrep.2019.04.060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594551PMC
May 2019

Cell Cycle: Abrogating Interphase/M Phase Bistability.

Curr Biol 2018 12;28(23):R1342-R1345

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853-2703, USA. Electronic address:

A new study reports the ability to generate cells caught in a 'no-man's land' between interphase and M phase by simultaneously disrupting feedback loops controlling the activities of the main mitotic driver Cdk1-cyclin B and its counteracting phosphatase PP2A-B55.
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http://dx.doi.org/10.1016/j.cub.2018.10.011DOI Listing
December 2018

Calcineurin-dependent Protein Phosphorylation Changes During Egg Activation in .

Mol Cell Proteomics 2019 03 26;18(Suppl 1):S145-S158. Epub 2018 Nov 26.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York. Electronic address:

In almost all animals studied to date, the crucial process of egg activation, by which an arrested mature oocyte transitions into an actively developing embryo, initiates with an increase in Ca in the oocyte's cytoplasm. This Ca rise sets off a series of downstream events, including the completion of meiosis and the dynamic remodeling of the oocyte transcriptome and proteome, which prepares the oocyte for embryogenesis. Calcineurin is a highly conserved phosphatase that is activated by Ca upon egg activation and that is required for the resumption of meiosis in ,, ascidians, and Drosophila. The molecular mechanisms by which calcineurin transduces the calcium signal to regulate meiosis and other downstream events are still unclear. In this study, we investigate the regulatory role of calcineurin during egg activation in ,. Using mass spectrometry, we quantify the phosphoproteomic and proteomic changes that occur during egg activation, and we examine how these events are affected when calcineurin function is perturbed in female germ cells. Our results show that calcineurin regulates hundreds of phosphosites and also influences the abundance of numerous proteins during egg activation. We find calcineurin-dependent changes in cell cycle regulators including Fizzy (Fzy), Greatwall (Gwl) and Endosulfine (Endos); in protein translation modulators including PNG, NAT, eIF4G, and eIF4B; and in important components of signaling pathways including GSK3β and Akt1. Our results help elucidate the events that occur during the transition from oocyte to embryo.
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http://dx.doi.org/10.1074/mcp.RA118.001076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6427240PMC
March 2019

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

Chromosoma 2018 12 18;127(4):489-504. Epub 2018 Aug 18.

Dipartimento di Biologia e Biotecnologie "C. Darwin", Sapienza, Università di Roma, Rome, Italy.

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.
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http://dx.doi.org/10.1007/s00412-018-0680-yDOI Listing
December 2018

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

J Biol Chem 2017 06 26;292(24):10026-10034. Epub 2017 Apr 26.

From the Department of Oral Biology, College of Dentistry, University of Nebraska Medical Center, Lincoln, Nebraska 68583 and

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. 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.
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http://dx.doi.org/10.1074/jbc.M117.778233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5473210PMC
June 2017

Ezrin activation by LOK phosphorylation involves a PIP-dependent wedge mechanism.

Elife 2017 04 21;6. Epub 2017 Apr 21.

Weill Institute for Molecular and Cell Biology, Cornell University, Ithaca, United States.

How cells specify morphologically distinct plasma membrane domains is poorly understood. Prior work has shown that restriction of microvilli to the apical aspect of epithelial cells requires the localized activation of the membrane-F-actin linking protein ezrin. Using an system, we now define a multi-step process whereby the kinase LOK specifically phosphorylates ezrin to activate it. Binding of PIP to ezrin induces a conformational change permitting the insertion of the LOK C-terminal domain to wedge apart the membrane and F-actin-binding domains of ezrin. The N-terminal LOK kinase domain can then access a site 40 residues distal from the consensus sequence that collectively direct phosphorylation of the appropriate threonine residue. We suggest that this elaborate mechanism ensures that ezrin is only phosphorylated at the plasma membrane, and with high specificity by the apically localized kinase LOK.
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http://dx.doi.org/10.7554/eLife.22759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5400502PMC
April 2017

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

Elife 2017 04 7;6. Epub 2017 Apr 7.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, United States.

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 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.
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http://dx.doi.org/10.7554/eLife.24665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5441869PMC
April 2017

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

Elife 2014 Mar 11;3:e01695. Epub 2014 Mar 11.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, United States.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3949306PMC
http://dx.doi.org/10.7554/eLife.01695DOI Listing
March 2014

The Drosophila RZZ complex - roles in membrane trafficking and cytokinesis.

J Cell Sci 2012 Sep 8;125(Pt 17):4014-25. Epub 2012 Jun 8.

Istituto Pasteur-Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Biologia e Biotecnologie, Sapienza, Università di Roma, P. le A. Moro 5, 00185 Roma, Italy.

The Zw10 protein, in the context of the conserved Rod-Zwilch-Zw10 (RZZ) complex, is a kinetochore component required for proper activity of the spindle assembly checkpoint in both Drosophila and mammals. In mammalian and yeast cells, the Zw10 homologues, together with the conserved RINT1/Tip20p and NAG/Sec39p proteins, form a second complex involved in vesicle transport between Golgi and ER. However, it is currently unknown whether Zw10 and the NAG family member Rod are also involved in Drosophila membrane trafficking. Here we show that Zw10 is enriched at both the Golgi stacks and the ER of Drosophila spermatocytes. Rod is concentrated at the Golgi but not at the ER, whereas Zwilch does not accumulate in any membrane compartment. Mutations in zw10 and RNAi against the Drosophila homologue of RINT1 (rint1) cause strong defects in Golgi morphology and reduce the number of Golgi stacks. Mutations in rod also affect Golgi morphology, whereas zwilch mutants do not exhibit gross Golgi defects. Loss of either Zw10 or Rint1 results in frequent failures of spermatocyte cytokinesis, whereas Rod or Zwilch are not required for this process. Spermatocytes lacking zw10 or rint1 function assemble regular central spindles and acto-myosin rings, but furrow ingression halts prematurely due to defective plasma membrane addition. Collectively, our results suggest that Zw10 and Rint1 cooperate in the ER-Golgi trafficking and in plasma membrane formation during spermatocyte cytokinesis. Our findings further suggest that Rod plays a Golgi-related function that is not required for spermatocyte cytokinesis.
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http://dx.doi.org/10.1242/jcs.099820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3482314PMC
September 2012

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

Genetics 2012 Aug 29;191(4):1181-97. Epub 2012 May 29.

Department of Biochemistry and Molecular Biology, Division of Reproductive Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, USA.

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.
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http://dx.doi.org/10.1534/genetics.112.140574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3416000PMC
August 2012

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

Mol Cell Biol 2012 Apr 21;32(8):1337-53. Epub 2012 Feb 21.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.

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.
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http://dx.doi.org/10.1128/MCB.06525-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3318580PMC
April 2012

Regulation of Greatwall kinase during Xenopus oocyte maturation.

Mol Biol Cell 2011 Jul 5;22(13):2157-64. Epub 2011 May 5.

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA.

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.
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http://dx.doi.org/10.1091/mbc.E11-01-0008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3128519PMC
July 2011

A novel role for greatwall kinase in recovery from DNA damage.

Cell Cycle 2010 Nov 14;9(21):4364-9. Epub 2010 Nov 14.

Howard Hughes Medical Institute, Department of Pharmacology, University of Colorado School of Medicine, Aurora, USA.

Activation of the DNA damage response (DDR) is critical for genomic integrity and tumor suppression. The occurrence of DNA damage quickly evokes the DDR through ATM/ATR-dependent signal transduction, which promotes DNA repair and activates the checkpoint to halt cell cycle progression. The "turn off" process of the DDR upon satisfaction of DNA repair, also known as "checkpoint recovery", involves deactivation of DDR elements, but the mechanism is poorly understood. Greatwall kinase (Gwl) has been identified as a key element in the G(2)/M transition and helps maintain M phase through inhibition of PP 2A/B55δ, the principal phosphatase for Cdk-phosphorylated substrates. Here we show that Gwl also promotes recovery from DNA damage and is itself directly inhibited by the DNA damage response (DDR). In Xenopus egg extracts, immunodepletion of Gwl increased the DDR to damaged DNA, whereas addition of wild type, but not kinase dead Gwl, inhibited the DDR. The removal of damaged DNA from egg extracts leads to recovery from checkpoint arrest and entry into mitosis, a process impaired by Gwl depletion and enhanced by Gwl overexpression. Moreover, activation of Cdk1 after the removal of damaged DNA is regulated by Gwl. Collectively, these results defines Gwl as a new regulator of the DDR, which plays an important role in recovery from DNA damage.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3055187PMC
http://dx.doi.org/10.4161/cc.9.21.13632DOI Listing
November 2010

Greatwall kinase protects mitotic phosphosites from barbarian phosphatases.

Proc Natl Acad Sci U S A 2010 Jul 30;107(28):12409-10. Epub 2010 Jun 30.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.

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http://dx.doi.org/10.1073/pnas.1006046107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2906557PMC
July 2010

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

Mol Biol Cell 2009 Nov 30;20(22):4777-89. Epub 2009 Sep 30.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.

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.
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http://dx.doi.org/10.1091/mbc.e09-07-0643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2777107PMC
November 2009

Roles of the Drosophila NudE protein in kinetochore function and centrosome migration.

J Cell Sci 2009 Jun 5;122(Pt 11):1747-58. Epub 2009 May 5.

Instituto Pasteur Fondazione Cenci Bolognetti, Dipartimento di Genetica e Biologia Molecolare, Sapienza, Università di Roma, 00185 Rome, Italy.

We examined the distribution of the dynein-associated protein NudE in Drosophila larval brain neuroblasts and spermatocytes, and analyzed the phenotypic consequences of a nudE null mutation. NudE can associate with kinetochores, spindles and the nuclear envelope. In nudE mutant brain mitotic cells, centrosomes are often detached from the poles. Moreover, the centrosomes of mutant primary spermatocytes do not migrate from the cell cortex to the nuclear envelope, establishing a new role for NudE. In mutant neuroblasts, chromosomes fail to congress to a tight metaphase plate, and cell division arrests because of spindle assembly checkpoint (SAC) activation. The targeting of NudE to mitotic kinetochores requires the dynein-interacting protein Lis1, and surprisingly Cenp-meta, a Drosophila CENP-E homolog. NudE is non-essential for the targeting of all mitotic kinetochore components tested. However, in the absence of NudE, the 'shedding' of proteins off the kinetochore is abrogated and the SAC cannot be turned off, implying that NudE regulates dynein function at the kinetochore.
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http://dx.doi.org/10.1242/jcs.041798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2684831PMC
June 2009

Identification of Drosophila mitotic genes by combining co-expression analysis and RNA interference.

PLoS Genet 2008 Jul 18;4(7):e1000126. Epub 2008 Jul 18.

Dipartimento di Genetica e Biologia Molecolare, Istituto di Biologia e Patologia Molecolari del CNR, Rome, Italy.

RNAi screens have, to date, identified many genes required for mitotic divisions of Drosophila tissue culture cells. However, the inventory of such genes remains incomplete. We have combined the powers of bioinformatics and RNAi technology to detect novel mitotic genes. We found that Drosophila genes involved in mitosis tend to be transcriptionally co-expressed. We thus constructed a co-expression-based list of 1,000 genes that are highly enriched in mitotic functions, and we performed RNAi for each of these genes. By limiting the number of genes to be examined, we were able to perform a very detailed phenotypic analysis of RNAi cells. We examined dsRNA-treated cells for possible abnormalities in both chromosome structure and spindle organization. This analysis allowed the identification of 142 mitotic genes, which were subdivided into 18 phenoclusters. Seventy of these genes have not previously been associated with mitotic defects; 30 of them are required for spindle assembly and/or chromosome segregation, and 40 are required to prevent spontaneous chromosome breakage. We note that the latter type of genes has never been detected in previous RNAi screens in any system. Finally, we found that RNAi against genes encoding kinetochore components or highly conserved splicing factors results in identical defects in chromosome segregation, highlighting an unanticipated role of splicing factors in centromere function. These findings indicate that our co-expression-based method for the detection of mitotic functions works remarkably well. We can foresee that elaboration of co-expression lists using genes in the same phenocluster will provide many candidate genes for small-scale RNAi screens aimed at completing the inventory of mitotic proteins.
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http://dx.doi.org/10.1371/journal.pgen.1000126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2537813PMC
July 2008

Roles of Greatwall kinase in the regulation of cdc25 phosphatase.

Mol Biol Cell 2008 Apr 16;19(4):1317-27. Epub 2008 Jan 16.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853-2703, USA.

We previously reported that immunodepletion of Greatwall kinase prevents Xenopus egg extracts from entering or maintaining M phase due to the accumulation of inhibitory phosphorylations on Thr14 and Tyr15 of Cdc2. M phase-promoting factor (MPF) in turn activates Greatwall, implying that Greatwall participates in an MPF autoregulatory loop. We show here that activated Greatwall both accelerates the mitotic G2/M transition in cycling egg extracts and induces meiotic maturation in G2-arrested Xenopus oocytes in the absence of progesterone. Activated Greatwall can induce phosphorylations of Cdc25 in the absence of the activity of Cdc2, Plx1 (Xenopus Polo-like kinase) or mitogen-activated protein kinase, or in the presence of an activator of protein kinase A that normally blocks mitotic entry. The effects of active Greatwall mimic in many respects those associated with addition of the phosphatase inhibitor okadaic acid (OA); moreover, OA allows cycling extracts to enter M phase in the absence of Greatwall. Taken together, these findings support a model in which Greatwall negatively regulates a crucial phosphatase that inhibits Cdc25 activation and M phase induction.
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http://dx.doi.org/10.1091/mbc.e07-11-1099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2291418PMC
April 2008

Developmental and cell cycle progression defects in Drosophila hybrid males.

Genetics 2007 Dec 18;177(4):2233-41. Epub 2007 Oct 18.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853-2703, USA.

Matings between D. melanogaster females and males of sibling species in the D. melanogaster complex yield hybrid males that die prior to pupal differentiation. We have reexamined a previous report suggesting that the developmental defects in these lethal hybrid males reflect a failure in cell proliferation that may be the consequence of problems in mitotic chromosome condensation. We also observed a failure in cell proliferation, but find in contrast that the frequencies of mitotic figures and of nuclei staining for the mitotic marker phosphohistone H3 in the brains of hybrid male larvae are extremely low. We also found that very few of these brain cells in male hybrids are in S phase, as determined by BrdU incorporation. These data suggest that cells in hybrid males are arrested in either the G(1) or G(2) phases of the cell cycle. The cells in hybrid male brains appear to be particularly sensitive to environmental stress; our results indicate that certain in vitro incubation conditions induce widespread cellular necrosis in these brains, causing an abnormal nuclear morphology noted by previous investigators. We also document that hybrid larvae develop very slowly, particularly during the second larval instar. Finally, we found that the frequency of mitotic figures in hybrid male larvae mutant for Hybrid male rescue (Hmr) is increased relative to lethal hybrid males, although not to wild-type levels, and that chromosome morphology in Hmr(-) hybrid males is also not completely normal.
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http://dx.doi.org/10.1534/genetics.107.079939DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2219493PMC
December 2007

Mitch a rapidly evolving component of the Ndc80 kinetochore complex required for correct chromosome segregation in Drosophila.

J Cell Sci 2007 Oct 25;120(Pt 20):3522-33. Epub 2007 Sep 25.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853-2703, USA.

We identified an essential kinetochore protein, Mitch, from a genetic screen in D. melanogaster. Mitch localizes to the kinetochore, and its targeting is independent of microtubules (MTs) and several other known kinetochore components. Animals carrying mutations in mitch die as late third-instar larvae; mitotic neuroblasts in larval brains exhibit high levels of aneuploidy. Analysis of fixed D. melanogaster brains and mitch RNAi in cultured cells, as well as video recordings of cultured mitch mutant neuroblasts, reveal that chromosome alignment in mitch mutants is compromised during spindle formation, with many chromosomes displaying persistent mono-orientation. These misalignments lead to aneuploidy during anaphase. Mutations in mitch also disrupt chromosome behavior during both meiotic divisions in spermatocytes: the entire chromosome complement often moves to only one spindle pole. Mutant mitotic cells exhibit contradictory behavior with respect to the spindle assembly checkpoint (SAC). Anaphase onset is delayed in untreated cells, probably because incorrect kinetochore attachment maintains the SAC. However, mutant brain cells and mitch RNAi cells treated with MT poisons prematurely disjoin their chromatids, and exit mitosis. These data suggest that Mitch participates in SAC signaling that responds specifically to disruptions in spindle microtubule dynamics. The mitch gene corresponds to the transcriptional unit CG7242, and encodes a protein that is a possible ortholog of the Spc24 or Spc25 subunit of the Ndc80 kinetochore complex. Despite the crucial role of Mitch in cell division, the mitch gene has evolved very rapidly among species in the genus Drosophila.
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http://dx.doi.org/10.1242/jcs.012112DOI Listing
October 2007

The Drosophila Lkb1 kinase is required for spindle formation and asymmetric neuroblast division.

Development 2007 Jun;134(11):2183-93

Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Genetica e Biologia Molecolare, Università di Roma La Sapienza, P.le A. Moro 5, 00185 Roma, Italy.

We have isolated lethal mutations in the Drosophila lkb1 gene (dlkb1), the homolog of C. elegans par-4 and human LKB1 (STK11), which is mutated in Peutz-Jeghers syndrome. We show that these mutations disrupt spindle formation, resulting in frequent polyploid cells in larval brains. In addition, dlkb1 mutations affect asymmetric division of larval neuroblasts (NBs); they suppress unequal cytokinesis, abrogate proper localization of Bazooka, Par-6, DaPKC and Miranda, but affect neither Pins/Galphai localization nor spindle rotation. Most aspects of the dlkb1 phenotype are exacerbated in dlkb1 pins double mutants, which exhibit more severe defects than those observed in either single mutant. This suggests that Dlkb1 and Pins act in partially redundant pathways to control the asymmetry of NB divisions. Our results also indicate that Dlkb1 and Pins function in parallel pathways controlling the stability of spindle microtubules. The finding that Dlkb1 mediates both the geometry of stem cell division and chromosome segregation provides novel insight into the mechanisms underlying tumor formation in Peutz-Jeghers patients.
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http://dx.doi.org/10.1242/dev.02848DOI Listing
June 2007

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

Curr Biol 2006 Jul;16(14):1441-6

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.

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.
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http://dx.doi.org/10.1016/j.cub.2006.06.024DOI Listing
July 2006

Greatwall kinase participates in the Cdc2 autoregulatory loop in Xenopus egg extracts.

Mol Cell 2006 Apr;22(1):83-91

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.

Mutations in the Drosophila gene encoding the serine-threonine protein kinase Greatwall have previously been shown to disrupt mitotic progression. To investigate Greatwall's mitotic function, we examined its behavior in Xenopus egg extracts. Greatwall is activated during mitosis by phosphorylation; in vitro evidence indicates that maturation promoting factor (MPF) is an upstream kinase. Conversely, depletion of Greatwall from mitotic extracts rapidly lowers MPF activity due to the accumulation of inhibitory phosphorylations on Cdc2 kinase. Greatwall depletion similarly prevents cycling extracts from entering M phase. The effects of Greatwall depletion can be rescued by the addition of either wild-type (wt) Greatwall or a noninhibitable form of Cdc2 kinase. These results demonstrate that Greatwall participates in an autoregulatory loop that generates and maintains sufficiently high MPF activity levels to support mitosis.
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http://dx.doi.org/10.1016/j.molcel.2006.02.022DOI Listing
April 2006

Twinstar, the Drosophila homolog of cofilin/ADF, is required for planar cell polarity patterning.

Development 2006 May 29;133(9):1789-97. Epub 2006 Mar 29.

Department of Molecular Cell and Developmental Biology, and Molecular Biology Institute, University of California at Los Angeles, Los Angeles, CA 90095, USA.

Planar cell polarity (PCP) is a level of tissue organization in which cells adopt a uniform orientation within the plane of an epithelium. The process of tissue polarization is likely to be initiated by an extracellular gradient. Thus, determining how cells decode and convert this graded information into subcellular asymmetries is key to determining how cells direct the reorganization of the cytoskeleton to produce uniformly oriented structures. Twinstar (Tsr), the Drosophila homolog of Cofilin/ADF (actin depolymerization factor), is a component of the cytoskeleton that regulates actin dynamics. We show here that various alleles of tsr produce PCP defects in the wing, eye and several other epithelia. In wings mutant for tsr, Frizzled (Fz) and Flamingo (Fmi) proteins do not properly localize to the proximodistal boundaries of cells. The correct asymmetric localization of these proteins instructs the actin cytoskeleton to produce one actin-rich wing hair at the distal-most vertex of each cell. These results argue that actin remodeling is not only required in the manufacture of wing hairs, but also in the PCP read-out that directs where a wing hair will be secreted.
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http://dx.doi.org/10.1242/dev.02320DOI Listing
May 2006

The putative Drosophila transcription factor woc is required to prevent telomeric fusions.

Mol Cell 2005 Dec;20(6):821-31

Istituto Pasteur-Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Genetica e Biologia Molecolare, Università di Roma La Sapienza, Roma, Italy.

Woc is a Drosophila zinc finger protein that shares homology with the human polypeptides ZNF261 and ZNF198 implicated in mental retardation and leukemia syndromes. We show that mutations in the woc gene cause frequent telomeric fusions in Drosophila brain cells. Woc localizes to all telomeres and most interbands of polytene chromosomes. In interbands, Woc precisely colocalizes with the initiating forms of RNA polymerase II (Pol II). To characterize the role of woc in telomere maintenance, we analyzed its relationships with Su(var)205, cav, atm, and rad50, four genes that prevent telomeric fusions; Su(var)205 and cav encode HP1 and HP1/ORC Associated Protein (HOAP), respectively. woc mutants displayed normal telomeric accumulations of both HP1 and HOAP, and mutations in cav, Su(var)205, atm, and rad50 did not affect Woc localization on polytene chromosome telomeres. Collectively, our results indicate that Woc is a transcription factor with a telomere-capping function independent of those of Su(var)205, cav, atm, and rad50.
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http://dx.doi.org/10.1016/j.molcel.2005.12.003DOI Listing
December 2005

Human Zwint-1 specifies localization of Zeste White 10 to kinetochores and is essential for mitotic checkpoint signaling.

J Biol Chem 2004 Dec 13;279(52):54590-8. Epub 2004 Oct 13.

CAS Key Laboratory for Structure Biology and Hefei National Laboratory for Physical Sciences at Microscale, Hefei 230027, China.

Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubules and the kinetochore, a multiprotein complex assembled onto centromeric DNA of the chromosome. Here we show that Zwint-1 is required and is sufficient for kinetochore localization of Zeste White 10 (ZW10) in HeLa cells. Zwint-1 specifies the kinetochore association of ZW10 by interacting with its N-terminal domain. Suppression of synthesis of Zwint-1 by small interfering RNA abolishes the localization of ZW10 to the kinetochore, demonstrating the requirement of Zwint-1 for ZW10 kinetochore localization. In addition, depletion of Zwint-1 affects no mitotic arrest but causes aberrant premature chromosome segregation. These Zwint-1-suppressed cells display chromosome bridge phenotype with sister chromatids inter-connected. Moreover, Zwint-1 is required for stable association of CENP-F and dynamitin but not BUB1 with the kinetochore. Finally, our studies show that Zwint-1 is a new component of the mitotic check-point, as cells lacking Zwint-1 fail to arrest in mitosis when exposed to microtubule inhibitors, yielding interphase cells with multinuclei. As ZW10 and Zwint-1 are absent from yeast, we reasoned that metazoans evolved an elaborate spindle checkpoint machinery to ensure faithful chromosome segregation in mitosis.
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http://dx.doi.org/10.1074/jbc.M407588200DOI Listing
December 2004

Feo, the Drosophila homolog of PRC1, is required for central-spindle formation and cytokinesis.

Curr Biol 2004 Sep;14(17):1569-75

Istituto Pasteur-Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia Molecolari del Consiglio Nazionale delle Ricerche, Dipartimento di Genetica e Biologia Molecolare, Università di Roma La Sapienza, P. le A. Moro 5, 00185 Roma, Italy.

We performed a functional analysis of fascetto (feo), a Drosophila gene that encodes a protein homologous to the Ase1p/PRC1/MAP65 conserved family of microtubule-associated proteins (MAPs). These MAPs are enriched at the spindle midzone in yeast and mammals and at the fragmoplast in plants, and are essential for the organization and function of these microtubule arrays. Here we show that the Feo protein is specifically enriched at the central-spindle midzone and that its depletion either by mutation or by RNAi results in aberrant central spindles. In Feo-depleted cells, late anaphases showed normal overlap of the antiparallel MTs at the cell equator, but telophases displayed thin MT bundles of uniform width instead of robust hourglass-shaped central spindles. These thin central spindles exhibited diffuse localizations of both the Pav and Asp proteins, suggesting that these spindles comprise improperly oriented MTs. Feo-depleted cells also displayed defects in the contractile apparatus that correlated with those in the central spindle; late anaphase cells formed regular contractile structures, but these structures did not constrict during telophase, leading to failures in cytokinesis. The phenotype of Feo-depleted telophases suggests that Feo interacts with the plus ends of central spindle MTs so as to maintain their precise interdigitation during anaphase-telophase MT elongation and antiparallel sliding.
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http://dx.doi.org/10.1016/j.cub.2004.08.054DOI Listing
September 2004

Three classes of genes mutated in colorectal cancers with chromosomal instability.

Cancer Res 2004 May;64(9):2998-3001

Sidney Kimmel Comprehensive Cancer Center and Howard Hughes Medical Institute at Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA.

Although most colorectal cancers are chromosomally unstable, the basis for this instability has not been defined. To determine whether genes shown to cause chromosomal instability in model systems were mutated in colorectal cancers, we identified their human homologues and determined their sequence in a panel of colorectal cancers. We found 19 somatic mutations in five genes representing three distinct instability pathways. Seven mutations were found in MRE11, whose product is involved in double-strand break repair. Four mutations were found among hZw10, hZwilch/FLJ10036, and hRod/KNTC, whose products bind to one another in a complex that localizes to kinetochores and controls chromosome segregation. Eight mutations were found in Ding, a previously uncharacterized gene with sequence similarity to the Saccharomyces cerevisiae Pds1, whose product is essential for proper chromosome disjunction. This analysis buttresses the evidence that chromosomal instability has a genetic basis and provides clues to the mechanistic basis of instability in cancers.
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http://dx.doi.org/10.1158/0008-5472.can-04-0587DOI Listing
May 2004

Greatwall kinase: a nuclear protein required for proper chromosome condensation and mitotic progression in Drosophila.

J Cell Biol 2004 Feb;164(4):487-92

Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853-2703, USA.

Mutations in the Drosophila gene greatwall cause improper chromosome condensation and delay cell cycle progression in larval neuroblasts. Chromosomes are highly undercondensed, particularly in the euchromatin, but nevertheless contain phosphorylated histone H3, condensin, and topoisomerase II. Cells take much longer to transit the period of chromosome condensation from late G2 through nuclear envelope breakdown. Mutant cells are also subsequently delayed at metaphase, due to spindle checkpoint activity. These mutant phenotypes are not caused by spindle aberrations, by global defects in chromosome replication, or by activation of a caffeine-sensitive checkpoint. The Greatwall proteins in insects and vertebrates are located in the nucleus and belong to the AGC family of serine/threonine protein kinases; the kinase domain of Greatwall is interrupted by a long stretch of unrelated amino acids.
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http://dx.doi.org/10.1083/jcb.200310059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2171981PMC
February 2004