Mars promotes dTACC dephosphorylation on mitotic spindles to ensure spindle stability

Microtubule-associated proteins (MAPs) ensure the fidelity of chromosome segregation by controlling microtubule (MT) dynamics and mitotic spindle stability. However, many aspects of MAP function and regulation are poorly understood in a developmental context. We show that mars, which encodes a Drosophila melanogaster member of the hepatoma up-regulated protein family of MAPs, is essential for MT stabilization during early embryogenesis. As well as associating with spindle MTs in vivo, Mars binds directly to protein phosphatase 1 (PP1) and coimmunoprecipitates from embryo extracts with minispindles and Drosophila transforming acidic coiled-coil (dTACC), two MAPs that function as spindle assembly factors. Disruption of binding to PP1 or loss of mars function results in elevated levels of phosphorylated dTACC on spindles. A nonphosphorylatable form of dTACC is capable of rescuing the lethality of mars mutants. We propose that Mars mediates spatially controlled dephosphorylation of dTACC, which is critical for spindle stabilization.     

Multiple protein phosphatases are required for mitosis in Drosophila

BACKGROUND: Approximately one-third of the Drosophila kinome has been ascribed some cell-cycle function. However, little is known about which of its 117 protein phosphatases (PPs) or subunits have counteracting roles. RESULTS: We investigated mitotic roles of PPs through systematic RNAi. We found that G(2)-M progression requires Puckered, the JNK MAP-kinase inhibitory phosphatase and PP2C in addition to string (Cdc25). Strong mitotic arrest and chromosome congression failure occurred after Pp1-87B downregulation. Chromosome alignment and segregation defects also occurred after knockdown of PP1-Flapwing, not previously thought to have a mitotic role. Reduction of several nonreceptor tyrosine phosphatases produced spindle and chromosome behavior defects, and for corkscrew, premature chromatid separation. RNAi of the dual-specificity phosphatase, Myotubularin, or the related Sbf "antiphosphatase" resulted in aberrant mitotic chromosome behavior. Finally, for PP2A, knockdown of the catalytic or A subunits led to bipolar monoastral spindles, knockdown of the Twins B subunit led to bridged and lagging chromosomes, and knockdown of the B' Widerborst subunit led to scattering of all mitotic chromosomes. Widerborst was associated with MEI-S332 (Shugoshin) and required for its kinetochore localization. CONCLUSIONS: We identify cell-cycle roles for 22 of 117 Drosophila PPs. Involvement of several PPs in G(2) suggests multiple points for its regulation. Major mitotic roles are played by PP1 with tyrosine PPs and Myotubularin-related PPs having significant roles in regulating chromosome behavior. Finally, depending upon its regulatory subunits, PP2A regulates spindle bipolarity, kinetochore function, and progression into anaphase. Discovery of several novel cell-cycle PPs identifies a need for further studies of protein dephosphorylation.     

Aurora A regulates the activity of HURP by controlling the accessibility of its microtubule-binding domain

HURP is a spindle-associated protein that mediates Ran-GTP-dependent assembly of the bipolar spindle and promotes chromosome congression and interkinetochore tension during mitosis. We report here a biochemical mechanism of HURP regulation by Aurora A, a key mitotic kinase that controls the assembly and function of the spindle. We found that HURP binds to microtubules through its N-terminal domain that hyperstabilizes spindle microtubules. Ectopic expression of this domain generates defects in spindle morphology and function that reduce the level of tension across sister kinetochores and activate the spindle checkpoint. Interestingly, the microtubule binding activity of this N-terminal domain is regulated by the C-terminal region of HURP: in its hypophosphorylated state, C-terminal HURP associates with the microtubule-binding domain, abrogating its affinity for microtubules. However, when the C-terminal domain is phosphorylated by Aurora A, it no longer binds to N-terminal HURP, thereby releasing the inhibition on its microtubule binding and stabilizing activity. In fact, ectopic expression of this C-terminal domain depletes endogenous HURP from the mitotic spindle in HeLa cells in trans, suggesting the physiological importance for this mode of regulation. We concluded that phosphorylation of HURP by Aurora A provides a regulatory mechanism for the control of spindle assembly and function.     

Decapentaplegic head capsule mutations disrupt novel peripodial expression controlling the morphogenesis of the Drosophila ventral head

Drosophila adult structures derive from imaginal discs, which are sacs with apposed epithelial sheets, the disc proper (DP) and the peripodial epithelium (PE). The Drosophila TGF-beta family member decapentaplegic (dpp) contributes to the development of adult structures through expression in all imaginal discs, driven by enhancers from the 3' cis-regulatory region of the gene. In the eye/antennal disc, there is 3' directed dpp expression in both the DP and PE associated with cell proliferation and eye formation. Here, we analyze a new class of dpp cis-regulatory mutations, which specifically disrupt a previously unknown region of dpp expression, controlled by enhancers in the 5' regulatory region of the gene and limited to the PE of eye/antennal discs. These are the first described Drosophila mutations that act by solely disrupting PE gene expression. The mutants display defects in the ventral adult head and alter peripodial but not DP expression of known dpp targets. However, apoptosis is observed in the underlying DP, suggesting that this peripodial dpp signaling source supports cell survival in the DP.     

HURP controls spindle dynamics to promote proper interkinetochore tension and efficient kinetochore capture

Through a functional genomic screen for mitotic regulators, we identified hepatoma up-regulated protein (HURP) as a protein that is required for chromosome congression and alignment. In HURP-depleted cells, the persistence of unaligned chromosomes and the reduction of tension across sister kinetochores on aligned chromosomes resulted in the activation of the spindle checkpoint. Although these defects transiently delayed mitotic progression, HeLa cells initiated anaphase without resolution of these deficiencies. This bypass of the checkpoint arrest provides a tumor-specific mechanism for chromosome missegregation and genomic instability. Mechanistically, HURP colocalized with the mitotic spindle in a concentration gradient increasing toward the chromosomes. HURP binds directly to microtubules in vitro and enhances their polymerization. In vivo, HURP stabilizes mitotic microtubules, promotes microtubule polymerization and bipolar spindle formation, and decreases the turnover rate of the mitotic spindle. Thus, HURP controls spindle stability and dynamics to achieve efficient kinetochore capture at prometaphase, timely chromosome congression to the metaphase plate, and proper interkinetochore tension for anaphase initiation.     

Dorso-ventral asymmetric functions of teashirt in Drosophila eye development depend on spatial cues provided by early DV patterning genes

The teashirt (tsh) gene has dorso-ventral (DV) asymmetric functions in Drosophila eye development: promoting eye development in dorsal and suppressing eye development in ventral by Wingless mediated Homothorax (HTH) induction [Development 129 (2002) 4271]. We looked for DV spatial cues required by tsh for its asymmetric functions. The dorsal Iroquois-Complex (Iro-C) genes and Delta (Dl) are required and sufficient for the tsh dorsal functions. The ventral Serrate (Ser), but not fringe (fng) or Lobe (L), is required and sufficient for the tsh ventral function. We propose that DV asymmetric function of tsh represents a novel tier of DV pattern regulation, which takes place after the spatial expression patterns of early DV patterning genes are established in the eye.     

Drosophila sticky/citron kinase is a regulator of cell-cycle progression, genetically interacts with Argonaute 1 and modulates epigenetic gene silencing

The sticky/citron kinase protein is a conserved regulator of cell-cycle progression from invertebrates to humans. While this kinase is essential for completion of cytokinesis, sticky/citron kinase phenotypes disrupting neurogenesis and cell differentiation suggest additional non-cell-cycle functions. However, it is not known whether these phenotypes are an indirect consequence of sticky mutant cell-cycle defects or whether they define a novel function for this kinase. We have isolated a temperature-sensitive allele of the Drosophila sticky gene and we show that sticky/citron kinase is required for histone H3-K9 methylation, HP1 localization, and heterochromatin-mediated gene silencing. sticky genetically interacts with Argonaute 1 and sticky mutants exhibit context-dependent Su(var) and E(var) activity. These observations indicate that sticky/citron kinase functions to regulate both actin-myosin-mediated cytokinesis and epigenetic gene silencing, possibly linking cell-cycle progression to heterochromatin assembly and inheritance of gene expression states.     

A Xenopus tribbles orthologue is required for the progression of mitosis and for development of the nervous system

The product of the Drosophila gene tribbles inhibits cell division in the ventral furrow of the embryo and thereby allows the normal prosecution of gastrulation. Cell division is also absent in involuting dorsal mesoderm during gastrulation in Xenopus, and to ask whether the two species employ similar mechanisms to coordinate morphogenesis and the cell cycle, we isolated a putative Xenopus homologue of tribbles which we call Xtrb2. Extensive cDNA cloning identified long and short forms of Xtrb2, termed Xtrb2-L and Xtrb2-S, respectively. Xtrb2 is expressed maternally and in mesoderm and ectoderm at blastula and gastrula stages. Later, it is expressed in dorsal neural tube, eyes, and cephalic neural crest. Time-lapse imaging of GFP-tagged Xtrb2-L suggests that during cell division, it is associated with mitotic spindles. Knockdown of Xtrb2 by antisense morpholino oligonucleotides (MOs) disrupted synchronous cell divisions during blastula stages, apparently as a result of delayed progression through mitosis and cytokinesis. At later stages, tissues expressing the highest levels of Xtrb2 were most markedly affected by morpholino knockdown, with perturbation of neural crest and eye development.     

Orbit, a novel microtubule-associated protein essential for mitosis in Drosophila melanogaster

We describe a Drosophila gene, orbit, that encodes a conserved 165-kD microtubule-associated protein (MAP) with GTP binding motifs. Hypomorphic mutations in orbit lead to a maternal effect resulting in branched and bent mitotic spindles in the syncytial embryo. In the larval central nervous system, such mutants have an elevated mitotic index with some mitotic cells showing an increase in ploidy. Amorphic alleles show late lethality and greater frequencies of hyperploid mitotic cells. The presence of cells in the hypomorphic mutant in which the chromosomes can be arranged, either in a circular metaphase or an anaphase-like configuration on monopolar spindles, suggests that polyploidy arises through spindle and chromosome segregation defects rather than defects in cytokinesis. A role for the Orbit protein in regulating microtubule behavior in mitosis is suggested by its association with microtubules throughout the spindle at all mitotic stages, by its copurification with microtubules from embryonic extracts, and by the finding that the Orbit protein directly binds to MAP-free microtubules in a GTP-dependent manner.     

Drosophila mars is required for organizing kinetochore microtubules during mitosis

Spindle assembly is essential for the equal distribution of genetic material to the daughter cells during mitosis. The process of spindle assembly is complicated and involves multiple levels of molecular regulation. It is generally accepted that mitotic spindles are emanated from the centrosomes and are assembled in the vicinity of chromosomes. However, the molecular mechanism involved in the spindle assembly during mitosis remains unclear. In this study, we have provided several lines of evidence to show that Drosophila Mars is required for the assembly and stabilization of kinetochore microtubules. In an immunocytochemical study, we show that Mars is mainly localized on the kinetochore microtubules during mitosis. Using RNA interference to deplete the Mars expression in Drosophila S2 cells resulted in the malformation of mitotic spindle that mainly lacked the kinetochore microtubules. The spindle defect resulted in mitotic delays by increasing the percentage of uncongressed chromosomes both in vitro and in vivo. In summary, this study has extended our previous study of Mars in cell cycle regulation and provided further evidence showing that Mars is required for the assembly of kinetochore microtubules.     

Differential requirement for the N-terminal catalytic domain of the DNA polymerase ε p255 subunit in the mitotic cell cycle and the endocycle

In Drosophila, the 255kDa catalytic subunit (dpolεp255) and the 58kDa subunit of DNA polymerase ε (dpolεp58) have been identified. The N-terminus of dpolεp255 carries well-conserved six DNA polymerase subdomains and five 3'→5' exonuclease motifs as observed with Polε in other species. We here examined roles of dpolεp255 during Drosophila development using transgenic fly lines expressing double stranded RNA (dsRNA). Expression of dpolεp255 dsRNA in eye discs induced a small eye phenotype and inhibited DNA synthesis, indicating a role in the G1-S transition and/or S-phase progression of the mitotic cycle. Similarly, expression of dpolεp255 dsRNA in the salivary glands resulted in small size and endoreplication defects, demonstrating a critical role in endocycle progression. In the eye disc, defects induced by knockdown of dpolεp255 were rescued by overexpression of the C-terminal region of dpolεp255, indicating that the function of this non-catalytic domain is conserved between yeast and Drosophila. However, this was not the case for the salivary gland, suggesting that the catalytic N-terminal region is crucial for endoreplication and its defect cannot be complemented by other DNA polymerases. In addition, several genetic interactants with dpolεp255 including genes related to DNA replication such as RFC, DNA primase, DNA polη, Mcm10 and Psf2 and chromatin remodeling such as Iswi were also identified.     

Mutation of the Apc1 homologue shattered disrupts normal eye development by disrupting G1 cell cycle arrest and progression through mitosis

The shattered1 (shtd1) mutation disrupts Drosophila compound eye structure. In this report, we show that the shtd1 eye defects are due to a failure to establish and maintain G1 arrest in the morphogenetic furrow (MF) and a defect in progression through mitosis. The observed cell cycle defects were correlated with an accumulation of cyclin A (CycA) and String (Stg) proteins near the MF. Interestingly, the failure to maintain G1 arrest in the MF led to the specification of R8 photoreceptor cells that undergo mitosis, generating R8 doublets in shtd1 mutant eye discs. We demonstrate that shtd encodes Apc1, the largest subunit of the anaphase-promoting complex/cyclosome (APC/C). Furthermore, we show that reducing the dosage of either CycA or stg suppressed the shtd1 phenotype. While reducing the dosage of CycA is more effective in suppressing the premature S phase entry in the MF, reducing the dosage of stg is more effective in suppressing the progression through mitosis defect. These results indicate the importance of not only G1 arrest in the MF but also appropriate progression through mitosis for normal eye development during photoreceptor differentiation.     

Drosophila crooked-neck protein co-fractionates in a multiprotein complex with splicing factors

The Drosophila crooked neck (crn) gene encodes an unusual TPR-containing protein whose function is essential for embryonic development. Homology with other TPR-proteins involved in cell cycle control, initially led to the proposal that Crn might play a critical role in regulation of embryonic cell divisions. Here, we show that Crn does not have a cell cycle function in the embryo. By using specific antibodies we also show that the Crn protein is a nuclear protein which localizes in "speckles" which could correspond to preferential localization of several other splicing factors. Fractionation of nuclear extracts on sucrose gradients revealed Crn in a 900 kDa multiproteic complex together with snRNPs, suggesting that Crn participates in the assembly of the splicing machinery in vivo.