Cell Cycle Regulation

[Fly 1:2, 125-131; March/April 2007]; ©2007 Landes Bioscience

Meeting Report

Cell Cycle Regulation We gathered in Philadelphia in balmy early March for the 48th Annual Drosophila Research Conference. It was a surprise to us that the session we had come to expect, ‘Mitosis, Meiosis and Cell Division’ was no longer on the program. Instead we found mitosis talks and posters in ‘Cell Division and Growth Control’, meiosis talks and posters in ‘Gametogenesis and Sex Determination’ and cytokinesis talks and posters in ‘Cytoskeleton and Cell Biology’. We, therefore, split up for maximal coverage and re‑grouped later for the Workshop on Cell Cycle Checkpoints. We apologize in advance for the brevity or omission of some reports, especially those on meiosis presented during regular sessions.

*Correspondence to: Tin Tin Su; Department of Molecular, Cellular and Developmental Biology; University of Colorado; 347 UCB Colorado Avenue; Boulder, Colorado 80309-0347 USA; Tel.: 303.735.3245; Fax: 303.492.7744; Email: [email protected]

Additional roles for Cyclin E

Cyclin E/Cdk2 promotes S phase, but also has additional roles in preventing r e‑replication of the genome and in coordinating histone synthesis to S phase. Two reports provide mechanistic insight into these additional roles of Cyclin E (Fig. 1). Karine Narbone from Mary Lilly’s laboratory at NIH offered a different explanation for an old observation that while a pulse of Cyclin E can activate DNA replication, continuous expression of Cyclin E inhibits it. In FLP‑out clones expressing RNAi constructs to deplete APC/C subunits in the salivary gland and ovarian follicle layer, Geminin and Cyclins A and B, which are substrates of APC/C‑directed proteolysis, accumulated. Lower ploidy was also observed, possibly due to inhibition of endoreplication. Expression of Cyclin E also resulted in accumulation of Geminin and cyclins, an effect suppressed by co‑expression of Fzr, an activator of APC/C. This is consistent with the idea that Cyclin E can inhibit APC/C.1 These results offer another explanation for the effect of continuous Cyclin E expression. In addition to blocking licensing of replication origins, via Cdk2 activity as proposed by previous reports, Cyclin E expression may also allow the accumulation of APC/C substrates that can block replication licensing. Taken together these data support the proposal that the APC/C is required beyond the mitotic/endocycle transition to promote endoreplication. Anne White from Bob Duronio’s group at the University of North Carolina, Chapel Hill, reported on how Cyclin E might coordinate histone synthesis with DNA replication. MPM2 is a widely used antibody that was raised originally against a HeLa cell mitotic extract and recognizes a phospho‑epitope on a number of mostly unknown proteins during mitosis in both fly and mammalian cells. In interphase Drosophila cells, nuclear MPM2 staining was shown previously to correlate with Cyclin E/Cdk2 activity.2 Duronio’s group reported that MPM2 staining, which appears as one or two nuclear foci in the syncytial embryo, colocalizes with the Histone Locus Body (HLB) and Lsm11. HLBs are nuclear structures associated with the histone locus that contain nascent histone transcripts, and Lsm11 is a member of U7 snRNP, which is involved in histone pre‑mRNA processing. MPM2 foci appear in nuclear division cycle 11 when histone transcription begins. The formation of MPM2 foci, which requires Cyclin E, does not require the histone locus, indicating independence from histone gene transcription. Previous work has shown that histone gene expression requires Cyclin E,3 so the role of Cyclin E in the formation of or activities within the HLB could account for this requirement.

OT D

Original manuscript submitted: 04/09/07 Revised manuscript submitted: 04/15/07 Manuscript accepted: 04/16/07

RIB

†Both authors contributed equally to this work.

IST

Department of Molecular, Cellular and Developmental Biology; University of Colorado; Boulder, Colorado USA

UT E

.

Kristin Garcia† Anita Wichmann† Tin Tin Su*

©

20

07

LA

ND

ES

BIO

SC

IEN

Drosophila, cell cycle, mitosis, cytokinesis, checkpoint, organogenesis

CE

Key words

.D

ON

Previously published online as a Fly E-publication: http://www.landesbioscience.com/journals/fly/article/4292

Regulation of E2F The E2F family of transcription factors directs the expression of genes necessary for S phase. Regulation of their activity provides a key mode of G1‑S regulation. Two reports point to proteolysis and splicing as means for regulation of E2F (Fig. 1).

www.landesbioscience.com

Fly

125

Cell Cycle Regulation

Figure 1. Meeting presentations on the regulation of E2F (boxed) and the cell cycle. Different types of cell cycles found during Drosophila development are depicted along with relevant presentations.

Shusaku Shibutani from Bob Duronio’s group at University of North Carolina, Chapel Hill, presented recently published findings on the regulation of E2F1 during the transition from G2‑regulated post‑blastoderm cell divisions to G1 arrest in embryonic cycle 17. Previous work showed that the Cdk2 inhibitor Dacapo (Dap) and Rbf1 are required to initiate G117 arrest and to maintain the repression of E2F1 targets during G117, respectively. Therefore, it was surprising to find that initial termination of E2F1 target gene expression that occurs during cycles 15 and 16 does not require Dap or Rbf1. Instead, S‑phase specific proteolysis of E2F1 may play a role.4 Maxim Frolov from the University of Illinois, Chicago described a recently published screen for modifiers of E2F1 mutant clones in the eye.5 Previous work showed that E2F1 mutants die as early stage larvae while E2F1, E2F2 double mutants die as late stage larvae or pupae. Thus, some of the phenotypes in E2F1 mutants may be attributed to the presence of the repressor form, E2F2. Consistent with this idea, a suppressor of the E2F1 mutant eye phenotype was found to encode a splicing factor needed for normal splicing of E2F2. The requirement for B52, an SR protein, was specific for E2F2 in that it was not needed for splicing of E2F1. Jun‑yuan Ji from Nick Dyson’s group at Massachusetts General Hospital reported on results from a dominant modifier genetic screen that provides insight into how E2F1 may communicate with 126

the transcriptional machinery. A screen for modifiers of rough eye or abnormal wing phenotypes that result from E2F1 knockdown by an inducible RNAi system identified Cdk8, a component of the transcriptional Mediator complex. Loss‑of‑function mutations in Cdk8 were found to suppress E2F1 dsRNA‑induced phenotypes in both the eye and wing. dsRNA knockdown of Cdk8 resulted in increased transcription of E2F1 target genes. Cdk8 and E2F1 proteins co‑immunoprecipitated in ectopic expression studies, and immunoprecipitated Cyclin C or Cdk8 was able to phosphorylate E2F1 in vitro. These results suggest that regulation by Cdk8 may counteract the transactivating activity of E2F1.

Syncytial embryos provide more insights into cell cycle regulation Embryonic syncytial division cycles occur in a common cytoplasm and are comprised primarily of S and M phases. The failure to coordinate S and M phases, such as the entry into M in the presence of ongoing S phase, results in mitotic chromosome segregation defects and embryonic lethality. Syncytial division phenotypes have been a powerful way to identify genes needed for coordination of S and M phases such as grapes (Chk1) and mei‑41 (ATR). A number of reports describe additional mutants with syncytial division defects, thus pointing to new genes with roles in S/M regulation (Fig. 1). Fly

2007; Vol. 1 Issue 2


Laura Lee’s group at Vanderbilt University presented work on two mutants, absent without leave (awol) and no poles (nopo), both of which are maternal effect lethal. Embryos from mutant mothers arrest during pre‑cortical syncytial divisions with mitotic spindle defects, such as barrel‑shaped spindles that lack centrosomes. These defects are rescued by mutations in chk2, indicating that the defects result from the ‘centrosome inactivation checkpoint’ that responds to DNA defects during mitosis. Jamie Rickmyre reported on mutants in awol, which encodes a Drosophila homolog of microcephalin (MCPH1). Mutations in human MCPH1 result in a form of autosomal recessive primary microcephaly. Drosophila MCPH1, like human MCPH1, contains BRCT domains, which are implicated in DNA repair and cell cycle control. awol mutations enhance the embryonic lethality of mei‑41 mutants, and MCPH1 levels are reduced in grp mutants. These data suggest that MCPH1 plays a role in the DNA replication checkpoint. Julie Merkle reported on mutants in nopo, which encodes a homolog of human TRIP (TRAF‑interacting protein) and contains a RING domain that resembles RING domains of known E3 ubiquitin ligases. Nopo interacts with the E2 conjugating enzyme Ben in a yeast 2‑hybrid assay; furthermore, ben mutants are maternal effect lethal and produce embryos with a nopo‑like phenotype, suggesting that Ben/Nopo forms an E2/E3 complex that regulates syncytial divisions. Exactly how Nopo and MCPH1 contribute to coordination of S and M phases remains to be determined. Vladic Mogila from Willis Li’s group at the University of Rochester Medical School presented recently published data that Drosophila MEK and ERK homologs function to coordinate S and M phases during syncytial cycles.6 This pathway can be activated upon ionizing radiation‑induced DNA damage but appears to be distinct from ATR/Chk1 pathway. Anne Royou from Bill Sullivan’s group at University of California, Santa Cruz, described how Grapes (Chk1) might coordinate nuclear and cytoplasmic events at entry into mitosis. Injection of purified Cyclin B into syncytial embryos led to premature nuclear envelope breakdown but did not advance chromosome condensation. Similarly, in embryos in which DNA synthesis was arrested and nuclei maintained in interphase by co‑injection of aphidicolin and cycloheximide, injection of Cyclin B led to Cdk1 activation only in the cytoplasm, as indicated by the dispersal of cortical myosin. However, the nuclear envelope in some of these embryos remained intact indicating that the nucleus remained protected from cytoplasmic Cdk1 activity. This protection is dependent on grapes (Chk1) since injection of Cyclin B into interphase‑arrested grp mutants resulted in activation of both cytoplasmic and nuclear Cdk1.

Mitosis and Cytokinesis Proper DNA segregation requires the appropriate coordination of mitosis and cytokinesis. In mitosis, the regulation of dynamic microtubules is key for proper mitotic spindle function. The position of the mitotic spindle, in addition to the interaction between microtubules and cortical actin, aids in appropriate cytokinesis. Several groups identified new proteins involved these processes, while other groups reported new roles for known proteins involved in mitosis and/or cytokinesis (Fig. 2). Shengjiang Tan from Daimark Bennett’s group at University of Oxford described the role of a protein phosphatase interacting subunit encoded by mars in mitotic spindle dynamics. mars mutants are female sterile due to embryonic lethality. Mitotic spindles in embryos from mars homozygous mothers show reduced tubulin and increased www.landesbioscience.com

phosphorylated D‑TACC (pD‑TACC) staining on the spindle. Mars physically interacts with microtubules and with D‑TACC, a protein that aids in stabilizing the minus‑end of microtubules at the centrosome. Mutation of the Aurora A phosphorylation site at S836 on D‑TACC to a non‑phosphorylatable residue rescued the embryonic lethality of mars mutants. Thus an essential function of Mars appears to be de‑phosphorylation of D‑TACC, possibly reversing the action of Aurora A. Reduced localization of pD‑TACC to the spindle may be necessary to allow for a more dynamic spindle. Sebastien Carreno from Francois Payre’s group at CNRS, Toulouse, identified a new role for Moesin in cytokinesis. Moesin is the only fly member of the Ezrin‑moesin‑radixin (ERM) family; ERM proteins are implicated in regulating cell morphology through actin‑membrane interactions in a signal‑dependent manner. Work presented here suggests a new role for an ERM protein in helping connect microtubules to cortical actin. Depletion of Moe by RNAi in S2 cells led to multinucleated cells with abnormal lobed shapes. During cytokinesis, phosphorylation of T559 on Moe by Ste2‑related kinase (Slk), as detected with a phospho‑specific antibody, is restricted to the constricting cell membranes at the cleavage furrow while non‑phosphorylated Moe remains associated to the poles. Similarly, Slk and possibly PIP2, a co‑factor for phosphorylation of Moe, are restricted to the cell equator during anaphase, which may account for spatial and temporal restriction of phosphorylation on Moe. Non‑phosphorylatable Moe mutants display destabilization of microtubule tips that abut the cortex while phospho‑mimic mutants have hyper‑stable microtubule ends. These results point to a role for Moe in stabilization of cortical tips of microtubules, specifically at the equator in late stages of mitosis. Microtubule‑cortex interactions are implicated in cleavage furrow formation during cytokinesis, and productive interactions are restricted to the cell equator, such that cell membrane constriction and cytokinesis occurs only at the equator. Moe may thus be a component of the regulatory mechanisms that restrict productive microtubule‑cortex interactions to the equator. Shubha Rao from Margarete Heck’s group at the University of Edinburgh reported on a genetic interactor of invadolysin which encodes a metalloprotease with roles in mitotic progression and cell migration.7 A second site non‑complementation screen identified non‑stop (not), a predicted ubiquitin protease involved in axon target recognition. not mutants phenocopy hypercondensed chromosomes seen in Invadolysin mutant larval neuroblasts. Ubiquitinated histone H2B protein levels appear to be mis‑regulated in not mutants, which may account for their chromosomal defect. Uta Wieland from David Glover’s group at University of Cambridge reported on cloning of merry‑go‑round (mgr), mutants in which larval neuroblasts show abnormal chromosome alignment. mgr encodes a member of a co‑chaperone complex that is involved in the folding of tubulin and actin in yeast. In electron micrographs, centrosomes in mgr RNAi‑treated cells show singlet microtubules instead of the usual doublets or triplets. RNAi of mgr or another putative member of this co‑chaperone complex, resulted in reduced centrosome number, mono‑centrosomal spindles, and collapsed spindles in S2 cells. These phenotypes, however, are quantitatively not as severe as knockdown of CCT‑Chaperonin complex, another tubulin folding complex that participates in later stages of protein folding. This difference suggests functional distinction for two complexes that act at different stages in what is thought to be the same pathway for protein folding. Nasser Rusan from Mark Peifer’s group at University of North Carolina, Chapel Hill, described the surprising behavior of Fly

127


Figure 2. Mitotic spindle and cytokinesis‑related processes that were represented by meeting presentations. (A) The mitotic spindle consists of dynamic microtubules (green) that nucleate from centrosomes composed of centrioles (black rectangles) and pericentriolar material (red). DNA (dark blue) aligns on the metaphase plate through microtubule interactions at the kinetochores (light blue); proper attachment of the spindle to kinetochores is monitored by the Spindle Assembly Checkpoint (SAC). (B) Cytokinesis involves the interaction of microtubule tips with the cell cortex to coordinate the formation of the cleavage furrow. (C) Asymmetric divisions that produce two unequal daughters, a neuroblast and a Ganglion Mother Cell (GMC) are aided by the spindle position, which is pre‑set by the centrosomes. GMCs shown are from previous divisions.

c entrosomes in larval neuroblasts (NB).8 NBs are stem cells that undergo asymmetric division to produce a large daughter NB and a smaller ganglion mother cell (GMC). Asymmetry in daughter cell size is aided by asymmetric positioning of the mitotic spindle and hence the division plane. The position of centrosomes to set up the spindle plays a key role in this process. Nasser and colleagues imaged centrosomes live and found that there is only one centrosome visible early in the cell cycle and the second one appears before mitosis about 130° from the first. This was contrary to the belief that when duplicated centrosomes separate to the opposite poles, they maintain typical centrosomal characteristics, such as the presence of Pericentriolar Material (PCM) and the ability to nucleate microtubules. Use of centriole versus centrosome markers show that centrioles do indeed separate and migrate to the opposite pole. However, while the stationary centrioles retain PCM, the migrating centrioles lose PCM until they are substantially migrated from the origin. Further, the stationary centrioles remain in the NB; it is the migrating centrioles that end up in the GMC. Thus, the asymmetry in the spindle is in place as early as immediately after the separation of stationary and migrating centrioles. Systematic searches for chromosomal proteins. MEI‑S332, a member of the shugoshin family of centromeric cohesion proteins, aids accurate chromosome segregation in mitosis and meiosis. Hannah Cohen from Terry Orr‑Weaver’s lab at MIT/Whitehead 128

Institute described a living cell microarray screen performed in collaboration with David Sabatini’s lab at the Whitehead Institute to identify genes that regulate proper localization of MEI‑S332. MEI‑S332 loads on centromeres at prophase and unloads at metaphase/anaphase transition. MEI‑S332 localization was analyzed in Drosophila Kc167 cells plated on glass slides that were spotted with 19,000 double‑stranded RNAs that target the annotated Drosophila genes. Localization analysis was performed with immunofluorescence imaging and computational software. Preliminary results indicate that this high‑throughput method can identify genes that, when depleted, prevent proper localization of MEI‑S332. Lucia Mentalova from Alvaro Tavares’s lab at Institute Gulbankian, Lisbon, presented work on the isolation and characterization of Drosophila kinetochore (KT) proteins. Although at least 60 KT proteins have been identified in budding yeast, there may be additional components of KTs that are yet to be identified in metazoa. To identify KT proteins, chromosomes were isolated by sucrose gradients from S2 cells blocked in mitosis. After enrichment of scaffold proteins by DNase treatment and sedimentation through a sucrose cushion, proteins were separated on 2D gels and identified by mass spectrometry. Eighty-two proteins were identified, including 16 previously identified KT components and 25 proteins with no obvious homologs. Four of the new proteins, detected by GFP tags, co‑localize with mitotic chromatin or with KTs. RNAi knockdown Fly



of one protein led to chromosome misalignment in metaphase and chromosome lagging in anaphase, thus demonstrating a functional role for a newly identified KT protein.

Coordinating cell proliferation with organogenesis Regulation of cell proliferation is key to organ size control during normal development and in tissue homeostasis after injury or during nutrient deprivation. Work from several groups addressed how cell proliferation is regulated in response to environmental and developmental cues (Fig. 1). Brent Wells from Laura Johnston’s group at Columbia University, College of Physicians and Surgeons, described the role of Drosophila p53 (dp53) in compensatory proliferation. The Johnston lab has previously implicated p53 in an initial cell cycle arrest, and a subsequent increase in cell proliferation that results from persistence of ‘undead’ cells. Ectopic co‑induction of a death inducer, such as Hid, and the caspase inhibitor, p35, results in cells that initiate the apoptosis program yet remain ‘undead’. This treatment alters proliferation throughout the disc, in both a cell autonomous and non‑autonomous manner. dp53 mutants fail to show altered proliferation, implicating p53 in compensatory proliferation.9 In new studies, another method was used to induce compensatory proliferation. Surgical removal of a portion of a larval imaginal disc leads to the formation of a blastema, a group of cells at the wound site that proliferate and differentiate to regenerate the disc. This process can be mimicked by ectopic activation of Wingless (Wg) signaling in specific cells of the prothoracic leg disc called the ‘weak point’. Wells and colleagues found that the efficiency of blastema formation that follows Wg induction was reduced in p53 mutants, suggesting that p53 is also needed for this mode of compensatory proliferation. Hannele Ruohola‑Baker from the University of Washington reports that germline stem cell (GSC) proliferation in the female ovary responds to nutrient through miRNA‑mediated repression of Dacapo/p21 (Dap). Previous work from the same group has shown that proliferation of GSCs require dicer‑1, which encodes a processing factor for production of mature miRNAs.10 In dicer‑1 mutants, GSCs stop dividing possibly due to upregulation of Dap, an inhibitor of Cyclin E/Cdk2 and of G1/S transition. Starvation of female flies results in a reduction of GSC numbers, and this reduction was found to require dap. Mutations in InR, the receptor for Insulin‑like Growth factor and a known target of nutrient sensing, resulted in a delay in G1/S transition in GSCs. These data led to the model that nutrient availability acts through InR to inhibit Dap in a Dicer‑dependent manner to allow proliferation of GSCs. Since Dicer‑1 is involved in miRNA processing, Ruohola‑Baker used a GFP‑Dap 3'UTR reporter construct to confirm that Dap is a target of miRNAs. The 3'UTR of Dap contained potential recognition sites for mir‑7 and bantam miRNAs. Like dicer‑1 mutants, mir‑7 mutants delay in G1/S as assayed by Cyclin E accumulation, while bantam is required for GSC maintenance. In parallel studies, Human ES cells are found to proliferate slowly if Dicer or Drosha, another processing factor for miRNAs, is knocked down. Thus miRNAs may also be required for proliferation of stem cells in human embryos. This system allowed the Ruohola‑Baker laboratory to screen for miRNAs that rescue the effect of depleting Dicer in add‑back experiments, and to identify miR195 in such a screen. Carla Lopes from Fernando Casares’s group at CABD, CSIC‑University Pablo de Olavide Seville, Spain, presented data on the role of the transcription factor Homothorax (Hth) in maintaining www.landesbioscience.com

roliferation in the eye imaginal disc. In the eye disc, the domain of p hth expression corresponds to the asynchronously proliferating cell population anterior to the morphogenetic furrow. These cells lose hth expression and enter mitosis in the so‑called first mitotic wave (FMW). Cells then become transiently arrested in G1 and either directly differentiate or undergo one round of mitosis prior to terminal differentiation. Clones of cells that are forced to express Hth posterior to the FMW show increased proliferation, as indicated by the number of cells with BrdU incorporation or phosphorylated Histone H3 staining, which is a mitotic marker. Also, FACS analyses show that ectopic Hth expression can increase the G2/M fraction of cells. The G2 delay is most likely due to the negative regulation of the expression of string/cdc25 by Hth. These data led to the model that Hth helps synchronize a large fraction of cells in G2, which can then enter FMW upon repression of hth and expression of string.

Cell cycle regulation in response to damage or dysfunction Stopping cell proliferation when necessary is as important as promoting proliferation. Work from several labs described cell cycle regulatory mechanisms that respond to meiotic problems, over‑replication of DNA, defective spindle assembly and mitochondrial dysfunction (Fig. 1). All but two of the following presentations were in the Cell Cycle Checkpoints Workshop and therefore did not have abstracts printed. The meiotic checkpoint in female oogenesis occurs in response to unrepaired DNA double strand breaks generated through recombination. Previous work has shown that this checkpoint acts through mei‑41 and chk2 to produce cell cycle arrest in prophase, abnormal karyosomes and D‑V patterning defects that render the egg inviable. This checkpoint, therefore, serves as a quality control mechanism to eliminate damaged eggs. Carla Klattenhoff from Bill Theurkauf ’s group at University of Massachusetts Medical Center, Wocester, followed up on their recently published work showing that mutations in the rasiRNA pathway activate the meiotic checkpoint.11 Repeat associated small interfering RNAs are enriched in the germline in flies and mouse and are associated with silencing of repeat sequences in centromeres and telomeres. Previous work showed that mutations in armitage and aubergine, which encode a helicase and Argonaute protein required for rasiRNA production, lead to DNA damage signaling and accumulation of H2AV foci, which are generally associated with DNA double strand breaks. New data, with previously reported genetic interactions, indicate that rasiRNA pathway mutations activate Mei-41 and ATM, which induce the Chk2‑dependent meiotic checkpoint. This is the first implication of ATM in the meiotic checkpoint, and suggests that rasiRNAs suppress DNA break formation in the germline. In a related talk, Vitor Barbosa from Ruth Lehmann’s group at HHMI/Skirball Institute and NYU reported on a screen for mutations that activate the meiotic checkpoint. By isolating mutants with defective dorso‑ventral egg shape pattern they identified cohiba, which encodes the fly homolog of budding yeast Pds5p. In other systems Pds5 colocalizes with chromosomal cohesins and is required for cohesion between sister chromosomes after replication. Barbosa found that checkpoint activation in dPds5 mutants is suppressed by loss of mei‑W68, which eliminates DSBs. This suggests that persistent DSBs may activate the checkpoint. Surprisingly, mutations in the ATR checkpoint kinase mei‑41 did not rescue checkpoint activation. One hypothesis is that dPds5 is not directly involved in Fly

129


DSB repair but maintains chromosome integrity independent of the ATR‑dependent checkpoint. Identification of checkpoint protein(s) that defects in dPds5 mutants signal through is currently underway. Brian Calvi from Syracuse University presented evidence that over‑replication can activate checkpoint responses. Over‑replication was driven by increased expression of double‑parked (fly ortholog of Cdt1) in diploid cells of larval imaginal discs and adult ovaries, which increased DNA content to 8N and beyond. This resulted in two types of checkpoint responses, cell cycle arrest or apoptosis with caspase activation. Although most apoptosis was dependent on Chk2 and p53, re‑replication also activated alternative, unknown cell death pathways. Re‑replicating cells had increased phosphorylation of the histone H2AV in repair foci, indicative of DNA damage and the induction of ATM/ATR activity. Evidence from other labs suggests that, in mammalian cells, Cdt1 activates similar checkpoint responses and may promote genome instability and cancer.12 Claudio Sunkel described recently published work that addressed the role of MAD2 in the spindle assembly checkpoint (SAC).13 Loss of MAD2 in S2 cells leads to accelerated progression through mitosis as indicated by a decrease in metaphase index and the failure to properly congress and segregate chromosomes. Additionally, the failure to arrest in mitosis upon microtubule depolymerization was observed, consistent with the conserved role for MAD2 in the SAC. Remarkably, all of these phenotypes can be rescued by a transient delay in mitosis using chemical proteosome inhibitors. When these delayed cells are subsequently released in the presence of a microtubule poison, the SAC is still intact, as shown by increased kinetochore (KT)‑associated BubR1 levels and intact cohesin complexes. These results indicate a Mad2‑independent SAC and suggest that Mad2 really plays a role in allowing enough time for the SAC proteins to localize to the KT. New data indicates that another SAC protein, ZW10, may also play a role in this KT‑independent mechanism to ensure proper mitotic progression. Depletion of ZW10 has the same effects as MAD2 depletion, and these effects are rescued with a transient arrest using a proteosome inhibitor. In MAD2‑depleted cells that have transiently arrested in mitosis, BubR1 is recruited onto the KTs. Depletion of BubR1 led to SAC failure but this could not be rescued by a transient mitotic arrest. These results suggest that activation of SAC relies on a MAD2‑dependent mechanism that provides time and a BubR1‑dependent mechanism that requires the assembly of SAC proteins onto the KT. Junyong Huang from Newcastle University, UK, reported that Fzy/Cdc20, an APC/C‑activator, still localizes to the KTs properly in MAD2 mutants but not in BubR1 mutants. These results are consistent with those from the Sunkel lab that identified distinct requirements for MAD2 and BubR1 in SAC. One aspect of the KT‑dependent mechanism that operates through BuBR1 may be recruitment to and inhibition of Fzy at the KTs, which would delay the metaphase‑anaphase transition. In related work Huang’s group also presented data on the potential role of phosphorylation on subcellular localization of an APC subunit, Cdc27. Mutation of prolines to alanines in 2 Cdk1 consensus sites disrupted the localization of Cdc27onto mitotic chromosomes in syncytial embryos. These mutations also lead to increased levels of Cyclin A and B, indicating reduced APC/C activity. Since a Cdc27 P304A P456A mutant was unable to rescue a Cdc27 mutant, these sites are important for proper Cdc27 chromosomal localization as well as APC/C function.

130

Joe Lipsick from Stanford University School of Medicine reported that clones of cells that are mutated for the Drosophila homolog of the proto‑oncogene Myb have reduced mRNA levels for genes encoding several SAC proteins, including Polo, Rod, BubR1 and MAD2. Myb is part of a large protein complex containing Rb and E2F, which may function to regulate the transcription of SAC genes. Reduction of mRNAs for SAC genes is obvious after only a few divisions in the absence of Myb, such as in small clones of mutant cells. In larger clones, which presumably result after several more divisions in the absence of Myb, there is yet another defect: partial phosphorylation of histone H3, which appears to be limited to heterochromatin and absent from euchromatin. Myb was shown to colocalize with euchromatin, so this result suggests that Myb is needed for H3 phosphorylation in euchromatin but not heterochromatin. Edward Owusu‑Ansah and Sudip Mandal from Utpal Banerjee’s group at the University of California, Los Angeles, described the regulation of G1 and S in response to mutations in components of the electron transport chain reactions in the mitochondria. Interestingly, mutations in complex I and complex IV enzymes of the electron transport chain both result in reduced BrdU incorporation but by different mechanisms. Previously published work from this group showed that mutations in a complex IV enzyme result in reduction of Cyclin E while the levels of Cdk2 inhibitor Dacapo and mitotic cyclins A and B remain unchanged.14 In contrast, clones of cells in the eye imaginal disc that lack complex I show elevated Dacapo while the level of Cyclin E remains normal. Their data suggest that complex I regulation of the cell cycle checkpoint requires elevated levels of Reactive Oxygen Species and involves a pathway that is distinct from that triggered by lowered ATP from Complex IV defects.

Conclusions We hope that these summaries illustrate the wealth of questions one could ask and the plethora of techniques one could use to answer these questions in Drosophila cell cycle research. From basic centrosome behavior to coordination of the cell cycle with organismal development, from systematic proteomics to the genetics of regeneration, the bug that we love continues to intrigue, educate and illuminate graduate students and seasoned principle investigators alike. References 1. Sigrist SJ, Lehner CF. Drosophila fizzy‑related down‑regulates mitotic cyclins and is required for cell proliferation arrest and entry into endocycles. Cell 1997; 90:671‑81. 2. Calvi BR, Lilly MA, Spradling AC. Cell cycle control of chorion gene amplification. Genes Dev 1998; 12:734‑44. 3. Lanzotti DJ, Kupsco JM, Marzluff WF, Duronio RJ. string(cdc25) and cyclin E are required for patterned histone expression at different stages of Drosophila embryonic development. Dev Biol 2004; 274:82‑93. 4. Shibutani S, Swanhart LM, Duronio RJ. Rbf1‑independent termination of E2f1‑target gene expression during early Drosophila embryogenesis. Development 2007; 134:467‑78. 5. Rasheva VI, Knight D, Bozko P, Marsh K, Frolov MV. Specific role of the SR protein splicing factor B52 in cell cycle control in Drosophila. Mol Cell Biol 2006; 26:3468‑77. 6. Mogila V, Xia F, Li WX. An intrinsic cell cycle checkpoint pathway mediated by MEK and ERK in Drosophila. Dev Cell 2006; 11:575‑82. 7. McHugh B, Krause SA, Yu B, Deans AM, Heasman S, McLaughlin P, Heck MM. Invadolysin: a novel, conserved metalloprotease links mitotic structural rearrangements with cell migration. J Cell Biol 2004; 167:673‑86. 8. Rusan NM, Peifer M. A role for a novel centrosome cycle in asymmetric cell division J Cell Bio 2007; 177:13-20. 9. Wells BS, Yoshida E, Johnston LA. Compensatory proliferation in Drosophila imaginal discs requires Dronc‑dependent p53 activity. Curr Biol 2006; 16:1606‑15. 10. Hatfield SD, Shcherbata HR, Fischer KA, Nakahara K, Carthew RW, Ruohola‑Baker H. Stem cell division is regulated by the microRNA pathway. Nature 2005; 435:974‑8.

Fly


Cell Cycle Regulation 11. Klattenhoff C, Bratu DP, McGinnis‑Schultz N, Koppetsch BS, Cook HA, Theurkauf WE. Drosophila rasiRNA pathway mutations disrupt embryonic axis specification through activation of an ATR/Chk2 DNA damage response. Dev Cell 2007; 12:45‑55. 12. Seo J, Chung YS, Sharma GG, Moon E, Burack WR, Pandita TK, Choi K. Cdt1 transgenic mice develop lymphoblastic lymphoma in the absence of p53. Oncogene 2005; 24:8176‑86. 13. Orr B, Bousbaa H, Sunkel CE. Mad2‑independent Spindle Assembly Checkpoint Activation and Controlled Metaphase‑Anaphase Transition in Drosophila S2 Cells. Mol Biol Cell 2007; 18:850‑63. 14. Mandal S, Guptan P, Owusu‑Ansah E, Banerjee U. Mitochondrial regulation of cell cycle progression during development as revealed by the tenured mutation in Drosophila. Dev Cell 2005; 9:843‑54.

www.landesbioscience.com

Fly

131