Proteomics of M-phase entry: 'Omen' vs. 'Omre', the battle for oocyte quality and beyond

The understanding of cell cycle regulation benefited greatly from omic approaches. Because the cell cycle engine relies heavily on proteins, proteomic methods play a key role in identification of cell cycle players. The proteomic approach delivers an enormous volume of data, but it often lacks comprehensiveness. To ensure the comprehensiveness of results the discovery of novel proteins must be followed by functional analysis. Using Xenopus laevis oocytes in two different proteomic screens, we have recently identified a number of proteins whose behavior suggested specific and unexpected roles in M-phase entry. Functional analysis of EP45 identified in one of these screens has shown that M-phase entry is stimulated by Oocyte-Maturation-ENhancer ('Omen') activity. The second screen suggests the presence of an antagonistic activity, which we call 'Omre' (Oocyte- -Maturation-REpressor). The equilibrium between Omen and Omre activities may determine the quality of oocytes and further embryo development via participation in making the decision whether to enter oocyte maturation. It remains an open question whether similar activities operate during mitotic divisions in embryonic and adult cells. Identifying such activities in somatic cells might impact on cancer treatments.     

Identification of Saccharomyces cerevisiae spindle pole body remodeling factors

The Saccharomyces cerevisiae centrosome or spindle pole body (SPB) is a dynamic structure that is remodeled in a cell cycle dependent manner. The SPB increases in size late in the cell cycle and during most cell cycle arrests and exchanges components during G1/S. We identified proteins involved in the remodeling process using a strain in which SPB remodeling is conditionally induced. This strain was engineered to express a modified SPB component, Spc110, which can be cleaved upon the induction of a protease. Using a synthetic genetic array analysis, we screened for genes required only when Spc110 cleavage is induced. Candidate SPB remodeling factors fell into several functional categories: mitotic regulators, microtubule motors, protein modification enzymes, and nuclear pore proteins. The involvement of candidate genes in SPB assembly was assessed in three ways: by identifying the presence of a synthetic growth defect when combined with an Spc110 assembly defective mutant, quantifying growth of SPBs during metaphase arrest, and comparing distribution of SPB size during asynchronous growth. These secondary screens identified four genes required for SPB remodeling: NUP60, POM152, and NCS2 are required for SPB growth during a mitotic cell cycle arrest, and UBC4 is required to maintain SPB size during the cell cycle. These findings implicate the nuclear pore, urmylation, and ubiquitination in SPB remodeling and represent novel functions for these genes.     

Membrane fusion proteins are required for oskar mRNA localization in the Drosophila egg chamber

We used a genetic screen in Drosophila to identify mutations which disrupt the localization of oskar mRNA during oogenesis. Based on the hypothesis that some cytoskeletal components which are required during the mitotic divisions will also be required for oskar mRNA localization during oogenesis, we designed the following genetic screen. We screened for P-element insertions in genes which slow down the blastoderm mitotic divisions. A secondary genetic screen was to generate female germ-line clones of these potential cell division cycle genes and to identify those which cause the mislocalization of oskar mRNA. We identified mutations in ter94 which disrupt the localization of oskar mRNA to the posterior pole of the oocyte. Ter94 is a member of the CDC48p/VCP subfamily of AAA proteins which are involved in homotypic fusion of the endoplasmic reticulum during mitosis. Consistent with the function of the yeast ortholog, ter94-mutant egg chambers are defective in the assembly of the endoplasmic reticulum. We tested whether other membrane biosynthesis genes are required for localizing oskar mRNA during oogenesis. We found that ovaries that are mutant for syntaxin-1a, rop, and synaptotagmin are also defective in oskar mRNA localization during oogenesis. We suggest a pathway for the role of membrane assembly proteins on oskar mRNA localization.     

A mosaic genetic screen for novel mutations affecting Drosophila neuroblast divisions

Background

The asymmetric segregation of determinants during cell division is a fundamental mechanism for generating cell fate diversity during development. In Drosophila, neural precursors (neuroblasts) divide in a stem cell-like manner generating a larger apical neuroblast and a smaller basal ganglion mother cell. The cell fate determinant Prospero and its adapter protein Miranda are asymmetrically localized to the basal cortex of the dividing neuroblast and segregated into the GMC upon cytokinesis. Previous screens to identify components of the asymmetric division machinery have concentrated on embryonic phenotypes. However, such screens are reaching saturation and are limited in that the maternal contribution of many genes can mask the effects of zygotic loss of function, and other approaches will be necessary to identify further genes involved in neuroblast asymmetric division.

Results

We have performed a genetic screen in the third instar larval brain using the basal localization of Miranda as a marker for neuroblast asymmetry. In addition to the examination of pupal lethal mutations, we have employed the MARCM (Mosaic Analysis with a Repressible Cell Marker) system to generate postembryonic clones of mutations with an early lethal phase. We have screened a total of 2,300 mutagenized chromosomes and isolated alleles affecting cell fate, the localization of basal determinants or the orientation of the mitotic spindle. We have also identified a number of complementation groups exhibiting defects in cell cycle progression and cytokinesis, including both novel genes and new alleles of known components of these processes.

Conclusion

We have identified four mutations which affect the process of neuroblast asymmetric division. One of these, mapping to the imaginal discs arrested locus, suggests a novel role for the anaphase promoting complex/cyclosome (APC/C) in the targeting of determinants to the basal cortex. The identification and analysis of the remaining mutations will further advance our understanding of the process of asymmetric cell division. We have also isolated a number of mutations affecting cell division which will complement the functional genomics approaches to this process being employed by other laboratories. Taken together, these results demonstrate the value of mosaic screens in the identification of genes involved in neuroblast division.     

A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen

To enable arrayed or pooled loss-of-function screens in a wide range of mammalian cell types, including primary and nondividing cells, we are developing lentiviral short hairpin RNA (shRNA) libraries targeting the human and murine genomes. The libraries currently contain 104,000 vectors, targeting each of 22,000 human and mouse genes with multiple sequence-verified constructs. To test the utility of the library for arrayed screens, we developed a screen based on high-content imaging to identify genes required for mitotic progression in human cancer cells and applied it to an arrayed set of 5,000 unique shRNA-expressing lentiviruses that target 1,028 human genes. The screen identified several known and approximately 100 candidate regulators of mitotic progression and proliferation; the availability of multiple shRNAs targeting the same gene facilitated functional validation of putative hits. This work provides a widely applicable resource for loss-of-function screens, as well as a roadmap for its application to biological discovery.     

Whole genome functional analysis identifies novel components required for mitotic spindle integrity in human cells

Background

The mitotic spindle is a complex mechanical apparatus required for accurate segregation of sister chromosomes during mitosis. We designed a genetic screen using automated microscopy to discover factors essential for mitotic progression. Using a RNA interference library of 49,164 double-stranded RNAs targeting 23,835 human genes, we performed a loss of function screen to look for small interfering RNAs that arrest cells in metaphase.

Results

Here we report the identification of genes that, when suppressed, result in structural defects in the mitotic spindle leading to bent, twisted, monopolar, or multipolar spindles, and cause cell cycle arrest. We further describe a novel analysis methodology for large-scale RNA interference datasets that relies on supervised clustering of these genes based on Gene Ontology, protein families, tissue expression, and protein-protein interactions.

Conclusion

This approach was utilized to classify functionally the identified genes in discrete mitotic processes. We confirmed the identity for a subset of these genes and examined more closely their mechanical role in spindle architecture.     

Genome-wide synthetic lethal screens identify an interaction between the nuclear envelope protein, Apq12p, and the kinetochore in Saccharomyces cerevisiae

The maintenance of genome stability is a fundamental requirement for normal cell cycle progression. The budding yeast Saccharomyces cerevisiae is an excellent model to study chromosome maintenance due to its well-defined centromere and kinetochore, the region of the chromosome and associated protein complex, respectively, that link chromosomes to microtubules. To identify genes that are linked to chromosome stability, we performed genome-wide synthetic lethal screens using a series of novel temperature-sensitive mutations in genes encoding a central and outer kinetochore protein. By performing the screens using different mutant alleles of each gene, we aimed to identify genetic interactions that revealed diverse pathways affecting chromosome stability. Our study, which is the first example of genome-wide synthetic lethal screening with multiple alleles of a single gene, demonstrates that functionally distinct mutants uncover different cellular processes required for chromosome maintenance. Two of our screens identified APQ12, which encodes a nuclear envelope protein that is required for proper nucleocytoplasmic transport of mRNA. We find that apq12 mutants are delayed in anaphase, rereplicate their DNA, and rebud prior to completion of cytokinesis, suggesting a defect in controlling mitotic progression. Our analysis reveals a novel relationship between nucleocytoplasmic transport and chromosome stability.     

Spindle assembly checkpoint regulates mitotic cell cycle progression during preimplantation embryo development

Errors in chromosome segregation or distribution may result in aneuploid embryo formation, which causes implantation failure, spontaneous abortion, genetic diseases, or embryo death. Embryonic aneuploidy occurs when chromosome aberrations are present in gametes or early embryos. To date, it is still unclear whether the spindle assembly checkpoint (SAC) is required for the regulation of mitotic cell cycle progression to ensure mitotic fidelity during preimplantation development. In this study, using overexpression and RNA interference (RNAi) approaches, we analyzed the role of SAC components (Bub3, BubR1 and Mad2) in mouse preimplantation embryos. Our data showed that overexpressed SAC components inhibited metaphase-anaphase transition by preventing sister chromatid segregation. Deletion of SAC components by RNAi accelerated the metaphase-anaphase transition during the first cleavage and caused micronuclei formation, chromosome misalignment and aneuploidy, which caused decreased implantation and delayed development. Furthermore, in the presence of the spindle-depolymerizing drug nocodazole, SAC depleted embryos failed to arrest at metaphase. Our results suggest that SAC is essential for the regulation of mitotic cell cycle progression in cleavage stage mouse embryos.     

A Genetic Screen Using the PiggyBac Transposon in Haploid Cells Identifies Parp1 as a Mediator of Olaparib Toxicity

Genetic perturbation screens have the potential to dissect a wide range of cellular phenotypes. Such screens have historically been difficult in diploid mammalian cells. The recent derivation of haploid embryonic stem cells provides an opportunity to cause loss of function mutants with a random mutagen in a mammalian cell with a normal genetic background. We describe an approach to genetic screens that exploits the highly active piggyBac transposon in haploid mammalian cells. As an example of haploid transposon (HTP) screening, we apply this approach to identifying determinants of cancer drug toxicity and resistance. In a screen for 6-thioguanine resistance we recovered components of the DNA mismatch repair pathway, a known requirement for toxicity. In a further screen for resistance to the clinical poly(ADP-ribose) polymerase (PARP) inhibitor olaparib we recovered multiple Parp1 mutants. Our results show that olaparib toxicity to normal cells is mediated predominantly via Parp1, and suggest that the clinical side effects of olaparib may be on target. The transposon mutant libraries are stable and can be readily reused to screen other drugs. The screening protocol described has several advantages over other methods such as RNA interference: it is rapid and low cost, and mutations can be easily reverted to establish causality.     

A genetic screen for components of the mammalian RNA interference pathway in Bloom-deficient mouse embryonic stem cells

Genetic screens performed in model organisms have helped identify key components of the RNA interference (RNAi) pathway. Recessive genetic screens have recently become feasible through the use of mouse embryonic stem (ES) cells that are Bloom's syndrome protein (Blm) deficient. Here, we developed and performed a recessive genetic screen to identify components of the mammalian RNAi pathway in Blm-deficient ES cells. Genome-wide mutagenesis using a retroviral gene trap strategy resulted in the isolation of putative homozygous RNAi mutant cells. Candidate clones were confirmed by an independent RNAi-based reporter assay and the causative gene trap integration site was identified using molecular techniques. Our screen identified multiple mutant cell lines of Argonaute 2 (Ago2), a known essential component of the RNAi pathway. This result demonstrates that true RNAi components can be isolated by this screening strategy. Furthermore, Ago2 homozygous mutant ES cells provide a null genetic background to perform mutational analyses of the Ago2 protein. Using genetic rescue, we resolve an important controversy regarding the role of two phenylalanine residues in Ago2 activity.     

A review of mitosis in the fission yeast Schizosaccharomyces pombe

Mitosis and cell division are the final events of the cell cycle, resulting in the precise segregation of chromosomes into two daughter cells. A highly controlled and accurate segregation of the chromosomes is required to ensure that each daughter cell receives a complete genome and remains viable. The fission yeast, Schizosaccharomyces pombe, is a unicellular eukaryotic organism which is particularly convenient for investigating these problems. It is very amenable to genetic analysis and its predominantly haploid life cycle has allowed the isolation of recessive temperature-sensitive mutants unable to complete the cell cycle. Classical genetic analysis of these mutants has been used to identify over 40 gene functions that are required for cell cycle progress in S. pombe. Many of these genes have now been cloned and sequenced and in some cases the encoded gene product has been identified. This approach, coupling classical and molecular genetics, allows identification of the molecules important in the mitotic processes and provides a means for establishing what functional roles they may play.     

A large-scale RNAi screen identifies functional classes of genes shaping synaptic development and maintenance

Neuronal circuit development and function require proper synapse formation and maintenance. Genetic screens are one powerful method to identify the mechanisms shaping synaptic development and stability. However, genes with essential roles in non-neural tissues may be missed in traditional loss-of-function screens. In an effort to circumvent this limitation, we used neuron-specific RNAi knock down in Drosophila and assayed the formation, growth, and maintenance of the neuromuscular junction (NMJ). We examined 1970 Drosophila genes, each of which has a conserved ortholog in mammalian genomes. Knock down of 158 genes in post-mitotic neurons led to abnormalities in the neuromuscular system, including misapposition of active zone components opposite postsynaptic glutamate receptors, synaptic terminal overgrowth and undergrowth, abnormal accumulation of synaptic material within the axon, and retraction of synaptic terminals from their postsynaptic targets. Bioinformatics analysis demonstrates that genes with overlapping annotated function are enriched within the hits for each phenotype, suggesting that the shared biological function is important for that aspect of synaptic development. For example, genes for proteasome subunits and mitotic spindle organizers are enriched among the genes whose knock down leads to defects in synaptic apposition and NMJ stability. Such genes play essential roles in all cells, however the use of tissue- and temporally-restricted RNAi indicates that the proteasome and mitotic spindle organizers participate in discrete aspects of synaptic development. In addition to identifying functional classes of genes shaping synaptic development, this screen also identifies candidate genes whose role at the synapse can be validated by traditional loss-of-function analysis. We present one such example, the dynein-interacting protein NudE, and demonstrate that it is required for proper axonal transport and synaptic maintenance. Thus, this screen has identified both functional classes of genes as well as individual candidate genes that are critical for synaptic development and will be a useful resource for subsequent mechanistic analysis of synapse formation and maintenance.     

A Highlights from MBoC Selection: Compartmentalized nodes control mitotic entry signaling in fission yeast

Cell cycle progression is coupled to cell growth, but the mechanisms that generate growth-dependent cell cycle progression remain unclear. Fission yeast cells enter into mitosis at a defined size due to the conserved cell cycle kinases Cdr1 and Cdr2, which localize to a set of cortical nodes in the cell middle. Cdr2 is regulated by the cell polarity kinase Pom1, suggesting that interactions between cell polarity proteins and the Cdr1-Cdr2 module might underlie the coordination of cell growth and division. To identify the molecular connections between Cdr1/2 and cell polarity, we performed a comprehensive pairwise yeast two-hybrid screen. From the resulting interaction network, we found that the protein Skb1 interacted with both Cdr1 and the Cdr1 inhibitory target Wee1. Skb1 inhibited mitotic entry through negative regulation of Cdr1 and localized to both the cytoplasm and a novel set of cortical nodes. Skb1 nodes were distinct structures from Cdr1/2 nodes, and artificial targeting of Skb1 to Cdr1/2 nodes delayed entry into mitosis. We propose that the formation of distinct node structures in the cell cortex controls signaling pathways to link cell growth and division.     

Ribosome synthesis-unrelated functions of the preribosomal factor Rrp12 in cell cycle progression and the DNA damage response

Given the high metabolic cost required to generate ribosomes, it has been assumed that proteins involved in ribosome synthesis might establish functional cross talk with other intracellular processes to efficiently couple ribosome production and cell growth. However, such interconnections have remained elusive due to the difficulty in separating the intra- and extraribosomal roles of ribosome biogenesis factors. Using a yeast functional screen, I have discovered that Rrp12, a conserved protein involved in ribosome maturation and export, plays roles in the cell cycle and the DNA damage response. These results indicate that Rrp12 participates in a karyopherin Kap121-dependent import route that is crucial for nuclear sequestration of ribonucleotide reductase subunits and, thereby, ensures the proper kinetics of deoxyribonucleotide production during the cell cycle. Within this route, Rrp12 acts as a cofactor important for the full functionality of Kap121. This activity is mechanistically different from the known roles of Rrp12 in ribosome biogenesis. I propose that the functional duality of Rrp12 may couple the control of ribosome production to the regulation of other cellular processes during cell cycle progression.     

Cell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle function

Correct assembly and function of the mitotic spindle during cell division is essential for the accurate partitioning of the duplicated genome to daughter cells. Protein phosphorylation has long been implicated in controlling spindle function and chromosome segregation, and genetic studies have identified several protein kinases and phosphatases that are likely to regulate these processes. In particular, mutations in the serine/threonine-specific Drosophila kinase polo, and the structurally related kinase Cdc5p of Saccharomyces cerevisae, result in abnormal mitotic and meiotic divisions. Here, we describe a detailed analysis of the cell cycle-dependent activity and subcellular localization of Plk1, a recently identified human protein kinase with extensive sequence similarity to both Drosophila polo and S. cerevisiae Cdc5p. With the aid of recombinant baculoviruses, we have established a reliable in vitro assay for Plk1 kinase activity. We show that the activity of human Plk1 is cell cycle regulated, Plk1 activity being low during interphase but high during mitosis. We further show, by immunofluorescent confocal laser scanning microscopy, that human Plk1 binds to components of the mitotic spindle at all stages of mitosis, but undergoes a striking redistribution as cells progress from metaphase to anaphase. Specifically, Plk1 associates with spindle poles up to metaphase, but relocalizes to the equatorial plane, where spindle microtubules overlap (the midzone), as cells go through anaphase. These results indicate that the association of Plk1 with the spindle is highly dynamic and that Plk1 may function at multiple stages of mitotic progression. Taken together, our data strengthen the notion that human Plk1 may represent a functional homolog of polo and Cdc5p, and they suggest that this kinase plays an important role in the dynamic function of the mitotic spindle during chromosome segregation.     

Systematic interpretation of genetic interactions using protein networks

Genetic interaction analysis,in which two mutations have a combined effect not exhibited by either mutation alone, is a powerful and widespread tool for establishing functional linkages between genes. In the yeast Saccharomyces cerevisiae, ongoing screens have generated >4,800 such genetic interaction data. We demonstrate that by combining these data with information on protein-protein, prote in-DNA or metabolic networks, it is possible to uncover physical mechanisms behind many of the observed genetic effects. Using a probabilistic model, we found that 1,922 genetic interactions are significantly associated with either between- or within-pathway explanations encoded in the physical networks, covering approximately 40% of known genetic interactions. These models predict new functions for 343 proteins and suggest that between-pathway explanations are better than within-pathway explanations at interpreting genetic interactions identified in systematic screens. This study provides a road map for how genetic and physical interactions can be integrated to reveal pathway organization and function.     

Ion channels and transporters in cancer. 1. Ion channels and cell proliferation in cancer

Progress through the cell mitotic cycle requires precise timing of the intrinsic molecular steps and tight coordination with the environmental signals that maintain a cell into the proper physiological context. Because of their great functional flexibility, ion channels coordinate the upstream and downstream signals that converge on the cell cycle machinery. Both voltage- and ligand-gated channels have been implicated in the control of different cell cycle checkpoints in normal as well as neoplastic cells. Ion channels mediate the calcium signals that punctuate the mitotic process, the cell volume oscillations typical of cycling cells, and the exocytosis of autocrine or angiogenetic factors. Other functions of ion channels in proliferation are still matter of debate. These may or may not depend on ion transport, as the channel proteins can form macromolecular complexes with growth factor and cell adhesion receptors. Direct conformational coupling with the cytoplasmic regulatory proteins is also possible. Derangement or relaxed control of the above processes can promote neoplasia. Specific types of ion channels have turned out to participate in the different stages of the tumor progression, in which cell heterogeneity is increased by the selection of malignant cell clones expressing the ion channel types that better support unrestrained growth. However, a comprehensive mechanistic picture of the functional relations between ion channels and cell proliferation is yet not available, partly because of the considerable experimental challenges offered by studying these processes in living mammalian cells. No doubt, such studies will constitute one of the most fruitful research fields for the next generation of cell physiologists.