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Asymmetric cell division (ACD) is a strategy for achieving cell diversity. Research carried out over the last two decades has shown that in some cell types that divide asymmetrically, mother and daughter centrosomes are noticeably different from one another in structure, behaviour, and fate, and that robust ACD depends upon centrosome function. Here, I review the latest advances in this field with special emphasis on the complex structure-function relationship of centrosomes with regards to ACD and on mechanistic insight derived from cell types that divide symmetrically but is likely to be relevant in ACD. I also include a comment arguing for the need to investigate the centrosome cycle in other cell types that divide asymmetrically.

With the aim of developing a genetic instability (GI) sensor in vivo we used the well-established Gal80/Gal4-UAS system combined with a visual GFP marker in Drosophila. We generated a collection of 25 Drosophila lines carrying GAL80 transgenes in different locations in all major chromosomes (X, Y, II, and III). We found low rates of GFP cells in epithelial tissues such as wing discs. In contrast, in larval brains, GFP positive clusters containing neural stem cells- also called neuroblasts (NBs)- and their offspring, were highly frequent. Using genetic and imaging-based approaches, we show that GFP NBs do not result from aneuploidy or mutations in the GAL80 gene, but rather by stochastic repression of GAL80 expression. We named this novel type of gene expression instability Illuminati. Importantly, Illuminati frequency is influenced by environmental and stress conditions. Further, we found that once established, Illuminati can be propagated over many cell cycles.

SUMMARYNeural stem cells (NSCs) divide asymmetrically to balance their self-renewal and differentiation, an imbalance in which can lead to NSC overgrowth and tumour formation. The functions of Parafibromin, a conserved tumour suppressor, in the nervous system are not established. Here, we demonstrate that Drosophila Parafibromin/Hyrax (Hyx) inhibits NSC overgrowth by governing cell polarity. Hyx is essential for the apicobasal polarization of NSCs, through its role in the asymmetric distribution of polarity proteins. hyx depletion results in the symmetric division of NSCs, leading to the formation of supernumerary NSCs in the larval brain. Importantly, we show that human Parafibromin can fully rescue NSC overgrowth and cell polarity defects in Drosophila hyx mutant brains. We have also discovered a novel role for Hyx in regulating the formation of interphase microtubule-organizing center and mitotic spindles in NSCs. Moreover, Hyx is required for the proper localization of a key centrosomal protein, Polo, and the microtubule-binding proteins Msps and D-TACC in dividing NSCs. Furthermore, Hyx directly regulates the polo expression in vitro. Altogether, our study provides the first evidence that the brain tumour suppressor-like role and polarity establishing functions of Hyx are mediated by its role in regulating microtubule growth and centrosomal assembly. The new paradigm that Parafibromin orchestrates cell polarization by regulating centrosomal assembly may be relevant to future studies on Parafibromin/HRPT2-associated cancers.

We have undertaken a study towards understanding the effect of ectopic expression of testis proteins in the soma in Drosophila. Here, we show that in the larval neuroepithelium, ectopic expression of the germline-specific component of the inner mitochondrial translocation complex tiny tim 2 (ttm2) brings about cell autonomous hyperplasia and extension of G2 phase. In the wing discs, cells expressing ectopic ttm2 upregulate Jun N-terminal kinase (JNK) signaling, present extended G2, become invasive, and elicit non-cell autonomous G2 extension and overgrowth of the wild-type neighboring tissue. Ectopic tomboy20, a germline-specific member of the outer mitochondrial translocation complex is also tumorigenic in wing discs. Our results demonstrate the tumorigenic potential of unscheduled expression of these two testis proteins in the soma. They also show that a unique tumorigenic event may trigger different tumor growth pathways depending on the tissular context.

Thioredoxins (Trxs) are ubiquitous enzymes that regulate the redox state in cells. In Drosophila, there are two germline-specific Trxs, Deadhead (Dhd) and TrxT. Both proteins belong to the L(3)mbt malignant brain tumor signature and to the MMS survival network of genes that mediate the cellular response to DNA damage. Dhd is a maternal protein required for early embryogenesis that promotes protamine-histone exchange in fertilized eggs and midblastula transition. TrxT is testis-specific and associates with the lampbrush loops of the Y chromosome.Here we present the first structures of Dhd and TrxT that unveil new features of these Thioredoxins. Dhd is highly positively charged, unusual in canonical Trxs. This positively charged surface can facilitate its approximation to DNA and to protamine oligomers, to promote chromatin remodeling. On the other hand, TrxT contains a C-terminal extension, mostly unstructured and highly flexible, which wraps the conserved core through a closed conformation. This extension partially covers the catalytic site and modulates the redox activity of the protein.The information provided by these structures can guide future work aimed at understanding how redox inputs modulate the initial steps of embryo development in Drosophila and may help in the design of molecular inhibitors through a structure-based approach.HighlightsWe have determined the first structures of the germline-specific Trxs Dhd and TrxT.Dhd has a highly positively charged surface that facilitates its approximation to DNA and protamine oligomers, to promote chromatin remodeling.TrxT contains a C-terminal extension, highly unusual in canonical Trxs, mostly unstructured and highly flexible.The TrxT C-terminal extension partially covers the catalytic site and modulates the redox activity of the protein.The differences observed in Thioredoxins can help in fine-tuning specific molecules to be active against selected insect species.

Centrobin homologues identified in different species localize on daughter centrioles. In Drosophila melanogaster sensory neurons, Centrobin (referred to as CNB in Drosophila) inhibits basal body function. These data open the question of CNB’s role in spermatocytes, where daughter and mother centrioles become basal bodies. In this study, we report that in these cells, CNB localizes equally to mother and daughter centrioles and is essential for C-tubules to attain the right position and remain attached to B-tubules as well as for centrioles to grow in length. CNB appears to be dispensable for meiosis, but flagellum development is severely compromised in Cnb mutant males. Remarkably, three N-terminal POLO phosphorylation sites that are critical for CNB function in neuroblasts are dispensable for spermatogenesis. Our results underpin the multifunctional nature of CNB that plays different roles in different cell types in Drosophila, and they identify CNB as an essential component for C-tubule assembly and flagellum development in Drosophila spermatogenesis.

Single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) are found at different rates in human cancer. To determine if these genetic lesions appear in Drosophila tumours we have sequenced the genomes of 17 malignant neoplasms caused by mutationsin l(3)mbt, brat, aurA, orlgl. We have found CNVs and SNPs in all the tumours. Tumour-linked CNVs range between 11 and 80 per sample, affecting between 92 and 1546 coding sequences. CNVs are in average less frequent inl(3)mbtthan inbratlines. Nearly half of the CNVs fall within the 10 to 100Kb range, all tumour samples contain CNVs larger that 100 Kb and some have CNVs larger than 1Mb. The rates of tumour-linked SNPs change more than 20-fold depending on the tumour type: late stagebrat,l(3)mbt, andaurAand lgl lines present median values of SNPs/Mb of exome of 0.16, 0.48, and 3.6, respectively. Higher SNP rates are mostly accounted for by C>A transversions, which likely reflect enhanced oxidative stress conditions in the affected tumours. Both CNVs and SNPs turn over rapidly. We found no evidence for selection of a gene signature affected by CNVs or SNPs in the cohort. Altogether, our results show that the rates of CNVs and SNPs, as well as the distribution of CNV sizes in this cohort of Drosophila tumours are well within the range of those reported for human cancer. Genome instability is therefore inherent to Drosophila malignant neoplastic growth at a variable extent that is tumour type dependent.AUTHOR SUMMARYDrosophila models of malignant growth can help to understand the molecular mechanisms of malignancy. These models are known to exhibit some of the hallmarks of cancer like sustained growth, immortality, metabolic reprogramming, and others. However, it is currently unclear if these fly models are affected by genome instability, which is another hallmark of many human malignant tumours. To address this issue we have sequenced and analysed the genomes of a cohort of seventeen fly tumour samples. We have found that genome instability is a common trait of Drosophila malignant tumours, which occurs at an extent that is tumour-type dependent, at rates that are similar to those of different human cancers.

Drosophila l(3)mbt malignant brain tumors present sexual dimorphism., The notable male predominance across many human cancer types remains unexplained. Here, we show that Drosophila l(3)mbt brain tumors are more invasive and develop as malignant neoplasms more often in males than in females. By quantitative proteomics, we have identified a signature of proteins that are differentially expressed between male and female tumor samples. Prominent among them is the conserved chromatin reader PHD finger protein 7 (Phf7). We show that Phf7 depletion reduces sex-dependent differences in gene expression and suppresses the enhanced malignant traits of male tumors. Our results identify potential regulators of sex-linked tumor dimorphism and show that these genes may serve as targets to suppress sex-linked malignant traits.

Drosophila Arl2 governs neuroblast asymmetric cell division through regulation of microtubule growth and localization of Msps to centrosomes., Asymmetric division of neural stem cells is a fundamental strategy to balance their self-renewal and differentiation. It is long thought that microtubules are not essential for cell polarity in asymmetrically dividing Drosophila melanogaster neuroblasts (NBs; neural stem cells). Here, we show that Drosophila ADP ribosylation factor like-2 (Arl2) and Msps, a known microtubule-binding protein, control cell polarity and spindle orientation of NBs. Upon arl2 RNA intereference, Arl2-GDP expression, or arl2 deletions, microtubule abnormalities and asymmetric division defects were observed. Conversely, overactivation of Arl2 leads to microtubule overgrowth and depletion of NBs. Arl2 regulates microtubule growth and asymmetric division through localizing Msps to the centrosomes in NBs. Moreover, Arl2 regulates dynein function and in turn centrosomal localization of D-TACC and Msps. Arl2 physically associates with tubulin cofactors C, D, and E. Arl2 functions together with tubulin-binding cofactor D to control microtubule growth, Msps localization, and NB self-renewal. Therefore, Arl2- and Msps-dependent microtubule growth is a new paradigm regulating asymmetric division of neural stem cells.

This protocol describes a method to allograft Drosophila larval tissue into adult fly hosts that can be used to assay the tumorigenic potential of mutant tissues. The tissue of interest is dissected, loaded into a fine glass needle and implanted into a host. Upon implantation, nontransformed tissues do not overgrow beyond their normal size, but malignant tumors grow without limit, are invasive and kill the host. By using this method, Drosophila malignant tumors can be transplanted repeatedly, for years, and therefore they can be aged beyond the short life span of flies. Because several hosts can be implanted using different pieces from a single tumor, the method also allows the tumor mass to be increased to facilitate further studies that may require large amounts of tissue (i.e., genomics, proteomics and so on). This method also provides an operational definition of hyperplastic, benign and malignant growth. The injection procedure itself requires only ∼1 d. Tumor development can then be monitored until the death of the implanted hosts.

Using transgenic RNAi technology, we have screened over 4000 genes to identify targets to inhibit malignant growth caused by the loss of function oflethal(3)malignant brain tumourinDrosophila in vivo. We have identified 131 targets, which belong to a wide range of gene ontologies. Most of these target genes are not significantly overexpressed in mbt tumours hence showing that, rather counterintuitively, tumour-linked overexpression is not a good predictor of functional requirement. Moreover, we have found that most of the genes upregulated in mbt tumours remain overexpressed in tumour-suppressed double-mutant conditions, hence revealing that most of the tumour transcriptome signature is not necessarily correlated with malignant growth. One of the identified target genes ismeiotic W68(mei-W68), theDrosophilaorthologue of the human cancer/testis geneSporulation-specific protein 11(SPO11), the enzyme that catalyses the formation of meiotic double-strand breaks. We show thatDrosophila mei-W68/SPO11drives oncogenesis by causing DNA damage in a somatic tissue, hence providing the first instance in which aSPO11orthologue is unequivocally shown to have a pro-tumoural role. Altogether, the results from this screen point to the possibility of investigating the function of human cancer relevant genes in a tractable experimental model organism likeDrosophila.

Loss of function of pins (partner of inscuteable) partially disrupts neuroblast (NB) polarity and asymmetric division, results in fewer and smaller NBs and inhibits Drosophila larval brain growth. Food deprivation also inhibits growth. However, we find that the combination of loss of function of pins and dietary restriction results in loss of NB asymmetry, overproliferation of Miranda-expressing cells, brain overgrowth and increased frequency of tumour growth on allograft transplantation. The same effects are observed in well-fed pins larvae that are mutant for pi3k (phosphatidylinositol 3-kinase) or exposed to the TOR inhibitor rapamycin. Thus, pathways that are sensitive to food deprivation and dependent on PI3K and TOR are essential to suppress tumour growth in Drosophila larval brains with compromised pins function. These results highlight an unexpected crosstalk whereby the normally growth-promoting, nutrient-sensing PI3K/TOR pathway suppresses tumour formation in neural stem cells with compromised cell polarity.

SummaryDrosophila neural stem cells, larval brain neuroblasts (NBs), align their mitotic spindles along the apical/basal axis during asymmetric cell division (ACD) to maintain the balance of self-renewal and differentiation. Here, we identified a protein complex composed of the tumor suppressor anastral spindle 2 (Ana2), a dynein light-chain protein Cut up (Ctp), and Mushroom body defect (Mud), which regulates mitotic spindle orientation. We isolated two ana2 alleles that displayed spindle misorientation and NB overgrowth phenotypes in larval brains. The centriolar protein Ana2 anchors Ctp to centrioles during ACD. The centriolar localization of Ctp is important for spindle orientation. Ana2 and Ctp localize Mud to the centrosomes and cell cortex and facilitate/maintain the association of Mud with Pins at the apical cortex. Our findings reveal that the centrosomal proteins Ana2 and Ctp regulate Mud function to orient the mitotic spindle during NB asymmetric division.

Old and newly synthesized centrosomes have different microtubule nucleating abilities and they contribute to cell polarity when they migrate to opposite poles during cell division. The asymmetric localization of epigenetic marks and kinetochore proteins could lead to the differential recognition of sister chromatids and the biased segregation of DNA strands to daughter cells during cell division. We propose that this asymmetric localization is linked to biased chromatid segregation, which might also be related to the acquisition of distinct cell fates after mitosis.

Polycomb Group (PcG) proteins silence critical developmental genes and modulate cell proliferation. Using the Drosophila melanogaster eye as a model system, we show that cells with mutations in the gene locus (ph) that encodes the PcG protein Polyhomeotic (PH) overproliferate and lose both the ability to differentiate and their normal polarity. They invade the neighboring tissues and, when combined with an activated form of the Ras proto-oncogene, they trigger the formation of metastases. PcG proteins bind to multiple genes in the Notch pathway and control their transcription as well as Notch signaling. The massive cell-autonomous overproliferation of ph mutant cell clones can be rescued by ectopic expression of a dominant negative form of Notch or by RNA interference (RNAi)-mediated repression of Notch. Conversely, overexpression of ph induces a small-eye phenotype that is rescued by activation of Notch signaling. These data show that ph is a tumor suppressor locus that controls cellular proliferation by silencing multiple Notch signaling components.

Cell polarity is inherent to animal development and requires microtubules. In essentially all non-terminally differentiated somatic and male germ-line animal cells, microtubule organisation is governed by centrosomes. Animal development without centrosomes would therefore seem inconceivable. The claim of flies without centrosomes may appear to challenge this notion. Does it?

Prefoldin is a molecular chaperone complex that regulates tubulin function in mitosis. Here, we show that Prefoldin depletion results in disruption of neuroblast polarity, leading to neuroblast overgrowth in Drosophila larval brains. Interestingly, co-depletion of Prefoldin and Partner of Inscuteable (Pins) leads to the formation of gigantic brains with severe neuroblast overgrowth, despite that Pins depletion alone results in smaller brains with partially disrupted neuroblast polarity. We show that Prefoldin acts synergistically with Pins to regulate asymmetric division of both neuroblasts and Intermediate Neural Progenitors (INPs). Surprisingly, co-depletion of Prefoldin and Pins also induces dedifferentiation of INPs back into neuroblasts, while depletion either Prefoldin or Pins alone is insufficient to do so. Furthermore, knocking down either α-tubulin or β-tubulin in pins- mutant background results in INP dedifferentiation back into neuroblasts, leading to the formation of ectopic neuroblasts. Overexpression of α-tubulin suppresses neuroblast overgrowth observed in prefoldin pins double mutant brains. Our data elucidate an unexpected function of Prefoldin and Pins in synergistically suppressing dedifferentiation of INPs back into neural stem cells.

The bead transfection method involves binding nucleic acids onto 3-microm-diameter paramagnetic beads, treating the beads with transfection reagent, and using them as scaffolds to direct transfection to individual cells or regions in a population. Typically, PCR products are used because they can be conveniently generated using biotinylated primers and can introduce site-directed mutations, without the need for cloning or plasmid purification. However, the method can be adapted to transfect plasmid DNA or RNA. The magnetic properties of the beads allows magnets to direct the loci of transfection in cell culture; magnetic arrays are built in cell culture chambers to allow multiple parallel transfections on the same microscope coverslip. The PCR reaction and transfection can be carried out in 1 d, and transfection results can be viewed in 24-48 h.

SummarySensory cilia are organelles that convey information to the cell from the extracellular environment. In vertebrates, ciliary dysfunction results in ciliopathies that in humans comprise a wide spectrum of developmental disorders [1–3]. In Drosophila, sensory cilia are found only in the neurons of type I sensory organs, but ciliary dysfunction also has dramatic consequences in this organism because it impairs the mechanosensory properties of bristles and chaetae and leads to uncoordination, a crippling condition that causes lethality shortly after eclosion [4–7]. The cilium is defined by the ciliary membrane, a protrusion of the cell membrane that envelops the core structure known as the axoneme, a microtubule array that extends along the cilium from the basal body. In vertebrates, basal body function requires centriolar distal and subdistal appendages and satellites. Because these structures are acquired through centriole maturation, only mother centrioles can serve as basal bodies. Here, we show that although centriole maturity traits are lacking in Drosophila, basal body fate is reserved to mother centrioles in Drosophila type I neurons. Moreover, we show that depletion of the daughter-centriole-specific protein Centrobin (CNB) enables daughter centrioles to dock on the cell membrane and to template an ectopic axoneme that, although structurally defective, protrudes out of the cell and is enveloped by a ciliary membrane. Conversely, basal body capability is inhibited in mother centrioles modified to carry CNB. These results reveal the crucial role of CNB in regulating basal body function in Drosophila ciliated sensory organs.

Drosophila larval neuroblasts (NBs) are an excellent model for asymmetric division and cell cycle studies in general. For decades, visualizing relevant structures like centrosomes, chromosomes, or the mitotic spindle relied exclusively on immunofluorescence on fix samples. More recently, improvements on sensitivity and acquisition speed of different confocal systems have made it possible to acquire time-resolved images of combined fluorescent reporters from single larval NBs. Here, we provide protocols to visualize centrosomes and other organelles from both primary cultures of isolated single NBs and ex vivo, whole-mounted larval brains.

Miranda, a highly coiled-coil protein, has been shown to be one of the key components necessary for the differential segregation of fate-determining factors during neurogenesis in Drosophila embryos. The multidomain protein Miranda, by segregating the transcription factor Prospero, guarantees the generation of cell diversity during the formation of the embryonic nervous system. While looking for new molecular components of the centrosome in Drosophila embryos, we have isolated a short isoform of Miranda. An antibody was raised directed against the central coiled-coil region of Miranda. and the pattern of expression of the protein was studied in details throughout Drosophila development. Here, we show that Miranda has a broad pattern of expression and is a rather ubiquitous molecule. Immunofluorescence on syncytial Drosophila embryos shows that Miranda has a dynamic redistribution and is associated with centrosomes. Electron microscopy on purified syncytial centrosomes shows that Miranda is located on the pericentriolar material along with gamma-tubulin. Taken all together, our data indicate for the first time that Miranda belongs to a growing class of proteins that concentrate at the centrosome in a cell-cycle and stage-specific manner. The observations that Miranda has a broad range of expression, as well as a dynamic and cell-cycle dependent subcellular localization, suggest that it may have alternative functions outside the embryonic nervous system. These potential new functions are discussed in this paper.

Fruit fly neuroblasts can either self-renew, rest or take on a specialized form, depending on the levels of a protein called Prospero.

A meeting on the regulation of cell division in Drosophila took place in Cortona. Italy, June 10–15, 2000. This is the second of a series that started in 1995 in Loch Ranoch. Scotland, UK. This meeting was organized by Maurizio Gatti (Rome, Italy) as an EMBO workshop and had additional financial support form the University of Rome ‘La Sapienza’ and the ‘Centro di Genetica Evoluzionistica del CNR’. ![][1] During the past 10 years, Drosophila has revealed itself as a powerful system for studying the cell cycle, a field traditionally dominated by yeast and Xenopus . There are a number of reasons for this, including the fact that Drosophila is a well‐established model system amenable to sophisticated genetic analysis and molecular biology techniques. In fact, it is actually the only genetically amenable, higher eukaryote that is being used extensively as a model system for cell‐cycle research. Furthermore, as a higher eukaryote, Drosophila uses much of the same cell cycle machinery that is also used in vertebrates. The similarities go beyond ultrastructural detail, as shown by the recently published analysis of the Drosophila genome sequence, which revealed that half of the fly protein sequences identified by the Drosophila genome project show significant similarity to mammalian proteins and that the fly has orthologues of 61% of human disease genes and 68% of the cancer genes that were surveyed (Rubin et al ., 2000). The same applies to cell division; analysis of the Drosophila genome confirms previous suggestions of strong parallelism between flies and human cell cycle regulators. Last, but not least, Drosophila is much more amenable than other classical cell‐cycle models to analysis with the new cell imaging techniques. ### Live imaging One of the most significant highlights of the meeting was the extensive use of live imaging, which is becoming commonplace in the study of the cell … [1]: /embed/graphic-1.gif

We have developed a rapid procedure termed Motif Trap (MT) to identify protein motifs that are able to target proteins to a distinct subcellular localisation in eukaryotic cells. By expressing random DNA fragments fused to green fluorescent protein (GFP), individual cells with the GFP localisation of interest are readily isolated allowing for the expressed DNA fragments to be cloned by RT-PCR. These can then be used to identify the corresponding full-length cDNAs. Using MT, we have identified patterns of GFP localisation which correspond to every major organelle and compartment. We have shown that MT is useful to identify new sequences that determine subcellular localisation as well as known targeting motifs.

After nearly a century of successful research, the value of the fly Drosophila melanogaster as a model system for genetic analysis is well established. In the past few years, Drosophila has also successfully been used for living‐organism‐based chemical screenings [1], [2]. Because it is still a new tool in this field, the value of flies for drug discovery is still under assessment and one of the recurrent and fair questions that remains open regards whether or not it is likely that compounds identified through screenings in flies are also going to be functional in mammals. To contribute to an answer, we have inverted the question and searched the literature for compounds that were originally known for their activity in human cells and were later shown to have the same molecular mechanism of action in Drosophila . We found that such compounds are not rare. Table EV1 shows a selection of 60 such compounds, including actin and microtubule poisons; inhibitors of DNA topoisomerases, kinases, and phosphatases; alkylating agents; …

Robust quantitative analyses of asymmetric division in certain cells in flies identify the major molecular players and, most interestingly, define a simple equation to explain this complex cellular process. See Letter p.280 Asymmetric cell division in which a cell produces two daughter cells with different cellular fates, is a fundamental process common to stem cells in development and beyond. Many fate determinants are partitioned at the cell cortex during asymmetric division, and in a range of cells a subset of signalling endosomes segregates unequally in the cytoplasm to mediate the distribution of Notch/Delta signalling molecules between the daughter cells. How this asymmetric distribution is achieved was unknown. Marcos Gonzalez-Gaitan and colleagues now show that, during asymmetric division, central spindle asymmetry is generated by the kinesin Klp10A and its antagonist Patronin, which in turn polarizes the direction of endosome motility. The authors were able to direct the endosome to the wrong cell by inverting the central spindle polarity using an antibody-mediated approach. The system described here is one that can target intracellular cargoes in general, and signalling endosomes in particular, to one of the daughter cells during asymmetric division.

The control of self-renewal and differentiation of neural stem and progenitor cells is a crucial issue in stem cell and cancer biology. Drosophila type II neuroblast lineages are prone to developing impaired neuroblast homeostasis if the limited self-renewing potential of intermediate neural progenitors (INPs) is unrestrained. Here, we demonstrate that Drosophila SWI/SNF chromatin remodeling Brahma (Brm) complex functions cooperatively with another chromatin remodeling factor, Histone deacetylase 3 (HDAC3) to suppress the formation of ectopic type II neuroblasts. We show that multiple components of the Brm complex and HDAC3 physically associate with Earmuff (Erm), a type II-specific transcription factor that prevents dedifferentiation of INPs into neuroblasts. Consistently, the predicted Erm-binding motif is present in most of known binding loci of Brm. Furthermore, brm and hdac3 genetically interact with erm to prevent type II neuroblast overgrowth. Thus, the Brm-HDAC3-Erm repressor complex suppresses dedifferentiation of INPs back into type II neuroblasts. DOI: http://dx.doi.org/10.7554/eLife.01906.001, eLife digest Stem cells show great promise for repairing damaged tissue, and maybe even generating new organs, but stem cell therapies will only be successful if researchers can understand and control the behaviour of stem cells in the lab. Neural stem cells or ‘neuroblasts’ from the brains of larval fruit flies have become a popular model for studying these processes, and one type of neuroblast—known as a ‘type II’ neuroblast—is similar to mammalian neural stem cells in many ways. When type II neuroblasts divide, they generate another neuroblast and a second cell called an intermediate neural progenitor (INP) cell. This progenitor cell then matures and undergoes a limited number of divisions to generate more INP cells and cells called ganglion mother cells. The process by which stem cells and INP cells become specific types of cells is known as differentiation. However, under certain circumstances, the INP cells can undergo the opposite process, which is called dedifferentiation, and become ‘ectopic neuroblasts’. This can give rise to tumors, so cells must employ a mechanism to prevent dedifferentiation. Researchers have known that a protein specifically expressed in INP cells called Earmuff is involved in this process, but many of the details have remained hidden. Now, Koe et al. have discovered that a multi-protein complex containing Earmuff and a number of other proteins—Brahma and HDAC3—have important roles in preventing dedifferentiation. All three proteins are involved in different aspects of gene expression: Earmuff is a transcription factor that controls the process by which the genes in DNA are transcribed to make molecules of messenger RNA; Brahma and HDAC3 are both involved in a process called chromatin remodeling. The DNA inside cells is packaged into a compact structure known as chromatin, and chromatin remodeling involves partially unpacking this structure so that transcription factors and other proteins can have access to the DNA. Koe et al. also showed that Earmuff, Brahma and HDAC3 combine to form a complex that prevents dedifferentiation. An immediate priority is to identify those genes whose expression is regulated by this complex in order to prevent dedifferentiation. DOI: http://dx.doi.org/10.7554/eLife.01906.002

abnormal spindle , a gene required for normal spindle structure and function in Drosophila melanogaster , lies immediately adjacent the gene tolloid at 96A/B. It encodes a 220-kD polypeptide with a predicted pI of 10.8. The recessive mutant allele asp 1 directs the synthesis of a COOH terminally truncated or internally deleted peptide of ∼124 kD. Wild-type Asp protein copurifies with microtubules and is not released by salt concentrations known to dissociate most other microtubule-associated proteins. The bacterially expressed NH 2 -terminal 512-amino acid peptide, which has a number of potential phosphorylation sites for p34 cdc2 and MAP kinases, strongly binds to microtubules. The central 579-amino acid segment of the molecule contains one short motif homologous to sequences in a number of actin bundling proteins and a second motif present at the calmodulin binding sites of several proteins. Immunofluorescence studies show that the wild-type Asp protein is localized to the polar regions of the spindle immediately surrounding the centrosome. These findings are discussed in relation to the known spindle abnormalities in asp mutants.

For decades, lower-model organisms such as Drosophila melanogaster have often provided the first glimpse into the mechanism of action of human cancer-related proteins, thus making a substantial contribution to elucidating the molecular basis of the disease. More recently, D. melanogaster strains that are engineered to recapitulate key aspects of specific types of human cancer have been paving the way for the future role of this 'workhorse' of biomedical research, helping to further investigate the process of malignancy, and serving as platforms for therapeutic drug discovery.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., Here we perform a large-scale study of the structural properties and the expression of proteins that constitute the human Centrosome. Centrosomal proteins tend to be larger than generic human proteins (control set), since their genes contain in average more exons (20.3 versus 14.6). They are rich in predicted disordered regions, which cover 57% of their length, compared to 39% in the general human proteome. They also contain several regions that are dually predicted to be disordered and coiled-coil at the same time: 55 proteins (15%) contain disordered and coiled-coil fragments that cover more than 20% of their length. Helices prevail over strands in regions homologous to known structures (47% predicted helical residues against 17% predicted as strands), and even more in the whole centrosomal proteome (52% against 7%), while for control human proteins 34.5% of the residues are predicted as helical and 12.8% are predicted as strands. This difference is mainly due to residues predicted as disordered and helical (30% in centrosomal and 9.4% in control proteins), which may correspond to alpha-helix forming molecular recognition features (α-MoRFs). We performed expression assays for 120 full-length centrosomal proteins and 72 domain constructs that we have predicted to be globular. These full-length proteins are often insoluble: Only 39 out of 120 expressed proteins (32%) and 19 out of 72 domains (26%) were soluble. We built or retrieved structural models for 277 out of 361 human proteins whose centrosomal localization has been experimentally verified. We could not find any suitable structural template with more than 20% sequence identity for 84 centrosomal proteins (23%), for which around 74% of the residues are predicted to be disordered or coiled-coils. The three-dimensional models that we built are available at http://ub.cbm.uam.es/centrosome/models/i​ndex.php, The authors gratefully acknowledge financial support from the Spanish Ministry of Science, grant CSD2006-00023, and from the Madrid Community, grant S2010/BMD-2305.

During interphase in Drosophila neuroblasts, the Centrobin (CNB)-positive daughter centriole retains pericentriolar material (PCM) and organizes an aster that is a key determinant of the orientation of cell division. Here we show that daughter centrioles depleted of CNB cannot fulfil this function whereas mother centrioles that carry ectopic CNB can. CNB co-precipitates with a set of centrosomal proteins that include γ-TUB, ANA2, CNN, SAS-4, ASL, DGRIP71, POLO and SAS-6. Following chemical inhibition of POLO or removal of three POLO phosphorylation sites present in CNB, the interphase microtubule aster is lost. These results demonstrate that centriolar CNB localization is both necessary and sufficient to enable centrioles to retain PCM and organize the interphase aster in Drosophila neuroblasts. They also reveal an interphase function for POLO in this process that seems to have co-opted part of the protein network involved in mitotic centrosome maturation.

Mutations in Drosophila merry-go-round (mgr) have been known for over two decades to lead to circular mitotic figures and loss of meiotic spindle integrity. However, the identity of its gene product has remained undiscovered. We now show that mgr encodes the Prefoldin subunit counterpart of human von Hippel Lindau binding-protein 1. Depletion of Mgr from cultured cells also leads to formation of monopolar and abnormal spindles and centrosome loss. These phenotypes are associated with reductions of tubulin levels in both mgr flies and mgr RNAi-treated cultured cells. Moreover, mgr spindle defects can be phenocopied by depleting β-tubulin, suggesting Mgr function is required for tubulin stability. Instability of β-tubulin in the mgr larval brain is less pronounced than in either mgr testes or in cultured cells. However, expression of transgenic β-tubulin in the larval brain leads to increased tubulin instability, indicating that Prefoldin might only be required when tubulins are synthesized at high levels. Mgr interacts with Drosophila von Hippel Lindau protein (Vhl). Both proteins interact with unpolymerized tubulins, suggesting they cooperate in regulating tubulin functions. Accordingly, codepletion of Vhl with Mgr gives partial rescue of tubulin instability, monopolar spindle formation, and loss of centrosomes, leading us to propose a requirement for Vhl to promote degradation of incorrectly folded tubulin in the absence of functional Prefoldin. Thus, Vhl may play a pivotal role: promoting microtubule stabilization when tubulins are correctly folded by Prefoldin and tubulin destruction when they are not.

Curing Insect Brain Tumors Mammalian tumors often show ectopic expression of genes that normally function only in the germ line. The possible contribution of cancer germline (CG) genes to malignancy is unknown. Janic et al. (p. 1824 ; see the Perspective by Wu and Ruvkun ) found that CG genes are also expressed in Drosophila brain tumors caused by mutants in the gene lethal (3) malignant brain tumor . Moreover, inactivation of some of these genes suppressed fly tumor growth. Because some Drosophila CG genes are orthologs of human CG genes, it is possible that inactivation of germline genes may show human tumor suppressor activity.

This unit describes a protocol for live imaging of Drosophila embryonic neural stem cellsusingtwo-photonmicroscopy.Comparedtotraditionalone-photonconfocalimaging,thistechnique renders higher-resolution optical sections from deeper within the embryo. It isideally suited to following embryonic neuroblasts located underneath the neuroepithelialcell layer for several rounds of cell division. Curr. Protoc. Stem Cell Biol. 13:1H.2.1-1H.2.9. C 2010 by John Wiley & Sons, Inc.Keywords: two-photon microscopy live imaging Drosophila embryo neuroblast INTRODUCTION This unit provides a detailed description of how to prepare and image live Drosophila embryonic neural stem cells using two-photon microscopy. Drosophila embryos consti-tute an excellent system for live microscopy (Cavey and Lecuit, 2008) and have beenused extensively to address the dynamics of different developmental processes. How-ever, relatively little live-imaging data exist on the behavior of embryonic neuroblasts(NBs), the

How a cell decides to self-renew or differentiate is a critical issue in stem cell and cancer biology. Atypical protein kinase C (aPKC) promotes self-renewal of Drosophila larval brain neural stem cells, neuroblasts. However, it is unclear how aPKC cortical polarity and protein levels are regulated. Here, we have identified a zinc-finger protein, Zif, which is required for the expression and asymmetric localization of aPKC. aPKC displays ectopic cortical localization with upregulated protein levels in dividing zif mutant neuroblasts, leading to neuroblast overproliferation. We show that Zif is a transcription factor that directly represses aPKC transcription. We further show that Zif is phosphorylated by aPKC both in vitro and in vivo. Phosphorylation of Zif by aPKC excludes it from the nucleus, leading to Zif inactivation in neuroblasts. Thus, reciprocal repression between Zif and aPKC act as a critical regulatory mechanism for establishing cell polarity and controlling neuroblast self-renewal.

Drosophila neuroblasts and mouse radial glial cells can divide asymmetrically to self-renew while producing differentiating daughter cells that contribute to brain growth. Intense research activity in the past few years has started to unveil some of the processes that govern asymmetric division in these two cell types. Here we discuss the case of centrosome asymmetry and the contribution of spindle orientation and non-spindle-related centrosome functions. Although still fragmentary, the emerging picture suggests that both notable parallelisms and striking differences apply. © 2010 Elsevier Ltd., BL is a postdoctoral fellow of the Research Foundation Flanders (FWO). IG is a holder of a CSIC JAE-predoctoral Fellowship. Work in E.M.’s laboratory is supported by grants BFU2007-60487 and CONSOLIDER CSD2007-00008.Work in C.G.’s laboratory is supported by E.U. and Spanish grants: ONCASYM-037398 FP6, BFU2006-05813, SGR2005 Generalitat de Catalunya, and Consolider CENTROSOME_3D CSD2006-23.

The Antarctic Convergence is the region of the Southern Ocean where the cold waters of the Circumpolar Current mingle with the warmer waters to the north. Below is the Antarctic continent.Nearly two years ago, a colleague of mine made me a once in a lifetime offer: to join a team that would spend seven weeks working at a research station in Antarctica, the kind of offer that one cannot refuse. And refuse I did not, but it was not an easy decision. To begin with, such a trip would make me unreachable to people chasing me for faculty meetings, grant reports, referee reports, annual reports… The thought was almost unbearable, but I felt that I could handle such isolation. Other potential sources of distress were the harsh Antarctic weather and the very short daylight that we would encounter – our trip would take place at the end of the Antarctic fall – but, again, having lived in Dundee (Scotland) for four years, these I also felt I could handle. However, there was something else.As anyone knows, Spain is a peninsula with a long and intense maritime tradition. Yet, I am from Madrid, a city located right in the middle of the central arid plateau, 400 km away from the nearest sea and 600 meters above sea level; as far away from the sea as you can be when you are in Spain. Not surprisingly, seafaring is not in my genes. And it just so happened that our destination could only be reached by sailing across the Drake Passage, renowned as one of the world's roughest stretches of ocean, and definitely not the best choice for a gentle initiation into sailing. Eventually, however, I did not allow this minor consideration to become an impediment, and on Sunday April 27th 2008 I went onboard R/V Laurence M. Gould, a 76 meter long, ice-strengthened research vessel (http://www.nsf.gov/od/opp/support/gould.jsp), for the four-day crossing of the Drake, from Punta Arenas to Antarctica. Interestingly, the mooring point of the Gould was not far from what remains of the old pier once used by Ernest Shackleton. I will only add to this point that the infamous waters lived up to my expectations; the trip South was shaky, and the trip back North was atrocious (http://www.youtube.com/watch?v=4sp72e_kejM).Our destination was Palmer Station (http://pal.lternet.edu/). Run by the US Antarctic Program, Palmer is located in Anvers Island, off the West coast of the Antarctic Peninsula (64.7 S, 64.0 W), and provides facilities for a range of research projects. Originally built by the US Navy, the station comprises two main buildings known as Bio Lab and GWR, and a number of smaller ones. Bio Lab houses the biological laboratories, together with services like the kitchen, dining room, communications centre, and weather room, as well as the bedrooms, still referred to as ‘berthing’ areas — a remnant of Palmer origins, like the concise and self-explanatory name of GWR, short for Garage, Warehouse, and Recreation areas. Nowadays, GWR also has its own berthing area. The smaller buildings include the aquarium, boathouse, dive locker building and others used for a number of geophysical science projects. Palmer is run on a communal basis and, besides everyone's specific duties as support personnel, scientists, surgeon, etc., all house chores like dishwashing, carpet cleaning, waste removal, window cleaning and the rest are shared without exception by everyone in the station, regardless of rank or experience.All aboard: R/V L.M. Gould anchored near Akademik Vernadsky Station (photograph taken from a zodiac approaching the vessel).View Large Image | View Hi-Res Image | Download PowerPoint SlideOur expedition was part of an NSF-funded project led by Professor Bill Detrich of Northeastern University, a marine biologist with ample experience on Antarctica (to whom I am indebted for this unique opportunity). Our goal was to collect material to study molecular determinants of cold adaptation of microtubule assembly and chaperonin-mediated protein folding in Antarctic fishes. The study would concentrate on two species of rockcod, Gobionotothen gibberifrons and Notothenia coriiceps, which amazingly develop, have mitosis, eat, mate and thrive in waters with the unappealing annual mean bottom temperature of −1.0°C. And getting those fishes was part of the fun. We collected them by benthic Otter trawl from the L.M. Gould in a number of expeditions that took us away from Palmer, sailing and trawling for a few days. Actually, the staff from the Gould did the trawling and we merely lent them a hand. These trips were really exciting. In one of them we crossed the Antarctic Circle — and got the corresponding certificate for it! In another of these trips we sailed, escorted by whales and penguins, along the Lemaire Channel and took the opportunity to pay a visit to the Ukrainian Antarctic Station, Akademik Vernadsky. From the fishes that we caught, my colleagues purified large quantities of the chaperonin CCT as well as putative cofactors, with which they are now trying to establish a functional very-low-temperature tubulin-folding in vitro assay. My part, more modest, consisted of purifying centrosomes with which, once back in my laboratory, I would carry out microtubule polymerisation assays at different temperatures using tubulin from the Antarctic fishes.Palmer Station: The entire Palmer community a few days before winter solstice (the author is the third from the left up in the balcony).View Large Image | View Hi-Res Image | Download PowerPoint SlideNaturally, before the trip I had some difficulties envisaging what work on the bench would be like in such a remote location, and I feared that the lack of any simple piece of equipment (rotors, columns, PAGE apparatus) could compromise the success of the entire project. My concern was groundless. Research facilities, both on board R/V Gould and at Palmer, turned out to be superb. I was also very impressed to see that all chemicals, antibodies, enzymes and small pieces of equipment that we had ordered — and the list was not short — were there when we arrived. This is a delicate part of conducting research in a place that is not on the main commercial routes: there is no next-day delivery in Antarctica and forgetting to order anything, no matter how trivial, can jeopardize the whole operation. No wonder then that such a list is thoroughly considered, revised, and supervised many months in advance, a job that was meticulously carried out by Bill's research technician Sandra Parker, whose contribution to the success of the expedition was paramount.And, if the research facilities were every bit as we needed them, the after-hours facilities were simply excellent and my colleague Jorge Cuellar (a specialist on CCT from the CNB in Madrid) and I made extensive use of them all. The gym, the sauna, the library, the video collection, the bar, and a hot tub overlooking the breaking front of the glacier, available round the clock, provided in-house entertainment with which to relax after work. At the weekends, weather permitting, sailing through brash ice in one of the station's zodiacs to visit the colonies of seals and birds in the nearby islands or hiking to the top of the nearby glacier were the most popular choices. Interestingly, at the top of the glacier there is a VLF receiver, maintained by researchers at Stanford University, which collects data for a project studying the effect that the approximately 1,000 lightning strikes that take place around the world per minute might have on global weather.At Palmer we enjoyed the company of another expedition, led by Professor Charles Amsler (University of Alabama at Birmingham, Alabama). Day in and day out his team, students, postdocs and professors alike, plunged into those nearly frozen waters that are home to a significant population of rather unfriendly leopard seals to collect samples for their studies on chemical interactions between organisms. We also overlapped, and shared lab and resources, with Professor John Postlethwait (University of Oregon, Eugene, Oregon) who carried out studies on a species of erythrocyteless icefish, a ‘natural knockout’ of the erythroid lineage that, indeed, breathes without haemoglobin.Needless to say, the entire trip was an astonishing experience that I thoroughly enjoyed. I would like to take this opportunity to thank both the entire staff at Palmer (special mention to the cooks Diane and Eric!) and the L.M. Gould crew who went to incredible lengths to make our life easier and facilitate our work. Incidentally, due to the excellent internet and telephone communication services at Palmer and on board the vessel, my dream of total isolation did not come true and unwanted requests for all sorts of reports kept coming. However, I learnt that saying something like “sorry, I am in Antarctica at the moment” works as an excellent deterrent. Try it!Hot and cold: Palmer provided great facilitioes for after-hours relaxation. The photograph shows the top of the ladder leading to the hot tub: plastic cups are allowed!View Large Image | View Hi-Res Image | Download PowerPoint Slide

A limited number of adult stem cells (SCs) maintain the homoestasis of different tissues through the lifetime of the individual by generating differentiating daughters and renewing themselves. Errors in the SC division rate or in the fine balance between self-renewal and differentiation might result in tissue overgrowth or depletion, two potentially lethal conditions. A few types of SCs have been identified in Drosophila. These include the SCs of the adult intestine and malpighian tubes, (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006; Singh et al., 2007), the prohematocytes that maintain the population of cells involved in the immunoresponse (Lanot et al., 2001; Lemaitre and Hoffmann, 2007), the SC of the follicle epithelia in the ovary (Nystul and Spradling, 2007), germ line SCs (GSCs) of both sexes (Fuller and Spradling, 2007) and neuroblasts (NBs), the fly neural SCs (Yu et al., 2006; Chia et al., 2008; Knoblich, 2008). Drosophila SCs have proved a fruitful model system to unveil some aspects of the molecular logic that sustains SC function. This review focuses on results obtained in the last few years from the study of NBs, particularly from the standpoint of the possible functional connection between asymmetric SC division and cancer.

Summary Genome instability (GI) and centrosomal alterations are common traits in human cancer [1, 2]. It is suspected that centrosome dysfunction may cause tumors by bringing about GI, but direct experimental proof is still lacking [3]. To explore the possible functional link between centrosome function and overgrowth, we have assayed the tumorigenic potential of a series of mutants that affect different centrosomal proteins in Drosophila . We have found that a significant number of such mutant conditions are tumorigenic in larval brain tissue, where self-renewing asymmetric division of neural stem cells is frequent, but not in symmetrically dividing epithelial cells. We have also found that mutations that increase GI without causing centrosome dysfunction are not tumorigenic in our assay. From these observations, we conclude that the tumors caused by centrosome dysfunction cannot be explained solely by the resulting genome instability. We propose that such tumors might be caused by impaired asymmetric division of neural stem cells [4]. These results show that centrosome loss, far from being innocuous, is a potentially dangerous condition in flies.

The dramatic cell shape changes during cytokinesis require the interplay between microtubules and the actomyosin contractile ring, and addition of membrane to the plasma membrane. Numerous membrane-trafficking components localize to the central spindle during cytokinesis, but it is still unclear how this machinery is targeted there and how membrane trafficking is coordinated with cleavage furrow ingression. Here we use an arf6 null mutant to show that the endosomal GTPase ARF6 is required for cytokinesis in Drosophila spermatocytes. ARF6 is enriched on recycling endosomes at the central spindle, but it is required neither for central spindle nor actomyosin contractile ring assembly, nor for targeting of recycling endosomes to the central spindle. However, in arf6 mutants the cleavage furrow regresses because of a failure in rapid membrane addition to the plasma membrane. We propose that ARF6 promotes rapid recycling of endosomal membrane stores during cytokinesis, which is critical for rapid cleavage furrow ingression.

Summary Background Centrosomes, the major organizers of the microtubule network in most animal cells, are composed of centrioles embedded in a web of pericentriolar material (PCM). Recruitment and stabilization of PCM on the centrosome is a centriole-dependent function. Compared to the considerable number of PCM proteins known, the molecular characterization of centrioles is still very limited. Only a few centriolar proteins have been identified so far in Drosophila , most related to centriole duplication. Results We have cloned asterless ( asl ) and found that it encodes a 120 kD highly coiled-coil protein that is a constitutive pancentriolar and basal body component. Loss of asl function impedes the stabilization/maintenance of PCM at the centrosome. In embryos deficient for Asl, development is arrested right after fertilization. Asl shares significant homology with Cep152, a protein described as a component of the human centrosome for which no functional data is yet available. Conclusions The cloning of asl offers new insight into the molecular composition of Drosophila centrioles and a possible model for the role of its human homolog. In addition, the phenotype of asl -deficient flies reveals that a functional centrosome is required for Drosophila embryo development.

Although they do not get cancer naturally, genetically manipulatedDrosophila melanogasterare a useful model for studying tumours. Recent results highlight the importance of asymmetric cell division and proper spindle alignment for preventing stem cells from giving rise to tumours. Recent genetic studies in flies have added further support to an increasing body of evidence that suggests that stem cells might be the cell-of-origin of certain tumours. Malfunction of the mechanisms that control the division of stem cells and the developmental fate of the two resulting daughters could be one of the initial events that steers cells into malignant transformation. These studies suggest a role for controlled spindle orientation in suppressing stem-cell overgrowth. In parallel, the machinery that drives asymmetry in stem cells has been further characterized, identifying new components and uncovering the unique, highly sophisticated behaviour of centrosomes in these cells.

SummaryStem cell asymmetric division requires tight control of spindle orientation. To study this key process, we have recorded Drosophila larval neural stem cells (NBs) engineered to express fluorescent reporters for microtubules, pericentriolar material (PCM), and centrioles. We have found that early in the cell cycle, the two centrosomes become unequal: one organizes an aster that stays near the apical cortex for most of the cell cycle, while the other loses PCM and microtubule-organizing activity, and moves extensively throughout the cell until shortly before mitosis when, located near the basal cortex, it recruits PCM and organizes the second mitotic aster. Upon division, the apical centrosome remains in the stem cell, while the other goes into the differentiating daughter. Apical aster maintenance requires the function of Pins. These results reveal that spindle orientation in Drosophila larval NBs is determined very early in the cell cycle, and is mediated by asymmetric centrosome function.

Two studies, one in this issue of Cell (Betschinger et al., 2006) and the other in Developmental Cell (Lee et al., 2006a) show that the cell-fate determinant Brain Tumor (Brat) suppresses self-renewal in one of the daughter cells that arise from the asymmetric division of a neural stem cell. This work suggests a mechanism by which loss of polarity in stem cells may lead to tumorigenesis.

Experimental approaches in Drosophila melanogaster over the last 20 years have played a fundamental role in elucidating the function, structure and molecular composition of the centrosome. However, quantitative data on the structure and function of the Drosophila centrosome are still lacking. This study uses, for the first time, whole mount electron microscopy in combination with negative staining on isolated centrosomes from the early Drosophila embryos to analyze its dimensions, structure and capacity to nucleate microtubules in vitro. We show that these organelles are on average 0.75 microm in diameter and have abundant pericentriolar material which often appears fibrillar and with bulbous protrusions. Corresponding to the abundant pericentriolar material, extensive microtubule nucleation occurs. Quantification of the number of microtubules nucleated showed that 50-300 active nucleation sites are present. We examined via electron microscopy immunogold labeling the distribution of gamma-tubulin, CNN, Asp and the MPM-2 epitopes that are phosphorylated through Polo and the Cdk1 kinase. The distribution of these proteins is homogeneous, with the MPM-2 epitopes exhibiting the highest density. In contrast, centrosomal subdomains are identified using a centriole marker to relate centrosome size to the centriole number by electron microscopy. In conclusion, we present a clear-cut technique assaying and quantifying the microtubule nucleation capacity and antigen distribution complementing molecular studies on centrosome protein complexes, cell organelle assembly and protein composition.

Loss of cell polarity and cancer are tightly correlated1, but proof for a causative relationship has remained elusive. In stem cells, loss of polarity and impairment of asymmetric cell division could alter cell fates and thereby render daughter cells unable to respond to the mechanisms that control proliferation2. To test this hypothesis, we generated Drosophila melanogaster larval neuroblasts containing mutations in various genes that control asymmetric cell division and then assayed their proliferative potential after transplantation into adult hosts. We found that larval brain tissue carrying neuroblasts with mutations in raps (also called pins), mira, numb or pros grew to more than 100 times their initial size, invading other tissues and killing the hosts in 2 weeks. These tumors became immortal and could be retransplanted into new hosts for years. Six weeks after the first implantation, genome instability and centrosome alterations, two traits of malignant carcinomas3,4, appeared in these tumors. Increasing evidence suggests that some tumors may be of stem cell origin5,6. Our results show that loss of function of any of several genes that control the fate of a stem cell's daughters may result in hyperproliferation, triggering a chain of events that subverts cell homeostasis in a general sense and leads to cancer.

High-throughput gene analysis would benefit from new approaches for delivering DNA or RNA into cells. Here we describe a simple system that allows any molecular biology laboratory to carry out multiple, parallel cell transfections on microscope coverslip arrays. By using magnetically defined positions and PCR product–coated paramagnetic beads, we achieved transfection in a variety of cell lines. Beads may be added to the cells at any time, allowing both spatial and temporal control of transfection. Because the beads may be coated with more than one gene construct, the method can be used to achieve cotransfection within single cells. Furthermore, PCR-generated mutants may be conveniently screened, bypassing cloning and plasmid purification steps. We illustrated the applicability of the method by screening combinatorial peptide libraries, fused to GFP, to identify previously unknown cellular localization motifs. In this way, we identified several localizing peptides, including structured localization signals based around the scaffold of a single C2H2 zinc finger.