Genetic instability from a single S phase after whole-genome duplication.

Diploid and stable karyotypes are associated with health and fitness in animals. By contrast, whole-genome duplications-doublings of the entire complement of chromosomes-are linked to genetic instability and frequently found in human cancers1-3. It has been established that whole-genome duplications fuel chromosome instability through abnormal mitosis4-8; however, the immediate consequences of tetraploidy in the first interphase are not known. This is a key question because single whole-genome duplication events such as cytokinesis failure can promote tumorigenesis9. Here we find that human cells undergo high rates of DNA damage during DNA replication in the first S phase following induction of tetraploidy. Using DNA combing and single-cell sequencing, we show that DNA replication dynamics is perturbed, generating under- and over-replicated regions. Mechanistically, we find that these defects result from a shortage of proteins during the G1/S transition, which impairs the fidelity of DNA replication. This work shows that within a single interphase, unscheduled tetraploid cells can acquire highly abnormal karyotypes. These findings provide an explanation for the genetic instability landscape that favours tumorigenesis after tetraploidization.

Illuminati: a form of gene expression plasticity in Drosophila neural stem cells.

While testing for genome instability in Drosophila as reported by unscheduled upregulation of UAS-GFP in cells that co-express GAL80 and GAL4, we noticed that, as expected, background levels were low in most developing tissues. However, GFP-positive clones were frequent in the larval brain. Most of these clones originated from central brain neural stem cells. Using imaging-based approaches and genome sequencing, we show that these unscheduled clones do not result from chromosome loss or mutations in GAL80. We have named this phenomenon 'Illuminati'. Illuminati is strongly enhanced in brat tumors and is also sensitive to environmental conditions such as food content and temperature. Illuminati is suppressed by Su(var)2-10, but it is not significantly affected by several modifiers of position effect variegation or Gal4::UAS variegation. We conclude that Illuminati identifies a previously unknown type of functional instability that may have important implications in development and disease.

Centrosome amplification can initiate tumorigenesis in flies.

Centrosome amplification is a common feature of many cancer cells, and it has been previously proposed that centrosome amplification can drive genetic instability and so tumorigenesis. To test this hypothesis, we generated Drosophila lines that have extra centrosomes in approximately 60% of their somatic cells. Many cells with extra centrosomes initially form multipolar spindles, but these spindles ultimately become bipolar. This requires a delay in mitosis that is mediated by the spindle assembly checkpoint (SAC). As a result of this delay, there is no dramatic increase in genetic instability in flies with extra centrosomes, and these flies maintain a stable diploid genome over many generations. The asymmetric division of the larval neural stem cells, however, is compromised in the presence of extra centrosomes, and larval brain cells with extra centrosomes can generate metastatic tumors when transplanted into the abdomens of wild-type hosts. Thus, centrosome amplification can initiate tumorigenesis in flies.

CHRONOCRISIS: When Cell Cycle Asynchrony Generates DNA Damage in Polyploid Cells.

Polyploid cells contain multiple copies of all chromosomes. Polyploidization can be developmentally programmed to sustain tissue barrier function or to increase metabolic potential and cell size. Programmed polyploidy is normally associated with terminal differentiation and poor proliferation capacity. Conversely, non-programmed polyploidy can give rise to cells that retain the ability to proliferate. This can fuel rapid genome rearrangements and lead to diseases like cancer. Here, the mechanisms that generate polyploidy are reviewed and the possible challenges upon polyploid cell division are discussed. The discussion is framed around a recent study showing that asynchronous cell cycle progression (an event that is named "chronocrisis") of different nuclei from a polyploid cell can generate DNA damage at mitotic entry. The potential mechanisms explaining how mitosis in non-programmed polyploid cells can generate abnormal karyotypes and genetic instability are highlighted.

Centrosomes: The good and the bad for brain development.

Centrosomes nucleate and organise the microtubule cytoskeleton in animal cells. These membraneless organelles are key structures for tissue organisation, polarity and growth. Centrosome dysfunction, defined as deviation in centrosome numbers and/or structural integrity, has major impact on brain size and functionality, as compared with other tissues of the organism. In this review, we discuss the contribution of centrosomes to brain growth during development. We discuss in particular the impact of centrosome dysfunction in Drosophila and mammalian neural stem cell division and fitness, which ultimately underlie brain growth defects.

The Janus soul of centrosomes: a paradoxical role in disease?

The centrosome is the main microtubule organizing center of animal cells. It contributes to spindle assembly and orientation during mitosis and to ciliogenesis in interphase. Numerical and structural defects in this organelle are known to be associated with developmental disorders such as dwarfism and microcephaly, but only recently, the molecular mechanisms linking centrosome aberrations to altered physiology are being elucidated. Defects in centrosome number or structure have also been described in cancer. These opposite clinical outcomes--arising from reduced proliferation and overproliferation respectively--can be explained in light of the tissue- and developmental-specific requirements for centrosome functions. The pathological outcomes of centrosome deficiencies have become clearer when considering its consequences. Among them, there are genetic instability (mainly aneuploidy, a defect in chromosome number), defects in the symmetry of cell division (important for cell fate specification and tissue architecture) and impaired ciliogenesis. In this review, we discuss the origins and the consequences of centrosome flaws, with particular attention on how they contribute to developmental diseases.

Consequences of Centrosome Dysfunction During Brain Development.

Development requires cell proliferation, differentiation and spatial organization of daughter cells to occur in a highly controlled manner. The mode of cell division, the extent of proliferation and the spatial distribution of mitosis allow the formation of tissues of the right size and with the correct structural organization. All these aspects depend on cell cycle duration, correct chromosome segregation and spindle orientation. The centrosome, which is the main microtubule-organizing centre (MTOC) of animal cells, contributes to all these processes. As one of the most structurally complex organs in our body, the brain is particularly susceptible to centrosome dysfunction. Autosomal recessive primary microcephaly (MCPH), primordial dwarfism disease Seckel syndrome (SCKS) and microcephalic osteodysplastic primordial dwarfism type II (MOPD-II) are often connected to mutations in centrosomal genes. In this chapter, we discuss the consequences of centrosome dysfunction during development and how they can contribute to the etiology of human diseases.

Never tear us apart--the importance of centrosome clustering.

The presence of more than two centrosomes (centrosome amplification) at the onset of mitosis has long been associated with multipolar spindle formation, and with the generation of genetic instability. However, in recent years, several studies have shown that a process termed 'centrosome clustering' actively contributes to bipolar division by promoting the gathering of extra centrosomes in two main poles. In this Commentary, we describe the main proteins that are involved in centriole duplication and discuss how centrosome amplification can be generated both in vitro and in vivo. We then summarize what is currently known about the processes that contribute to bipolar spindle formation when extra centrosomes are present, and which forces contribute to this process. Finally, we discuss how extra centrosomes might contribute to tumorigenesis, giving emphasis to the role of centrosome amplification in promoting genetic instability.

Drosophila Ajuba is not an Aurora-A activator but is required to maintain Aurora-A at the centrosome.

The LIM-domain protein Ajuba localizes at sites of epithelial cell-cell adhesion and has also been implicated in the activation of Aurora-A (Aur-A). Despite the expected importance of Ajuba, Ajuba-deficient mice are viable, which has been attributed to functional redundancy with the related LIM-domain protein LIMD1. To gain insights into the function of Ajuba, we investigated its role in Drosophila, where a single gene (jub) encodes a protein closely related to Ajuba and LIMD1. We identified a key function in neural stem cells, where Jub localizes to the centrosome. In these cells, mutation in jub leads to centrosome separation defects and aberrant mitotic spindles, which is a phenotype similar to that of aur-A mutants. We show that in jub mutants Aur-A activity is not perturbed, but that Aur-A recruitment and maintenance at the centrosome is affected. As a consequence the active kinase is displaced from the centrosome. On the basis of our studies in Drosophila neuroblasts, we propose that a key function of Ajuba, in these cells, is to maintain active Aur-A at the centrosome during mitosis.

Experimental Approaches to Generate and Isolate Human Tetraploid Cells.

Cancer cells are frequently affected by large-scale chromosome copy number changes, such as polyploidy or whole chromosome aneuploidy, and thus understanding the consequences of these changes is important for cancer research. In the past, it has been difficult to study the consequences of large-scale genomic changes, especially in pure isogenic populations. Here, we describe two methods to generate tetraploid cells induced either by cytokinesis failure or mitotic slippage. These treatments result in mixed population of diploids and tetraploids that can be analyzed directly. Alternatively, tetraploid populations can be established by single cell clone selection or by fluorescence activated cell sorting. These methods enable to analyze and compare the consequences of whole-genome doubling between the parental cell line, freshly arising tetraploid cells, and post-tetraploid aneuploid clones.

Short-term molecular consequences of chromosome mis-segregation for genome stability.

Chromosome instability (CIN) is the most common form of genome instability and is a hallmark of cancer. CIN invariably leads to aneuploidy, a state of karyotype imbalance. Here, we show that aneuploidy can also trigger CIN. We found that aneuploid cells experience DNA replication stress in their first S-phase and precipitate in a state of continuous CIN. This generates a repertoire of genetically diverse cells with structural chromosomal abnormalities that can either continue proliferating or stop dividing. Cycling aneuploid cells display lower karyotype complexity compared to the arrested ones and increased expression of DNA repair signatures. Interestingly, the same signatures are upregulated in highly-proliferative cancer cells, which might enable them to proliferate despite the disadvantage conferred by aneuploidy-induced CIN. Altogether, our study reveals the short-term origins of CIN following aneuploidy and indicates the aneuploid state of cancer cells as a point mutation-independent source of genome instability, providing an explanation for aneuploidy occurrence in tumors.

Centrioles in flies: the exception to the rule?

Centrioles and basal bodies are MT based structures that present a highly conserved ninefold symmetry. Centrioles can be found at the core of the centrosome where they participate in PCM recruitment and organization, contributing to cytoplasmic MT nucleation. Basal bodies are normally located closely to the plasma membrane where they are responsible for axoneme assembly to form structures such as cilia or flagella. While it is well accepted that these organelles have important roles in cell and tissue organization, their contribution to certain phases of animal development is still not entirely established. Here we review the role of centrosomes and cilia in Drosophila melanogaster and briefly discuss the implications of these findings to other model organisms.

The centrosome opens the way to mitosis.

During mitosis, the interaction between chromosomes and microtubules requires nuclear envelope disassembly in prophase. Two articles in this issue of Developmental Cell show that centrosomes have a role in promoting nuclear envelope breakdown (Hachet et al., 2007; Portier et al., 2007). Surprisingly, the role of the centrosome in this process is independent of its role as a microtubule nucleation organelle. Instead, the centrosome seems to act as a spatial regulator for the activation of the Aurora A kinase.

Microvilli-derived extracellular vesicles carry Hedgehog morphogenic signals for Drosophila wing imaginal disc development.

Morphogens are secreted molecules that regulate and coordinate major developmental processes, such as cell differentiation and tissue morphogenesis. Depending on the mechanisms of secretion and the nature of their carriers, morphogens act at short and long range. We investigated the paradigmatic long-range activity of Hedgehog (Hh), a well-known morphogen, and its contribution to the growth and patterning of the Drosophila wing imaginal disc. Extracellular vesicles (EVs) contribute to Hh long-range activity; however, the nature, the site, and the mechanisms underlying the biogenesis of these vesicular carriers remain unknown. Here, through the analysis of mutants and a series of Drosophila RNAi-depleted wing imaginal discs using fluorescence and live-imaging electron microscopy, including tomography and 3D reconstruction, we demonstrate that microvilli of the wing imaginal disc epithelium are the site of generation of small EVs that transport Hh across the tissue. Further, we show that the Prominin-like (PromL) protein is critical for microvilli integrity. Together with actin cytoskeleton and membrane phospholipids, PromL maintains microvilli architecture that is essential to promote its secretory function. Importantly, the distribution of Hh to microvilli and its release via these EVs contribute to the proper morphogenesis of the wing imaginal disc. Our results demonstrate that microvilli-derived EVs are carriers for Hh long-range signaling in vivo. By establishing that members of the Prominin protein family are key determinants of microvilli formation and integrity, our findings support the view that microvilli-derived EVs conveying Hh may provide a means for exchanging signaling cues of high significance in tissue development and cancer.

A catalog of numerical centrosome defects in epithelial ovarian cancers.

Centrosome amplification, the presence of more than two centrosomes in a cell is a common feature of most human cancer cell lines. However, little is known about centrosome numbers in human cancers and whether amplification or other numerical aberrations are frequently present. To address this question, we have analyzed a large cohort of primary human epithelial ovarian cancers (EOCs) from 100 patients. We found that rigorous quantitation of centrosome number in tumor samples was extremely challenging due to tumor heterogeneity and extensive tissue disorganization. Interestingly, even if centrosome clusters could be identified, the incidence of centrosome amplification was not comparable to what has been described in cultured cancer cells. Surprisingly, centrosome loss events where a few or many nuclei were not associated with centrosomes were clearly noticed and overall more frequent than centrosome amplification. Our findings highlight the difficulty of characterizing centrosome numbers in human tumors, while revealing a novel paradigm of centrosome number defects in EOCs.

Centrosomes in disease: how the same music can sound so different?

Centrosomes are the major microtubule organizing center of animal cells. Centrosomes contribute to timely bipolar spindle assembly during mitosis and participate in the regulation of other processes such as polarity establishment and cell migration. Centrosome numbers are tightly controlled during the cell cycle to ensure that mitosis is initiated with only two centrosomes. Deviations in centrosome number or structure are known to impact cell or tissue homeostasis and can impact different processes as diverse as proliferation, death or disease. Interestingly, defects in centrosome number seem to culminate with common responses, which depend on p53 activation even in different contexts such as development or cancer. p53 is a tumor suppressor gene with essential roles in the maintenance of genetic stability normally stimulated by various cellular stresses. Here, we review current knowledge and discuss how defects in centrosome structure and number can lead to different human pathologies.

Topical Delivery of Niacinamide to Skin Using Hybrid Nanogels Enhances Photoprotection Effect.

Niacinamide (NIA) has been widely used in halting the features of ageing by acting as an antioxidant and preventing dehydration. NIA's physicochemical properties suggest difficulties in surpassing the barrier imposed by the stratum corneum layer to reach the target in the skin. To improve cutaneous delivery of NIA, a hybrid nanogel was designed using carrageenan and polyvinylpyrrolidone polymers combined with jojoba oil as a permeation enhancer. Three different types of transethosomes were prepared by the thin-film hydration method, made distinct by the presence of either an edge activator or a permeation enhancer, to allow for a controlled delivery of NIA. Formulations were characterized by measurements of size, polydispersity index, zeta potential, encapsulation efficiency, and loading capacity, and by evaluating their chemical interactions and morphology. Skin permeation assays were performed using Franz diffusion cells. The hybrid hydrogels exhibited robust, porous, and highly aligned macrostructures, and when present, jojoba oil changed their morphology. Skin permeation studies with transethosomes-loaded hydrogels showed that nanogels per se exhibit a more controlled and enhanced permeation, in particular when jojoba oil was present in the transethosomes. These promising nanogels protected the human keratinocytes from UV radiation, and thus can be added to sunscreens or after-sun lotions to improve skin protection.

Overexpressing centriole-replication proteins in vivo induces centriole overduplication and de novo formation.

BACKGROUND: Centrosomes have important roles in many aspects of cell organization, and aberrations in their number and function are associated with various diseases, including cancer. Centrosomes consist of a pair of centrioles surrounded by a pericentriolar matrix (PCM), and their replication is tightly regulated. Here, we investigate the effects of overexpressing the three proteins known to be required for centriole replication in Drosophila-DSas-6, DSas-4, and Sak. RESULTS: By directly observing centriole replication in living Drosophila embryos, we show that the overexpression of GFP-DSas-6 can drive extra rounds of centriole replication within a single cell cycle. Extra centriole-like structures also accumulate in brain cells that overexpress either GFP-DSas-6 or GFP-Sak, but not DSas-4-GFP. No extra centrioles accumulate in spermatocytes that overexpress any of these three proteins. Most remarkably, the overexpression of any one of these three proteins results in the rapid de novo formation of many hundreds of centriole-like structures in unfertilized eggs, which normally do not contain centrioles. CONCLUSIONS: Our data suggest that the levels of centriolar DSas-6 determine the number of daughter centrioles formed during centriole replication. Overexpression of either DSas-6 or Sak can induce the formation of extra centrioles in some tissues but not others, suggesting that centriole replication is regulated differently in different tissues. The finding that the overexpression of DSas-4, DSas-6, or Sak can rapidly induce the de novo formation of centriole-like structures in Drosophila eggs suggests that this process results from the stabilization of centriole-precursors that are normally present in the egg.