Simple Metazoan Research Articles

MALT-1 shortens lifespan by inhibiting autophagy in the intestine of C. elegans.

The caspase-like protease MALT1 promotes immune responses and oncogenesis in mammals by activating the transcription factor NF-κB. MALT1 is remarkably conserved from mammals to simple metazoans devoid of NF-κB homologs, like the nematode C. elegans. To discover more ancient, NF-κB -independent MALT1 functions, we analysed the phenotype of C. elegans upon silencing of MALT-1 expression systemically or in a tissue-specific manner. MALT-1 silencing in the intestine caused a significant increase in life span, whereas intestinal overexpression of MALT-1 shortened life expectancy. Interestingly, MALT-1-deficient animals showed higher constitutive levels of autophagy in the intestine, which were particularly evident in aged or starved nematodes. Silencing of the autophagy regulators ATG-13, BEC-1 or LGG-2, but not the TOR homolog LET-363, reversed lifespan extension caused by MALT-1 deficiency. These findings suggest that MALT-1 limits the lifespan of C. elegans by acting as an inhibitor of an early step of autophagy in the intestine.

A toolkit for assembly of targeting clones for C. elegans transgenesis.

Transgenic worms are a key resource for C. elegans researchers dissecting molecular pathways using this simple metazoan model system. Transgenes provide an avenue to visualize developmental events, cellular processes as well as real-time signal events in live animals using genetically encoded sensors. Generation of these tools has become increasingly efficient with the advent of numerous integration methods including transposon, CRISPR and recombinase-mediated integration. A growing limitation in transgene production is the assembly of the targeting constructs used to direct insertion of sequences into the genome. Here we present a toolkit that facilitates rapid assembly of complex reporters using a Golden Gate (GG) cloning-based approach. Co-assembly of one to eight DNA segments into an integration vector can be routinely obtained at high efficiency using a library of entry plasmids. The toolkit consists of 20 SapI GG entry vectors and 100 SapI GG insert plasmids containing a variety of promoters, FPs, tags, linkers, ORFs, 3' UTRs and numerous components for bipartite expression systems that can be mixed to create a huge repertoire of reporter constructs. The assembly process also works well with PCR products and 5' phosphorylated double stranded oligonucleotides, and such DNAs can be used to supply novel genes, promoters, and tags into the pipeline. In addition, the toolkit also provides a series of 12 empty BsaI -based GG assembly vectors that facilitate the construction of additional SapI GG plasmids containing novel inserts. A manual outlining the entire approach is provided as an appendix as well as a Microsoft® Excel based assembly tool which allows the user to choose individual inserts among the libraries of clones at each position in the assembly template and output an annotated sequence. The assembly process can easily be multiplexed and is typically over 90% efficient. The approach is sufficiently efficient to make microinjection rather than clone generation the limiting factor in transgene generation.

Intercellular communication and the organization of simple multicellular animals.

Animal cells are amazing examples of decentralized systems: By interchanging information about their position and internal state, cells coordinate their behavior and organize themselves in time and space. Examples of this behavior are the development of an embryo or of an organoid. In this work we have asked which are the "rules of intercellular relationship" that allow the organization of an abstract cell collective into structures similar to simple metazoans, without being specific about the (molecular, cellular or physical) nature of the processes involved. To do so, we have used a computational modeling approach following a modified version of the "Swarmalator" concept introduced by O'Keeffe, Hong and Strogatz (2017): a collection of interacting particles ("swarmalators"), each of which defined by a position in space and an internal state (a phase). The key feature is that swarmalators are coupled, so that their position and internal state are both affected by the position and state of all other swarmalators. This model can be easily analogized to biological systems, with "cells" being the swarmalators, and their phase the cell's internal state or "cell type". With this model we explore the conditions (represented by the coupling parameters) that would allow the organization of a multicellular "bioswarmer" and its dynamics along a sort of life cycle. Originally developed in 2D, we implement the model in 3D as well. We describe how changing the strength of intercellular communication can alter the structure and differentiation state of the bioswarmer, how internal polarization can arise and trigger collective directed migration, or how partly erasing the cellular memory of cell state is critical to allow bioswarmers to transit through different states. In addition, we show that the size of a multicellular ensemble might control the differentiation of its constituent cells without changing its rules of relationship.

Time-off-pick Assay to Measure Caenorhabditis elegans Motility.

Caenorhabditis elegans is a simple metazoan that is often used as a model organism to study various human ailments with impaired motility phenotypes, including protein conformational diseases. Numerous motility assays that measure neuro-muscular function have been employed using C. elegans . Here, we describe "time-off-pick" (TOP), a novel assay for assessing motility in C. elegans . TOP is conducted by sliding an eyebrow hair under the mid-section of the worm and counting the number of seconds it takes for the worm to crawl completely off. The time it takes for the worm to crawl off the eyebrow hair is proportional to the severity of its motility defect. Other readouts of motility include crawling or swimming phenotypes, and although widely established, have some limitations. For example, worms that are roller mutants are less suitable for crawling or swimming assays. We demonstrated that our novel TOP assay is sensitive to age-dependent changes in motility, thus, providing another more inclusive method to assess motor function in C. elegans . Graphical abstract: Conceptual overview of the "time-off-pick" (TOP) assay. Various C. elegans models exhibit age-dependent defects in motility. The time it takes for a worm to crawl off of an eyebrow pick that is slid under its mid-section is measured in TOP seconds. A greater TOP is indicative of a greater motility defect. Eventually, worms with phenotypes that lead to paralysis will not be able to leave the pick.

Measurements of Innate Immune Function in C. elegans.

The microscopic nematode Caenorhabditis elegans has emerged as a powerful system to characterize evolutionarily ancient mechanisms of pathogen sensing, innate immune activation, and protective host responses. Experimentally, C. elegans can be infected with a wide variety of human pathogens, as well as with natural pathogens of worms that were isolated from wild-caught nematodes. Here, we focus on an experimental model of bacterial pathogenesis that utilizes the human opportunistic bacterial pathogen Pseudomonas aeruginosa and present an algorithm that can be used to study mechanisms of immune function in nematodes. An initial comparison of the susceptibility of a C. elegans mutant to P. aeruginosa infection with its normal lifespan permits an understanding of a mutant's effect on pathogen susceptibility in the context of potential pleotropic consequences on general worm fitness. Assessing the behavior of nematodes in the presence of P. aeruginosa can also help determine if a gene of interest modulates pathogen susceptibility by affecting the host's ability to avoid a pathogen. In addition, quantification of the pathogen load in the C. elegans intestine during infection, characterization of immune effector transcription that are regulated by host defense pathways and an initial assessment of tissue specificity of immune gene function can refine hypotheses about the mechanism of action of a gene of interest. Together, these protocols offer one approach to characterize novel host defense mechanisms in a simple metazoan host.

Placozoa

Schierwater & DeSalle introduce the enigmatic phylum Placozoa.

Comparative Genomics of the Zic Family Genes.

Zic family genes encode five C2H2-type zinc finger domain-containing proteins that have many roles in animal development and maintenance. Recent phylogenetic analyses showed that Zic family genes are distributed in metazoans (multicellular animals), except Porifera (sponges) and Ctenophora (comb jellies). The sequence comparisons revealed that the zinc finger domains were absolutely conserved among the Zic family genes. Zic zinc finger domains are similar to, but distinct from those of the Gli, Glis, and Nkl gene family, and these zinc finger protein families are proposed to have been derived from a common ancestor gene. The Gli-Glis-Nkl-Zic superfamily and some other eukaryotic zinc finger proteins share a tandem CWCH2 (tCWCH2) motif, a hallmark for inter-zinc finger interaction between two adjacent C2H2 zinc fingers. In Zic family proteins, there exist additional evolutionally conserved domains known as ZOC and ZFNC, both of which may have appeared before cnidarian-bilaterian divergence. Comparison of the exon-intron boundaries in the Zic zinc finger domains revealed an intron (A-intron) that was absolutely conserved in bilaterians (metazoans with bilateral symmetry) and a placozoan (a simple nonparasitic metazoan). In vertebrates, there are five to seven Zic paralogs among which Zic1, Zic2, and Zic3 are generated through a tandem gene duplication and carboxy-terminal truncation in a vertebrate common ancestor, sharing a conserved carboxy-terminal sequence. Several hypotheses have been proposed to explain the Zic family phylogeny, including their origin, unique features in the first and second zinc finger motif, evolution of the nuclear localization signal, significance of the animal taxa-selective degeneration, gene multiplication in the vertebrate lineage, and involvement in the evolutionary alteration of the animal body plan.

Multilayered Reprogramming in Response to Persistent DNA Damage in C. elegans

SummaryDNA damage causally contributes to aging and age-related diseases. Mutations in nucleotide excision repair (NER) genes cause highly complex congenital syndromes characterized by growth retardation, cancer susceptibility, and accelerated aging in humans. Orthologous mutations in Caenorhabditis elegans lead to growth delay, genome instability, and accelerated functional decline, thus allowing investigation of the consequences of persistent DNA damage during development and aging in a simple metazoan model. Here, we conducted proteome, lipidome, and phosphoproteome analysis of NER-deficient animals in response to UV treatment to gain comprehensive insights into the full range of physiological adaptations to unrepaired DNA damage. We derive metabolic changes indicative of a tissue maintenance program and implicate an autophagy-mediated proteostatic response. We assign central roles for the insulin-, EGF-, and AMPK-like signaling pathways in orchestrating the adaptive response to DNA damage. Our results provide insights into the DNA damage responses in the organismal context.

A simple answer to complex questions: Caenorhabditis elegans as an experimental model for examining the DNA damage response and disease genes.

The genetic information is constantly challenged by genotoxic attacks. DNA repair mechanisms evolved early in evolution and recognize and remove the various lesions. A complex network of DNA damage responses (DDR) orchestrates a variety of physiological adaptations to the presence of genome instability. Erroneous repair or malfunctioning of the DDR causes cancer development and the accumulation of DNA lesions drives the aging process. For understanding the complex DNA repair and DDR mechanisms it is pivotal to employ simple metazoan as model systems. The nematode Caenorhabditis elegans has become a well-established and popular experimental organism that allows dissecting genome stability mechanisms in dynamic and differentiated tissues and under physiological conditions. We provide an overview of the distinct advantages of the nematode system for studying DDR and provide a range of currently applied methodologies.

Apical and basal epitheliomuscular F-actin dynamics duringHydrabud evagination

ABSTRACTBending of 2D cell sheets is a fundamental morphogenetic mechanism during animal development and reproduction. A critical player driving cell shape during tissue bending is the actin cytoskeleton. Much of our current knowledge about actin dynamics in whole organisms stems from studies of embryonic development in bilaterian model organisms. Here, we have analyzed actin-based processes during asexual bud evagination in the simple metazoan Hydra. We created transgenic Hydra strains stably expressing the actin marker Lifeact-GFP in either ectodermal or endodermal epitheliomuscular cells. We then combined live imaging with conventional phalloidin staining to directly follow actin reorganization. Bending of the Hydra epithelial double layer is initiated by a group of epitheliomuscular cells in the endodermal layer. These cells shorten their apical-basal axis and arrange their basal muscle processes in a circular configuration. We propose that this rearrangement generates the initial forces to bend the endoderm towards the ectoderm. Convergent tissue movement in both epithelial layers towards the centre of evagination then leads to elongation and extension of the bud along its new body axis. Tissue movement into the bud is associated with lateral intercalation of epithelial cells, remodelling of apical septate junctions, and rearrangement of basal muscle processes. The work presented here extends the analysis of morphogenetic mechanisms beyond embryonic tissues of model bilaterians.

DNA damage responses and stress resistance: Concepts from bacterial SOS to metazoan immunity

The critical need for species preservation has driven the evolution of mechanisms that integrate stress signals from both exogenous and endogenous sources. Past research has been largely focused on cell-autonomous stress responses; however, recently their systemic outcomes within an organism and their implications at the ecological and species levels have emerged. Maintenance of species depends on the high fidelity transmission of the genome over infinite generations; thus, many pathways exist to monitor and restore the integrity of the genome and to coordinate DNA repair with other cellular processes, such as cell division and growth. The specifics of these DNA damage responses (DDRs) vary vastly but some general themes are conserved from ancient organisms, such as bacteria and archaea, to humans. Despite decades of research, however, DDRs still have many layers of complexity and some surprises left to be discovered. One of the most interesting current research topics is the link between DNA damage and stress resistance: the outcomes of DDRs can protect the organism from other secondary challenges. At this time, these types of responses are best characterized in bacteria and the simple metazoan model, Caenorhabditis elegans, but it is becoming clear that similar processes also exist in higher organisms.

The Remarkably Diverse Family of T-Box Factors in Caenorhabditis elegans.

The nematode Caenorhabditis elegans is a simple metazoan animal that is widely used as a model to understand the genetic control of development. The completely sequenced C. elegans genome contains 22 T-box genes, and they encode factors that show remarkable diversity in sequence, DNA-binding specificity, and function. Only three of the C. elegans T-box factors can be grouped into the conserved subfamilies found in other organisms, while the remaining factors are significantly diverged and unlike those in most other animals. While some of the C. elegans factors can bind canonical T-box binding elements, others bind and regulate target gene expression through distinct sequences. The nine genetically characterized T-box factors have varied functions in development and morphogenesis of muscle, hypodermal tissues, and neurons, as well as in early blastomere fate specification, cell migration, apoptosis, and sex determination, but the functions of most of the C. elegans T-box factors have not yet been extensively characterized. Like T-box factors in other animals, interaction with a Groucho-family corepressor and posttranslational SUMOylation have been shown to affect C. elegans T-box factor activity, and it is likely that additional mechanisms affecting T-box factor activity will be discovered using the effective genetic approaches in this organism.

The regulation of thermal stress induced apoptosis in corals reveals high similarities in gene expression and function to higher animals.

Recent studies suggest that controlled apoptotic response provides an essential mechanism, enabling corals to respond to global warming and ocean acidification. However, the molecules involved and their functions are still unclear. To better characterize the apoptotic response in basal metazoans, we studied the expression profiles of selected genes that encode for putative pro- and anti-apoptotic mediators in the coral Stylophora pistillata under thermal stress and bleaching conditions. Upon thermal stress, as attested by the elevation of the heat-shock protein gene HSP70’s mRNA levels, the expression of all studied genes, including caspase, Bcl-2, Bax, APAF-1 and BI-1, peaked at 6–24 h of thermal stress (hts) and declined at 72 hts. Adversely, the expression levels of the survivin gene showed a shifted pattern, with elevation at 48–72 hts and a return to basal levels at 168 hts. Overall, we show the quantitative anti-apoptotic traits of the coral Bcl-2 protein, which resemble those of its mammalian counterpart. Altogether, our results highlight the similarities between apoptotic networks operating in simple metazoans and in higher animals and clearly demonstrate the activation of pro-cell survival regulators at early stages of the apoptotic response, contributing to the decline of apoptosis and the acclimation to chronic stress.

Longevity through DNA damage tolerance

Longevity through DNA damage tolerance

Specific Conserved C-terminal Amino Acids of Caenorhabditis elegans HMP-1/α-Catenin Modulate F-actin Binding Independently of Vinculin*

Stable intercellular adhesions formed through the cadherin-catenin complex are important determinants of proper tissue architecture and help maintain tissue integrity during morphogenetic movements in developing embryos. A key regulator of this stability is α-catenin, which connects the cadherin-catenin complex to the actin cytoskeleton. Although the C-terminal F-actin-binding domain of α-catenin has been shown to be crucial for its function, a more detailed in vivo analysis of discrete regions and residues required for actin binding has not been performed. Using Caenorhabditis elegans as a model system, we have characterized mutations in hmp-1/α-catenin that identify HMP-1 residues 687-742 and 826-927, as well as amino acid 802, as critical to the localization of junctional proximal actin during epidermal morphogenesis. We also find that the S823F transition in a hypomorphic allele, hmp-1(fe4), decreases actin binding in vitro. Using hmp-1(fe4) animals in a mutagenesis screen, we were then able to identify 11 intragenic suppressors of hmp-1(fe4) that revert actin binding to wild-type levels. Using homology modeling, we show that these amino acids are positioned at key conserved sites within predicted α-helices in the C terminus. Through the use of transgenic animals, we also demonstrate that HMP-1 residues 315-494, which correspond to a putative mechanotransduction domain that binds vinculin in vertebrate αE-catenin, are not required during epidermal morphogenesis but may aid efficient recruitment of HMP-1 to the junction. Our studies are the first to identify key conserved amino acids in the C terminus of α-catenin that modulate F-actin binding in living embryos of a simple metazoan.

Molecular cross-talk between sponge host and associated microbes

Marine organisms especially those that live sessile, as sponges, are well known to have specific relationships with a great variety of microorganisms including bacteria and fungi. As most simple metazoan phylum, the Porifera, which emerged first during the transition from the non-Metazoa to the Metazoa from the common ancestor, comprise wide arrays of recognition molecules, both for Gram-negative bacteria and for Gram-positive bacteria as well as for fungi. They react specifically with effector molecules to inhibit or kill the invading microorganisms. The elicitation and the subsequent effector reactions of the sponges towards these microbes are outlined. However, besides of the elimination of bacteria and fungi, some of those taxa are kept as symbionts of the sponges, allowing them, for example, to accumulate the essential element manganese or to synthesize carotinoids. The sponges produce low-molecular-weight bioactive compounds, secondary metabolites, to eliminate the microorganisms. In addition, they are armed with cationic antimicrobial peptides allowing them to defend against invasive microorganisms and, in parallel, to kill or repel also metazoan invaders. The broad range of chemically and functionally different compounds qualifies the Porifera as the most important animal phylum to be exploited as a source for the isolation of new potential drugs. First molecular biological strategies have been outlined to obtain those compounds in a sustainable way, by producing them recombinantly.

Introducing Worm

In 1963 Sydney Brenner proposed to study the development of multi-cellular organisms using a genetic approach in a simple animal. He settled on a small, transparent nematode, Caenorhabditis elegans, which develops quickly and is anatomically simple with less than a thousand somatic cells. Almost half a century later there are many more researchers studying C. elegans than there are somatic cells in this animal and the areas of research have expanded to cover many aspects of cell and developmental biology, behavior, physiology and more recently evolutionary and population biology. Pioneering studies have led to Nobel prizes for six C. elegans researchers in the last decade, illustrating the importance of the discoveries made by studying this relatively simple metazoan. C. elegans researchers have always maintained a strong sense of community and this collegiality is also visible in the collection and dissemination of research results. Wormbase, the main database integrating molecular, genetic and phenotypic data on genes and genomes, has a long history of collecting and presenting data contributed by researchers independently of official publications. This information-sharing spirit is also apparent in the “Worm Breeder’s Gazette”, which provides an informal way to report a variety of interesting results without official peer-review process. Despite this collective intelligence, the current size of the worm field makes it difficult to keep up with all the information. More and more interesting findings never make it into official publications, in part because it can be frustrating to see a manuscript through the publication process. This new journal can help to reverse this trend, insuring that information gets out to the community in a timely manner. Worm aims to provide a straightforward and quick publication process. Editors and reviewers are active researchers of the same model organism with an intimate knowledge of its advantages and limitations at the experimental level. Time and again, discoveries from nematode researchers have implications for other researchers working on a seemingly unrelated question. Worm invites submissions from all areas of research using C. elegans and other nematodes. Sometimes results are intriguing, but do not provide immediate “mechanistic insights”. Nevertheless they might be of immediate interest to the research community. Worm offers the opportunity to submit such results as short communications. More and more studies now cross the borders of traditional disciplines, so that they might not be suitable for topic-oriented journals. Worm offers an opportunity to present exciting results irrespective of the topic of research. Worm also publishes techniques or methods papers of relevance to nematode researchers. While C. elegans currently is the most prominent nematode, it is certainly not the only one studied. With more and more genome sequence information available, research on other nematodes is likely to expand even further. We encourage all nematode researchers to consider Worm as a journal for the dissemination of their discoveries. We are optimistic that this journal will provide a place for nematode researchers to submit some of their most interesting findings. I hope you will enjoy the first issue and keep this journal in mind not just as reader, but also as author.

In vivo imaging of basement membrane movement: ECM patterning shapes Hydra polyps.

Growth and morphogenesis during embryonic development, asexual reproduction and regeneration require extensive remodeling of the extracellular matrix (ECM). We used the simple metazoan Hydra to examine the fate of ECM during tissue morphogenesis and asexual budding. In growing Hydra, epithelial cells constantly move towards the extremities of the animal and into outgrowing buds. It is not known, whether these tissue movements involve epithelial migration relative to the underlying matrix or whether cells and ECM are displaced as a composite structure. Furthermore, it is unclear, how the ECM is remodeled to adapt to the shape of developing buds and tentacles. To address these questions, we used a new in vivo labeling technique for Hydra collagen-1 and laminin, and tracked the fate of ECM in all body regions of the animal. Our results reveal that Hydra 'tissue movements' are largely displacements of epithelial cells together with associated ECM. By contrast, during the evagination of buds and tentacles, extensive movement of epithelial cells relative to the matrix is observed, together with local ECM remodeling. These findings provide new insights into the nature of growth and morphogenesis in epithelial tissues.

Abstract C6: Asymmetry in chromatin patterns in all cancer daughter cells

Abstract After the implementation of updates to the 120-year-old tissue biopsy process, newly achieved tissue detail and clarity revealed a distinct asymmetry of daughter cells and chromatin; this discovery has been observed across numerous disciplines and holds the potential to profoundly affect the research and subsequent development of treatments for every form of cancer. By using a reengineered tissue processing methodology to achieve higher levels of histological detail, the pattern of chromatin packaging was then analyzed at 1000–1600x. These phylogenetic and histological observances indicated a predominant binary feature of chromatin in daughter cells. Once identified, these differences were then interrogated with antibodies against modified histone tails. Anti-phophorylated histone H1 clearly demonstrates a distinction between chromosome sets beginning in prophase through to metaphase and telaphase. Conversely, H4 acetylated at K16 exhibit dissimilarity only in the interphase chromatin. Within these interphase cells, the nuclei of the slightly smaller daughter cells reveal more coarsely clumped chromatin both on the nuclear membrane and inside the nucleus. The cytoplasm of daughter cells also displays unique differences in reference to the degree of granulation as well as with protein expression. The same characteristics were examined in protozoans, diatoms, dictyostylium, simple metazoans, insects, and animals. This process is always present in malignancies, where the more undifferentiated tumors display greater levels of dissimilarity. With the advent of time-lapse fluorescence real-time microscopy, it becomes apparent that these variations between daughter cells are actually indicative of pairing. By altering the fundamental understanding of cancer cell biology, approaches to cancer research and treatment can then be appropriately modified. The knowledge that cancer cell division is asymmetric and daughter cells are non-identical in terms of their chromatin and gene expression affects the understanding of cancer cell behavior. In practice, cell cancer lines are poor equivalents for cancer research because cancer is dimorphic and heterogeneous in the tissue slides. Therefore, future advances in cancer treatments may stem from the awareness of both daughter phenotypes. All current concepts on cell biology, cell division, and chromatin are based on framework, understanding, and tools developed in the 19th century. After incorporating vital updates to this 120-year-old methodology, processed tissue is nearly artifact free and features deeper detail because tissue can be cut down to 0.5μm. Ultimately, these improvements allow for the observation of light and dark cells of ultra-structure under the light microscope. The foundation of these findings was established through extensive series of micrograph images and antibody-interrogated experiments, the full impact of which can only be conveyed on the poster. Given recent advances in technology and histological processing, it is now necessary to re-examine the fundamental concepts of chromatin and cellular operating systems because recognizing the existence of a cellular binary operating system has the potential to profoundly impact research in cancer biology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C6.

Characterization of Nme6-like gene/protein from marine sponge Suberites domuncula

Nucleoside diphosphate kinases (NDPKs) are evolutionarily conserved enzymes involved in many biological processes such as metastasis, proliferation, development, differentiation, ciliary functions, vesicle transport and apoptosis in vertebrates. Biochemical mechanisms of these processes are still largely unknown. Sponges (Porifera) are simple metazoans without tissues, closest to the common ancestor of all animals. They changed little during evolution and probably provide the best insight into the metazoan ancestors' genomic features. The purpose of this study was to address structural and functional properties of group II Nme6 gene/protein ortholog from the marine sponge Suberites domuncula, Nme6, in order to elucidate its evolutionary history. Sponge Nme6 gene and promoter were sequenced and analysed with various bioinformatical tools. Nme6 and Nme6Δ31 proteins were produced in E. coli strain BL21 and NDPK activity was measured using a coupled pyruvate kinase-lactate dehydrogenase assay. Subcellular localization in human tumour cells was examined by confocal scanning microscopy. Our results show that the sponge Nme6 compared to human Nme6 does not possess NDPK activity, does not localize in mitochondria at least in human cells although it has a putative mitochondrial signal sequence, lacks two recent introns that comprise miRNAs and have different transcriptional binding sites in the promoter region. Therefore, we conclude that the structure of Nme6 gene has changed during metazoan evolution possibly in correlation with the function of the protein.