Origine et évolution du noyau cellulaire / Origin and Evolution of the eucaryotic nucleus
Colloque organisé par Patrick Forterre du 8 au 13 juillet 2004
Juliette Azimzadeh, Eric Bapteste, Philip Bell, Michel Bornens, Céline Brochier, Valérie Doye, Patrick Forterre, John Fuerst, Eric Gilson, Simonetta Gribaldo, Gaspar Jekely, Andrew H. Knoll, Eugene V. Koonin, Jacomine Krijnse-Locker, Purificacion Lopez-Garcia, David Moreira, Elisabeth Pennisi, David Penny, Hervé Philippe, David Prangishvili, Peter Shaw, Luis Villarreal
Le colloque qui s’est tenu à la fondation des Treilles sur l’origine et l’évolution du noyau cellulaire, du 9 au 13 juillet 2004, a réuni 22 participants, qui ont tous participé à la totalité du colloque, dont Elisabeth Pennisi, journaliste de la revue Science. Les participants comprenaient des scientifiques de haut niveau appartenant à plusieurs nationalités, mais aussi plusieurs chercheurs Européens de niveau post-doctorants, dont plusieurs femmes. Environ un tiers des participants ne s’étaient jamais rencontré, quatre seulement avaient précédemment été invité aux Treilles.
L’objectif de départ a été atteint (regrouper des évolutionnistes et des spécialistes de la biologie moléculaire du noyau). De l’avis unanime, les participants ont été enthousiasmés par cette rencontre. Il est clairement apparu que le sujet venait à maturité, grâce aux données récentes de la génomique.
De nombreux résultats non publiés ont été présentés, en particulier, plusieurs analyses phylogénétiques de larges ensembles de protéines nucléaires (protéines du pore, du nucléole, etc.). Les participants sont tombés d’accord sur le fait que le dernier ancêtre commun des eucaryotes possédait déjà un noyau très évolué. Pour expliquer son origine, des scénarios élaborés ont été proposés par les partisans de l’hypothèse autogène (différenciation du noyau à partir de structures membranaires intracellulaires) et par les partisans de l’hypothèse endosymbiotique (évolution du noyau à partir d’un symbiont de type Archaea). Une nouvelle hypothèse, l’origine virale du noyau, a été discutée pour la première fois en détail par plusieurs intervenants. Le moment fort du colloque a été la conférence de John Fuerst (Australie) sur le noyau présent chez les Planctomycètes (bactéries !). Des photos en microscopie électronique montrent la présence de pores nucléaires sur la membrane de ce noyau bactérien, et des homologues de protéines nucléaires eucaryotes ont été mises en évidence dans le génome de deux Planctomycétes récemment séquencés. Ce résultat refute définitivement sur le plan évolutif la dichotomie eucaryote/procaryote, déjà mise à mal par les travaux de Carl Woese dans les années 1970-1980.
De nombreuses collaborations entre évolutionnistes et biologistes moléculaires ont été initiées aux Trelles et devraient se concrétiser dans les années à venir. Un article sur le colloque a été publié dans la revue Science (Pennisi E., The birth of the Nucleus, Science, 305, 766-768, 2004).
The meeting held in Les Treilles from July 9 to July 13 2004 on the origin and evolution of the cellular nucleus was the third of a series devoted to molecular and cellular evolution, with an emphasis on early life history. The first one, held in 1996 and entitled « the last common ancestor and beyond » tried, at the dawn of the genomic era (the first genome was sequenced in 1995) to picture the last common ancestor to all cellular living beings. No consensus about this entity was reached, except for the designation of a new name, LUCA (Last Universal Common Ancestor) that has now become popular and widely used, both in the press and the scientific community. The second meeting, held in 2000, was devoted to viruses, their origin, evolution and biodiversity. It played an important role in the great comeback that viruses are now having in evolutionary studies. Once excluded from the universal tree of life, viruses are now growingly considered as bona fide critical players in the great game of evolution (as it will become apparent in this third meeting compte rendu). The meeting of this year was devoted to another of the great mysteries of the history of life: the origin and evolution of the cellular nucleus. The main difference with the 1996 meeting on LUCA is that we have now about 200 completely sequenced genomes from the three domains of life, and a revival of microbial ecology, which has highlighted new surprising microbes and microbial communities. It is thus a really exciting time to tackle with a new vision several questions that were previously considered seriously only by a few theoreticians, and with very little hard data to deal with.
The question of the origin of the cell nucleus is intimately linked to the quest of the origin of eucaryotic cells (our lineage) hence, with the quest of our own origins. Before starting to report about the meeting itself, let me thus revisit briefly the traditional notion of division of the living world in Eucaryotes and Procaryotes. The basic distinction is simple: procaryotic cells (meaning cells before the nucleus) have no nucleus, whilst eucaryotic cells (meaning cells with a true nucleus) have one. This makes a big difference in a few fundamental mechanisms responsible for the way genetic information is processed in either type of cells. In Procaryotes, organisms composed of procaryotic cells, the genetic material (DNA) is directly in contact with the protein-synthesizing machinery, whereas in Eucaryotes, organisms composed of eucaryotic cells, the DNA is separated from the cytoplasm by a double membrane (the nuclear envelope). Accordingly, whilst the transcription of DNA into messenger RNA is coupled to protein synthesis in Procaryotes, these two processes are uncoupled in Eucaryotes. The eucaryotic mRNA must therefore be transported from the nucleus to the cytoplasm prior to its translation on the ribosomes. This transfer occurs through complex cylindrical protein structures embedded into the nuclear double-membrane: the nuclear pores. To further complicate the picture, most eucaryotic mRNA are not directly transcribed as such, but have to maturate from primary transcripts, sometimes containing huge amounts of non coding sequences. This maturation involves, among other steps, the removal of most of these non-coding sequences (introns) by the spliceosome (a nucleoprotein complex bigger than the ribosome) and various chemical modifications. Finally, beside the nucleus, eucaryotic cells have one or two types of organelles (mitochondria and chloroplasts), an elaborated system of internal membranes (endoplasmic reticulum, Golgi apparatus), and a complex cytoskeleton. Procaryotes thus appear much simpler than Eucaryotes. Furthermore, the two major eucaryotic organelles, mitochondria and chloroplasts, originated from ancient bacteria (Procaryotes). As a consequence, it is usually considered that Procaryotes preceded Eucaryotes during evolution (in agreement with their name) and that they correspond to the most ancestral forms of life still present on our planet.
The Eucaryote/Procaryote dichotomy was originally proposed by Chatton in 1937, and later on popularized as a general classification scheme by Stanier and van Niel in 1962. It was embraced by microbiologists and molecular biologists alike, who loved the idea of classifying all living things based on an apparently fundamental divide, deeply rooted at the cellular and molecular levels. The evolutionary scenario behind this dichotomy, from simple Procaryotes to complex Eucaryotes, seemed reasonable to most scientists, with the exception of only a few skeptics (see below). However, the eucaryote/procaryote dichotomy eventually turned out to be highly misleading in evolutionary terms. As shown by Carl Woese in the late seventies, the living world is in fact divided into three cellular lineages (or domains), not two. This new division is fundamentally based on the existence of three different types of mechanisms for DNA replication, transcription and translation in the living world. Two of the three domains (the Archaea and the Bacteria) are composed of organisms that are exclusively Procaryotes (but see the end of this report for a surprise), whilst all members of the third domain (Eucarya) are Eucaryotes, as implied by its name!
The three-domains concept, now widely accepted by the biological community, leaves open one critical question: how did the Eucaryotic cell type of organization come into being? Over the last years, several authors have put forward a number of hypotheses, at times totally contradictory, in order to explain the origin of the Eucaryotes and of their main distinguishing feature: the nucleus. These hypotheses have to take into account the observation that Archaea, although Procaryotes in term of cellular organization, are more similar to Eucaryotes than to Bacteria when looking at their informational mechanisms: DNA replication, transcription and translation. For many authors, the three domains originated from a primitive entity (a progenote sensu Woese) and the features common to Archaea and Eucaryotes testify for a late separation of these two domains (as compared to their common separation from Bacteria). In such an hypothesis, the nucleus originated autogenously in an ancestral pre-eucaryotic lineage (the urcaryote, sensu Woese) that was sister group to Archaea. Alternatively, a popular hypothesis is that Eucaryotes originated from an ancient association (fusion, endosymbiosis, or syntrophy) between a bacterium and an archaeon. In these scenarios, the nucleus evolved from the archaeal component of this association, much like mitochondria and chloroplasts evolved from ancient endosymbiotic bacteria. In opposition to these ‘Procaryotes-first’ theories, a few authors have suggested that the nucleus appeared instead very early in evolution, and was lost in Bacteria and Archaea (in that case, procaryotes should be renamed postcaryotes!). This hypothesis implies that features common to Archaea and Eucaryotes are primitive instead of derived. Proponents of this scenario think that LUCA was a quite complex entity, whose molecular mechanisms were reminiscent of eucaryotic ones, and that the pathway from LUCA to Procaryotes involved much streamlining and reductive evolution. In order to explain why the bacterial versions of informational proteins are so different from the archaeal/eucaryotic ones, they propose that the bacterial lineage was particularly fast-evolving. Finally, several authors have recently proposed a radically new hypothesis to explain the origin of the nucleus. They suggest that the ancestor of the nucleus was a DNA virus, resembling present-day large DNA viruses that replicate in the cytoplasm of eucaryotic cells. This idea seems a priori counter-intuitive, as viruses are usually considered as byproducts of evolution. However, recent data (discussed below) have changed this way of thinking, making way for viruses in the universal tree of life as major actors in early life evolution.
It is striking that researchers who have put forward these various hypotheses on the origin of the nucleus are often theoreticians or bioinformaticians who have never directly worked on the nucleus at the bench. Conversely, the many researchers (molecular biologists, biochemists, geneticists, cytologists) who carry on day-by-day experimental studies on the nucleus, have usually a very descriptive point of view, and are too often scarcely interested in the issue of nucleus and Eucaryote origins. Most of the time, these researchers simply take advantage of the latest and apparently most appealing evolutionary hypothesis to interpret their results, instead of looking at their experimental findings for clues favoring a particular evolutionary scenario (or eventually proposing new ones). The main goal of the meeting held at the Fondation des Treilles was precisely to get together these two scientific communities: on one side the evolutionists who have recently proposed new and original hypotheses on the origin of the nucleus, and on the other side a selection of scientists actively involved in the most recent advances into nuclear function and structure in different eucaryotic species. This has been obviously a success, even if in terms of number, the balance was on the evolutionist side. The two communities have perfectly interacted, starting several new active collaborations. The timing of the meeting was also good, given that the debate on the origin of the nucleus was recently boosted by new hypotheses made by several of the participants, as well as by progresses in various related scientific domains (for instance in the reconstruction of ancient phylogenies). More importantly, the sequencing of several eucaryotic genomes over the last two years, coupled to preliminary proteomic analyses, allow us for the first time to tackle the issue of the origin of the nucleus from a comparative genomics perspective.
In the following account of the meeting, I will focus on the evolutionary hypotheses proposed by the participants and on experimental data directly relevant to the main topic of the meeting, bypassing for reasons of space many exciting descriptions of nuclear molecular biology that have been extensively reviewed in recent literature. I will purposely give a personal and subjective view of the meeting (I cannot be totally neutral on this point), but trying to keep a balance between the various proposed scenarios and the different aspects discussed during this meeting.
As a start, Peter Shaw gave a broad overview about the structure and physiology of the nucleus, and summarized all aspects of nuclear biology. More detailed state of the art presentations were later on focused on the nucleolus (Peter Shaw), chromosome structure, with an emphasis on telomers (Eric Gilson), and the nuclear pores (Valerie Doye). Michel Bornens and Juliette Azimzadeh gave an overview of the centrosome, a specific eucaryotic structure that, although located outside of the nucleus, tightly coordinates chromosome division and the cell cycle. These various talks, intermixed with those given by evolutionists, all portrayed the nucleus as a highly complex and dynamic structure (a short video even reminded us that the nucleus actively moves inside the cell). Many talks indeed emphasized the integration of the various components of the eucaryotic cells, in particular the tight coupling between various nuclear structures and the cytoskeleton. An important point for the issue of the origin of the nucleus is that its « double-membrane » is actually a single membrane folded onto itself, and derived from the membrane of the endoplasmic reticulum. Another major feature of the nucleus is the nuclear pore complex that controls the traffic of all macromolecules between the nucleoplasm of the nucleus and the cytoplasm of the cell. Nuclear pores are astonishing structures, with a size equal to that of 30 ribosomes! The problem of the origin of nuclear pores is intriguing, since, as pointed by several participants, the existence of nuclear pore complexes should have predated the enclosure of the chromosome by the nuclear membrane. The mechanism of centrosome duplication is also fascinating and mysterious. The centrosome per se is not present in all eucaryotes, but it is one of the many manifestations of the basal body, a universal eucaryotic structure related to the cytoskeleton. Michel Bornens emphasized the selective value of such a structure that couples predation, cell division and locomotion.
In their presentations, the speakers systematically compared the different nuclear components in animals, plants and fungi (more precisely in the most common model systems for these three kingdoms: Homo sapiens (or Mus musculus or Drosophila), the plant Arabidopsis thaliana, and the yeast Saccharomyces cerevisiae. Many nuclear structures are conserved between animals, plants and fungi, but some are quite variable, indicating that the nucleus is also a dynamic evolutionary entity. This raised a critical question: is it possible to infer from such comparison a picture of the nucleus in ancient eucaryotes? The answer, just a few years ago, would have been no, because we had no idea of the structure of the nucleus in the majority of eucaryotic groups. Recent phylogenetic analyses based on the concatenation of conserved proteins have indeed indicated that eucaryotes can be roughly assigned to eight phyla, and the most common model organisms all belong to only two of them (Plantae and Opisthokonts, the latter grouping animals and true fungi, as well as several groups of unicellulars). However, the recent exhaustive identification, by proteomic analysis, of nuclear proteins in model organisms, together with the sequencing of several eucaryotic genomes from phyla ‘without experimental model organisms’, makes now possible a direct approach to the problem: looking in these genomes for homologues of nuclear proteins identified in yeast, human, and plants.
Several participants presented phylogenomic analyses focused on a particular nuclear structure, such as the nucleolus (David Moreira), the nuclear pore complex (Eugene Koonin, Celine Brochier and Eric Bapteste) or the spliceosome (David Penny). These analyses, which combine the systematic search of these proteins in all completely sequenced genomes and the construction of the corresponding phylogenetic trees, are preliminary for several reasons. First, the available sampling of eucaryotic genomes is still largely discontinuous (several phyla have so far no representatives in the list of completely sequenced eucaryotic genomes). Secondly, as clearly shown by Celine Brochier and Eric Bapteste in the case of nuclear pores, the rate of evolution of many nuclear pore proteins differ extremely from one to the other. Some of them evolve so rapidly that it is difficult to recognize yeast and human homologues, although both of them belong to the same Eucaryotic group (the Opisthokonts). Accordingly, some nuclear proteins that appear to be missing in a given protist lineage might be actually there, but simply impossible to detect in the absence of structural information. Finally, a number of putatively nuclear proteins indicated by proteomic analyses are probably false positives, as indicated by the results presented at the meeting by Peter Shaw. His laboratory is involved in the experimental validation of proteomic data, which identified 270 putative nucleolar proteins in the plant Arabidopsis. Candidate proteins are systematically labeled by a fluorescent dye in vivo to infer their intracellular localization. The first outcome of this study indicates that 10% of the 100 « nucleolar » proteins tested are in fact cytoplasmic. In a parallel work of molecular phylogenetics, David Moreira identified 14 proteins of possible bacterial origin amongst the minimal set of 133 « nucleolar proteins » common to human and yeast. Could it be that these bacterial proteins correspond to the « cytoplasmic contaminants » detected by the work of Peter Shaw? This is a clear case for close collaboration between cell biologists and molecular phylogeneticists.
Despite the recent accumulation of data from eucaryotic genome projects, we still don’t have a reliable history of the eucaryotic domain, i.e. we don’t know the order of emergence of the eight phyla now recognized. This was clearly shown by Hervé Philippe, who pointed out the difficulties in dealing with ancient phylogenies (in particular because of the great variability in evolutionary rates for a given protein in different lineages) and argued for the necessity of improving current phylogenetic reconstruction methods. He presented a case study in which he showed how only very sophisticated approaches allowed recovery of the monophyly of all eucaryotes containing chloroplasts surrounded by two membranes (i.e. green plants, red algae and Glaucocystophytes), indicating a single primary endosymbiotic event at the origin of this organelle. This result was only achieved by the selection of the best presently available phylogenetic methods and by the careful removal of rapidly evolving proteins from the dataset. Simonetta Gribaldo and Eric Bapteste pointed out that, given such difficulties in recovering ancient divergences in the eucaryotic tree, complementary phylogenetic signal should be looked for, possibly in shared ancestral gene losses between major lineages.
There was anyway a consensus at the meeting that, from comparative genomic analyses, it was possible to infer the presence of an already elaborated nucleus in the Last Eucaryotic Common Ancestor (LECA). The LECA would have already contained mitochondria, a nucleolus (with a minimal set of 144 proteins), a nuclear pore complex (with all different major structures present today), basal bodies, telomers, a complex spliceosome, and a sex cycle with meiosis. For example, David Penny identified 74 spliceosomal proteins probably present in the LECA. This result clearly indicates that “spliceosomal” introns were already present at that time. Gaspar Jékely also identified in the LECA a complete set of proteins involved in various signal transduction pathways (small GTPases), crucial for the formation of the cytoskeleton, and the control of cell cycle. LECA was thus most likely a complex eucaryote quite similar to its present-day descendents. The presence of a well developed cytoskeleton in this organism supports the popular notion that Eucaryotes evolved from a predator type of cell.
In agreement with the idea of a complex LECA, Eugene Koonin inferred from comparative global genomic analyses that its genome encoded for at least 4000 proteins (more than some present-day eucaryotes, such as the microsporidium Encephalitozoon cuniculi!). Eukaryotic genomes are characterized by the presence of many paralogous proteins (homologous proteins that diverged from each other following gene duplication). Many of the proteins present in LECA already belong to families of paralogous proteins; this allowed Eugene Koonin and coworkers to reconstruct an even more ancestral genome, by retaining only one protein for each paralogous family. They assumed that this organism was the First (specific) Eucaryotic Common Ancestor (FECA). Interestingly, many essential genes, which have duplicated during the evolution from FECA to LECA (i.e. before the diversification of the eukaryotic domain) have never duplicated thereafter, whilst others have only duplicated after the divergence of the different major eucaryotic lineages. This suggests that the mode of eucaryotic evolution shifted dramatically after LECA.
Is it possible to have an idea of the time when the nucleus came into being? Andrew Knoll tried to partly answer this difficult question by reviewing present knowledge of the fossil record of ancient eucaryotic microfossils. The discovery of clear fossils of Red Algae (Bangiophytes) dated at 1.2 Gy indicates that the split between the main eucaryotic phyla derived from LECA occurred earlier than this time. Andrew Knoll suggests that eucaryotes harbouring mitochondria could have been selected after the first documented rise of oxygen at 2.3 – 2.4 Gy. This indicates that LECA (already containing a fully evolved nucleus) could have originated anytime between 2.3 and 1.2 Gy. Dating based on molecular phylogenetic analyses performed by Hervé Philippe favour the earlier date. In any case, the precise timing of appearance of the nucleus seems actually out of reach considering the scarcity of the fossil record and the current technical limitations of molecular analyses.
If it’s not known WHEN the nucleus appeared on the stage, is it possible to know WHY? Once more, the answer is elusive; several suggestions have been made in the past (protection of the genetic material against damaging agents, better quality control of messenger RNA) but without real convincing arguments. It was noticed for instance by one participant that most DNA-damaging agents can in fact freely travel through nuclear pores. Thus we don’t know if there is (or if there was) a clear selective advantage for the presence of a nucleus, or if the appearance of the nucleus was « simply an accident » in life history. In any case, we would like to know HOW it happened! This was the reason for our gathering and the answer could also help to understand the WHEN and WHY.
I will first discuss the endosymbiotic hypotheses that have been at the forefront these recent years. Several authors have proposed for a long time that Eucaryotes originated via the mixing of several procaryotic lineages. In recent years, this hypothesis was revived by the outcome of comparative genomic analyses showing that, in addition to genes of archaeal affinity, Eucaryotic genomes harbor many genes of bacterial origin without a clear affiliation to a-proteobacteria (hence not of obvious mitochondrial origin). Recent versions of endosymbiotic hypotheses (including the “ring of life” paper published in Nature after our meeting) differ from each other regarding the mechanism of the endosymbiosis, as well as the nature of the archaeal symbionts and of the bacterial hosts, but in all cases involve an archaeon as the ancestor of the nucleus and a bacterium as the ancestor of the cytoplasm.
The more elaborated of these hypotheses was proposed in 1998 by Purificación López-García and David Moreira. They specifically suggested that the eucaryotic emerged from a syntrophic symbiosis between a methanogenic archaeon and a hydrogen-producing d-proteobacterium (a myxobacterium). The nucleus would be a reminiscent of the methanogenic archaeon. In the postulated syntrophic relationships, the archaeon would have used the hydrogen excreted by the proteobacterium to produce methane and other reduced carbon compounds useful for the bacterium. Based on this hypothesis, they rightly predicted the existence of methanotroph anaerobic d-proteobacteria, that were indeed discovered two years later. Microbial consortia in which methanogenic archaea and d-proteobacteria live in syntrophy indeed exist today in anoxic environments, with archaeal cells surrounded by bacterial cells.
At our meeting, Purificación López-García and David Moreira developed further their endosymbiotic hypothesis by assuming that the nucleolus could be a relic of the cytoplasm from the archaeal symbiont. They have been looking for genomic data supporting their hypothesis which predicts that eucaryotic proteins of archaeal affinity should be more closely related to methanogens than to other archaea, and that eucaryotic proteins of bacterial affinity should be specifically related either to alpha proteobacteria (if they originated from the mitochondrial endosymbiont) or to delta proteobacteria (if they originated from the bacterial metabolic partner). They found a few examples of such proteins, but this was not enough to convince the opponents of their hypothesis. Hervé Philippe stressed the point that most eucaryotic proteins of archaeal affinity do not branch from within a particular archaeal lineage, as predicted by the endosymbiosis hypothesis, but are outgroups to their archaeal homologues, as predicted by the “urkaryote” hypothesis. He also noticed that, taking into account the problem of phylogenetic reconstruction for ancient divergences, all eucaryotic proteins with bacterial affinity could well be of mitochondrial origin.
Several participants also remarked that endosymbiosis hypotheses do not explain the existence of the previously mentioned 2000 eucaryotic-specific genes that were already present in LECA and have no counterparts in Procaryotes. To answer this criticism, Purificación López-García and David Moreira suggest that the merging event at the origin of the eucaryotic lineage led to a dramatic increase in the evolutionary rates of many proteins, and in the relaxation of evolutionary constraints, leading to an explosion of molecular innovations. Patrick Forterre and Herve Philippe questioned this argument, noticing that in known cases of endosymbiosis (mitochondria and chloroplasts, secondary endosymbiosis of red algae) there was no dramatic alteration in the rate of evolution of proteins encoded by the symbiont. Hervé Philippe also argued that the endosymbiosis of Cyanobacteria did not lead to a drastic modification in the sequences of plant macromolecules. They finally remark that endosymbiosis usually induce a reduction in the genome size of the symbiont and an expansion of the genome size of the host, whereas the endosymbiosis scenarios for the origin of the nucleus assume exactly the opposite (including the complete disappearance of the host genome).
In the framework of endosymbiosis, one has also to explain the origin of present-day nuclear membrane and nuclear pores. After the merging of the bacterium and the archaeon, the proto-nucleus should have been enclosed by two superimposed membranes, the outer one of bacterial origin and the inner one of archaeal origin. Several participants made the point that both mitochondria and chloroplasts are indeed surrounded by such double membranes and that multiple endosymbioses lead to multiple membrane layers. It is thus unclear in the endosymbiotic hypotheses why the two membranes of the proto-nucleus were finally replaced in modern cells by a single bacterial-like nuclear membrane folded onto itself?
Gáspár Jékely, has recently proposed a new version of the more ancient autogenous hypothesis, specifically based on his phylogenomic analysis of the superfamily of small GTPases, the so called G-proteins. These proteins are typical of eucaryotic systems (phagocytosis, movement of the cytoskeleton, formation of the nuclear envelope, formation of the cilium, cell signaling). In the scenario proposed by Gáspár Jékely, the first step in the emergence of the nucleus was the formation of a primitive endoplasmic reticulum by invagination of the cytoplasmic membrane. This was triggered by the recruitment of kinesins by the ancestral G-protein to couple the cytoplasmic membrane to a primitive cytoskeleton. The next step was the appearance of the Ran proteins in the course of duplication and diversification of the G-protein family. Gáspár Jékely argued that the nucleus could not have originated before the “invention” of Ran proteins, because they are critical to the formation of the nucleus in modern cells. In particular, Ran facilitates the recruitment of nucleoporins to the chromatin and the nuclear membrane from the endoplasmic reticulum.
Interestingly, the G-proteins superfamily can be divided into seven subfamilies (including the Ran one), based on sequence comparison. These subfamilies would have diverged from a single G-protein ancestor present in the FECA. Phylogenetic analysis indicates that at least one member of each of them was already present in the LECA and that Ran appeared after the first duplication of the ancestral G-protein gene. If the idea suggested by Gáspár Jékely is correct (no Ran no nucleus), this would mean that the FECA had no nucleus (an important implication) and that the nucleus originated in the evolutionary lineage leading from FECA to LECA !
Gáspár Jékely suggests that the autogenous origin of the nucleus occurred in a bacterium without a cell wall. This would solve the « membrane problem » because, in this case, we have a continuity between the ancestral bacterial membrane and the present « bacterial-like » membranes of the nucleus and endoplasmic reticulum systems. However, as noticed by Purificación López-García, there is now a problem with the informational proteins, since the model does not explain why the ancestral bacterial-like proteins became archaeal like in present-day eucaryotes! To solve this conundrum, Patrick Forterre suggested that the scenario proposed by Gáspár Jékely occurred in a now extinct cellular lineage (Woese’s urkaryote) with bacterial-like membranes and archaeal-like informational proteins.
The origin of nuclear pores remains a mystery both in the endosymbiosis and the autogenous hypotheses. As noticed by Eugene Koonin, there is no archaeal dominance (over Bacteria) for components of the nuclear pore complex, in striking contrast with most other nuclear components. There is also no detectable similarity between nucleoporins and proteins involved in bacterial transport and secretion. In fact, most nucleoporins have no obvious homologues in Archaea or Bacteria. This could be related to the observation that mechanisms to transport RNA across membranes are presently unknown in Procaryotes.
For a long time, the autogenous and the endosymbiotic hypotheses were the only two competing scenarios to explain the origin of the eucaryotic nucleus. However, in 2001, two authors independently proposed an original new hypothesis, i.e. the viral eukaryogenesis hypothesis. They both suggested that eucaryotes originated from a large DNA-virus related to Poxviruses. This provocative hypothesis was welcome by scientists who have tried during the last years to include viruses in the universal tree of life. One of the two proponents of the viral eucaryogenesis theory, Philip Bell, was present at Les Treilles and summarized the arguments supporting this idea. In particular, he made the point that viruses have evolved a number of complex molecular devices to translocate RNA or DNA through cellular membranes. Such devices could have served as starting points for the origin of the nuclear pore complex.
Philip Bell also mentioned in favor of the viral hypothesis several features of the Poxviruses cell cycle and molecular biology that are indeed reminiscent of the eucaryotic nucleus biology, such as replication in the cytoplasm, and similar maturation of their messenger RNA (capping and polyadenylation). Furthermore, Poxviruses and their relatives encode several homologues of eucaryotic nuclear proteins that in phylogenetic trees come out as outgroups to all eucaryotic proteins (suggesting a very ancient relationship).
The Poxvirus cell cycle was described in detail at the meeting by Jacomine Krijnse-Locker, an expert in the molecular biology of this virus. She pointed out that early viral genes are transcribed inside the virus core and that viral mRNAs are transferred from the viral core to the cytoplasm by an unknown mechanism (possibly reminiscent of the extrusion of mRNA from nuclear pores!). Even more strikingly, once uncoated, the viral DNA is replicated in «mininuclei» which are formed by the recruitment of the endoplasmic reticulum and intermediate filaments around viral DNA, a process strikingly similar to the formation of the nuclear membrane. As in the case of the nucleus, DNA replication only occurs once the mininuclei are fully assembled, and the mininuclei are disassembled only after completion of DNA replication (both processes being regulated by protein phosphorylation). Finally, Poxvirus membranes originate also from the endoplasmic reticulum, a property shared by the nucleus and other members of the LCDV family (Large Cytoplasmic DNA Viruses).
Shortly after the meeting, the publication of the sequence of the giant Mimivirus by Claverie and colleagues was welcome by proponent of the viral eukaryogenesis hypothesis as another discovery in line with this hypothesis. The mimivirus is an NCLDV whose genome is more than three time as bif as the genome of some small parasitic bacteria. The size of the Mimivirus viral factory is in fact quite similar to the size of a large eukaryotic nucleus.
The classical view is that Poxviruses, Mimivirus and other LCDV viruses have adapted their molecular biology and cell cycle to those of their eucaryotic hosts. However, as noticed by Luis Villarreal, many other viruses successfully infect eucaryotic cells by using completely different strategies. The proponents of the viral eucaryogenesis theory thus argue that all characteristic features of LCDV indeed originated in these viruses, and were later on transferred to the host. A final exciting touch to the viral eucaryogenesis hypothesis was given by Luis Villarreal when he briefly reported the existence of infectious nuclei in red algae, i.e. nuclei that are able to migrate from one cell to another and to replicate into the new recipient cell!
In its original version, the authors of the viral eucaryogenesis theory (and Philip Bell at the meeting) suggested specifically that the nucleus originated from the persistent infection of a wall-less methanogenic archaeon by an archaeal virus related to present-day Poxviruses. The idea of a Poxvirus infecting an archaeon could appear a priori bizarre. However, David Prangishvili described at the meeting several viruses from hyperthermophilic Archaea with features previously observed only in eucaryotic cells, such as telomere-like repeats (TTGGA) at the end of their chromosomes. One of them also exhibits Poxvirus-like features such as a linear DNA genome with covalently closed termini and replication involving a resolvase to segregate daughter molecules. Interestingly, most archaeal viruses do not kill their host but live in a carrier state, giving persistent infection, as expected for the ancestor of the nucleus in the viral eucaryogenesis hypothesis. One can therefore imagine the existence of a not yet discovered large DNA virus infecting a methanogen, as required by the scenario proposed by Philip Bell.
However, if the viral ancestor of the nucleus infected a methanogen, why was the archaeal membrane of this methanogen replaced later by the bacterial type of membrane present in the modern nucleus? Again, Patrick Forterre noticed that this problem can be solved by postulating that the host of the nucleus ancestor was an urkaryote with a bacterial-like membrane (in terms of lipid composition). The viral eucaryogenesis theory was indeed welcome by Patrick Forterre, who suggested that viruses played a main role in the origin and evolution of DNA genomes. His theory is that the different steps in the transition from RNA to DNA (reduction of the ribose, replacement of uridine by thymidine, retro-transcription of the genetic message from RNA to DNA) were triggered by the invention of new viral RNA/DNA-modifying enzymes used by viruses to protect their genomes from the nucleases of the host. If DNA and DNA replication mechanisms evolved in the viral world (within different DNA viruses lineages), one may imagine that DNA was later on transferred from viruses to cells, replacing their ancestral cellular RNA genomes. Patrick Forterre speculated at the meeting that such transfer might have occurred three times independently in three different RNA cells to give rise to the present-day three domains of life. This would explain why critical parts of the DNA replication machineries are non-homologous between Bacteria and Archaea as well as the existence of critical differences between DNA replication mechanisms in Archaea and Eucarya (such as domain-specific DNA polymerases). In this scenario, the origin of the eucaryotic nucleus could be a large DNA virus infecting an RNA cell with bacterial-like lipids and archaeal ribosomes!
The viral scenario has problems of its own. Eugene Koonin noticed that there is no obvious viral gene in the list of 2000 specific proteins of the putative « urkaryote ». However, Philip Bell and Patrick Forterre showed a few examples of phylogenetic analyses suggesting that DNA replication or RNA processing proteins could have been transferred early on from viruses to cells. Unfortunately, these ancient phylogenies are poorly resolved and often difficult to interpret. The viral branches in many phylogenies are very long, and it is not clear if this is due to an early divergence from cellular sequences or to an accelerated rate of evolution in the viral lineage (or both).
The last day of the meeting turned out to be really exciting. It has been known for little more than ten years that a poorly studied group of planktonic bacteria, the Planctomycetales, has complex systems of intracellular membranes and that some taxa, such as Gemmata obscuriglobus, have their DNA enclosed into something that strikingly resembles to a nuclear membrane. Surprisingly, these ‘nucleated bacteria’ have attracted little attention, their nucleus being considered unrelated to the eucaryotic one. This did not even change when Celine Brochier and Hervé Philippe published two years ago in Nature a paper on bacterial phylogeny, suggesting that Planctomycetales may represent the earliest divergence in the bacterial tree. However, this situation will now probably evolve rapidly after the last findings that were presented at the Treilles meeting. John Fuerst indeed showed us electron microscopic pictures that indicate the presence of pore-like structures in the nuclear membrane of G. obscuriglobus. Finally, John Fuerst claimed that he could identify genes encoding putative homologues of several eucaryotic nuclear proteins in the genome of Gemmata obscuriglobus, one of two recently sequenced members of the Planctomycetales, suggesting that the nuclei of Planctomycetales and Eucaryotes may be homologues. Building on this hypothesis, John Fuerst proposed at the meeting that the ancestor of these nuclei was already present in the LUCA, and proposes a root of the universal tree of life in the eucaryotic branch, in agreement with phylogenetic analyses published by Hervé Philippe a few years ago.
John Fuerst also related to the meeting the discovery by Ute Hentschel’s group at University of Wurzburg of a new phylum of nucleated bacteria, the Poribacteria, which are symbionts of sponges. Recent phylogenic analyses suggest that Planctomycetales and Poribacteria are evolutionary related, and form a monophyletic bacterial supergroup, together with Chlamydiae and Verrumicrobiales. The genomes of Planctomycetales and Chlamydiae contain a higher number of eucaryotic-like genes than other Bacteria, and close relatives of eucaryotic tubulins have been recently identified in Verrumicrobiales. All these data suggest that these Bacteria diverged earlier than other Bacteria and retained ancestral characteristics (such as the nucleus) that became fixed in Eucarya.
The evolutionary relationships between the eucaryotic nucleus and the nucleus of Planctomycetales will be for sure a hot topic in the near future. The similarities noticed by John Fuerst between some Planctomycetes proteins and eucaryotic nucleoporins are weak and certainly disputable (e.g. they were not detected by Eugene Koonin in a recent in silico analysis published after the meeting). The problem will be probably solved only by the purification of Planctomycetes nuclear pores and the structural comparison of their proteins with nucleoporins. If the homology is proven, all present-day theories for the origin of the nucleus will have to be revisited.
As noticed by Purificación López-García, an important step will be to determine whether the nucleus is an ancestral character in the bacterial super-group combining Planctomycetales with Porribacteria, Chlamydiae and Verrumicrobiales. This should be feasible via a combination of comparative genomics and molecular phylogenetics analyses. If the answer is yes, this will be a strong argument in favor of a nucleated LUCA. However, if the answer is no, Planctomycetes and Poribacteria will still be fascinating groups of Bacteria allowing us to follow up the emergence of a nucleus in a well defined group of organisms. In both cases, the outcome will be exciting and highly relevant to our origins.
After so much new information and lively discussions on conflicting theories, all participants enjoyed the relaxing last afternoon with informal talks around the sunny terrace that makes a meeting in Les Treilles such an unforgettable experience. David Penny and Eugene Koonin tried to summarize the meeting “conclusions”, quite a difficult task. Apart for a consensus about the complex nature of the last eucaryotic common ancestor (LECA), there was no common theory in view for the origin of the nucleus. It was not surprising that proponents of a particular theory were not ready to give up and found new arguments to counter their opponents. But things are ongoing, you need time to change your mind or to integrate some radically new facts or proposals in your current way of thinking. In the long term, the collision of alternative views is the only way to be sure to produce really innovative thinking. All experimentalists present at the meeting were delighted by this exchange of ideas. They were often not aware of some of the theories (and facts) exposed and, besides continuing to collect hard data, they could well be happy in the future to produce new theories of their own.
We concluded our meeting by an exciting exercise: devising a research program to study the origin of the nucleus: how to test alternative theories, how to get possible missing pieces that are required to complete the puzzle? All participants put their suggestions on papers and we compared and combined our ideas. Everybody agreed on the fact that we need to push further the exploration of microbial world biodiversity at both the cellular and viral levels. We probably still don’t know organisms that could hold important clues about the origin of eucaryotic cells. There is possibly somewhere an odd organism whose discovery will shake all present theories (John Fuerst for instance claimed that some nucleated “bacteria” that live as symbionts in sponges could well be in fact nucleated archaea!). Exploration of the microbial diversity should go on both at the organismal and at the genomic levels. Complete genome sequences are absolutely required to make sense of the biology of an organism and to place this organism in an historical context. We need in particular more genomes of Myxobacteria (to test syntrophu model), of Planctomycetes and relatives, of Protists (with representatives of all major eucaryotic phyla), of Archaea (including archaeal viruses and representatives of novel archaeal phyla) and finally of more giant viruses (yet to be discovered). We also need to improve our methods for phylogenetic reconstruction in order to get a clear-cut view of the history of each of the three domains, to identify more precisely (if possible) the origin of transferred genes and to get better insights on the evolutionary relationships between cellular and viral proteins. At the bench level, we definitely need to dissect the molecular biology of many more nuclei from various eucaryotes. In particular, we should get as much additional data as possible on the nuclei of many protists representative of the various major eucaryotic phyla now recognized by the evolutionists.
Finally, one should start to think more (and think about experiments) about such big eucaryotic mysteries as the origin of the spliceosome, of the basal bodies and their strange duplication mechanism, or else of the spectacular sex-related meiotic mechanism. Real sex (not to be confused with “simple” chromosome recombination pathways) is specific to Eucaryotes and could well be born with them. There is definitely a close link between sex and the nucleus whose origin is far from clear and could hold the key to the solution of our enigma (so I am already thinking to a future meeting in Les Treilles on the origin of sex!). The last word of the present meeting was finally, as usual, to thank La Fondation des Treilles for providing such a great opportunity to tackle fundamental questions in an awe inspiring setting. This setting is characterized by open spaces, great landscapes with no visual barriers . This explains why it helps so much to break our own limitations and favor profound and intuitive thinking.