Nuclear envelope expansion in budding yeast is independent of cell growth and does not determine nuclear volume.

Most cells exhibit a constant ratio between nuclear and cell volume. The mechanism dictating this constant ratio and the nuclear component(s) that scale with cell size are not known. To address this, we examined the consequences to the size and shape of the budding yeast nucleus when cell expansion is inhibited by down-regulating components of the secretory pathway. We find that under conditions where cell size increase is restrained, the nucleus becomes bilobed, with the bulk of the DNA in one lobe and the nucleolus in the other. The formation of bilobed nuclei is dependent on fatty acid and phospholipid synthesis, suggesting that it is associated with nuclear membrane expansion. Bilobed nuclei appeared predominantly after spindle pole body separation, suggesting that nuclear envelope expansion follows cell-cycle cues rather than cell size. Importantly, cells with bilobed nuclei had the same nuclear:cell volume ratio as cells with round nuclei. Therefore, the bilobed nucleus could be a consequence of continued NE expansion as cells traverse the cell cycle without an accompanying increase in nuclear volume due to the inhibition of cell growth. Our data suggest that nuclear volume is not determined by nuclear envelope availability but by one or more nucleoplasmic factors.

Non-essential MCM-related proteins mediate a response to DNA damage in the archaeon Methanococcus maripaludis.

The single minichromosome maintenance (MCM) protein found in most archaea has been widely studied as a simplified model for the MCM complex that forms the catalytic core of the eukaryotic replicative helicase. Organisms of the order Methanococcales are unusual in possessing multiple MCM homologues. The Methanococcus maripaludis S2 genome encodes four MCM homologues, McmA-McmD. DNA helicase assays reveal that the unwinding activity of the three MCM-like proteins is highly variable despite sequence similarities and suggests additional motifs that influence MCM function are yet to be identified. While the gene encoding McmA could not be deleted, strains harbouring individual deletions of genes encoding each of the other MCMs display phenotypes consistent with these proteins modulating DNA damage responses. M. maripaludis S2 is the first archaeon in which MCM proteins have been shown to influence the DNA damage response.

The yeast polo kinase Cdc5 regulates the shape of the mitotic nucleus.

Abnormal nuclear size and shape are hallmarks of aging and cancer. However, the mechanisms regulating nuclear morphology and nuclear envelope (NE) expansion are poorly understood. In metazoans, the NE disassembles prior to chromosome segregation and reassembles at the end of mitosis. In budding yeast, the NE remains intact. The nucleus elongates as chromosomes segregate and then divides at the end of mitosis to form two daughter nuclei without NE disassembly. The budding yeast nucleus also undergoes remodeling during a mitotic arrest; the NE continues to expand despite the pause in chromosome segregation, forming a nuclear extension, or "flare," that encompasses the nucleolus. The distinct nucleolar localization of the mitotic flare indicates that the NE is compartmentalized and that there is a mechanism by which NE expansion is confined to the region adjacent to the nucleolus. Here we show that mitotic flare formation is dependent on the yeast polo kinase Cdc5. This function of Cdc5 is independent of its known mitotic roles, including rDNA condensation. High-resolution imaging revealed that following Cdc5 inactivation, nuclei expand isometrically rather than forming a flare, indicating that Cdc5 is needed for NE compartmentalization. Even in an uninterrupted cell cycle, a small NE expansion occurs adjacent to the nucleolus prior to anaphase in a Cdc5-dependent manner. Our data provide the first evidence that polo kinase, a key regulator of mitosis, plays a role in regulating nuclear morphology and NE expansion.

Putting molecules in their place.

Each class of microscope is limited to imaging specific aspects of cell structure and/or molecular organization. However, imaging the specimen by complementary microscopes and correlating the data can overcome this limitation. Whilst not a new approach, the field of correlative imaging is currently benefitting from the emergence of new microscope techniques. Here we describe the correlation of cryogenic fluorescence tomography (CFT) with soft X-ray tomography (SXT). This amalgamation of techniques integrates 3D molecular localization data (CFT) with a high-resolution, 3D cell reconstruction of the cell (SXT). Cells are imaged in both modalities in a near-native, cryopreserved state. Here we describe the current state of the art in correlative CFT-SXT, and discuss the future outlook for this method.

Nuclear division: giving daughters their fair share.

How do nuclear components, apart from chromosomes, partition equally to daughter nuclei during mitosis? In Schizosaccharomyces japonicus, the conserved LEM-domain nuclear envelope protein Man1 ensures the formation of identical daughter nuclei by coupling nuclear pore complexes to the segregating chromosomes.

The dynamic nature of the nuclear envelope: lessons from closed mitosis.

In eukaryotes, chromosomes are encased by a dynamic nuclear envelope. In contrast to metazoans, where the nuclear envelope disassembles during mitosis, many fungi including budding yeast undergo "closed mitosis," where the nuclear envelope remains intact throughout the cell cycle. Consequently, during closed mitosis the nuclear envelope must expand to accommodate chromosome segregation to the two daughter cells. A recent study by Witkin et al. in budding yeast showed that if progression through mitosis is delayed, for example due to checkpoint activation, the nuclear envelope continues to expand despite the block to chromosome segregation. Moreover, this expansion occurs at a specific region of the nuclear envelope- adjacent to the nucleolus- forming an extension referred to as a "flare." These observations raise questions regarding the regulation of nuclear envelope expansion both in budding yeast and in higher eukaryotes, the mechanisms confining mitotic nuclear envelope expansion to a particular region and the possible consequences of failing to regulate nuclear envelope expansion during the cell cycle.

The budding yeast nuclear envelope adjacent to the nucleolus serves as a membrane sink during mitotic delay.

The mechanisms that dictate nuclear shape are largely unknown. Here we screened the budding yeast deletion collection for mutants with abnormal nuclear shape. A common phenotype was the appearance of a nuclear extension, particularly in mutants in DNA repair and chromosome segregation genes. Our data suggest that these mutations led to the abnormal nuclear morphology indirectly, by causing a checkpoint-induced cell-cycle delay. Indeed, delaying cells in mitosis by other means also led to the appearance of nuclear extensions, whereas inactivating the DNA damage checkpoint pathway in a DNA repair mutant reduced the fraction of cells with nuclear extensions. Formation of a nuclear extension was specific to a mitotic delay, because cells arrested in S or G2 had round nuclei. Moreover, the nuclear extension always coincided with the nucleolus, while the morphology of the DNA mass remained largely unchanged. Finally, we found that phospholipid synthesis continued unperturbed when cells delayed in mitosis, and inhibiting phospholipid synthesis abolished the formation of nuclear extensions. Our data suggest a mechanism that promotes nuclear envelope expansion during mitosis. When mitotic progression is delayed, cells sequester the added membrane to the nuclear envelope associated with the nucleolus, possibly to avoid disruption of intranuclear organization.

Shaping the nucleus: factors and forces.

Take a look at a textbook illustration of a cell and you will immediately be able to locate the nucleus, which is often drawn as a spherical or ovoid shaped structure. But not all cells have such nuclei. In fact, some disease states are diagnosed by the presence of nuclei that have an abnormal shape or size. What defines nuclear shape and nuclear size, and how does nuclear geometry affect nuclear function? While the answer to the latter question remains largely unknown, significant progress has been made towards understanding the former. In this review, we provide an overview of the factors and forces that affect nuclear shape and size, discuss the relationship between ER structure and nuclear morphology, and speculate on the possible connection between nuclear size and its shape. We also note the many interesting questions that remain to be explored.

Shuttle vector system for Methanococcus maripaludis with improved transformation efficiency.

We have identified an open reading frame and DNA element that are sufficient to maintain shuttle vectors in Methanococcus maripaludis. Strain S0001, containing ORF1 from pURB500 integrated into the M. maripaludis genome, supports a significantly smaller shuttle vector, pAW42, and a 7,000-fold increase in transformation efficiency for pURB500-based vectors.

Major players on the microbial stage: why archaea are important.

As microbiology undergoes a renaissance, fuelled in part by developments in new sequencing technologies, the massive diversity and abundance of microbes becomes yet more obvious. The Archaea have traditionally been perceived as a minor group of organisms forced to evolve into environmental niches not occupied by their more 'successful' and 'vigorous' counterparts, the bacteria. Here we outline some of the evidence gathered by an increasingly large and productive group of scientists that demonstrates not only that the Archaea contribute significantly to global nutrient cycling, but also that they compete successfully in 'mainstream' environments. Recent data suggest that the Archaea provide the major routes for ammonia oxidation in the environment. Archaea also have huge economic potential that to date has only been fully realized in the production of thermostable polymerases. Archaea have furnished us with key paradigms for understanding fundamentally conserved processes across all domains of life. In addition, they have provided numerous exemplars of novel biological mechanisms that provide us with a much broader view of the forms that life can take and the way in which micro-organisms can interact with other species. That this information has been garnered in a relatively short period of time, and appears to represent only a small proportion of what the Archaea have to offer, should provide further incentives to microbiologists to investigate the underlying biology of this fascinating domain.

Bacterial and eukaryotic systems collide in the three Rs of Methanococcus.

Methanococcus maripaludis S2 is a methanogenic archaeon with a well-developed genetic system. Its mesophilic nature offers a simple system in which to perform complementation using bacterial and eukaryotic genes. Although information-processing systems in archaea are generally more similar to those in eukaryotes than those in bacteria, the order Methanococcales has a unique complement of DNA replication proteins, with multiple MCM (minichromosome maintenance) proteins and no obvious originbinding protein. A search for homologues of recombination and repair proteins in M. maripaludis has revealed a mixture of bacterial, eukaryotic and some archaeal-specific homologues. Some repair pathways appear to be completely absent, but it is possible that archaeal-specific proteins could carry out these functions. The replication, recombination and repair systems in M. maripaludis are an interesting mixture of eukaryotic and bacterial homologues and could provide a system for uncovering novel interactions between proteins from different domains of life.

An archaeal order with multiple minichromosome maintenance genes.

In eukaryotes, a complex of six highly related minichromosome maintenance (MCM) proteins is believed to function as the replicative helicase. Until recently, systems for exploring the molecular mechanisms underlying eukaryotic MCM function have been biochemically intractable. To overcome this, molecular studies of MCM function have been carried out using MCM homologues from the archaea. Archaeal MCM systems studied to date possess a single functional MCM, which forms a homohexameric complex that displays DNA binding, ATPase and helicase activities. We have identified an archaeal order that possesses multiple MCM homologues. blast searches of available Methanococcales genomes reveal that members of this order possess between two and eight MCM homologues. Phylogenetic analysis suggests that an ancient duplication in the Methanococcales gave rise to two major groups of MCMs. One group contains Methanococcus maripaludis S2 McmD and possesses a conserved C-terminal insert similar to one observed in eukaryotic MCM3, while the other group contains McmA, -B and -C. Analysis of the genome context of MCMs in the latter group indicates that these genes could have arisen from phage-mediated events. When co-expressed in Escherichia coli, the four MCMs from M. maripaludis co-purify, indicating the formation of heteromeric complexes in vitro. The presence of homologues from both groups in all Methanococcales indicates that there could be functionally important differences between these proteins and that Methanococcales MCMs may therefore provide an interesting additional model for eukaryotic MCM function.

Conformational flexibility and molecular interactions of an archaeal homologue of the Shwachman-Bodian-Diamond syndrome protein.

BACKGROUND: Defects in the human Shwachman-Bodian-Diamond syndrome (SBDS) protein-coding gene lead to the autosomal recessive disorder characterised by bone marrow dysfunction, exocrine pancreatic insufficiency and skeletal abnormalities. This protein is highly conserved in eukaryotes and archaea but is not found in bacteria. Although genomic and biophysical studies have suggested involvement of this protein in RNA metabolism and in ribosome biogenesis, its interacting partners remain largely unknown. RESULTS: We determined the crystal structure of the SBDS orthologue from Methanothermobacter thermautotrophicus (mthSBDS). This structure shows that SBDS proteins are highly flexible, with the N-terminal FYSH domain and the C-terminal ferredoxin-like domain capable of undergoing substantial rotational adjustments with respect to the central domain. Affinity chromatography identified several proteins from the large ribosomal subunit as possible interacting partners of mthSBDS. Moreover, SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiments, combined with electrophoretic mobility shift assays (EMSA) suggest that mthSBDS does not interact with RNA molecules in a sequence specific manner. CONCLUSION: It is suggested that functional interactions of SBDS proteins with their partners could be facilitated by rotational adjustments of the N-terminal and the C-terminal domains with respect to the central domain. Examination of the SBDS protein structure and domain movements together with its possible interaction with large ribosomal subunit proteins suggest that these proteins could participate in ribosome function.

Methanococcus maripaludis: an archaeon with multiple functional MCM proteins?

There are a large number of proteins involved in the control of eukaryotic DNA replication, which act together to ensure DNA is replicated only once every cell cycle. Key proteins involved in the initiation and elongation phases of DNA replication include the MCM (minchromosome maintenance) proteins, MCM2-MCM7, a family of six related proteins believed to act as the replicative helicase. Genome sequencing has revealed that the archaea possess a simplified set of eukaryotic replication homologues. The complexity of the DNA replication machinery in eukaryotes has led to a number of archaeal species being adapted as model organisms for the study of the DNA replication process. Most archaea sequenced to date possess a single MCM homologue that forms a hexameric complex. Recombinant MCMs from several archaea have been used in the biochemical characterization of the protein, revealing that the MCM complex has ATPase, DNA-binding and -unwinding activities. Unusually, the genome of the methanogenic archaeon Methanococcus maripaludis contains four MCM homologues, all of which contain the conserved motifs required for function. The availability of a wide range of genetic tools for the manipulation of M. maripaludis and the relative ease of growth of this organism in the laboratory makes it a good potential model for studying the role of multiple MCMs in DNA replication.