Cell biology of yeast zygotes, from genesis to budding.

The zygote is the essential intermediate that allows interchange of nuclear, mitochondrial and cytosolic determinants between cells. Zygote formation in Saccharomyces cerevisiae is accomplished by mechanisms that are not characteristic of mitotic cells. These include shifting the axis of growth away from classical cortical landmarks, dramatically reorganizing the cell cortex, remodeling the cell wall in preparation for cell fusion, fusing with an adjacent partner, accomplishing nuclear fusion, orchestrating two steps of septin morphogenesis that account for a delay in fusion of mitochondria, and implementing new norms for bud site selection. This essay emphasizes the sequence of dependent relationships that account for this progression from cell encounters through zygote budding. It briefly summarizes classical studies of signal transduction and polarity specification and then focuses on downstream events.

Nuclear import of aristaless-related homeobox protein via its NLS1 regulates its transcriptional function.

Nucleocytoplasmic transport of transcription factors is essential in eukaryotes. We previously reported the presence of two functional NLSs in the homeodomain protein, aristaless-related homeobox (Arx) protein, which is a key transcriptional repressor of LMO1, SHOX2, and PAX4 during development. NLS2, that overlaps the homeodomain, is recognized directly by multiple importin ßs, but not by importin αs. In this study, we found that the N-terminal NLS1 of Arx is targeted by multiple importin α proteins, including importin α3 and α5. Both in vivo and in vitro assays demonstrated that nuclear import of Arx via NLS1 is mediated by the importin α/ß pathway. Mutagenesis analysis indicated that two basic amino acids, (84)K and (87)R, are essential to the function of NLS1, and that their mutation prevents interactions of Arx with importin αs. Interestingly, inhibition of nuclear import of Arx via NLS1 clearly attenuates its ability of transcriptional repression, suggesting that nuclear import of Arx via NLS1 contributes to its transcriptional function.

Karyopherins in nuclear transport of homeodomain proteins during development.

Homeodomain proteins are crucial transcription factors for cell differentiation, cell proliferation and organ development. Interestingly, their homeodomain signature structure is important for both their DNA-binding and their nucleocytoplasmic trafficking. The accurate nucleocytoplasmic distribution of these proteins is essential for their functions. We summarize information on (a) the roles of karyopherins for import and export of homeoproteins, (b) the regulation of their nuclear transport during development, and (c) the corresponding complexity of homeoprotein nucleocytoplasmic transport signals. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.

Production of nascent ribosome precursors within the nucleolar microenvironment of Saccharomyces cerevisiae.

35S rRNA transcripts include a 5'-external transcribed spacer followed by rRNAs of the small and large ribosomal subunits. Their processing yields massive precursors that include dozens of assembly factor proteins. In Saccharomyces cerevisiae, nucleolar assembly factors form 2 coaxial layers/volumes around ribosomal DNA. Most of these factors are cyclically recruited from a latent state to an operative state, and are extensively conserved. The layers match, at least approximately, known subcompartments found in higher eukaryotic cells. ∼80% of assembly factors are essential. The number of copies of these assembly factors is comparable to the number of nascent transcripts. Moreover, they exhibit "isoelectric balance," with RNA-binding candidate "nucleator" assembly factors being notably basic. The physical properties of pre-small subunit and pre-large subunit assembly factors are similar, as are their 19 motif signatures detected by hierarchical clustering, unlike motif signatures of the 5'-external transcribed spacer rRNP. Additionally, many assembly factors lack shared motifs. Taken together with the progression of rRNP composition during subunit maturation, and the realization that the ribosomal DNA cable is initially bathed in a subunit-nonspecific assembly factor reservoir/microenvironment, we propose a "3-step subdomain assembly model": Step (1): predominantly basic assembly factors sequentially nucleate sites along nascent rRNA; Step (2): the resulting rRNPs recruit numerous less basic assembly factors along with notably basic ribosomal proteins; Step (3): rRNPs in nearby subdomains consolidate. Cleavages of rRNA then promote release of rRNPs to the nucleoplasm, likely facilitated by the persistence of assembly factors that were already associated with nucleolar precursors.

Pronounced and extensive microtubule defects in a Saccharomyces cerevisiae DIS3 mutant.

Subunits of the RNA processing exosome assemble into structurally distinct protein complexes that function in disparate cellular compartments and RNA metabolic pathways. Here, in a genetic, cell biological and transcriptomic analysis, we examined the role of Dis3, an essential polypeptide with endo- and 3'→5' exo-ribonuclease activity, in cell cycle progression. We present several lines of evidence that perturbation of DIS3 affects microtubule (MT) localization and structure in Saccharomyces cerevisiae. Cells with a DIS3 mutant: (a) accumulate anaphase and pre-anaphase mitotic spindles; (b) exhibit spindles that are misorientated and displaced from the bud neck; (c) harbour elongated spindle-associated astral MTs; (d) have an increased G1 astral MT length and number; and (e) are hypersensitive to MT poisons. Mutations in the core exosome genes RRP4 and MTR3 and the exosome cofactor gene MTR4, but not other exosome subunit gene mutants, also elicit MT phenotypes. RNA deep sequencing analysis (RNA-seq) shows broad changes in the levels of cell cycle- and MT-related transcripts in mutant strains. Collectively, the data presented in this study suggest an evolutionarily conserved role for Dis3 in linking RNA metabolism, MTs and cell cycle progression.

The nucleolus as a polarized coaxial cable in which the rDNA axis is surrounded by dynamic subunit-specific phases.

In ribosomal DNA (rDNA) repeats, sequences encoding small-subunit (SSU) rRNA precede those encoding large-subunit (LSU) rRNAs. Processing the composite transcript and subunit assembly requires >100 subunit-specific nucleolar assembly factors (AFs). To investigate the functional organization of the nucleolus, we localized AFs in S. cerevisiae in which the rDNA axis was "linearized" to reduce its dimensionality, thereby revealing its coaxial organization. In this situation, rRNA synthesis and processing continue. The axis is embedded in an inner layer/phase of SSU AFs that is surrounded by an outer layer/phase of LSU AFs. When subunit production is inhibited, subsets of AFs differentially relocate between the inner and outer layers, as expected if there is a cycle of repeated relocation whereby "latent" AFs become "operative" when recruited to nascent subunits. Recognition of AF cycling and localization of segments of rRNA make it possible to infer the existence of assembly intermediates that span between the inner and outer layers and to chart the cotranscriptional assembly of each subunit. AF cycling also can explain how having more than one protein phase in the nucleolus makes possible "vectorial 2-phase partitioning" as a driving force for relocation of nascent rRNPs. Because nucleoplasmic AFs are also present in the outer layer, we propose that critical surface remodeling occurs at this site, thereby partitioning subunit precursors into the nucleoplasm for post-transcriptional maturation. Comparison to observations on higher eukaryotes shows that the coaxial paradigm is likely to be applicable for the many other organisms that have rDNA repeats.

Evolutionary and transcriptional analysis of karyopherin beta superfamily proteins.

In eukaryotes, karyopherin beta superfamily proteins mediate nucleocytoplasmic transport of macromolecules. We investigated the evolutionary and transcriptional patterns of these proteins using bioinformatics approaches. No obvious homologs were found in prokaryotes, but an extensive set of beta-karyopherin proteins was found in yeast. Among 14 beta-karyopherins of Saccharomyces cerevisiae, eight corresponded to their human orthologs directly without diversification, two were lost, and the remaining four proteins exhibited gene duplications by different mechanisms. We also identified beta-karyopherin orthologs in Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Xenopus tropicalis, Gallus gallus, and Mus musculus. beta-Karyopherins were ubiquitously but nonuniformly expressed in distinct cells and tissues. In yeast and mice, the titer of some beta-karyopherin transcripts appeared to be regulated both during the cell cycle and during development. Further virtual analysis of promoter binding elements suggested that the transcription factors SP1, NRF-2, HEN-1, RREB-1, and nuclear factor Y regulate expression of most beta-karyopherin genes. These findings emphasize new mechanisms in functional diversification of beta-karyopherins and regulation of nucleocytoplasmic transport.

Immobility, inheritance and plasticity of shape of the yeast nucleus.

BACKGROUND: Since S. cerevisiae undergoes closed mitosis, the nuclear envelope of the daughter nucleus is continuous with that of the maternal nucleus at anaphase. Nevertheless, several constitutents of the maternal nucleus are not present in the daughter nucleus. The present study aims to identify proteins which impact the shape of the yeast nucleus and to learn whether modifications of shape are passed on to the next mitotic generation. The Esc1p protein of S. cerevisiae localizes to the periphery of the nucleoplasm, can anchor chromatin, and has been implicated in targeted silencing both at telomeres and at HMR. RESULTS: Upon increased Esc1p expression, cell division continues and dramatic elaborations of the nuclear envelope extend into the cytoplasm. These "escapades" include nuclear pores and associate with the nucleolus, but exclude chromatin. Escapades are not inherited by daughter nuclei. This exclusion reflects their relative immobility, which we document in studies of prezygotes. Moreover, excess Esc1p affects the levels of multiple transcripts, not all of which originate at telomere-proximal loci. Unlike Esc1p and the colocalizing protein, Mlp1p, overexpression of selected proteins of the inner nuclear membrane is toxic. CONCLUSION: Esc1p is the first non-membrane protein of the nuclear periphery which - like proteins of the nuclear lamina of higher eukaryotes - can modify the shape of the yeast nucleus. The elaborations of the nuclear envelope ("escapades") which appear upon induction of excess Esc1p are not inherited during mitotic growth. The lack of inheritance of such components could help sustain cell growth when parental nuclei have acquired potentially deleterious characteristics.

Nucleolar asymmetry and the importance of septin integrity upon cell cycle arrest.

Cell cycle arrest can be imposed by inactivating the anaphase promoting complex (APC). In S. cerevisiae this arrest has been reported to stabilize a metaphase-like intermediate in which the nuclear envelope spans the bud neck, while chromatin repeatedly translocates between the mother and bud domains. The present investigation was undertaken to learn how other features of nuclear organization are affected upon depletion of the APC activator, Cdc20. We observe that the spindle pole bodies and the spindle repeatedly translocate across the narrow orifice at the level of the neck. Nevertheless, we find that the nucleolus (organized around rDNA repeats on the long right arm of chromosome XII) remains in the mother domain, marking the polarity of the nucleus. Accordingly, chromosome XII is polarized: TelXIIR remains in the mother domain and its centromere is predominantly located in the bud domain. In order to learn why the nucleolus remains in the mother domain, we studied the impact of inhibiting rRNA synthesis in arrested cells. We observed that this fragments the nucleolus and that these fragments entered the bud domain. Taken together with earlier observations, the restriction of the nucleolus to the mother domain therefore can be attributed to its massive structure. We also observed that inactivation of septins allowed arrested cells to complete the cell cycle, that the alternative APC activator, Cdh1, was required for completion of the cell cycle and that induction of Cdh1 itself caused arrested cells to progress to the end of the cell cycle.

Polyglutamine Tract Expansion Increases S-Nitrosylation of Huntingtin and Ataxin-1.

Expansion of the polyglutamine (polyQ) tract in the huntingtin (Htt) protein causes Huntington's disease (HD), a fatal inherited movement disorder linked to neurodegeneration in the striatum and cortex. S-nitrosylation and S-acylation of cysteine residues regulate many functions of cytosolic proteins. We therefore used a resin-assisted capture approach to identify these modifications in Htt. In contrast to many proteins that have only a single S-nitrosylation or S-acylation site, we identified sites along much of the length of Htt. Moreover, analysis of cells expressing full-length Htt or a large N-terminal fragment of Htt shows that polyQ expansion strongly increases Htt S-nitrosylation. This effect appears to be general since it is also observed in Ataxin-1, which causes spinocerebellar ataxia type 1 (SCA1) when its polyQ tract is expanded. Overexpression of nitric oxide synthase increases the S-nitrosylation of normal Htt and the frequency of conspicuous juxtanuclear inclusions of Htt N-terminal fragments in transfected cells. Taken together with the evidence that S-nitrosylation of Htt is widespread and parallels polyQ expansion, these subcellular changes show that S-nitrosylation affects the biology of this protein in vivo.

Visual experience regulates transient expression and dendritic localization of fragile X mental retardation protein.

Fragile X syndrome is the most common form of inherited mental retardation and is caused by the loss of function of the Fragile X mental retardation protein (FMRP). FMRP is an RNA binding protein thought to play a key role in protein synthesis-dependent synaptic plasticity. The regulation of FMRP expression itself is also likely to be an important control point in this process. Here we used dark-reared/light-exposed rats to determine the role of experience in regulating FMRP levels in the visual cortex. We find that FMRP levels increase in the cell bodies and dendrites of visual cortical neurons after as little as 15 min of light exposure. Remarkably, FMRP expression in these neurons returns to baseline levels by 30 min of light exposure. These changes were post-transcriptional because the FMR1 mRNA levels remained constant over this time period. A transient increase in FMRP levels was also observed in synaptic fractions prepared from visual cortices of light-exposed animals. In contrast, alpha-calcium/calmodulin-dependent kinase II expression showed a sustained upregulation under these conditions. Finally, the increase in FMRP expression was inhibited by blockade of NMDA receptors. This tight temporal-spatial regulation suggests that FMRP plays a dynamic role in a distinct epoch of experience-dependent synaptic plasticity.

The calcium-binding protein p54/NEFA is a novel luminal resident of medial Golgi cisternae that traffics independently of mannosidase II.

A new Golgi resident, p54, has been demonstrated in several eukaryotic species and in multiple organs. Based on Triton X-114 partition, carbonate extraction and trypsin protection assays, p54 behaved as an extrinsic membrane protein, facing the luminal compartment. p54 was purified by two-dimensional electrophoresis and identified by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry as NEFA, a calcium-binding protein (Barnikol-Watanabe et al., 1994, Biol. Chem. Hoppe Seyler, 375, 497-512). By immunofluorescence, p54/NEFA essentially colocalized with the medial Golgi marker mannosidase II, and did not overlap with the cis-Golgi marker p58, nor with the trans-Golgi network (TGN) marker TGN38. By immuno-electron microscopy, p54/NEFA localized in the medial cisternae and in Golgi-associated vesicles. p54/NEFA remained associated with mannosidase II despite Golgi disruption by nocodazole, caffeine, or, to some extent, potassium depletion (a new procedure to induce Golgi disassembly), but the two markers rapidly dissociated upon brefeldin A treatment and at metaphase, and reassociated upon drug removal and at the end of anaphase. Since p54/NEFA is a peripheral luminal membrane constituent, its distinct trafficking from the transmembrane marker mannosidase II suggests a novel Golgi retention mechanism, by strong association of this soluble protein with another integral transmembrane resident.

The axis of progression of disease.

Starting with genetic or environmental perturbations, disease progression can involve a linear sequence of changes within individual cells. More often, however, a labyrinth of branching consequences emanates from the initial events. How can one repair an entity so fine and so complex that its organization and functions are only partially known? How, given the many redundancies of metabolic pathways, can interventions be effective before the last redundant element has been irreversibly damaged? Since progression ultimately proceeds beyond a point of no return, therapeutic goals must target earlier events. A key goal is therefore to identify early changes of functional importance. Moreover, when several distinct genetic or environmental causes converge on a terminal phenotype, therapeutic strategies that focus on the shared features seem unlikely to be useful - precisely because the shared events lie relatively downstream along the axis of progression. We therefore describe experimental strategies that could lead to identification of early events, both for cancer and for other diseases.

Non-apoptotic toxicity of Pseudomonas aeruginosa toward murine cells.

Although P. aeruginosa is especially dangerous in cystic fibrosis (CF), there is no consensus as to how it kills representative cell types that are of key importance in the lung. This study concerns the acute toxicity of the sequenced strain, PAO1, toward a murine macrophage cell line (RAW 264.7). Toxicity requires brief contact with the target cell, but is then delayed for more than 12 h. None of the classical toxic effectors of this organism is required and cell death occurs without phagocytosis or acute perturbation of the actin cytoskeleton. Apoptosis is not required for toxicity toward either RAW 264.7 cells or for alveolar macrophages. Transcriptional profiling shows that encounter between PAO1 and RAW 264.7 cells elicits an early inflammatory response, followed by growth arrest. As an independent strategy to understand the mechanism of toxicity, we selected variant RAW 264.7 cells that resist PAO1. Upon exposure to P. aeruginosa, they are hyper-responsive with regard to classical inflammatory cytokine production and show transient downregulation of transcripts that are required for cell growth. They do not show obvious morphologic changes. Although they do not increase interferon transcripts, when exposed to PAO1 they dramatically upregulate a subset of the responses that are characteristic of exposure to g-interferon, including several guanylate-binding proteins. The present observations provide a novel foundation for learning how to equip cells with resistance to a complex challenge.

Flow cytometry-based purification of S. cerevisiae zygotes.

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) (1). S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions (2). Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function. Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells (3-8). Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated. Reports of purification of yeast zygotes describe protocols based on their sedimentation properties (9); however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Comparative and evolutionary aspects of macromolecular translocation across membranes.

Membrane barriers preserve the integrity of organelles of eukaryotic cells, yet the genesis and ongoing functions of the same organelles requires that their limiting membranes allow import and export of selected macromolecules. Multiple distinct mechanisms are used for this purpose, only some of which have been traced to prokaryotes. Some can accommodate both monomeric and also large heterooligomeric cargoes. The best characterized of these is nucleocytoplasmic transport. This synthesis compares the unidirectional and bidirectional mechanisms of macromolecular transport of the endoplasmic reticulum, mitochondria, peroxisomes and the nucleus, calls attention to the powerful experimental approaches which have been used for their elucidation, discusses their regulation and evolutionary origins, and highlights relatively unexplored areas.

The roles of multiple importins for nuclear import of murine aristaless-related homeobox protein.

Nuclear import of proteins with nuclear localization signals (NLSs) is mediated by shuttling carriers, the importins. Some cargoes display more than a single NLS, and among these are homeodomain proteins such as Arx, which is critical for development of multiple tissues. Arx has two functional NLSs. The present studies show that several pathways can import Arx via its NLS2, which is within its DNA binding homeodomain. Using an in vitro nuclear import assay, we show that import of Arx via NLS2 can be mediated by importin beta1, importin 9, or importin 13, with binding being strongest to importin beta1. All binding is sensitive to RanGTP. Experiments based on precise domain deletions indicate that NLS2 binds impbeta1, imp9, and imp13 and includes both an importin binding subdomain and a regulatory subdomain with arginine residues being important for function. Moreover, Arx can be co-precipitated with these importins when NLS2 is present. Although nuclear import of Arx can be mediated by these three importin betas, importin beta1 seems to play the major role judging from in vivo small interfering RNA ablations and the in vitro import assay. This is the first evidence to show the role of importin beta1 in nuclear import of paired-type homeodomain proteins. We propose a novel and possibly quite general mechanism for nuclear import of paired-type homeodomain proteins which is critical for development.

Interaction between importin 13 and myopodin suggests a nuclear import pathway for myopodin.

Importin 13 is a member of the importin beta superfamily of nuclear transport proteins and is expressed in multiple tissues at high levels both in humans and rodents, including fetal lung, brain, and heart. In order to elucidate potential functions of imp13 in the heart, we have used rat imp13 as bait to screen a human heart cDNA library and identified an interaction with the C-terminal peptide of myopodin (a.a. 360-698), an actin-bundling protein, associated with tumor-suppressor activity that localizes to both the cytoplasm and the nucleus. We have used GST-pull down assays and co-immunoprecipitation experiments to demonstrate an interaction between imp13 and full-length myopodin and observed that RanGTP dissociates the myopodin-imp13 complex. In studies of cultured cells, we show that both imp13 siRNA and a C-terminal fragment of imp13 protein prevent nuclear localization of myopodin. We, therefore, conclude that imp13 functions in myopodin import and we suggest that the regulation of these events is critical for normal and abnormal cellular differentiation.

Ribosomal RNA in Alzheimer disease is oxidized by bound redox-active iron.

Oxidative modification of cytoplasmic RNA in vulnerable neurons is an important, well documented feature of the pathophysiology of Alzheimer disease. Here we report that RNA-bound iron plays a pivotal role for RNA oxidation in vulnerable neurons in Alzheimer disease brain. The cytoplasm of hippocampal neurons showed significantly higher redox activity and iron(II) staining than age-matched controls. Notably, both were susceptible to RNase, suggesting a physical association of iron(II) with RNA. Ultrastructural analysis further suggested an endoplasmic reticulum association. Both rRNA and mRNA showed twice the iron binding as tRNA. rRNA, extremely abundant in neurons, was considered to provide the greatest number of iron binding sites among cytoplasmic RNA species. Interestingly, the difference of iron binding capacity disappeared after denaturation of RNA, suggesting that the higher order structure may contribute to the greater iron binding of rRNA. Reflecting the difference of iron binding capacity, oxidation of rRNA by the Fenton reaction formed 13 times more 8-hydroxyguanosine than tRNA. Consistent with in situ findings, ribosomes purified from Alzheimer hippocampus contained significantly higher levels of RNase-sensitive iron(II) and redox activity than control. Furthermore, only Alzheimer rRNA contains 8-hydroxyguanosine in reverse transcriptase-PCR. Addressing the biological significance of ribosome oxidation by redox-active iron, in vitro translation with oxidized ribosomes from rabbit reticulocyte showed a significant reduction of protein synthesis. In conclusion these results suggest that rRNA provides a binding site for redox-active iron and serves as a redox center within the cytoplasm of vulnerable neurons in Alzheimer disease in advance of the appearance of morphological change indicating neurodegeneration.