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.

A zygote-based assay to evaluate intranuclear shuttling in .

It is often necessary to learn whether macromolecules occupy a fixed place in cells. This protocol makes it possible to learn whether individual nucleolar proteins in remain in place or depart from and return to the nucleolus. The protocol uses early zygotes in which parental nucleoli are separate for at least one hour.

Cell cycle arrest of in conjunction with labeling of the cell wall.

can be arrested in metaphase by depleting Cdc20. We describe (1) how to achieve this arrest and verify it, (2) how to label cell surface glycans covalently to distinguish mother from bud, and (3) how to detect the nucleolus and learn that it remains in the mother domain upon arrest. For complete details on the use and execution of this protocol, please refer to Tartakoff et al.

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.

Delayed Encounter of Parental Genomes Can Lead to Aneuploidy in .

We have investigated an extreme deviation from the norm of genome unification that occurs during mating in the yeast, This deviation is encountered when yeast that carry a mutation of the spindle pole body protein, Kar1, are mated with wildtype cells. In this case, nuclear fusion is delayed and the genotypes of a fraction of zygotic progeny suggest that chromosomes have "transferred" between the parental nuclei in zygotes. This classic, yet bizarre, occurrence is routinely used to generate aneuploid (disomic) yeast.

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.

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.

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.

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.

Sequential logic of polarity determination during the haploid-to-diploid transition in Saccharomyces cerevisiae.

In many organisms, the geometry of encounter of haploid germ cells is arbitrary. In Saccharomyces cerevisiae, the resulting zygotes have been seen to bud asymmetrically in several directions as they produce diploid progeny. What mechanisms account for the choice of direction, and do the mechanisms directing polarity change over time? Distinct subgroups of cortical "landmark" proteins guide budding by haploid versus diploid cells, both of which require the Bud1/Rsr1 GTPase to link landmarks to actin.

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 βs, but not by importin αs.

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.

Septin-containing barriers control the differential inheritance of cytoplasmic elements.

Fusion of haploid cells of Saccharomyces cerevisiae generates zygotes. We observe that the zygote midzone includes a septin annulus and differentially affects redistribution of supramolecular complexes and organelles. Redistribution across the midzone of supramolecular complexes (polysomes and Sup35p-GFP [PSI+]) is unexpectedly delayed relative to soluble proteins; however, in [psi-] × [PSI+] crosses, all buds eventually receive Sup35p-GFP [PSI+].

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).

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.

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.

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 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.

Nuclear fusion and genome encounter during yeast zygote formation.

When haploid cells of Saccharomyces cerevisiae are crossed, parental nuclei congress and fuse with each other. To investigate underlying mechanisms, we have developed assays that evaluate the impact of drugs and mutations. Nuclear congression is inhibited by drugs that perturb the actin and tubulin cytoskeletons.

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.

Covert genetic selections to optimize phenotypes.

In many high complexity systems (cells, organisms, institutions, societies, economies, etc.), it is unclear which components should be regulated to affect overall performance. To identify and prioritize molecular targets which impact cellular phenotypes, we have developed a selection procedure ("SPI"-single promoting/inhibiting target identification) which monitors the abundance of ectopic cDNAs.

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.

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.

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.