Improved gene editing and fluorescent-protein tagging in using a Golden Gate-based CRISPR-Cas9 plasmid system.

CRISPR-Cas9 systems can be used for precise genome editing in filamentous fungi, including . However, current CRISPR-Cas9 systems for rely on relatively complex or multi-step cloning methods to build a plasmid expressing both Cas9 and an sgRNA targeting a genomic locus. In this study we improve on existing plasmid-based CRISPR-Cas9 systems for by creating an extremely simple-to-use CRISPR-Cas9 system for genome editing.

Microtubule-independent movement of the fission yeast nucleus.

Movement of the cell nucleus typically involves the cytoskeleton and either polymerization-based pushing forces or motor-based pulling forces. In the fission yeast , nuclear movement and positioning are thought to depend on microtubule polymerization-based pushing forces. Here, we describe a novel, microtubule-independent, form of nuclear movement in fission yeast.

Identification of 15 New Bypassable Essential Genes of Fission Yeast.

Every organism has a different set of genes essential for its viability. This indicates that an organism can become tolerant to the loss of an essential gene under certain circumstances during evolution, via the manifestation of 'masked' alternative mechanisms. In our quest to systematically uncover masked mechanisms in eukaryotic cells, we developed an extragenic suppressor screening method using haploid spores deleted of an essential gene in the fission yeast Schizosaccharomyces pombe.

Reconstitution of Microtubule Nucleation In Vitro Reveals Novel Roles for Mzt1.

Microtubule (MT) nucleation depends on the γ-tubulin complex (γ-TuC), in which multiple copies of the heterotetrameric γ-tubulin small complex (γ-TuSC) associate to form a ring-like structure (in metazoans, γ-tubulin ring complex; γ-TuRC) [1-7]. Additional conserved regulators of the γ-TuC include the small protein Mzt1 (MOZART1 in human; GIP1/1B and GIP2/1A in plants) [8-13] and proteins containing a Centrosomin Motif 1 (CM1) domain [10, 14-19]. Many insights into γ-TuC regulators have come from in vivo analysis in fission yeast Schizosaccharomyces pombe.

Fission Yeast NDR/LATS Kinase Orb6 Regulates Exocytosis via Phosphorylation of the Exocyst Complex.

NDR/LATS kinases regulate multiple aspects of cell polarity and morphogenesis from yeast to mammals. Fission yeast NDR/LATS kinase Orb6 has been proposed to control cell polarity by regulating the Cdc42 guanine nucleotide exchange factor Gef1. Here, we show that Orb6 regulates polarity largely independently of Gef1 and that Orb6 positively regulates exocytosis.

Local and global Cdc42 guanine nucleotide exchange factors for fission yeast cell polarity are coordinated by microtubules and the Tea1-Tea4-Pom1 axis.

The conserved Rho-family GTPase Cdc42 plays a central role in eukaryotic cell polarity. The rod-shaped fission yeast has two Cdc42 guanine nucleotide exchange factors (GEFs), Scd1 and Gef1, but little is known about how they are coordinated in polarized growth. Although the microtubule cytoskeleton is normally not required for polarity maintenance in fission yeast, we show here that when function is compromised, disruption of microtubules or the polarity landmark proteins Tea1, Tea4 or Pom1 leads to disruption of polarized growth.

Exportin Crm1 is repurposed as a docking protein to generate microtubule organizing centers at the nuclear pore.

Non-centrosomal microtubule organizing centers (MTOCs) are important for microtubule organization in many cell types. In fission yeast , the protein Mto1, together with partner protein Mto2 (Mto1/2 complex), recruits the γ-tubulin complex to multiple non-centrosomal MTOCs, including the nuclear envelope (NE). Here, we develop a comparative-interactome mass spectrometry approach to determine how Mto1 localizes to the NE.

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Technology in Fission Yeast.

Shotgun proteomics combined with stable isotope labeling by amino acids in cell culture (SILAC) is a powerful approach to quantify proteins and posttranslational modifications across the entire proteome. SILAC technology in must cope with the "arginine conversion problem," in which isotope-labeled arginine is converted to other amino acids. This can be circumvented by either using stable isotope-marked lysine only (as opposed to the more standard lysine/arginine double labeling) or using yeast genetics to create strains that only very inefficiently convert arginine.

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-Based Quantitative Proteomics and Phosphoproteomics in Fission Yeast.

Modern mass spectrometry (MS)-based approaches are capable of identifying and quantifying thousands of proteins and phosphorylation events in a single biological experiment. Here we present a (phospho)proteomic workflow based on in-solution proteome digestion of samples labeled by stable isotope labeling by amino acids in cell culture (SILAC) and phosphopeptide enrichment using strong cation exchange (SCX) and TiO chromatographies. These procedures are followed by high-accuracy MS measurement on an Orbitrap mass spectrometer and subsequent bioinformatic processing using MaxQuant software.

Construction, Growth, and Harvesting of Fission Yeast Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Strains.

Stable isotope labeling by amino acids in cell culture (SILAC) enables the relative quantification of protein amounts and posttranslational modifications in complex biological samples through the use of stable heavy isotope-labeled amino acids. Here we describe methods for the application of SILAC to fission yeast using either labeled lysine or a combination of labeled lysine and labeled arginine. The latter approach is more complicated than the use of labeled lysine alone but may yield a more comprehensive (phospho)proteomic analysis.

Remodeling of the Fission Yeast Cdc42 Cell-Polarity Module via the Sty1 p38 Stress-Activated Protein Kinase Pathway.

The Rho family GTPase Cdc42 is a key regulator of eukaryotic cellular organization and cell polarity [1]. In the fission yeast Schizosaccharomyces pombe, active Cdc42 and associated effectors and regulators (the "Cdc42 polarity module") coordinate polarized growth at cell tips by controlling the actin cytoskeleton and exocytosis [2-4]. Localization of the Cdc42 polarity module to cell tips is thus critical for its function.

Mto2 multisite phosphorylation inactivates non-spindle microtubule nucleation complexes during mitosis.

Microtubule nucleation is highly regulated during the eukaryotic cell cycle, but the underlying molecular mechanisms are largely unknown. During mitosis in fission yeast Schizosaccharomyces pombe, cytoplasmic microtubule nucleation ceases simultaneously with intranuclear mitotic spindle assembly. Cytoplasmic nucleation depends on the Mto1/2 complex, which binds and activates the γ-tubulin complex and also recruits the γ-tubulin complex to both centrosomal (spindle pole body) and non-centrosomal sites.

Deletion of Genes Encoding Arginase Improves Use of "Heavy" Isotope-Labeled Arginine for Mass Spectrometry in Fission Yeast.

The use of "heavy" isotope-labeled arginine for stable isotope labeling by amino acids in cell culture (SILAC) mass spectrometry in the fission yeast Schizosaccharomyces pombe is hindered by the fact that under normal conditions, arginine is extensively catabolized in vivo, resulting in the appearance of "heavy"-isotope label in several other amino acids, most notably proline, but also glutamate, glutamine and lysine. This "arginine conversion problem" significantly impairs quantification of mass spectra. Previously, we developed a method to prevent arginine conversion in fission yeast SILAC, based on deletion of genes involved in arginine catabolism.

Pom1 regulates the assembly of Cdr2-Mid1 cortical nodes for robust spatial control of cytokinesis.

Proper division plane positioning is essential to achieve faithful DNA segregation and to control daughter cell size, positioning, or fate within tissues. In Schizosaccharomyces pombe, division plane positioning is controlled positively by export of the division plane positioning factor Mid1/anillin from the nucleus and negatively by the Pom1/DYRK (dual-specificity tyrosine-regulated kinase) gradients emanating from cell tips. Pom1 restricts to the cell middle cortical cytokinetic ring precursor nodes organized by the SAD-like kinase Cdr2 and Mid1/anillin through an unknown mechanism.

Activation of the γ-tubulin complex by the Mto1/2 complex.

The multisubunit γ-tubulin complex (γ-TuC) is critical for microtubule nucleation in eukaryotic cells, but it remains unclear how the γ-TuC becomes active specifically at microtubule-organizing centers (MTOCs) and not more broadly throughout the cytoplasm. In the fission yeast Schizosaccharomyces pombe, the proteins Mto1 and Mto2 form the Mto1/2 complex, which interacts with the γ-TuC and recruits it to several different types of cytoplasmic MTOC sites. Here, we show that the Mto1/2 complex activates γ-TuC-dependent microtubule nucleation independently of localizing the γ-TuC.

Characterization of Mug33 reveals complementary roles for actin cable-dependent transport and exocyst regulators in fission yeast exocytosis.

Although endocytosis and exocytosis have been extensively studied in budding yeast, there have been relatively few investigations of these complex processes in the fission yeast Schizosaccharomyces pombe. Here we identify and characterize fission yeast Mug33, a novel Tea1-interacting protein, and show that Mug33 is involved in exocytosis. Mug33 is a Sur7/PalI-family transmembrane protein that localizes to the plasma membrane at the cell tips and to cytoplasmic tubulovesicular elements (TVEs).

Microtubule stabilization in vivo by nucleation-incompetent γ-tubulin complex.

Although the fission yeast Schizosaccharomyces pombe contains many of the γ-tubulin ring complex (γ-TuRC)-specific proteins of the γ-tubulin complex (γ-TuC), several questions about the organizational state and function of the fission yeast γ-TuC in vivo remain unresolved. Using 3×GFP-tagged γ-TuRC-specific proteins, we show here that γ-TuRC-specific proteins are present at all microtubule organizing centers in fission yeast and that association of γ-TuRC-specific proteins with the γ-tubulin small complex (γ-TuSC) does not depend on Mto1, which is a key regulator of the γ-TuC. Through sensitive imaging in mto1Δ mutants, in which cytoplasmic microtubule nucleation is abolished, we unexpectedly found that γ-TuC incapable of nucleating microtubules can nevertheless associate with microtubule minus-ends in vivo.

Fission yeast Mto1 regulates diversity of cytoplasmic microtubule organizing centers.

Microtubule nucleation by the γ-tubulin complex occurs primarily at centrosomes, but more diverse types of microtubule organizing centers (MTOCs) also exist, especially in differentiated cells. Mechanisms generating MTOC diversity are poorly understood. Fission yeast Schizosaccharomyces pombe has multiple types of cytoplasmic MTOCs, and these vary through the cell cycle.

A catalytic role for Mod5 in the formation of the Tea1 cell polarity landmark.

Many systems regulating cell polarity involve stable landmarks defined by internal cues. In the rod-shaped fission yeast Schizosaccharomyces pombe, microtubules regulate polarized vegetative growth via a landmark involving the protein Tea1. Tea1 is delivered to cell tips as packets of molecules associated with growing microtubule ends and anchored at the plasma membrane via a mechanism involving interaction with the membrane protein Mod5.

New and old reagents for fluorescent protein tagging of microtubules in fission yeast; experimental and critical evaluation.

The green fluorescent protein (GFP) has become a mainstay of in vivo imaging in many experimental systems. In this chapter, we first discuss and evaluate reagents currently available to image GFP-labeled microtubules in the fission yeast Schizosaccharomyces pombe, with particular reference to time-lapse applications. We then describe recent progress in the development of robust monomeric and tandem dimer red fluorescent proteins (RFPs), including mCherry, TagRFP-T, mOrange2, mKate, and tdTomato, and we present data assessing their suitability as tags in S.

A genetic engineering solution to the "arginine conversion problem" in stable isotope labeling by amino acids in cell culture (SILAC).

Stable isotope labeling by amino acids in cell culture (SILAC) provides a straightforward tool for quantitation in proteomics. However, one problem associated with SILAC is the in vivo conversion of labeled arginine to other amino acids, typically proline. We found that arginine conversion in the fission yeast Schizosaccharomyces pombe occurred at extremely high levels, such that labeling cells with heavy arginine led to undesired incorporation of label into essentially all of the proline pool as well as a substantial portion of glutamate, glutamine, and lysine pools.

Inexpensive synthetic-based matrix for both conventional and rapid purification of protein A- and tandem affinity purification-tagged proteins.

Immunoglobulin G (IgG)-Sepharose is often used for purification of protein A- and tandem affinity purification (TAP)-tagged proteins from eukaryotic cells, but because it is based on an agarose matrix, it is not always optimal for all proteins. Synthetic matrices such as IgG-Dynabeads have improved properties over IgG-Sepharose but are generally expensive. Here we describe the preparation and properties of an IgG matrix based on Fractogel EMD beads.

Improved tools for efficient mapping of fission yeast genes: identification of microtubule nucleation modifier mod22-1 as an allele of chromatin- remodelling factor gene swr1.

Fission yeast genes identified in genetic screens are usually cloned by transformation of mutants with plasmid libraries. However, for some genes this can be difficult, and positional cloning approaches are required. The mutation swi5-39 reduces recombination frequency in homozygous crosses and has been used as a tool in mapping gene position (Schmidt, 1993).