Characterization of Bud3 domains sufficient for bud neck targeting in .

The cytoskeleton serves a diverse set of functions in both multi- and unicellular organisms, including movement, transport, morphology, cell division and cell signalling. The septin family of cytoskeletal proteins are found within all fungi and metazoans and can generate three-dimensional scaffolds that promote membrane curvature, serve as physical barriers and coordinate cell cycle checkpoints. In budding yeast, the septins organize into polymerized filaments that decorate the division site between mother and daughter cells during mitosis; assembly of this structure at the 'bud neck' is critical for completion of cytokinesis and execution of numerous other cellular events.

Analysis of a Cas12a-based gene-drive system in budding yeast.

The discovery and adaptation of CRISPR/Cas systems within molecular biology has provided advances across biological research, agriculture and human health. Genomic manipulation through use of a CRISPR nuclease and programmed guide RNAs has become a common and widely accessible practice. The identification and introduction of new engineered variants and orthologues of Cas9 as well as alternative CRISPR systems such as the type V group have provided additional molecular options for editing.

Reconstructed evolutionary history of the yeast septins Cdc11 and Shs1.

Septins are GTP-binding proteins conserved across metazoans. They can polymerize into extended filaments and, hence, are considered a component of the cytoskeleton. The number of individual septins varies across the tree of life-yeast (Saccharomyces cerevisiae) has seven distinct subunits, a nematode (Caenorhabditis elegans) has two, and humans have 13.

Analysis of CRISPR gene drive design in budding yeast.

Control of biological populations remains a critical goal to address the challenges facing ecosystems and agriculture and those posed by human disease, including pests, parasites, pathogens and invasive species. A particular architecture of the CRISPR/Cas biotechnology - a gene drive - has the potential to modify or eliminate populations on a massive scale. Super-Mendelian inheritance has now been demonstrated in both fungi and metazoans, including disease vectors such as mosquitoes.

Mathematical modeling of self-contained CRISPR gene drive reversal systems.

There is a critical need for further research into methods to control biological populations. Numerous challenges to agriculture, ecological systems, and human health could be mitigated by the targeted reduction and management of key species (e.g.

Modulating CRISPR gene drive activity through nucleocytoplasmic localization of Cas9 in .

Background: The bacterial CRISPR/Cas genome editing system has provided a major breakthrough in molecular biology. One use of this technology is within a nuclease-based gene drive. This type of system can install a genetic element within a population at unnatural rates.

Development of a multi-locus CRISPR gene drive system in budding yeast.

The discovery of CRISPR/Cas gene editing has allowed for major advances in many biomedical disciplines and basic research. One arrangement of this biotechnology, a nuclease-based gene drive, can rapidly deliver a genetic element through a given population and studies in fungi and metazoans have demonstrated the success of such a system. This methodology has the potential to control biological populations and contribute to eradication of insect-borne diseases, agricultural pests, and invasive species.

Gene drive inhibition by the anti-CRISPR proteins AcrIIA2 and AcrIIA4 in Saccharomyces cerevisiae.

Given the widespread use and application of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas gene editing system across many fields, a major focus has been the development, engineering and discovery of molecular means to precisely control and regulate the enzymatic function of the Cas9 nuclease. To date, a variety of Cas9 variants and fusion assemblies have been proposed to provide temporally inducible and spatially controlled editing functions. The discovery of a new class of 'anti-CRISPR' proteins, evolved from bacteriophage in response to the prokaryotic nuclease-based immune system, provides a new platform for control over genomic editing.

Some assembly required: Contributions of Tom Stevens' lab to the V-ATPase field.

Tom Stevens' lab has explored the subunit composition and assembly of the yeast V-ATPase for more than 30 years. Early studies helped establish yeast as the predominant model system for study of V-ATPase proton pumps and led to the discovery of protein splicing of the V-ATPase catalytic subunit. The Vma phenotype, characteristic of loss-of-V-ATPase activity in yeast was key in determining the enzyme's subunit composition via yeast genetics.

Tuning CRISPR-Cas9 Gene Drives in .

Control of biological populations is an ongoing challenge in many fields, including agriculture, biodiversity, ecological preservation, pest control, and the spread of disease. In some cases, such as insects that harbor human pathogens (, malaria), elimination or reduction of a small number of species would have a dramatic impact across the globe. Given the recent discovery and development of the CRISPR-Cas9 gene editing technology, a unique arrangement of this system, a nuclease-based "gene drive," allows for the super-Mendelian spread and forced propagation of a genetic element through a population.

Yeast Still a Beast: Diverse Applications of CRISPR/Cas Editing Technology in .

The recent discovery and use of CRISPR/Cas9 gene editing technology has provided new opportunities for scientific research in many fields of study including agriculture, genetic disorders, human disease, biotechnology, and basic biological research. The ability to precisely target DNA sequences and either remove, modify, or replace genetic sequences provides a new level of control in nearly all eukaryotic organisms, including budding yeast. Given the many discoveries made in over the past decades spanning genetics, cell biology, and biochemistry, as well as the development of new technologies that have allowed high throughput screening, robotic automation, and a platform for synthetic genome engineering, the yeast community has also started to recognize the utility and complementary nature of CRISPR-based methodologies.

Method for Multiplexing CRISPR/Cas9 in Using Artificial Target DNA Sequences.

Genome manipulation has become more accessible given the advent of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) editing technology. The Cas9 endonuclease binds a single stranded (single guide) RNA (sgRNA) fragment that recruits the complex to a corresponding genomic target sequence where it induces a double stranded break. Eukaryotic repair systems allow for the introduction of exogenous DNA, repair of existing mutations, or deletion of endogenous gene products.

CRISPR-UnLOCK: Multipurpose Cas9-Based Strategies for Conversion of Yeast Libraries and Strains.

continues to serve as a powerful model system for both basic biological research and industrial application. The development of genome-wide collections of individually manipulated strains (libraries) has allowed for high-throughput genetic screens and an emerging global view of this single-celled Eukaryote. The success of strain construction has relied on the innate ability of budding yeast to accept foreign DNA and perform homologous recombination, allowing for efficient plasmid construction () and integration of desired sequences into the genome.

TOR Complex 2-Regulated Protein Kinase Fpk1 Stimulates Endocytosis via Inhibition of Ark1/Prk1-Related Protein Kinase Akl1 in Saccharomyces cerevisiae.

Depending on the stress, plasma membrane alterations activate or inhibit yeast target of rapamycin (TOR) complex 2, which, in turn, upregulates or downregulates the activity of its essential downstream effector, protein kinase Ypk1. Through phosphorylation of multiple substrates, Ypk1 controls many processes that restore homeostasis. One such substrate is protein kinase Fpk1, which is negatively regulated by Ypk1.

Septin-Associated Protein Kinases in the Yeast .

Septins are a family of eukaryotic GTP-binding proteins that associate into linear rods, which, in turn, polymerize end-on-end into filaments, and further assemble into other, more elaborate super-structures at discrete subcellular locations. Hence, septin-based ensembles are considered elements of the cytoskeleton. One function of these structures that has been well-documented in studies conducted in budding yeast is to serve as a scaffold that recruits regulatory proteins, which dictate the spatial and temporal control of certain aspects of the cell division cycle.

Detection of protein-protein interactions at the septin collar in Saccharomyces cerevisiae using a tripartite split-GFP system.

Various methods can provide a readout of the physical interaction between two biomolecules. A recently described tripartite split-GFP system has the potential to report by direct visualization via a fluorescence signal the intimate association of minimally tagged proteins expressed at their endogenous level in their native cellular milieu and can capture transient or weak interactions. Here we document the utility of this tripartite split-GFP system to assess in living cells protein-protein interactions in a dynamic cytoskeletal structure-the septin collar at the yeast bud neck.

Coordinate action of distinct sequence elements localizes checkpoint kinase Hsl1 to the septin collar at the bud neck in Saccharomyces cerevisiae.

Passage through the eukaryotic cell cycle requires processes that are tightly regulated both spatially and temporally. Surveillance mechanisms (checkpoints) exert quality control and impose order on the timing and organization of downstream events by impeding cell cycle progression until the necessary components are available and undamaged and have acted in the proper sequence. In budding yeast, a checkpoint exists that does not allow timely execution of the G2/M transition unless and until a collar of septin filaments has properly assembled at the bud neck, which is the site where subsequent cytokinesis will occur.

mCAL: A New Approach for Versatile Multiplex Action of Cas9 Using One sgRNA and Loci Flanked by a Programmed Target Sequence.

Genome editing exploiting CRISPR/Cas9 has been adopted widely in academia and in the biotechnology industry to manipulate DNA sequences in diverse organisms. Molecular engineering of Cas9 itself and its guide RNA, and the strategies for using them, have increased efficiency, optimized specificity, reduced inappropriate off-target effects, and introduced modifications for performing other functions (transcriptional regulation, high-resolution imaging, protein recruitment, and high-throughput screening). Moreover, Cas9 has the ability to multiplex, i.

Assembly, molecular organization, and membrane-binding properties of development-specific septins.

Septin complexes display remarkable plasticity in subunit composition, yet how a new subunit assembled into higher-order structures confers different functions is not fully understood. Here, this question is addressed in budding yeast, where during meiosis Spr3 and Spr28 replace the mitotic septin subunits Cdc12 and Cdc11 (and Shs1), respectively. In vitro, the sole stable complex that contains both meiosis-specific septins is a linear Spr28-Spr3-Cdc3-Cdc10-Cdc10-Cdc3-Spr3-Spr28 hetero-octamer.

Complex Ligation Using Homologous Recombination and High-efficiency Plasmid Rescue from .

The protocols presented here allow for the facile generation of a wide variety of complex multipart DNA constructs (tagged gene products, gene fusions, chimeric proteins, and other variants) using homologous recombination and ligation in budding yeast (). This method is straightforward, efficient and cost-effective, and can be used both for vector creation and for subsequent one-step, high frequency integration into a chromosomal locus in yeast. The procedure utilizes PCR with extended oligonucleotide "tails" of homology between multiple fragments to allow for reassembly in yeast in a single transformation followed by a method for highly efficient plasmid extraction from yeast (for transformation into bacteria).

The Carboxy-Terminal Tails of Septins Cdc11 and Shs1 Recruit Myosin-II Binding Factor Bni5 to the Bud Neck in Saccharomyces cerevisiae.

Unlabelled: Septins are a conserved family of GTP-binding proteins that form heterooctameric complexes that assemble into higher-order structures. In yeast, septin superstructure at the bud neck serves as a barrier to separate a daughter cell from its mother and as a scaffold to recruit the proteins that execute cytokinesis. However, how septins recruit specific factors has not been well characterized.

Comprehensive Genetic Analysis of Paralogous Terminal Septin Subunits Shs1 and Cdc11 in Saccharomyces cerevisiae.

Septins are a family of GTP-binding proteins considered to be cytoskeletal elements because they self-assemble into filaments and other higher-order structures in vivo. In budding yeast, septins establish a diffusion barrier at the bud neck between a mother and daughter cell, promote membrane curvature there, and serve as a scaffold to recruit other proteins to the site of cytokinesis. However, the mechanism by which any septin engages a partner protein has been unclear.

Sorting of the yeast vacuolar-type, proton-translocating ATPase enzyme complex (V-ATPase): identification of a necessary and sufficient Golgi/endosomal retention signal in Stv1p.

Subunit a of the yeast vacuolar-type, proton-translocating ATPase enzyme complex (V-ATPase) is responsible for both proton translocation and subcellular localization of this highly conserved molecular machine. Inclusion of the Vph1p isoform causes the V-ATPase complex to traffic to the vacuolar membrane, whereas incorporation of Stv1p causes continued cycling between the trans-Golgi and endosome. We previously demonstrated that this targeting information is contained within the cytosolic, N-terminal portion of V-ATPase subunit a (Stv1p).

Evolution of increased complexity in a molecular machine.

Many cellular processes are carried out by molecular 'machines'-assemblies of multiple differentiated proteins that physically interact to execute biological functions. Despite much speculation, strong evidence of the mechanisms by which these assemblies evolved is lacking. Here we use ancestral gene resurrection and manipulative genetic experiments to determine how the complexity of an essential molecular machine--the hexameric transmembrane ring of the eukaryotic V-ATPase proton pump--increased hundreds of millions of years ago.