Improved Blue, Green, and Red Fluorescent Protein Tagging Vectors for S. cerevisiae

Lee, Sidae; Lim, Wendell A.; Thorn, Kurt S.

doi:10.1371/journal.pone.0067902

Cited by 207 publications

(231 citation statements)

References 37 publications

Supporting

Mentioning

225

Contrasting

Order By: Relevance

“…All media were generated as previously described (Ho and Burgess 2011;Lui et al 2013). Gene knockouts and fluorescent tagging were constructed using standard tailed PCR based gene replacement and tagging techniques (Longtine et al 1998;Goldstein and McCusker 1999;Sheff and Thorn 2004;Lee et al 2013). All gene knockouts were confirmed by PCR and new alleles were confirmed by DNA sequencing.…”

Section: Strainsmentioning

confidence: 99%

“…Chromosome spreads were prepared according to Rockmill (2009) and imaged using a Nikon Structured Illumination super-resolution microscope. The fluorophores imaged in this paper were DAPI, eGFP, mRuby2, and mCherry (Sheff and Thorn 2004;Lee et al 2013). Primary antibodies used in this study include rabbit polyclonal antibody to GFP (Noldus Information Technology NB600-308; 1:2000 dilution), mouse monoclonal antibody to mCherry (World Lab ATB-T5604; 1:2000 dilution).…”

Section: Spotting Assay For Vegetative Growthmentioning

confidence: 99%

See 1 more Smart Citation

The Nucleoporin Nup2 Contains a Meiotic-Autonomous Region that Promotes the Dynamic Chromosome Events of Meiosis

Chu

Gromova²,

Newman³

et al. 2017

Genetics

View full text Add to dashboard Cite

Meiosis is a specialized cellular program required to create haploid gametes from diploid parent cells. Homologous chromosomes pair, synapse, and recombine in a dynamic environment that accommodates gross chromosome reorganization and significant chromosome motion, which are critical for normal chromosome segregation. In Saccharomyces cerevisiae, Ndj1 is a meiotic telomere-associated protein required for physically attaching telomeres to proteins embedded in the nuclear envelope. In this study, we identified additional proteins that act at the nuclear periphery from meiotic cell extracts, including Nup2, a nonessential nucleoporin with a known role in tethering interstitial chromosomal loci to the nuclear pore complex. We found that deleting NUP2 affects meiotic progression and spore viability, and gives increased levels of recombination intermediates and products. We identified a previously uncharacterized 125 aa region of Nup2 that is necessary and sufficient for its meiotic function, thus behaving as a meiotic autonomous region (MAR). Nup2-MAR forms distinct foci on spread meiotic chromosomes, with a subset overlapping with Ndj1 foci. Localization of Nup2-MAR to meiotic chromosomes does not require Ndj1, nor does Ndj1 localization require Nup2, suggesting these proteins function in different pathways, and their interaction is weak or indirect. Instead, several severe synthetic phenotypes are associated with the nup2D ndj1D double mutant, including delayed turnover of recombination joint molecules, and a failure to undergo nuclear divisions without also arresting the meiotic program. These data suggest Nup2 and Ndj1 support partially overlapping functions that promote two different levels of meiotic chromosome organization necessary to withstand a dynamic stage of the eukaryotic life cycle.

show abstract

Section: Strainsmentioning

confidence: 99%

Section: Spotting Assay For Vegetative Growthmentioning

confidence: 99%

The Nucleoporin Nup2 Contains a Meiotic-Autonomous Region that Promotes the Dynamic Chromosome Events of Meiosis

Chu

Gromova²,

Newman³

et al. 2017

Genetics

View full text Add to dashboard Cite

show abstract

“…Due to the observed PSM defect in sps1D cells, we reexamined sfGFPSps1 localization and detected a dim, transient localization at the PSM ( Figure S2). sfGFP folds and matures quickly but photobleaches rapidly (Lee et al 2013;Slubowski et al 2015). The PSM localization of sfGFP-Sps1 fades to undetectable levels within 3 sec of epifluorescent illumination, making it difficult to observe.…”

Section: Sps1 Has a Dynamic Localization During Sporulationmentioning

confidence: 99%

“…Standard genetic methods were used to create strains unless otherwise noted (Rose and Fink 1990). Epitope-tagged strains and gene knockout strains were created using PCR-mediated integration as previously described (Longtine et al 1998;Lee et al 2013). Primers and plasmids used in this study are listed in Table S3 and Table S4.…”

mentioning

confidence: 99%

Timely Closure of the Prospore Membrane Requires SPS1 and SPO77 in Saccharomyces cerevisiae

et al. 2016

View full text Add to dashboard Cite

During sporulation in Saccharomyces cerevisiae, a double lipid bilayer called the prospore membrane is formed de novo, growing around each meiotic nucleus and ultimately closing to create four new cells within the mother cell. Here we show that SPS1, which encodes a kinase belonging to the germinal center kinase III family, is involved in prospore membrane development and is required for prospore membrane closure. We find that SPS1 genetically interacts with SPO77 and see that loss of either gene disrupts prospore membrane closure in a similar fashion. Specifically, cells lacking SPS1 and SPO77 produce hyperelongated prospore membranes from which the leading edge protein complex is not removed from the prospore membrane in a timely fashion. The SPS1/ SPO77 pathway is required for the proper phosphorylation and stability of Ssp1, a member of the leading edge protein complex that is removed and degraded when the prospore membrane closes. Genetic dissection of prospore membrane closure finds SPS1 and SPO77 act in parallel to a previously described pathway of prospore membrane closure that involves AMA1, an activator of the meiotic anaphase promoting complex.KEYWORDS prospore membrane; meiotic exit; anaphase promoting complex; sporulation; cytokinesis; germinal center kinase B IOLOGICAL membranes provide a barrier for inhibiting the flow of materials between a cell and its surroundings and for compartmentalizing the contents of the various organelles within a cell. The size and shape of membranes are central to their functions. Membranes are essential for many fundamental cellular processes including ion balance, energy generation, and secretion. In cell division, membrane dynamics are particularly important, where they act to segregate the contents of the cellular progeny.The process of cell division differs among cells. Most commonly, animal cells divide through a mechanism requiring the assembly of an actin-based contractile ring at the division site (reviewed in Green et al. 2012). This actomyosin ring contracts and matures, ultimately leading to scission of the membrane necks mediated by the ESCRTIII (endosomal sorting complex required for transport III) complex. However, other variations of cytokinesis occur: cellularization during early Drosophila embryogenesis, which requires the growth of membranes around the nuclei before the contractile event (Lee and Harris 2014) and cell division in plants, which requires the secretion of vesicles to the division plane to form the phragmoplast, which will eventually separate the two daughter cells (reviewed in Jürgens 2005). Actin is not always involved in cytokinesis; although prokaryotic cells have actin-like filaments (reviewed in Carballido-López 2006), these filaments are not used for cytokinesis (Pollard and Wu 2010). Similarly, cytokinesis in Trypanosoma brucei (a bikont eukaryote) does not require actin but seems to utilize microtubules (Wheeler et al. 2013). In the budding yeast Saccharomyces cerevisiae, an actin-based contractile ring plays a role in ...

show abstract

“…Finally, SNAP-SiMPull tags allow the fluorescent properties of the tagged molecule (e.g., the wavelength of emission) to be altered by addition of differently colored fluorophores to the cell lysate rather than by expression of different protein-FP fusions. This is particularly advantageous in cases where different FP fusions have been shown to impact protein function in different ways such as mislocalization of RFP but not GFP-tagged proteins (Lee et al 2013).…”

Section: Discussionmentioning

confidence: 99%

Rapid isolation and single-molecule analysis of ribonucleoproteins from cell lysate by SNAP-SiMPull

Rodgers

Paulson

Hoskins

2015

RNA

View full text Add to dashboard Cite

Large macromolecular complexes such as the spliceosomal small nuclear ribonucleoproteins (snRNPs) play a variety of roles within the cell. Despite their biological importance, biochemical studies of snRNPs and other machines are often thwarted by practical difficulties in the isolation of sufficient amounts of material. Studies of the snRNPs as well as other macromolecular machines would be greatly facilitated by new approaches that enable their isolation and biochemical characterization. One such approach is single-molecule pull-down (SiMPull) that combines in situ immunopurification of complexes from cell lysates with subsequent single-molecule fluorescence microscopy experiments. We report the development of a new method, called SNAP-SiMPull, that can readily be applied to studies of splicing factors and snRNPs isolated from whole-cell lysates. SNAP-SiMPull overcomes many of the limitations imposed by conventional SiMPull strategies that rely on fluorescent proteins. We have used SNAP-SiMPull to study the yeast branchpoint bridging protein (BBP) as well as the U1 and U6 snRNPs. SNAP-SiMPull will likely find broad use for rapidly isolating complex cellular machines for single-molecule fluorescence colocalization experiments.

show abstract

Improved Blue, Green, and Red Fluorescent Protein Tagging Vectors for S. cerevisiae

Cited by 207 publications

References 37 publications

The Nucleoporin Nup2 Contains a Meiotic-Autonomous Region that Promotes the Dynamic Chromosome Events of Meiosis

The Nucleoporin Nup2 Contains a Meiotic-Autonomous Region that Promotes the Dynamic Chromosome Events of Meiosis

Timely Closure of the Prospore Membrane Requires SPS1 and SPO77 in Saccharomyces cerevisiae

Rapid isolation and single-molecule analysis of ribonucleoproteins from cell lysate by SNAP-SiMPull

Contact Info

Product

Resources

About