Using rapid freeze and freeze-substitution for the preparation of yeast cells for electron microscopy and three-dimensional analysis

Giddings, Thomas H.; O’Toole, Eileen; Morphew, Mary K.; Mastronarde, David N.; McIntosh, J. Richard; Winey, Mark

doi:10.1016/s0091-679x(01)67003-1

Cited by 90 publications

(67 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cells for EM were prepared as described previously (Giddings et al 2001). Thin sections were viewed on a Philips CM10 electron microscope (Philips Electronic Instruments Co.) and recorded by using a Gatan digital camera and Digital Micrograph Software package (Gatan Inc.).…”

Section: Electron Microscopymentioning

confidence: 99%

Virus-like particles of the Ty3 retrotransposon assemble in association with P-body components

Beliakova-Bethell¹,

Beckham²,

Giddings³

et al. 2005

RNA

Self Cite

137

View full text Add to dashboard Cite

Retroviruses and retrotransposons assemble intracellular immature core particles around a RNA genome, and nascent particles collect in association with membranes or as intracellular clusters. How and where genomic RNA are identified for retrovirus and retrotransposon assembly, and how translation and assembly processes are coordinated is poorly understood. To understand this process, the subcellular localization of Ty3 RNA and capsid proteins and virus-like particles was investigated. We demonstrate that mRNAs, proteins, and virus-like particles of the yeast Ty3 retrotransposon accumulate in association with cytoplasmic P-bodies, which are sites of mRNA translation repression, storage, and degradation. Deletions of genes encoding P-body proteins decreased Ty3 transposition and caused changes in the pattern of Ty3 foci, underscoring the biological significance of the association of Ty3 virus-like protein components and P-bodies. These results suggest the hypothesis that P-bodies may serve to segregate translation and assembly functions of the Ty3 genomic RNA to promote assembly of virus-like particles. Because Ty3 has features of a simple retrovirus and P-body functions are conserved between yeast and metazoan organisms, these findings may provide insights into host factors that facilitate retrovirus assembly.

show abstract

Section: Electron Microscopymentioning

confidence: 99%

Virus-like particles of the Ty3 retrotransposon assemble in association with P-body components

Beliakova-Bethell¹,

Beckham²,

Giddings³

et al. 2005

RNA

Self Cite

137

View full text Add to dashboard Cite

show abstract

“…Cells were fixed, processed, and imaged as described previously (Giddings et al, 2001). Twenty-four sfi1 cells were analyzed very thoroughly by serial section.…”

Section: Electron Microscopymentioning

confidence: 99%

“…Electron microscopy reveals duplicated but unseparated SPBs in the sfi1 C-terminal mutants. Cells were presynchronized cells in G1 with ␣-factor and released at 37°C for 3 h. Cells were high-pressure frozen, and then they were freeze-substituted in 2% osmium tetroxide and 0.1% uranyl acetate (see Giddings et al, 2001 tion results in a doubling of the bridge length, and the Sfi1 C termini are now positioned at the center of the bridge (Figure 7). This model was strongly supported by immuno-EM analysis of strains that expressed Sfi1p with either N-or C-terminal GFP tags: the N terminus was positioned next to the junction between the SPB and the proximal end of the half-bridge, whereas the C-terminal tag was found to be close to the distal end, or in the center of the bridge in paired SPBs (Li et al, 2006).…”

Section: Multiple Functions For Sfi1pmentioning

confidence: 99%

Novelsfi1Alleles Uncover Additional Functions for Sfi1p in Bipolar Spindle Assembly and Function

Anderson

Prudden

Prochnik

et al. 2007

MBoC

Self Cite

View full text Add to dashboard Cite

A variety of spindle and kinetochore defects have been shown to induce a mitotic delay through activation of the spindle checkpoint. With the aim of identifying novel mitotic defects we carried out a mad1 synthetic lethal screen in budding yeast. In this screen, four novel alleles of sfi1 were isolated. SFI1 is an essential gene, previously identified through its interaction with centrin/CDC31 and shown to be required for spindle pole body (SPB) duplication. The new mutations were all found in the C-terminal domain of Sfi1p, which has no known function, but it is well conserved among budding yeasts. Analysis of the novel sfi1 mutants, through a combination of light and electron microscopy, revealed duplicated SPBs <0.3 m apart. Importantly, these SPBs have completed duplication, but they are not separated, suggesting a possible defect in splitting of the bridge. We discuss possible roles for Sfi1p in this step in bipolar spindle assembly.

show abstract

“…Aliquots of cells expressing Ppc89-GFP were prepared for EM as described previously (Giddings et al, 2001). Briefly, cells were harvested by vacuum filtration onto 0.45-m Millipore filters and cryofixed by high-pressure freezing in a HPM-010 (BAL-TEC/RMC, Tucson, AZ).…”

Section: Electron Microscopymentioning

confidence: 99%

Ppc89 Links Multiple Proteins, Including the Septation Initiation Network, to the Core of the Fission Yeast Spindle-Pole Body

Rosenberg

Tomlin

McDonald

et al. 2006

MBoC

View full text Add to dashboard Cite

The spindle-pole body (SPB), the yeast analog of the centrosome, serves as the major microtubule (MT) organizing center in the yeast cell. In addition to this central function, the SPB organizes and concentrates proteins required for proper coordination between the nuclear-division cycle and cytokinesis. For example, the Schizosaccharomyces pombe septationinitiation network (SIN), which is responsible for initiating actomyosin ring constriction and septation, is assembled at the SPB through its two scaffolding components, Sid4 and Cdc11. In an effort to identify novel SIN interactors, we purified Cdc11 and identified by mass spectrometry a previously uncharacterized protein associated with it, Ppc89. Ppc89 localizes constitutively to the SPB and interacts directly with Sid4. A fusion between the N-terminal 300 amino acids of Sid4 and a SPB targeting domain of Ppc89 supplies the essential function of Sid4 in anchoring the SIN. ppc89⌬ cells are inviable and exhibit defects in SPB integrity, and hence in spindle formation, chromosome segregation, and SIN localization. Ppc89 overproduction is lethal, resulting primarily in a G2 arrest accompanied by massive enlargement of the SPB and increased SPB MT nucleation. These results suggest a fundamental role for Ppc89 in organization of the S. pombe SPB.

show abstract

Using rapid freeze and freeze-substitution for the preparation of yeast cells for electron microscopy and three-dimensional analysis

Cited by 90 publications

References 36 publications

Virus-like particles of the Ty3 retrotransposon assemble in association with P-body components

Virus-like particles of the Ty3 retrotransposon assemble in association with P-body components

Novelsfi1Alleles Uncover Additional Functions for Sfi1p in Bipolar Spindle Assembly and Function

Ppc89 Links Multiple Proteins, Including the Septation Initiation Network, to the Core of the Fission Yeast Spindle-Pole Body

Contact Info

Product

Resources

About