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*CIL – Cell Image Library accession number. Please use this to reference an image.

CIL:13863*  Cite 

In tem1Δ cells expressing a Bfa1-Tem1 fusion protein, Cdc15-3HA (red) loads prematurely onto spindle pole bodies during metaphase as determined by spindle morphology (tubulin, green) and nuclear morphology (DAPI, blue). The Bfa1-Tem1 fusion protein is constitutively and asymmetrically targeted to spindle pole bodies. Compare with control image CIL# 13864. Image is Fig 3C, bottom panels, in J Cell Biol. (2011) 192: 599-614. Images in Fig 3 include CIL# 13862, 13863, 13864, 13865, 13866, 13867.

Technical Details

Cells (MATa tem1::KanMX ura3::eGFP-BFA1–TEM1::URA3 cdc15::CDC15-3HA), grown in rich media with glucose, were fixed for 15 min in 3.7% formaldehyde and 0.1 M potassium phosphate buffer, pH 6.4. Cells were then washed twice with 0.1 M potassium phosphate buffer, pH 6.4, and resuspended in 1.2 M sorbitol in 0.12 M K2HPO4/0.033 M citric acid, pH 5.9. Fixed cells were digested with 0.1 mg/ml zymolyase-100T (US Biological) and 1/10 volume of glusulase (PerkinElmer) at 30C for 15 min, washed once, and resuspended in 1.2 M sorbitol in 0.12 M K2HPO4/0.033 M citric acid, pH 5.9. Primary antibodies were anti-HA monoclonal antibody (HA.11; 1:500) and anti-tubulin (Abcam; 1:500). Secondary antibodies were: anti-mouse Cy3 (for HA) and anti-rat FITC (for tubulin). Cells were resuspended in DAPI (1 mg/ml). Imaging was performed at 25C using a Leica DM6000 microscope equipped with a 100x/1.40 NA oil immersion objective lens, A4, L5, and TX2 filters, and a digital CCD camera (DFC350, Leica). Pictures were processed with LAS AF (leica) and ImageJ software.

Biological Sources
NCBI Organism Classification
Saccharomyces cerevisiae
Cell Line
Cellular Component
spindle pole body
spindle pole body
tubulin complex
Mauricio Valerio-Santiago
Fernando Monje-Casas
J Cell Biol. (2011) 192: 599-614.
Digital Object Identifier (DOI)
Archival Resource Key (ARK)
Grouping This image is part of a group.
Sample Preparation
formaldehyde fixed tissue
Relation To Intact Cell
whole mounted tissue
Spatial Axis Image Size Pixel Size
X 187px 0.0642µm
Y 187px 0.0642µm