Journal: The Journal of Cell Biology

Article Title: Mps3p is a novel component of the yeast spindle pole body that interacts with the yeast centrin homologue Cdc31p

doi: 10.1083/jcb.200208169

Figure Lengend Snippet: Mps3p is required early in G1 for SPB duplication. Wild-type (SLJ001) and mps3-1 mutant (SLJ910) cells were synchronized in G1 by treatment with 1 μg/ml α-factor for 4 h at 23°C and then released into fresh media at 23°C or 36°C. Samples from each culture 1, 2, and 3 h after release from α-factor (αf) were removed and analyzed. Asynchronous cells (A) are shown as controls. (A) Flow cytometric analysis of DNA content indicated that mps3-1 mutants at 36°C do not arrest during the first mitosis. The biphasic peaks in wild-type cells represent cells with G1 (1N) and G2/M (2N) DNA content. mps3-1 mutants in G1 and G2/M have a 2N and 4N DNA content, respectively. (B) For each sample, cells were examined by indirect immunofluorescence microscopy. At 1 and 3 h after release, the number of bipolar (black bars) and monopolar (white bars) spindles was determined by counting the number of Tub4p signals per cell ( n = 200). Numbers are averages based on three independent experiments. (C) Indirect immunofluorescence images from mps3-1 mutants released for 1 and 3 h from α-factor at 36°C show that Mps3p function is not required for SPB duplication and bipolar spindle formation during the first mitosis, but that Mps3p is required for SPB duplication and bipolar spindle formation during the second cell cycle. The mitotic spindle was detected using anti-tubulin antibodies (green), SPBs were visualized with anti-Tub4p antibodies (red), and DNA was stained with DAPI (blue). Bars, 5 μm.

Article Snippet: The following primary antibody dilutions were used: microtubules, 1:500 rat anti-tubulin antibody YOL 1/34 (Accurate Chemical & Scientific Corp.); SPBs, 1:500 affinity-purified anti-Tub4p antibodies (a gift of T. Stearns, Stanford University, Palo Alto, CA); Mps3–GFP, 1:5,000 affinity-purified anti-GFP polyclonal antibodies (CLONTECH Laboratories, Inc.); and Cdc31p, 1:500 affinity-purified anti-Cdc31p antibodies.

Techniques: Mutagenesis, Immunofluorescence, Microscopy, Staining

Journal: The Journal of Cell Biology

Article Title: Mps3p is a novel component of the yeast spindle pole body that interacts with the yeast centrin homologue Cdc31p

doi: 10.1083/jcb.200208169

Figure Lengend Snippet: Mps3p is a component of the SPB. Wild-type diploid cells (SLJ126) and homozygous diploids containing MPS3–GFP (SLJ915) were grown to mid-log phase at 23°C. (A) Cells were harvested, and lysates were analyzed by Western blotting with anti-GFP antibodies. (B) Paraformaldehyde-fixed wild-type and Mps3p–GFP cells were stained with DAPI to visualize the DNA (blue), anti-tubulin antibodies to recognize microtubules (red), and anti-GFP antibodies to localize Mps3p–GFP (green, arrows). The merged image shows that Mps3p–GFP localizes to discrete foci at the poles of the mitotic spindle (yellow). Bar, 5 μm. (C and D) Mps3p–GFP localization was also analyzed in living cells by epifluorescence microscopy. Mps3p–GFP (green) appears as one to two foci coincident with the DNA (blue). In addition, a fraction of Mps3p–GFP was also detected at the nuclear envelope (D, arrows). Bar, 5 μm. (E) Localization of Mps3p–GFP to the SPB was confirmed by immuno-EM using anti-GFP antibodies and colloidal gold–conjugated secondary antibodies. Thin sections of two different cells are shown. The SPB is embedded in the nuclear envelope (NE), which separates the nucleus (N) and cytoplasm (C). Microtubules (MTs) can be see on the nuclear side of the SPB. Arrowheads indicate the position of the gold particles that recognize Mps3p–GFP. A total of 37 SPBs were analyzed, and the distribution of gold particles to the central plaque (CP), outer plaque (OP), inner plaque (IP), bridge (B), and nuclear envelope (NE) is indicated. In some sections, localization to a discrete SPB substructure could not be determined (SPB). Bar, 0.1 μm.

Article Snippet: The following primary antibody dilutions were used: microtubules, 1:500 rat anti-tubulin antibody YOL 1/34 (Accurate Chemical & Scientific Corp.); SPBs, 1:500 affinity-purified anti-Tub4p antibodies (a gift of T. Stearns, Stanford University, Palo Alto, CA); Mps3–GFP, 1:5,000 affinity-purified anti-GFP polyclonal antibodies (CLONTECH Laboratories, Inc.); and Cdc31p, 1:500 affinity-purified anti-Cdc31p antibodies.

Techniques: Western Blot, Staining, Epifluorescence Microscopy

Journal: The Journal of Cell Biology

Article Title: Mps3p is a novel component of the yeast spindle pole body that interacts with the yeast centrin homologue Cdc31p

doi: 10.1083/jcb.200208169

Figure Lengend Snippet: Mps3p interacts with Cdc31p. (A) Bacterial extracts from cells expressing GST, GST–Kar1p, GST–Mps3p-Ct, or nothing (−) were electrophoretically separated, and proteins were transferred to nitrocellulose. Expression of GST and the GST fusion proteins was detected by Western blotting with anti-GST antibodies (left). Membranes were also probed with purified 6His–Cdc31p labeled with the infrared dye Alexa ® 680 in a gel overlay assay to determine if proteins in each extract were capable of binding Cdc31p (right). (B) 2μ plasmids containing MPS3 or CDC31 or the empty vector were transformed into mps3-1 (SLJ910). Cells were grown in media lacking uracil overnight at 23°C to a density of ∼4 OD 600 U/ml, serially diluted fourfold, and spotted onto SD URA − plates. Plates were incubated at 23°C or 36°C for 3 d. (C) The mps3-1 mutant (SLJ775) was crossed to strains containing mutations in CDC31 ( cdc31-2 , SLJ809, and CDC31-16 , SLJ906). After loss of plasmids in the heterozygous diploid, p URA3-MPS3 (pSJ140) was retransformed, and cells were sporulated at 23°C, dissected, and analyzed. Progeny from tetratype spores were grown overnight at 23°C to a density of ∼5 OD 600 U/ml, serially diluted fourfold, and spotted onto SD URA − or 5-fluoro-orotic acid plates. Plates were incubated for 3 d at 23°C or 30°C. (D) The mps3-1 mutant (SLJ775) was crossed to the kar1- Δ 17 mutant (SLJ843), and localization of Cdc31p in progeny from a tetratype tetrad was analyzed after a 4-h shift to 36°C. Cells were stained with anti-Cdc31p (red) and anti-tubulin (green) antibodies and with DAPI (blue) to visualize DNA. Arrows point to the diminished Cdc31p signal in mps3-1 and kar1- Δ 17 . No Cdc31p staining was observed in mps3-1 kar1- Δ 17 . The graph summarizes three independent localization experiments using two independently derived tetrads ( n = 200 in each sample). Bars, 5 μm.

Article Snippet: The following primary antibody dilutions were used: microtubules, 1:500 rat anti-tubulin antibody YOL 1/34 (Accurate Chemical & Scientific Corp.); SPBs, 1:500 affinity-purified anti-Tub4p antibodies (a gift of T. Stearns, Stanford University, Palo Alto, CA); Mps3–GFP, 1:5,000 affinity-purified anti-GFP polyclonal antibodies (CLONTECH Laboratories, Inc.); and Cdc31p, 1:500 affinity-purified anti-Cdc31p antibodies.

Techniques: Expressing, Western Blot, Purification, Labeling, Overlay Assay, Binding Assay, Plasmid Preparation, Transformation Assay, Incubation, Mutagenesis, Staining, Derivative Assay