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. 2010 Nov 1;191(3):493–503. doi: 10.1083/jcb.201006092

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© 2010 Moore et al.

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Figure 7. — The accumulation of Elm1 at the bud neck promotes its function in the SPC. (A) The localization of Elm1 to the bud neck requires its carboxy-terminal region. Tandem RFP/tdimer2 was integrated behind codon 420 of ELM1. Cells also express GFP-Tub1. Images were collected on a wide-field microscope. Strain: yJC6854. (B) Kin4 activity is supported by overexpressed elm1Δ420 or chimeric Bni4-elm1Δ420. Strains with high-copy plasmids containing KIN4 under the control of a galactose-inducible promoter and 10-fold dilution series were spotted onto media selective for plasmid retention and containing either galactose to induce expression or glucose to inhibit expression. Strains containing empty vector are shown as controls. Strains: wild type (wt), yJC2295; elm1Δ, yJC5254; elm1Δ420-tdimer2, yJC6849; P_GAL1-elm1Δ420-tdimer2, yJC7286; BNI4-elm1Δ420-tdimer2, yJC7292; and KCC4-elm1Δ420-tdimer2, yJC7293. Plasmids: pGAL-KIN4, pBJ1651 and vector, pBJ216. (C) Multi-MTOC assay for SPC function. Mother compartment is indicated by light gray bars, and multiple MTOCs resulting from checkpoint failure are indicated by dark gray bars. Values are the means of 10 counts of at least 50 cells from two separate experiments. Error bars are the standard error of the means. Strains: dyn1Δ, yJC5603; dyn1Δ elm1Δ, yJC7079; dyn1Δ P_GAL1-elm1Δ420-tdimer2, yJC7299,7300; dyn1Δ elm1Δ BNI4-elm1Δ420-tdimer2, yJC7296,7297; and dyn1Δ elm1Δ KCC4-elm1Δ420-tdimer2, yJC7298. (D) Bni4-elm1Δ420-tdimer2 localizes to the bud neck when the mitotic spindle is in the mother compartment. Cells are dyn1Δ mutants that also express GFP-Tub1. Images were collected on a wide-field microscope. Arrowhead points to Bni4-elm1Δ420-tdimer2 at the bud neck. Strain: yJC7296. (E) Quantification of fluorescence intensities for Elm1-tdimer2 and Bni4-elm1Δ420-tdimer2 across the bud neck. Fluorescence intensities were measured (see Materials and methods) in cells with mitotic spindles in the mother based on GFP-tubulin. Plotted values are the means from 12 cells for each strain. Solid lines indicate outlines of cells. Strains: Elm1-tdimer2, yJC6852 and Bni4-elm1Δ420-tdimer2, yJC7296. a.u., arbitrary units. Bars, 2 µm.