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. 2015 Apr 27;209(2):247–259. doi: 10.1083/jcb.201408118

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© 2015 Li et al.

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Figure 2. — Localization of Ndj1 to SPB depends on Mps3. (A) Colocalization of Ndj1 and Mps3 during yeast meiosis. Time-lapse live-cell microscopy was performed as in Fig. 1 G. Strain HY3881 was used. Projected images of eight z sections are shown. Ndj1 was tagged with GFP (green), Mps3 with RFP (red). Time 0 is defined as the point of SPB separation. (B) Immunoblot showing the depletion of Mps3 protein in P_CLB2-MPS3 cells (HY3911) during meiosis. Note that in wild-type cells (HY3871), Mps3 peaks around 4 h after induction of meiosis, then appears to be modified and degraded during meiosis. (C) Localization of Ndj1 in P_CLB2-MPS3 cells (HY3911). Live-cell microscopy was performed as in A. SPB was marked by Spc42-RFP (red). Note that Ndj1 (green) fails to form a focus at the SPB. Quantification of Ndj1 localization to the SPB is shown to the right. The data shown are from a single representative experiment out of four repeats. For the experiment shown, n = 200. (D and E) Localization of Mps3-GFP in wild-type (HY4418) and ndj1Δ (HY4419) cells during meiosis. Time-lapse microscopy was performed as in A. Mps3 is tagged with GFP (green), Tub4 with RFP (red). Time 0 is defined as the point of SPB separation. Note that Mps3 localizes to the SPB in both strains (arrows). (F) Ndj1 localization in csm4Δ cells. Strains HY4086 (wild-type) and HY4852 (csm4Δ) were used. Live-cell microscopy was performed as in A, and three continuous z sections are shown. Ndj1-GFP, green; Tub4-RFP, red. (G) Csm4 localization in meiotic cells (HY4383). A GFP-CSM4 allele was used to localize Csm4 (green) in meiotic cells. Note that Csm4 does not form a focus at the SPB, as determined by Tub4-RFP (red). Bars, 2 µm.