γ-Tubulin Is Required for Proper Recruitment and Assembly of Kar9–Bim1 Complexes in Budding Yeast

Abstract

Microtubule plus-end-interacting proteins (؉TIPs) promote the dynamic interactions between the plus ends (؉ends) of astral microtubules and cortical actin that are required for preanaphase spindle positioning. Paradoxically, ؉TIPs such as the EB1 orthologue Bim1 and Kar9 also associate with spindle pole bodies (SPBs), the centrosome equivalent in budding yeast. Here, we show that deletion of four C-terminal residues of the budding yeast ␥-tubulin Tub4 (tub4-⌬dsyl) perturbs Bim1 and Kar9 localization to SPBs and Kar9-dependant spindle positioning. Surprisingly, we find Kar9 localizes to microtubule ؉ends in tub4-⌬dsyl cells, but these microtubules fail to position the spindle when targeted to the bud. Using cofluorescence and coaffinity purification, we show Kar9 complexes in tub4-⌬dsyl cells contain reduced levels of Bim1. Astral microtubule dynamics is suppressed in tub4-⌬dsyl cells, but it are restored by deletion of Kar9. Moreover, Myo2-and F-actin-dependent dwelling of Kar9 in the bud is observed in tub4-⌬dsyl cells, suggesting defective Kar9 complexes tether microtubule ؉ends to the cortex. Overproduction of Bim1, but not Kar9, restores Kar9-dependent spindle positioning in the tub4-⌬dsyl mutant, reduces cortical dwelling, and promotes Bim1-Kar9 interactions. We propose that SPBs, via the tail of Tub4, promote the assembly of functional ؉TIP complexes before their deployment to microtubule ؉ends.

Post-translational Modifications Regulate Assembly of Early Spindle Orientation Complex in Yeast

“…Leisner et al (18) showed that sumoylation occurs during metaphase and that Kar9p(4R) fails to localize to only one of the spindle pole bodies. It was further shown that the Bim1p-Kar9p complex assembles before loading onto the microtubules via the spindle pole bodies (37). Here, we have demonstrated that Kar9p sumoylation is important for complex formation with Bim1p.…”

Regulation of Glia Number in Drosophila by Rap/Fzr, an Activator of the Anaphase-Promoting Complex, and Loco, an RGS Protein

“…Significance and P-values were derived using Student's t-test. For high-intensity fluorescent images involving densely packed tissue, the Volocity software uses voxel intensity measurements to quantify cell number (Cuschieri et al 2006). For larval brains stained with anti-Dachshund antibodies, confocal image stacks were acquired using the same settings for all samples, with a gain of 815.…”

Spc24 and Stu2 Promote Spindle Integrity When DNA Replication Is Stalled

“…After 30 min., samples were taken every 15 min and fixed in 70% ethanol. Stu2-VFP in fixed cells (Figure 7) was imaged with a WaveFX spinning disk confocal microscope (Quorum Technologies, Guelph, Ontario, Canada) as described previously (Cuschieri et al, 2006) without agar pads. Optical sections (0.5 m) were acquired through a Ϯ2.5-m z-plane (total of 5.0-m stack) by using Volocity 3DM acquisition software (Improvision, Conventry, United Kingdom).…”

“…Cells were mounted on a prewarmed 30°C heated stage and allowed to equilibrate for 15 min before imaging. Multichannel four-dimensional imaging of Spc29-CFP and Stu2-VFP fluorescent fusion proteins was performed using a WaveFX spinning disk confocal system (Quorum Technologies) as described previously (Cuschieri et al, 2006). A Tokai Hit stage warmer was used to shift cells from 25 to 30°C, image acquisition commenced 15 min after the stage reached 30°C.…”