Fig. 7
DyneinmMTBD exhibits reduced affinity for She1 in vitro and is She1-insensitive in vivo. a Schematic representation of the experimental setup used in b. b Relative recruitment of She1-TMR by monomeric GFP–dynein331 or GFP–dynein331 mMTBD. Different points reflect the mean fluorescence intensity values (along with standard deviations) for She1-TMR (fixed at 40 nM) vs. increasing concentrations (0–30 nM for wild-type and 0–100 nM for mMTBD) of indicated GFP–dynein331. c Two-hybrid assay demonstrating an interaction between the yeast derived dyneinMTBD and She1 (Methods). d Cartoon representation of the localization of full-length dynein heavy chain (Dyn1) in either wild-type (left) or She1-overexpressing cells (right). Note that the mechanism for plus end localization of dynein (which is MTBD-independent51) is distinct from that by which dynein binds along the length of astral microtubules upon She1 overexpression (MTBD-dependent; Supplementary Fig. 6). e, f Representative images of GAL1p:SHE1 cells expressing mRuby2-Tub1 (α-tubulin) and either Dyn1-3YFP (e) or Dyn1mMTBD-3YFP (f). Cells were grown to mid-log phase in SD media supplemented with raffinose (uninduced; −galactose) or galactose plus raffinose (induced for 3.5 h; +galactose) and then mounted on agarose pads for confocal fluorescence microscopy (blue arrows, plus end foci; blue arrowheads, cortical foci; red arrows, astral microtubule decoration; red arrowhead, cytoplasmic focus). Foci were identified in two-color movies and scored accordingly (scale bars, 1 µm). g, h Dynein-mediated spindle movements were quantitated in hydroxyurea (HU)-arrested kar9∆ cells with indicated DYN1 and SHE1 alleles. Cells were arrested with HU for 2.5 h, and then mounted on agarose pads containing HU for confocal fluorescence microscopy. Full Z-stacks of GFP-labeled microtubules (GFP-Tub1) were acquired every 10 s for 10 min. Cells with buds of at least 2.5 µm in diameter were chosen for analysis. Graphs depicting the number of dynein-mediated spindle movements (g) and the fraction of such events in which the spindle traversed the bud neck (h; in which the spindle midpoint crossed the bud neck) for the indicated yeast strains are shown (error bars, standard error of proportion; n ≥ 43 cells; n ≥ 155 events). P-values were calculated using a two-tailed unpaired t test
