Mitotic spindle positioning in Saccharomyces cerevisiae is accomplished by antagonistically acting microtubule motor proteins

Abstract

Proper positioning of the mitotic spindle is often essential for cell division and differentiation processes. The asymmetric cell division characteristic of budding yeast, Saccharomyces cerevisiae, requires that the spindle be positioned at the mother-bud neck and oriented along the mother-bud axis. The single dynein motor encoded by the S. cerevisiae genome performs an important but nonessential spindle-positioning role. We demonstrate that kinesin-related Kip3p makes a major contribution to spindle positioning in the absence of dynein. The elimination of Kip3p function in dyn1Delta cells severely compromised spindle movement to the mother-bud neck. In dyn1Delta cells that had completed positioning, elimination of Kip3p function caused spindles to mislocalize to distal positions in mother cell bodies. We also demonstrate that the spindle-positioning defects exhibited by dyn1 kip3 cells are caused, to a large extent, by the actions of kinesin- related Kip2p. Microtubules in kip2Delta cells were shorter and more sensitive to benomyl than wild-type, in contrast to the longer and benomyl-resistant microtubules found in dyn1Delta and kip3Delta cells. Most significantly, the deletion of KIP2 greatly suppressed the spindle localization defect and slow growth exhibited by dyn1 kip3 cells. Likewise, induced expression of KIP2 caused spindles to mislocalize in cells deficient for dynein and Kip3p. Our findings indicate that Kip2p participates in normal spindle positioning but antagonizes a positioning mechanism acting in dyn1 kip3 cells. The observation that deletion of KIP2 could also suppress the inviability of dyn1Delta kar3Delta cells suggests that kinesin-related Kar3p also contributes to spindle positioning.

Figure 2

Binucleate cell production and growth properties of motor mutants. (A) dyn1Δ kip3Δ cells form binucleate cells at 26°C. The arrowhead points to a cell with an aberrant nuclear morphology. (B) Quantitation of binucleate cells produced at 26 and 12°C. For A and B, cells of the indicated genotypes were grown in rich media to log phase at 26°C and stained with DAPI or shifted to 12°C for 24 h before staining. Binucleate cells are those in which two or more nuclear DNA masses could be visualized in one cell body. Each value represents the average of three experiments in which 200 cells were examined. Error bars indicate the SEM. (C) Cells of the indicated genotypes were spotted onto solid media and incubated at the temperature noted. Two spots for each combination are shown, with the second equal to a 40-fold dilution of the first. Yeast strains used: wild-type (MAY589), kip3Δ (MAY4517), dyn1Δ (MAY4434), kip2Δ (MAY2038), dyn1Δ kip3Δ (MAY4544), kip2Δ kip3Δ (MAY4716), dyn1Δ kip2Δ (MAY4566), and dyn1Δ kip2Δ kip3Δ (MAY4560).

Figure 2

Binucleate cell production and growth properties of motor mutants. (A) dyn1Δ kip3Δ cells form binucleate cells at 26°C. The arrowhead points to a cell with an aberrant nuclear morphology. (B) Quantitation of binucleate cells produced at 26 and 12°C. For A and B, cells of the indicated genotypes were grown in rich media to log phase at 26°C and stained with DAPI or shifted to 12°C for 24 h before staining. Binucleate cells are those in which two or more nuclear DNA masses could be visualized in one cell body. Each value represents the average of three experiments in which 200 cells were examined. Error bars indicate the SEM. (C) Cells of the indicated genotypes were spotted onto solid media and incubated at the temperature noted. Two spots for each combination are shown, with the second equal to a 40-fold dilution of the first. Yeast strains used: wild-type (MAY589), kip3Δ (MAY4517), dyn1Δ (MAY4434), kip2Δ (MAY2038), dyn1Δ kip3Δ (MAY4544), kip2Δ kip3Δ (MAY4716), dyn1Δ kip2Δ (MAY4566), and dyn1Δ kip2Δ kip3Δ (MAY4560).

Figure 2

Binucleate cell production and growth properties of motor mutants. (A) dyn1Δ kip3Δ cells form binucleate cells at 26°C. The arrowhead points to a cell with an aberrant nuclear morphology. (B) Quantitation of binucleate cells produced at 26 and 12°C. For A and B, cells of the indicated genotypes were grown in rich media to log phase at 26°C and stained with DAPI or shifted to 12°C for 24 h before staining. Binucleate cells are those in which two or more nuclear DNA masses could be visualized in one cell body. Each value represents the average of three experiments in which 200 cells were examined. Error bars indicate the SEM. (C) Cells of the indicated genotypes were spotted onto solid media and incubated at the temperature noted. Two spots for each combination are shown, with the second equal to a 40-fold dilution of the first. Yeast strains used: wild-type (MAY589), kip3Δ (MAY4517), dyn1Δ (MAY4434), kip2Δ (MAY2038), dyn1Δ kip3Δ (MAY4544), kip2Δ kip3Δ (MAY4716), dyn1Δ kip2Δ (MAY4566), and dyn1Δ kip2Δ kip3Δ (MAY4560).

Figure 5

Benomyl resistance of motor mutants. Cells of the indicated genotypes were spotted to solid rich media containing the concentrations of benomyl indicated. Plates were incubated at 26°C for 3 d. Strains used are the same as in Fig. 2.

Figure 1

KIP2 is responsible for the growth defects of dyn1Δ kip3Δ and dyn1Δ kar3Δ cells. Wild-type (MAY591), dyn1Δ kip3Δ kip2Δ (MAY4560), and dyn1Δ kar3Δ kip2Δ (MAY4761) yeast strains were transformed with the KIP2-containing plasmid pFC50 and with a vector control (pRS317). Transformation plates were incubated at 26°C for 2 d.

Figure 3

Antitubulin immunofluorescence microscopy of motor mutants. Cells were synchronized with hydroxyurea in rich media at 26°C, fixed with formaldehyde, and processed for antitubulin immunofluorescence microscopy. Strains used are the same as in Fig. 2. Bar (top right), 2 μm.

Figure 4

Quantitation of microtubule phenotypes. Digitized images of the cells from Fig. 3 were analyzed for microtubule characteristics. (A) The percent of spindles with no visible cytoplasmic microtubules (cMTs). Values were determined from scoring all cells in four microscope fields (∼40 cells per field) and averaging. (B) The lengths of spindles (pole-to-pole distance) and cMTs were measured (see Materials and Methods). Values equal the average of between 94 to 166 measurements. For both A and B, error bars indicate the SEM.

Figure 6

Loss of Kip3p function in dynein-deficient cells caused spindle-positioning defects. Cell cultures of the indicated genotypes were synchronized with α-factor at 26°C and then shifted to 37°C. At the time points indicated, samples were removed and stained with DAPI. The top panel indicates the percent of cells that were large budded with the nucleus positioned away from the neck (defined as those in which the closest distance between the nucleus and the neck was greater than one half of the diameter of the entire nuclear DNA mass). The bottom panel indicates the percent of cells that were large budded with two DAPI-staining masses located within one cell body. •, dyn1Δ kip3Δ (pkip3-14) (MAY4921); □, dyn1Δ kip2Δ kip3Δ (pkip3-14) (MAY5009); ▴, dyn1Δ kip3Δ (pKIP3) (MAY4924); ♦, dyn1Δ kip2Δ kip3Δ (pKIP3) (MAY5008); ▪, wild-type (MAY589).

Figure 7

The spindle and nucleus were drawn away from the mother–bud neck after loss of Kip3p function in the absence of Dyn1p. Cell cycle progression of dyn1Δ kip3Δ (pkip3-14) strain (MAY4921) was arrested by growth in the presence of hydroxyurea at 26°C. The culture was then shifted to 37°C for 90 min. Samples taken before (top row) and after (bottom row) the temperature shift were processed for antitubulin immunofluorescence (left column) and DAPI staining (right column).

Figure 8

Kip2p caused nuclear mislocalization in cells deficient for dynein and Kip3p. (A) Quantitation of large-budded cells with mislocalized nuclei after hydroxyurea arrest at 26°C and then a shift to 37°C for the indicated times (see Fig. 7). Strains used were the same as in Fig. 6. (B) Quantitation of large-budded cells with mislocalized nuclei caused by galactose-induced expression of KIP2. Cells were synchronized with hydroxyurea and then switched to media containing galactose to induce expression of KIP2. Samples were analyzed at the indicated times after the addition of galactose. ▵, dyn1Δ kip2Δ kip3Δ (P_GAL-KIP2) (MAY4819); ✖, dyn1Δ kip2Δ kip3Δ (P_GAL-vector) (MAY4820); ○, wild-type (P_GAL-KIP2) (MAY4816); ⋄, wild-type (P_GAL-vector) (MAY4817).

Figure 9

Proposed functional relationship for the Kip2p, Kip3p, Kar3p, and dynein motors. The antagonistic actions of these motors leads to normal spindle positioning. Kip2p is proposed to antagonize the actions of Kip3p and Kar3p because deletion of KIP2 suppressed dyn1Δ kip3Δ and dyn1Δ kar3Δ, but not kip3Δ kar3Δ. Kip3p and Kar3p share a single arrow since they appear to overlap for an essential function that cannot be accomplished by dynein. Dynein is proposed to act in a distinct pathway leading to spindle positioning, although it is possible that it is antagonized by Kip2p as well.