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Molecular Linkage Underlying Microtubule Orientation Toward Cortical Sites in Yeast

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Science  24 Mar 2000:
Vol. 287, Issue 5461, pp. 2257-2259
DOI: 10.1126/science.287.5461.2257

Abstract

Selective microtubule orientation toward spatially defined cortical sites is critical to polarized cellular processes as diverse as axon outgrowth and T cell cytotoxicity. In yeast, oriented cytoplasmic microtubules align the mitotic spindle between mother and bud. The cortical marker protein Kar9 localizes to the bud tip and is required for the orientation of microtubules toward this region. Here, we show that Kar9 directs microtubule orientation by acting through Bim1, a conserved microtubule-binding protein. Bim1 homolog EB1 was originally identified through its interaction with adenomatous polyposis coli (APC) tumor suppressor, raising the possibility that an APC-EB1 linkage orients microtubules in higher cells.

The orientation of microtubules toward defined cortical sites in eukaryotic cells is a common theme in the regulation of nuclear position, the targeting of secretion, the navigation of growth cones, and the alignment of mitotic spindles. However, identification of the molecular means by which microtubules associate with spatially defined sites on the cortex has remained elusive. Budding yeast must align the mitotic spindle in parallel to the mother-bud axis to segregate one nucleus to each progeny cell. It is thought that cytoplasmic microtubules are selectively oriented toward the bud and that the forces generated by microtubule motors and microtubule shortening produce the movement and alignment of the spindle (1–5). The budding yeast KAR9 was genetically defined as being required for spindle alignment (6,7), and the Kar9 protein localizes to the tips of buds and mating projections, two positions toward which microtubules become oriented (7). No microtubule binding has been shown for Kar9.

To determine the mechanism by which microtubules are oriented toward the bud tip in yeast, a two-hybrid screen was performed with Kar9. One interacting protein was Bim1, which was isolated in a two-hybrid screen against α-tubulin (BIM, binds tomicrotubules) (8). Bim1 exhibited strong and specific interactions with Kar9 (Fig. 1A). Although direct binding of Bim1 to microtubules has not yet been reported, Bim1 does associate with the microtubule cytoskeleton and affects microtubule dynamics in yeast (8, 9). Bim1 may also be a component of a checkpoint pathway that monitors mitotic spindle alignment (10).

Figure 1

Specific binding between Kar9 and Bim1. (A) Two-hybrid interaction between Kar9 fused to the Gal4 DNA binding domain (DBD) and Bim1 fused to the Gal4 activation domain (AD) were measured by standard methods (27). Numbers represent units of β-galactosidase activity. (B) GST-Bim1 precipitates Kar9 in vitro. Interactions between the indicated GST fusion proteins and Kar9 or luciferase were detected by autoradiography (28). (C) GST-Kar9 precipitates Bim1 from crude yeast lysates. Extracts from strains with either myc-tagged Bim1 or a myc-tagged control protein, Ynl240c, were incubated with GST, GST-Kar9, or GST-Hof1 (28). Myc-tagged proteins were detected by immunoblotting. B, myc-tagged Bim1; Y, myc-tagged control Ynl240c. (D) Kar9 and Bim1 coprecipitate in yeast extracts. Extracts of strains expressing GST-Kar9 and either myc-tagged Bim1 or myc-tagged Ynl240c were precipitated with glutathione sepharose beads and were immunoblotted with antibodies to myc (29).

Specific binding was observed between a fusion protein made of glutathione S-transferase and Bim1 (GST-Bim1), which was purified fromEscherichia coli, and Kar9 that was synthesized and radiolabeled in vitro (Fig. 1B). GST-Bim1 bound to radiolabeled Kar9 protein and showed no affinity for the control protein luciferase. A control fusion protein, GST-Hof1, showed no affinity for Kar9. In further experiments, a GST-Kar9 fusion protein bound to the epitope-tagged Bim1 in crude yeast extracts (Fig. 1C). Finally, GST-Kar9 and tagged Bim1 coprecipitated from extracts of yeast expressing both proteins (Fig. 1D).

Genetic evidence has suggested that spindle alignment in yeast is produced by two independent pathways: one defined by KAR9and the other defined by dynein and dynactin components. Because the pathways overlap in directing spindle alignment, the combination of mutations between pathways leads to a complete block in spindle alignment and to lethality (7, 11). When mutations affecting components of the same pathway are combined, the double-mutant phenotype is no more severe than either of the single-mutant phenotypes. Binding between Kar9 and Bim1 suggests that Bim1 is a component of the Kar9 pathway. Although bim1 mutations are lethal in combination with mutations affecting the dynactin component (act5), this genetic interaction has been interpreted to indicate that Bim1 is part of a checkpoint that protects the cell from dividing prematurely if a defect in spindle alignment occurs (10). An alternative view is that Bim1 plays a mechanistic role in microtubule orientation and spindle alignment. This mechanistic role is consistent with the observations of Schwartz et al., who reported that bim1 mutants exhibit a microtubule orientation defect during mating, where cells are arrested in G1 and a mitotic spindle checkpoint would be irrelevant (8). Although the two proposed roles for Bim1 are not mutually exclusive, if Bim1 is a component of a general checkpoint that monitors spindle alignment, then bim1 should be lethal in combination with kar9, a mutant that has a spindle alignment defect. Unlike the lethal bim1act5 combination, thebim1kar9 double mutant was viable and had a growth rate identical to that of the bim1 or kar9 single mutant (12). Analyzed quantitatively, the bim1 andkar9 mutants had similar defects in spindle movement and alignment, and importantly the phenotype of the bim1kar9double mutant was no more severe than the phenotypes of the single mutants (Fig. 2, A and B). In a control experiment, combiningkar9 with a mutation in BIK1, which has similar genetic properties to bim1 and encodes a microtubule-binding protein (13), leads to a double-mutant phenotype which is enhanced in severity as compared to the phenotypes of the single mutants (7, 12). The finding that the kar9 andbim1 mutations are not additive in terms of phenotype suggests that their products act in the same pathway or work together as a complex.

Figure 2

kar9 and bim1 mutants show similar defects in spindle migration and alignment whereas double mutants show no enhancement in the severity of their phenotype. (A) Effects of kar9 and bim1 mutations on nuclear migration. In large budded cells, undivided nuclei were scored as being in the bud-proximal (solid bar) or bud-distal (open bar) hemisphere of the mother cell (30). (B) Large budded cells with elongated or divided nuclei were scored as to whether the nuclear or spindle alignment had (solid bar) or had not (open bar) occurred properly (30).

Kar9 localizes to a cap at the distal tip of the bud and to cytoplasmic microtubules in yeast (7). Bim1 also localizes to microtubules with some preference for their distal ends (8, 9). When overexpressed, Kar9 was detected in association with cytoplasmic microtubules (Fig. 3A). This association was abolished in abim1-null mutant. According to the view that Kar9 acts on microtubules through Bim1, Bim1 should be able to recruit Kar9 to microtubules in vitro. Kar9 did not readily associate with microtubules in vitro (Fig. 3, B and C). However, the addition of purified Bim1 promoted Kar9 co-sedimentation with microtubules. Thus, Kar9 appears to interact with cytoplasmic microtubules through Bim1.

Figure 3

The association of Kar9 with microtubules is dependent on BIM1 in vivo and in vitro.(A) Kar9-GFP, microtubules (MT), and DNA as seen in wild-type and bim1Δ cells (31). The association of Kar9 with microtubules, observed in wild-type cells, was abolished in a bim1 mutant. (B) GST-Bim1 sediments (P) with microtubules in a standard microtubule pelleting assay, but remains in the supernatant (S) in the absence of microtubules (32). (C) Bim1 recruits Kar9 to microtubules. In vitro–translated Kar9 efficiently co-pellets with microtubules only when added in combination with Bim1 (32, 33).

Overexpression of KAR9 often resulted in the aberrant translocation of the entire nucleus and spindle into the bud (Fig. 4). To test the view that Kar9 acts through Bim1, we examined the extent of this gain-of-function phenotype in a bim1-null mutant and a bik1-null control mutant. When KAR9 was overexpressed, 28% of cells with large buds had nuclei translocated into the bud. Thebim1-null mutation reversed this phenotype to a low level (2%), the same level of nuclear translocation observed beforeKAR9 expression, while the bik1-null mutation had little effect (32%).

Figure 4

Overexpression of KAR9 leads to excessive nuclear migration, which is abolished by mutations inBIM1. (A) A wild-type cell is shown with its nucleus positioned at the mother-bud neck. A cell overexpressingKAR9 is shown with the nucleus completely translocated into the bud. (B) The percentage of large budded cells with the nucleus completely translocated into the bud is shown in the indicated strains with or without KAR9 overexpression.n > 300 cells (31, 33).

We propose the following mechanism by which microtubules are oriented toward the bud tip. The exploratory movements of individual microtubules search the cytoplasm for stabilization sites (3, 4,14). When a growing microtubule extends into the bud, the interaction between cortical Kar9 and microtubule-bound Bim1 stabilizes the microtubule tip. Forces from motors and/or microtubule shortening act on this microtubule tether to align the spindle. Bim1 belongs to the EB1 family of microtubule-binding proteins. EB1 was identified through its binding to the adenomatous polyposis coli (APC) tumor suppressor protein, defects of which contribute to colon cancer (15–17). APC localizes to cortical sites associated with microtubule stabilization, supporting the possibility of a linkage analogous to Kar9-Bim1 whereby APC acts through EB1 to orient microtubules to spatially defined cortical sites (18).

  • * To whom correspondence should be addressed. E-mail: chant{at}fas.harvard.edu

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