MT–Bud6p dynamic interactions in kar9Δ cells
We have previously studied the dynamic behavior of astral MT–cortex interactions in wild-type cells expressing GFP-Bud6 and GFP-Tub1. This analysis showed a high incidence of MT interactions at GFP-Bud6 cortical sites throughout the cell cycle. Moreover, shrinkage of MTs at the cortex occurs at Bud6p sites. This mode of interaction is abolished in bud6
Δ cells and is therefore Bud6p-dependent (Segal et al., 2002
To evaluate the involvement of Kar9p in Bud6p-associated MT capture, we undertook a comparative analysis of wild-type versus kar9Δ cells coexpressing GFP-Bud6 and GFP-α tubulin (Tub1p) fusions. Interactions were studied along the cell cycle divided arbitrarily into three stages based on spindle pathway landmarks and the program of Bud6p localization ( A).
Figure 1. Dynamic behavior of cortical Bud6p–astral MT interactions in kar9Δ cells. (A) Landmark events in Bud6p-mediated spindle orientation in wild-type cells. Solid black arrows show operational definition of cell cycle stages used in this work. (more ...)
The kar9Δ mutation did not affect MT–Bud6p contacts from mitotic exit to generation of a new budding site (ME to BE: 54.7%, n = 1105 in kar9Δ vs. 53.8%, n = 557 in wild type, B). Once the Bud6p ring at the previous division site disassembled and accumulation began at the prebud site, MTs reoriented to this new area of capture ( A). In addition, interactions at Bud6p sites occurred at wild-type frequencies from onset of anaphase to mitotic exit in kar9Δ cells ( B, SE to ME: 76.1%, n = 822 in kar9Δ vs. 78.1%, n = 420 in wild type) and MT shrinkage at Bud6p sites was unperturbed ( B, open boxes within black bars).
Figure 2. Bud6p–astral MT interactions in kar9Δ cells. Selected frames from representative time-lapse series analyzed in B, illustrating dynamic Bud6p-astral MT interactions in kar9Δ GFP:BUD6 GFP:TUB1 cells. (A) After mitotic exit (more ...)
In contrast, kar9Δ cells exhibited a marked decrease in interactions with Bud6p decorated areas after bud emergence ( B, BE to SE). Cells showed repeated MT interactions with the bud tip in small-budded cells (100% cells within 10 min from bud emergence, n = 32). Yet, as the bud continued to grow, cells failed to maintain MTs oriented in the bud ( B, arrowheads). As a result, MTs spent significant time probing the mother cell cortex away from Bud6p marked regions. However, once GFP-Bud6 decorated the bud neck, MTs resumed interactions with this discrete area (, arrows). In spite of the reduction in Bud6p–MT contacts in S phase, kar9Δ cells still showed MT shrinkage at Bud6p sites ( B).
Bud6p–astral MT interactions during anaphase in kar9Δ cells occurred at wild-type levels. These interactions contributed to spindle positioning even if anaphase began within the mother cell ( E).
The correlation between MT–Bud6p interactions and the duration of cortical contacts previously reported in wild-type cells (Segal et al., 2002
) was still apparent in kar9
Δ cells. On average, interactions at Bud6p sites lasted 2.2 ± 1.2 min (n
= 75) from mitotic exit to bud emergence, 2.0 ± 0.3 min (n
= 42) from bud emergence to spindle assembly and increased to 3.0 ± 1.8 min (n
= 92) from spindle elongation to mitotic exit. In contrast, interactions away from Bud6p sites lasted 0.6 ± 0.2 min (n
= 112). These values were in agreement with those in wild-type cells (for review see Segal et al., 2002
Together, these results confirmed that a kar9Δ mutation did not alter the dynamic characteristics of cortical Bud6p–MT interactions, instead, it perturbed the maintenance of astral MT orientation toward the bud from late bud emergence through spindle assembly and thus, indirectly, decreased the incidence of capture at Bud6p sites.
Analysis of Kar9p-driven MT delivery to the bud in wild-type or bud6Δ cells
We then examined wild-type or bud6Δ cells expressing CFP-Tub1 and Kar9-GFP to determine the dynamic behavior of Kar9p bound to MTs, the effect on astral MT orientation to cell cortex areas and the relationship to MT-driven SPB movement toward the bud.
Decoration of MTs by Kar9-GFP was initiated by recruitment at the SPB in most cases, both in wild-type or bud6Δ cells (93.5%, n = 113 MTs and 96.2%, n = 104, respectively). Kar9p was detected at both SPBs at onset of spindle assembly (14 of 16 time-lapse series spanning spindle assembly) but was clearly asymmetric in spindles longer than 1.2 ± 0.2 μm (). Kar9p traveled along MTs toward the plus or minus end. In addition, Kar9p moved while fixed at the plus end of a growing or shrinking MT ( A). These modes of dynamic behavior occurred significantly in bud6Δ cells. However, the bud6Δ mutation slightly reduced Kar9p translocation along persistent MTs ( A, black and gray bars in wild type vs. bud6Δ).
Figure 3. Association of Kar9-GFP to SPBs during spindle assembly in wild-type cells. Selected frames of time-lapse series of wild-type cells expressing Kar9-GFP (overlaid in green) and CFP-Tub1 (red) showing Kar9p recruitment at both SPBs during the assembly of (more ...)
Figure 4. Kar9p dynamic behavior in wild-type versus bud6Δ cells. (A) Kar9p dynamic behavior in association with MTs in wild type (107 cells recorded) or bud6Δ (99 cells recorded). Transits along MTs or movement at the MT plus end were as described (more ...)
The distribution of cortical interactions involving Kar9p-bound MTs by cell compartment was altered in bud6Δ cells. In general, MTs decorated by Kar9p were already directed toward the bud or became oriented when Kar9p occupied the plus end (, B–D). A bud6Δ mutation markedly decreased interactions confined to the bud neck ( A, open portion of bars for each mode of dynamic behavior). Thus, Kar9p directed MTs to the bud, yet, Bud6p appeared to dictate bud neck capture.
Of all Kar9p-bound MTs in wild-type cells, 31% (n = 113 MTs) were associated with SPB movement toward the bud (24% in bud6Δ cells, n = 104 MTs).
To correlate the direction of movement with Kar9p dynamic behavior, all events involving Kar9p return to the spindle pole were scored for coupled SPB movement toward the cortex. As shown in A, there was no correlation between the direction of SPB movement and shrinkage of Kar9p-bound MTs in wild-type or bud6Δ cells (< 7%, n > 120). In fact, the SPB remained stationary relative to the cortex in the majority of Kar9p returns to the pole.
Figure 5. Analysis of SPB movement coupled to Kar9p-bound MTs. (A) SPB movement associated with Kar9p return to the pole in wild-type or bud6Δ cells was categorized as described in Materials and methods. Movement coupled to all Kar9p returns to the pole (more ...)
In wild-type cells, Kar9p dynamic behavior in the absence of SPB movement was confined to the vicinity of the bud neck within the mother ( B). This was associated with angular movement of MTs of constant length similar to previously reported Kar9p or Myo2p-dependent transports (Hwang et al., 2003
; Liakopoulos et al., 2003
). Alternatively, Kar9p-bound MTs shrunk away from the cortex without causing SPB movement ( C, arrow). In conclusion, Kar9p did not mediate SPB movement via changes in MT length. This was in contrast with, spindle orientation associated with cortical Bud6p relying primarily on MT shrinkage.
Δ cells SPBs were initially present away from the bud and became quickly repositioned as a Kar9p-bound MT moved toward the bud without observable shrinkage, presumably, along an actin cable (, arrows). This type of processive MT movements toward the bud were absent in bud6
Δ cells (n
> 150 MTs). Thus, a bud6
Δ mutant supported Kar9p-dependent MT orientation and relied on long-range Kar9p-bound transport to compensate for lack of early MT–cortex interactions with the new bud and the inability to mobilize SPBs by MT shrinkage (Segal et al., 2002
Figure 6. Kar9p-mediated MT transport in a bud6Δ cell. Selected frames from a time-lapse series showing Kar9p-mediated orientation of the SPB in a bud6Δ cell expressing CFP-Tub1 and Kar9-GFP. Overlays of CFP-Tub1 (red) and Kar9-GFP (green) images (more ...)
Spindle orientation in bud6Δ kar9Δ or bud6Δ dyn1Δ mutants
Genetic analysis of spindle orientation has assigned motor activities and cortical determinants to putative early and late pathways required for spindle position (Heil-Chapdelaine et al., 1999
). Kar9p and dynein are regarded as key components of the “early” and “late” pathways, respectively. To further assess the contribution of Bud6p in these pathways, the phenotypes resulting from double mutant combinations bud6
Δ or bud6
, dynein heavy chain; Eshel et al., 1993
; Li et al., 1993
) were characterized.
Astral MT behavior and spindle orientation defects observed in bud6
Δ cells demonstrated the additive impact of deleting BUD6
over a single kar9
Δ mutation. Early orientation of astral MTs toward the emerging bud was abolished as in single bud6
Δ mutants (not depicted; Segal et al., 2002
), whereas spindle positioning in the double mutant was markedly impaired relative to either single mutant ( and ).
Time-lapse analysis of spindle behavior at anaphase onset in bud6, kar9, and bud6kar9 mutants
Figure 7. Spindle orientation phenotypes in bud6Δ kar9Δ cells. Selected frames from representative time-lapse series showing spindle orientation defects in bud6Δ kar9Δ GFP:TUB1 cells. For quantitative information see . (A) (more ...)
A range of phenotypes in bud6Δ kar9Δ cells highlighted the contribution of Bud6p to cortical capture in the absence of Kar9p.
First, retention of the spindle at the bud neck was compromised causing transient positioning within the bud ( A, 17.0–18.5 min). This phenotype was as prevalent (26%, n = 39 cells recorded) as in bud6Δ cells and never observed in kar9Δ cells ().
Second, onset of spindle elongation along the mother-bud axis was markedly reduced (33%, n
= 39), possibly due to the additional absence of MT interactions with the bud neck ( B and see last section of Results describing A). This contrasted with kar9
Δ cells (), in which MT interactions with the bud neck still contribute toward orientation (Segal et al., 2000b
; and see last section of Results describing A). Initial spindle elongation within the mother cell in kar9
Δ cells was followed by dynein-driven positioning of the spindle part way through anaphase ( B) as in kar9
Δ cells (Segal et al., 2000b
; Yeh et al., 2000
Figure 9. Effect of decreased MT turnover on orientation of MT–cortex interactions in kar9Δ cells. (A) Astral MT–cortex interactions in the indicated strains expressing GFP-Tub1 (number of cells recorded: 81 wild type, 77 kar9Δ, (more ...)
Third, aberrant loss of spindle orientation in mid-anaphase of bud6Δ kar9Δ mutants ( C, 7.5–9.5 min), as observed in 13% (n = 39) of cells recorded (), underscored Bud6p-dependent capture in anaphase. This indicated that dynein-driven events might be impaired yet sufficient to sustain viability of bud6Δ kar9Δ cells.
We also determined the effects of deleting BUD6
on spindle phenotypes of dyn1
Δ cells, with particular focus on anaphase. In wild-type cells, the “fast phase” of spindle elongation ( A, 0–2.5 min and 6.0–10 min) is typically coupled to translocation of the SPBd
into the bud (Yeh et al., 1995
). SPB translocation is delayed in dyn1
Δ mutants, although spindle elongation still begins along the mother-bud axis ( and B). Spindles only became overtly misaligned during anaphase in 9% of cells (n
= 35) after initial elongation along the mother-bud axis ( C).
Figure 8. Spindle defects in bud6Δ dyn1Δ cells. (A) Spindle elongation and translocation of the SPBd into the bud are coupled in early anaphase of wild-type cells. Representative time-lapse series showing correct insertion of the spindle at the (more ...)
Spindle behavior in bud6, dyn1, and bud6dyn1 mutants
In contrast, bud6Δ dyn1Δ mutants already showed a mild preanaphase spindle orientation defect and concomitant reduction of spindle elongation along the mother-bud axis (). Spindle alignment could be rectified during early anaphase, presumably, through Kar9p-directed astral MT delivery to the bud ( D).
Impaired MT capture at the bud neck in dyn1
Δ cells led to loss of polarity of mid-anaphase spindles held within the mother cell, as astral MTs emerging from both poles entered the bud ( E; for review see Yeh et al., 2000
). In asynchronous populations, only 60% (n
= 302) of late anaphase spindles in dyn1
Δ cells retained apparent polarity (only one SPB connected to the bud) in contrast to 90% (n
= 210) in dyn1
Δ cells ().
Finally, bud6Δ dyn1Δ cells containing misaligned anaphase spindles within the mother failed to restrain mitotic exit. Progression of the spindle pathway past spindle disassembly was sufficiently frequent to be recorded by real-time microscopy ( F, 6.0–10.0 min). Accordingly, asynchronous bud6Δ dyn1Δ cultures contained 10% of cells exhibiting supernumerary SPBs (). Such cells were not present in bud6Δ kar9Δ or single bud6Δ, kar9Δ, or dyn1Δ mutant populations (n = 1,000 cells).
The observed interactions between bud6
Δ and kar9
Δ or dyn1
Δ mutations stress the separate contributions of Kar9p and Bud6p to spindle orientation. In addition, the impact of Bud6p-dependent MT capture at the bud neck for correct spindle dynamics and progression (Segal et al., 2000b
) was further demonstrated by the more complex spindle defects of bud6
Δ or bud6
Δ mutants during anaphase.
Effect of reduced MT turnover on spatial distribution of MT–cortex interactions in kar9Δ cells
The observed dynamic behavior of Kar9p was difficult to reconcile with the proposed role for Kar9p in cortical capture of MTs. Kar9p kept a fixed distance from the SPB during MT tracking along actin cables, and frequently decorated MTs already reaching within the bud. Moreover, the phenotype of kar9Δ cells after bud emergence ( B), suggested that Kar9p might additionally control MT dynamic behavior to promote persistence of MTs in the bud. This raised the possibility that the requirement for Kar9p could be bypassed by a decrease in MT instability. Under these conditions, MT capture coincident with cortical Bud6p in the bud might prevail and suppress the spindle orientation defect of kar9Δ mutants.
To address this question, time-lapse analysis was performed to compare wild-type, kar9
Δ, and kar9
cells for the distribution of MT–cortex interactions by cell compartment from bud emergence to assembly of ~2-μm-long spindles ( A). The tub2C354S
allele was introduced to reduce MT dynamics (Gupta et al., 2002
Wild-type cells exhibited a characteristic distribution of MT–cortex interactions in the mother, the bud and at the bud neck. These interactions ( A) led to correctly oriented preanaphase spindles, which initiated elongation along the mother-bud axis (). In kar9Δ cells, interactions within the bud were selectively decreased ( A). As a result, correct alignment at anaphase onset was reduced to 60% (n = 25; ).
Effect of tub2C354S on spindle alignment in kar9Δ cells
Introduction of the tub2C354S allele in the kar9Δ mutant restored astral MT–cortex interactions with the bud () and significantly improved preanaphase spindle orientation (, C–E; ). Astral MTs became oriented toward the bud ( B) and continued to interact with the bud cortex until anaphase (, B–E). Elongation of the spindle along the mother-bud axis was increased to 86% of anaphase cells recorded (n = 22; ).
The tub2C354S allele could not correct spindle orientation in kar9Δ bud6Δ cells (; ). The kar9Δ bud6Δ tub2C354S mutant initiated anaphase along the mother-bud axis in only 25% of cells recorded (n = 40). This highlighted the importance of Bud6p in directing MT capture at the incipient bud cortex. Accordingly, tub2C354S failed to change bud6Δ mutant phenotypes ( A and ).
These data were validated by separately scoring preanaphase spindle orientation in asynchronous cultures of all strains studied by time-lapse analysis ().
Thus, decreased MT turnover suppressed spindle orientation defects of kar9
Δ cells, in a Bud6p-dependent manner. This result confirmed the participation of Bud6p in MT capture at the cell cortex and pointed to an additional role for Kar9p in control of MT dynamic behavior. Indeed, Kar9p-bound MTs entering the bud underwent repeated cycles of recovery (unpublished data). Such cycles were confirmed by time-lapse analysis of wild-type cells expressing GFP-Tub1 (at least 2 cycles in 60% of MTs entering the bud, n
= 135) but were markedly decreased in a kar9
Δ mutant in which 97% of MTs entering the bud (n
= 105) underwent a single cycle of growth and shrinkage past the bud neck (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200407167/DC1