APC-mutant Cells Are Defective in Disassembly of the Actomyosin Ring
To determine if APC activity helps control cytokinesis, we examined cells defective in APC function. Cells with cytokinesis defects form chains of cell bodies that remain connected even after brief sonication (Ko et al., 2007
). About 3% of exponentially growing wild-type cells were present in clusters of ≥3 cell bodies, whereas in a cdh1
Δ strain, the number of clustered cells rose threefold to 9% (Supplemental Figure S1). Thus, APCCdh1
activity appears to be required for efficient cytokinesis.
To further explore APCCdh1
function in cytokinesis, we analyzed the localization of GFP-tagged cytokinesis proteins in fixed cdh1
Δ cells. We found that the localizations of many proteins (Cdc12, Hof1, Bni1, Bnr1, Cyk3, Mob1, Myo2, and Tus1) were not detectably affected by deletion of CDH1
(data not shown). However, the behavior of the type II myosin Myo1 was strikingly altered. In a wild-type strain, Myo1-GFP was not detectable in cells that had recently completed cytokinesis, as marked by the absence of a Myo1 ring and the presence of large actin patches at the bud neck (Bi et al., 1998
; A). However, in cdh1
Δ cells at the same stage, multiple Myo1-GFP patches were typically present at the neck and elsewhere in the cell and did not colocalize with actin patches (B).
Figure 1. Cdh1 is required for normal disassembly of the actomyosin ring. Myo1-GFP was visualized in wild-type (A and C; strain GT049) and cdh1Δ (B and D; strain GT052) cells. (A and B) Cells were fixed with ethanol and stained with TRITC-phalloidin (red) (more ...)
We next analyzed the localization dynamics of Myo1-GFP by time-lapse microscopy. In wild-type cells, the Myo1 ring appeared at the time of bud emergence and remained at the bud neck until cytokinesis, when it contracted to a single dot in an average time of 5.9 ± 0.9 min (C; ). Within 10 min after the end of contraction, the Myo1 dot completely disappeared, suggesting that the actomyosin ring had fully disassembled. In cdh1Δ cells, the Myo1 ring also appeared at bud emergence and later contracted to a single dot with similar kinetics (5.8 ± 1.3 min; D; ). However, the Myo1 dot did not disappear after the completion of ring contraction. Instead, Myo1-GFP patches remained visible at the site of cytokinesis and elsewhere in the cell (D). More extended time-lapse observations showed that these patches could persist throughout G1 and only disappeared as the new Myo1 ring formed in the next cell cycle (Supplemental Figure S2). Many of the persistent Myo1 patches were highly mobile. We tracked their movements and saw that in ~63% of the cells, a Myo1 patch traveled toward the bud neck and appeared to merge with another patch at that site (D, asterisks). In most cases, Myo1 patch movement toward the neck occurred exclusively in the daughter cell. Patch movement was not seen in cells treated with the actin-depolymerizing agent LAT-A, suggesting that these movements depend on F-actin (data not shown).
Ring contraction and disassembly in selected strains
To better quantitate the cdh1Δ phenotype, we counted the numbers of cells with one or more Myo1-GFP patches visible somewhere in the cell at times >10 min after the completion of ring contraction. Only 8% of wild-type cells, but 100% of cdh1Δ cells, contained these Myo1 patches (; ). In almost all of the cdh1Δ cells observed, the Myo1 patches remained visible for the duration of the time-lapse observations. We then counted the maximum number of Myo1-GFP patches visible in each cell at any time from 10 min after the end of ring contraction until the end of the time-lapse series. More than 90% of cdh1Δ cells contained two or more patches, whereas none of the wild-type cells had more than one ().
Figure 2. Myo1, Mlc2, Mlc1, and Iqg1 exhibit ring-disassembly defects in APC mutant cells. Strains GT049 (MYO1-GFP), GT052 (MYO1-GFP cdh1Δ), GT104 (MLC2-GFP), GT110 (MLC2-GFP cdh1Δ), GT208 (GFP-MLC1), GT209 (GFP-MLC1 cdh1Δ), GT050 (IQG1-GFP (more ...)
The results described above were obtained in the W303 strain background. Similar results were obtained with strains in the S288C background (see , below).
To confirm that holoenzyme APC activity, and not just Cdh1, is required for Myo1-ring disassembly, we analyzed cells lacking all APC activity. Because the APC is normally essential for viability, we used a background (pds1
) in which the APC is not essential (Thornton and Toczyski, 2003
) and compared cells that were otherwise wild type to those lacking Apc2, an essential subunit of the APC holoenzyme. In the former strain, as in wild-type cells, the Myo1-GFP signal disappeared within 10 min after the end of ring contraction (Supplemental Figure S3A). In the apc2
Δ strain, Myo1 patches persisted for ≥10 min after the end of ring contraction (Supplemental Figure S3B), supporting the hypothesis that the APC, with its activator Cdh1, is required for proper disassembly of Myo1-containing structures after actomyosin-ring contraction.
APCCdh1 Is Required for Myosin Light-Chain and Iqg1 Disassembly
We next investigated whether the APC also regulates the disassembly of other actomyosin-ring components. The “regulatory” myosin light-chain Mlc2 binds to the IQ2 motif of myosin (Luo et al., 2004
), and in wild-type cells, its behavior was similar to that of Myo1: the Mlc2-GFP ring contracted in an average time of 5.1 ± 1.2 min (A; ) and disappeared within 10 min after the completion of ring contraction in 88% of cells (). In cdh1
Δ cells, the rate of ring contraction was similar (6.9 ± 1.7 min), but Mlc2-GFP patches persisted in all cells for ≥10 min after the end of ring contraction, and most cells contained ≥3 such patches ( and B; ).
Mlc1, the “essential” myosin light chain, is a multifunctional protein that associates with Myo1 during cytokinesis and with the type V myosin, Myo2, on secretory vesicles throughout most of the cell cycle (Stevens and Davis, 1998
; Wagner et al., 2002
; Luo et al., 2004
). Because cells carrying Mlc1 tagged at its chromosomal locus were inviable (perhaps because GFP-tagged Mlc1 has reduced function), we analyzed the behavior of GFP-Mlc1 after expression under control of the MET
promoter. In wild-type cells, the GFP-Mlc1 ring contracted in an average time of 5.7 ± 1.1 min (Supplemental Figure S4A; ). As described previously (Wagner et al., 2002
), GFP-Mlc1 did not disappear after ring contraction but remained visible in all cells >10 min after the end of ring contraction (; Supplemental Figure S4A), presumably reflecting its association with Myo2. In cdh1
Δ cells, GFP-Mlc1 contracted with wild-type kinetics (6.2 ± 1.6 min; Supplemental Figure S4B; ). As in wild type, GFP-Mlc1 foci remained visible >10 min after the end of ring contraction in all cells, but the number of cells with ≥3 such foci increased from 57% in wild type to 93% in cdh1
Δ cells (; Supplemental Figure S4B). These data suggest that APCCdh1
activity is involved in disassembling the Myo1-associated subpopulation of Mlc1 complexes after actomyosin-ring contraction.
Finally, we analyzed the IQGAP protein Iqg1, which interacts with the actomyosin ring through an association with Mlc1 (Boyne et al., 2000
; Shannon and Li, 2000
). In wild-type cells, the Iqg1-GFP ring contracted in an average time of 4.6 ± 1.0 min (C; ), and an Iqg1-GFP patch remained visible >10 min after the end of ring contraction in just one of 11 cells examined (). In cdh1
Δ cells, the rate of ring contraction was similar (5.3 ± 1.5 min; D; ), but two or more Iqg1-GFP patches remained visible in all cells >10 min after the end of ring contraction ( and D). We conclude that Iqg1-ring disassembly, like that of Myo1, Mlc2, and probably Mlc1, depends on APCCdh1
Myo1 Colocalizes with Mlc2, Iqg1, and Mlc1 in cdh1Δ Cells
Because Myo1, Mlc2, Mlc1, and Iqg1 are all defective in dispersal in apc mutant cells, we used time-lapse microscopy of double-labeled cells to investigate whether these proteins colocalize in the same patches after ring contraction. In a cdh1Δ strain expressing both Mlc2-GFP and Myo1 tagged with mCherry (Myo1-Cherry), we observed colocalization of the two proteins at all time points in each of eight cells examined (A; Supplemental Figure S5; Supplemental Table S1). Similarly, Iqg1-GFP and Myo1-Cherry colocalized at all time points in each of 14 cells examined (B; Supplemental Figure S6; Supplemental Table S1).
Figure 4. Myo1 colocalizes with Mlc2, Mlc1, and Iqg1, but not with Sec2, in cdh1Δ cells. cdh1Δ strains expressing Myo1-Cherry (red) and a GFP-tagged protein were examined by time-lapse microscopy (see Supplemental Figures S5–S8); representative (more ...)
As expected, cells coexpressing GFP-Mlc1 and Myo1-Cherry presented a more complicated picture. GFP-Mlc1 patches were detectable in just eight of 11 cells examined (probably because of variable expression from the MET promoter). In these cells, all Myo1-Cherry patches colocalized with GFP-Mlc1 at every time point (C; Supplemental Figure S7; Supplemental Table S1), but some faint GFP-Mlc1 patches did not colocalize with Myo1-Cherry (C, asterisks) and presumably represented Myo2-associated protein (see above). To explore this further, we investigated whether Myo1-Cherry colocalized with the secretory-vesicle protein Sec2-GFP. We observed partial colocalization in only one of eight cells undergoing cytokinesis (D; Supplemental Figure S8; Supplemental Table S1). These results are consistent with the other evidence that there are both secretory-vesicle– and actomyosin-ring–associated pools of Mlc1 and that the latter, but not the former, dissociates after cytokinesis in an APCCdh1-dependent manner.
APCCdh1 Involvement in Ring Disassembly during Contraction
The actomyosin ring has long been thought to disassemble progressively as it contracts (Schroeder, 1972
). Consistent with this model, the total fluorescence intensity of GFP-tagged ring components appeared to decline during contraction in wild-type cells (C and , A and C). This decline seemed less pronounced in cells lacking Cdh1 (D and , B and D). Quantitative measurements of Myo1-GFP fluorescence confirmed that cdh1
Δ cells have a significant defect in the normal decline in ring fluorescence (A). Thus, APCCdh1
is not just required for ring disassembly after contraction but also for the disassembly that occurs during contraction.
To explore further the relationships between ring contraction and APCCdh1
-mediated disassembly, we analyzed ring behavior in cells treated with LAT-A. The absence of filamentous actin in these cells is known to prevent Myo1-ring contraction, and the Myo1 ring gradually disassembles in late mitosis, at about the time that cytokinesis would normally occur (Bi et al., 1998
). In agreement with these results, we found that Myo1-GFP disappeared within 10 min of septin-ring splitting in 10 of 10 wild-type cells observed (B, top). In contrast, in cdh1
Δ cells, the Myo1-GFP ring remained visible for the duration of the experiment, for as long as 76 min after septin-ring splitting, in all 19 cells observed (B, bottom). APCCdh1
is thus required for ring disassembly even when contraction is prevented.
Defective Completion of Septation in cdh1Δ Cells
We used electron microscopy to explore further the functions of the APC in cytokinesis. In wild-type cells, ingression of the cleavage furrow takes place concomitantly with actomyosin-ring contraction and formation of the chitinous primary septum of the cell wall (visible as an electron-lucent line in electron micrographs; Vallen et al., 2000
; Roh et al., 2002
; Cabib, 2004
). The process is completed by fusion of the invaginating membranes in the center of the neck and formation of a smooth, continuous disk of primary septum; secondary septa are then deposited on both sides of the primary septum (A). In a cdh1
Δ mutant, the early stages of cytokinesis and septum formation appeared normal, but the completion of septation was often strikingly abnormal. Among 95 cells examined in which some secondary-septum formation had occurred but cell separation had not begun, 26 (27%) exhibited one of the three types of defective septal structures shown in B. Careful examination of an isogenic wild-type strain (YEF473A) revealed that a few cells (five of 100 examined) had similar but less pronounced abnormalities.
Figure 6. Defects in the final stage of primary-septum formation in cdh1Δ and IQG1Δ42 cells. Strains YEF473A (A, wild type), KO255 (B, cdh1Δ), and GT124 (C, IQG1Δ42) were examined by electron microscopy after growth at 24°C; (more ...)
Removal of Several Known APC Targets Does Not Promote Actomyosin-Ring Disassembly
Because the APC is an E3 ubiquitin-protein ligase that mediates the destruction of its substrates by the 26S proteasome, the evidence presented above suggests that the destruction of one or more APC substrates is required for the disassembly of the actomyosin ring during and after its contraction. Clb2, Hsl1, Ase1, Fin1, and Spo12 are all substrates of APCCdh1
(Juang et al., 1997
; Burton and Solomon, 2001
; Shah et al., 2001
; Woodbury and Morgan, 2007
). If the degradation of any one of these substrates is required for actomyosin-ring disassembly, then deletion of the corresponding gene might rescue the disassembly defect in cdh1
Δ cells. We constructed strains lacking both Cdh1 and one of the candidate substrates and monitored Myo1-GFP disassembly by time-lapse microscopy. However, we observed no rescue of the ring-disassembly defect: in each strain, Myo1-GFP foci persisted for >10 min in 100% of cells in which Myo1 had completed contraction ().
The Polo-like protein kinase Cdc5 is another APCCdh1
substrate that is involved in regulating cytokinesis. Because Cdc5 is essential for viability, we could not easily test the effects of deleting CDC5
. Instead, we analyzed Myo1 behavior in cells expressing an APC-resistant form of Cdc5 that lacks amino acids 5-70 (Charles et al., 1998
; Shirayama et al., 1998
). In these cells, the Myo1-GFP ring contracted with normal kinetics (6.1 ± 1.5 min; ), and Myo1-GFP patches were not present at times >10 min after the completion of ring contraction (Supplemental Figure S9). Thus, the stabilization of Cdc5 alone is not sufficient to block actomyosin-ring disassembly.
A major function of the APC is to trigger the destruction of mitotic cyclins and thereby reduce Cdk1 activity in late mitosis. Thus, it seemed possible that elevated Cdk1 activity in a cdh1
Δ cell might inhibit actomyosin-ring disassembly. Although this possibility seemed unlikely, because the Clb-Cdk1 inhibitor Sic1 accumulates in late mitosis and suppresses Cdk1 activity in cdh1
Δ cells (Schwab et al., 1997
), we tested it further by overexpressing SIC1
Δ cells and analyzing Myo1-GFP ring behavior. Sic1 overproduction did not rescue the defect in actomyosin-ring disassembly, as 95% of the cells contained one or (commonly) more Myo1-GFP patches at times ≥10 min after the completion of ring contraction (Supplemental Figure S10; ). Taken together with the results of CLB2
deletion (see above), this result suggests strongly that APCCdh1
does not promote actomyosin-ring disassembly by reducing Cdk1 activity.
APCCdh1-dependent Degradation of Iqg1 Contributes to Actomyosin-Ring Disassembly
We recently identified Iqg1 as an APCCdh1
substrate (Ko et al., 2007
). As Iqg1 is required to recruit actin to the bud neck and to initiate actomyosin-ring contraction, we speculated that its APC-dependent degradation might contribute to actomyosin-ring disassembly. We therefore monitored Myo1-GFP in cells carrying an APC-resistant mutant form of Iqg1, Iqg1Δ42, which lacks the first 42 amino acids that contain its APC-recognition sequence. Single Myo1-GFP patches were observed at times ≥10 min after the completion of ring contraction in 65% of cells (n = 20), compared with 9% of wild-type cells (n = 22; A). Thus, the degradation of Iqg1 appears to contribute to actomyosin-ring disassembly. However, the fact that the IQG1
mutant does not fully phenocopy the cdh1
Δ mutant suggests that Iqg1 is not the only APCCdh1
substrate whose degradation is important for ring disassembly.
Electron-microscopic observations supported these conclusions. In 18% (n = 55) of IQG1Δ42 cells examined at the stage when septum formation was largely complete, we observed defects in septum completion that were similar to those seen in cdh1Δ cells (C; cf. B). In accord with the fluorescence-microscopy observations, the abnormalities in IQG1Δ42 cells typically appeared less severe than those in cdh1Δ cells.
To further assess the importance of Iqg1 destruction in actomyosin-ring disassembly, we analyzed Myo1 behavior in iqg1
Δ cells. The Myo1-GFP signal was far less intense in these cells than in wild type, so that the results were less clear than in our other experiments. Consistent with earlier observations (Shannon and Li, 1999
), Myo1 ring contraction was not apparent in cells lacking Iqg1; instead, the Myo1 ring faded away over a period of ~15 min (B, top). Few if any Myo1-GFP patches were seen ≥10 min later. In cells lacking both Iqg1 and Cdh1, the Myo1-GFP signal was more readily detected, and it faded away over a period of ~15–25 min, sometimes after a slow constriction (presumably reflecting the slow closure of the neck by secondary septal material in iqg1
Δ cells: Nishihama and Pringle, unpublished results; B, bottom). Ten minutes later, persistent Myo1-GFP patches were observed in 59% of cdh1
Δ cells (n = 61), compared with 100% of cdh1
Δ cells, and the maximum number of patches per cell was also substantially decreased (A). This partial rescue of the cdh1
Δ phenotype by the iqg1
Δ mutation is consistent with the hypothesis that APC-dependent destruction of Iqg1 contributes to actomyosin-ring disassembly.
The APC Collaborates with Mlc2 in Ring Disassembly
A defect in actomyosin-ring disassembly has also been observed in cells lacking Mlc2 (Luo et al., 2004
). To determine if Mlc2 and Cdh1 promote ring disassembly by the same or distinct mechanisms, we compared the disassembly defect in an mlc2
Δ double mutant to the defects in the single mutants. In mlc2
Δ single mutant cells, we did not observe a significant defect: as in wild-type, myosin patches were visible in only ~8% of cells (n = 12) at times ≥10 min after the end of ring contraction (A). However, the delay in myosin-ring disassembly observed by Luo et al. (2004)
was only 2–8 min after the end of ring contraction, and our methods may not be sensitive enough to detect this defect.
To examine the double mutant, we first used time-lapse microscopy. By this assay, its phenotype was similar to that of the cdh1Δ single mutant (A; n = 23). We were concerned, however that our time-lapse protocol might underestimate the long-term accumulation of Myo1 patches, because this protocol scores only those cells in which the completion of ring contraction occurred at some point during the 30-min recorded time frame. Thus, we performed an additional experiment in which we scored an exponential-phase population for the number of Myo1-GFP patches per cell in all cells that had completed ring contraction but not cell separation. With this assay, we found that Myo1-GFP patches accumulated to a twofold higher level in the double mutant than in the cdh1Δ single mutant (C). We also found that the double-mutant population contained more clustered cells than either single mutant (18 vs. 5 and 9%), indicating a higher rate of cytokinesis failures (Supplemental Figure S1). Taken together, the data indicate that the mlc2Δ cdh1Δ double mutant has a more severe phenotype than either of the single mutants, suggesting that Mlc2 and Cdh1 promote actomyosin-ring disassembly by mechanisms that are at least partially distinct.
Because deletion of IQG1 partially rescued the defect in actomyosin-ring disassembly in cdh1Δ cells, we next tested whether stabilization of Iqg1 in the absence of Mlc2 would affect ring disassembly. In mlc2Δ IQG1Δ42 cells, Myo1-GFP patches did not disappear after ring contraction in any of the 14 cells examined (A), a phenotype much more severe than that of the mlc2Δ (or IQG1Δ42) single mutant. In terms of the numbers of patches per cell, the double-mutant phenotype was less severe than that of a cdh1Δ single mutant (A). Nonetheless, the observation that an APC-resistant Iqg1 exacerbates the phenotype of an mlc2Δ mutant further supports the conclusion that APCCdh1 and Mlc2 promote actomyosin-ring disassembly by mechanisms that are at least partially distinct.