Assembly of an Actin Ring at the Mother–Bud Neck Late in the Cell Cycle and Its Contraction After Anaphase
During experiments done for other purposes, we serendipitously observed that brief incubation of fixed cells with low concentrations of rhodamine-phalloidin allowed the clear and consistent visualization of a distinct actin ring structure in a subset of the cells in an asynchronous population (Fig. , cells 3–6
). Although such a structure had been observed previously (Adams and Pringle, 1984
; Kilmartin and Adams, 1984
), it had been difficult to visualize consistently using the usual staining techniques. Thus, it had remained unclear whether a distinct actin ring structure was normally formed during a particular (perhaps brief) stage of the cell cycle or whether the structures visualized might only represent occasional fortuitous arrangements of the well known actin cables and/or patches. With the lower phalloidin concentration used here, the cables and patches are less strongly stained, and the actin ring is much easier to see. Although it is not clear why the ring should be preferentially stained at lower phalloidin concentrations, it is clear from the work described here and from recent work by others (Epp and Chant, 1997
; Lippincott and Li, 1998
; see Discussion) that the ring is indeed a distinct structure and not an artifact.
Figure 1 Formation and contraction of an actin ring during the cell cycle. Cells of strain YEF473 growing exponentially in SC medium were triple-stained for F-actin (A), tubulin (B), and DNA (C) as described in Materials and Methods. Cells shown are in G1 (more ...)
To characterize the timing of actin ring appearance and its behavior during the cell cycle, wild-type cells were triple-stained to visualize F-actin, tubulin, and DNA. Actin rings were observed in ~5% of the cells in asynchronous populations of two different strains (YEF473 and M-238) grown in SC liquid medium at 23°C (>700 cells counted per strain). Rings were observed only in late-anaphase or post-anaphase cells with fully separated chromosomes (Fig. , compare cells 1 and 2 with cells 3–6) and were seen in nearly all such cells. The diameter of the actin ring was not identical in every cell. The largest rings (~1 μm-diam) were consistently observed in cells with fully elongated spindles that were intact (Fig. , cell 3) or just beginning to disassemble (Fig. , cell 4). Smaller rings were seen in cells with disassembling or disassembled spindles (Fig. , cells 5 and 6). Thus, the actin ring assembles near the end of anaphase and appears to contract during or after spindle disassembly.
Assembly of a Myo1p Ring Early in the Cell Cycle and Its Contraction After Anaphase
The behavior of the actin ring was reminiscent of that of the contractile actomyosin ring involved in animal cell cytokinesis. To ask if the yeast actin ring also contains a type II myosin, we fused GFP-encoding sequences to the COOH-terminal end of MYO1, which encodes the only type II myosin in S. cerevisiae. The tagged Myo1p was expressed from its own promoter at its normal chromosomal locus and appeared to be fully functional by several criteria, including the normal morphology and cell separation of the strain harboring the tagged gene (see also below, and note that the strain background in which the tagging was done was that in which deletion of MYO1 produced the more conspicuous phenotype). We then performed time-lapse analysis of living cells expressing Myo1p–GFP. Newborn unbudded cells did not display localized Myo1p (Fig. , 00:00). Before bud emergence, a ring of Myo1p assembled at one pole of the cell (Fig. , 07:59; that these structures were indeed rings was verified by observations on cells in suspension), and a bud emerged from that site ~5–6 min later (Fig. , 13:10). As the bud grew, the ring of Myo1p remained visible at the mother–bud neck and retained a constant diameter of ~1 ± 0.1 μm (n = 6) (Fig. , 18:10 and 49:27; Fig. A, 00:00 and 02:59) until nuclear division. (The timing of nuclear division is difficult to discern in Fig. but was clear in other series.) The Myo1p ring then contracted to a point and disappeared (Fig. , A, 02: 59–09:59, and B). Contraction of the ring took 7–9 min from the initiation of contraction to the disappearance of the Myo1p-GFP signal. After contraction of the Myo1p ring, a septum visible by DIC microscopy formed rapidly (Fig. A, 9:59), and cell separation (detected as a rotation of the daughter cell relative to the mother; e.g., Fig. A, 19:10) occurred ~10 min later.
Figure 2 Formation of the Myo1p–GFP ring relative to bud emergence. Cells of strain YEF1698 growing exponentially in SC medium were spotted onto SC medium containing 25% gelatin and observed by time-lapse photomicroscopy as described in Materials and (more ...)
Figure 3 Contraction of the Myo1p–GFP ring late in the cell cycle. Time-lapse analysis of strain YEF1698 was performed as in Fig. . (A) Pairs of GFP fluorescence and DIC images were recorded at the indicated times from a cell positioned (more ...)
Coincidence of the Actin and Myo1p Rings
To examine whether the actin and Myo1p rings were part of the same structure, exponentially growing cells expressing Myo1p–GFP were fixed with ice-cold 70% ethanol and stained with rhodamine-phalloidin and bisBenzimide. Before bud emergence, actin patches clustered at the presumptive bud site together with the Myo1p ring (Fig. , cell 1), but no actin ring was detected. In budded preanaphase cells, the Myo1p ring was visible at the neck, while actin patches were concentrated in the bud (Fig. , cell 2). In late-anaphase and post-anaphase cells, the Myo1p and actin rings appeared to coincide at the neck (Fig. , cells 3 and 4). In all cells with a detectable actin ring (25 cells scored), the diameters of the Myo1p and actin rings were similar (Fig. , cells 3 and 4). Thus, the actin ring assembles at the site of the preexisting Myo1p ring, and the actomyosin ring then contracts to a point and disappears. Using the Myo1p–GFP as a guide in these preparations, it was possible to observe smaller actin rings that had previously escaped detection and that represented the final stages of ring contraction; including these structures, ~9% of the cells (n = 373) in an asynchronous population contained an actomyosin ring. In addition, ~7% of the cells in the same population contained clusters of actin patches symmetrically disposed on both sides of the neck; none of these cells contained a Myo1p ring (Fig. , cells 5 and 6). This suggests that the actomyosin ring contracts and disappears before the actin patches congregate at the neck.
Figure 4 Coincidence of the actin and Myo1p rings. Cells of strain YEF1698 growing exponentially in YM-P medium were fixed with ice-cold 70% ethanol (refer to Materials and Methods) and then stained for F-actin (A) and DNA (C). The GFP signal was also recorded (more ...)
Dependence of Actin Ring Assembly on Myo1p
To determine whether Myo1p is important for the formation or contraction of the actin ring, we generated MYO1 deletion strains in two genetic backgrounds (refer to Materials and Methods). These strains were viable, and their phenotypes are described in more detail below. We were unable to detect any cells containing actin rings in either strain YEF1820 or JMY1318 (>1,000 cells scored in each case). Examples of myo1Δ cells with fully elongated spindles are shown in Fig. , D–F; wild-type cells at this stage of the cell cycle almost always contained a detectable actin ring (Fig. , A–C). Thus, Myo1p appears to be required for formation of the actin ring. Myo1p–GFP supported actin ring formation and contraction (see above), indicating that it was functional in these regards.
Figure 5 Dependence of actin ring formation on Myo1p. Cells of wild-type strain YEF473 (A–C) and myo1Δ/myo1Δ strain YEF1820 (D–F) growing exponentially in SC medium were triple-stained for F-actin (A and D), tubulin (B and E), (more ...)
Dependence of Myo1p Ring Contraction, but Not of Myo1p Ring Formation or Maintenance, on F-actin
To determine whether F-actin is important for the maintenance or contraction of the Myo1p ring, we treated exponentially growing cells of a Myo1p–GFP-expressing strain with 200 μM LAT-A and observed them by time-lapse video microscopy. At this concentration, LAT-A caused the loss of all detectable F-actin within 10 min (Fig. , B
) (Ayscough et al., 1997
). Most budded and some unbudded cells retained a strong Myo1p–GFP signal after LAT-A treatment (Fig. A, 03:38
). In five small-budded cells observed, the intensity of the Myo1p– GFP ring did not change significantly during 1–4.5 h of filming (Fig. A, right-hand cell
, and data not shown), indicating that maintenance of the Myo1p ring does not require F-actin. In contrast, observations on large-budded cells revealed that the Myo1p–GFP ring disappeared following anaphase (nuclei visualized by DIC microscopy), but that it did so without contracting (Fig. A, left-hand cell
). In the seven cells observed, the disappearance of the Myo1p ring took ~8 min, which was very similar to the time required for contraction of the ring when F-actin was present (see above). During its disappearance in the LAT-A–treated cells, the Myo1p–GFP ring appeared to remain constant in diameter at ~1 ± 0.1 μm (n
= 7), indicating that F-actin is essential for contraction of the Myo1p ring.
Figure 6 Dependence of Myo1p ring contraction, but not of Myo1p ring maintenance, on F-actin. (A) Cells of strain YEF1698 growing exponentially in SC medium were treated with 200 μM LAT-A for 10 min and then spotted onto solid SC/25% gelatin medium (more ...)
To determine whether F-actin is essential for the initial formation of the Myo1p ring at the presumptive bud site, we isolated stationary phase cells of a Myo1p–GFP-expressing strain and inoculated them into fresh medium in the presence or absence of LAT-A. In the absence of LAT-A, most cells assembled detectable Myo1p–GFP rings (Fig. , A, open circles
, and B
) and budded (data not shown) during the 4-h time course. In the presence of LAT-A, cells did not form buds over the course of the experiment, but they did form Myo1p–GFP rings (Fig. , A
, closed circles
, and C
). However, ring formation was significantly delayed: at 2 h, only 9% of the cells contained detectable Myo1p–GFP rings, compared with 41% of the cells incubated in the absence of LAT-A. In contrast, other (actin-independent) markers of the presumptive bud site appear with normal kinetics when cells are treated with LAT-A under these conditions (Ayscough et al., 1997
), indicating that such treatment does not produce a nonspecific delay in reentry into the cell cycle. Thus, it appears that F-actin is not essential for the initial assembly of the Myo1p ring but does contribute to the efficiency and timing of ring formation.
Figure 7 Formation of Myo1p–GFP rings in the absence of F-actin. Unbudded cells of strain YEF1698 were isolated as described in Materials and Methods and reinoculated into fresh YM-P medium in the absence or presence of 200 μM LAT-A. (A) Time-course (more ...)
Septin Dependence of Myo1p Ring Formation and Maintenance
To determine whether the formation and/or maintenance of the Myo1p ring requires the septins, we generated a diploid strain homozygous both for MYO1–GFP
and for the temperature-sensitive cdc12-6
septin mutation. Cells were fixed and processed for visualization of Myo1p–GFP or the septin Cdc11p during exponential growth at 23°C or 1 h after a shift to the restrictive temperature of 37°C. At 23°C, most cells displayed normal septin localization to the presumptive bud site or the bud neck, but some cells had aberrantly elongated buds and/or lacked detectable septin staining at the bud neck (Fig. C
), indicating a partial defect even at 23°C, as observed previously for other cdc12-6
strains (Adams, 1984
). At this temperature, Myo1p–GFP rings were seen in 43% of the cells (Fig. A
= 207). However, after a shift to 37°C for 1 h, no septin staining or Myo1p–GFP rings were detected (Fig. , B
= 229). For comparison, wild-type cells expressing Myo1p– GFP showed a detectable Myo1p–GFP ring in 71% of cells (n
= 215) at 23°C and 65% of cells (n
= 234) at 37°C. Thus, assembly and maintenance of the Myo1p ring appear to require septin function.
Figure 8 Dependence of Myo1p–GFP ring formation and maintenance on the septins. Cells of strain YEF1798 were fixed with 70% ethanol (refer to Materials and Methods) and processed for the observation of Myo1p-GFP (A and B) or fixed with formaldehyde and (more ...)
Cytokinesis and Cell Separation in myo1 Mutants
Previous studies using disruptions or partial deletions of MYO1
suggested that loss of Myo1p function caused a partially penetrant defect in cytokinesis and/or cell separation associated with aberrant septum deposition (Watts et al., 1987
; Rodriguez and Paterson, 1990
; Brown, 1997
; Rodriguez, J., personal communication). To examine further the function of Myo1p, we generated complete or nearly complete deletions of the MYO1
coding region in two different strain backgrounds. The myo1Δ
cells displayed growth defects relative to wild-type cells that were variable but relatively mild in one strain background and variable but relatively severe in the other. In particular, diploid strain JMY1318 grew nearly as well as wild type, and although haploid myo1Δ
segregants from this diploid formed colonies of variable size, many of these were comparable to wild-type colonies. In contrast, diploid strain YEF1820 grew significantly more slowly than wild type, and haploid myo1Δ
segregants in this strain background were either inviable or formed small or medium-sized colonies. Much of this variability in colony size appeared to be epigenetic and/or to reflect differences in the efficiency of spore germination or the initial outgrowth of single cells, because in both strain backgrounds, restreaking cells from a single colony resulted again in a range of colony sizes.
Phenotypes at the single cell level were also somewhat variable. In strain JMY1318, many cells appeared normal, but there were also chains of cells (Fig. B) suggestive of a partially penetrant cytokinesis or cell separation defect. Light sonication readily separated the cells (Fig. C), indicating that cytokinesis had in fact been completed. The defect was more severe in strain YEF1820, which formed longer chains (Fig. D) that were resistant to sonication (data not shown). However, most cells could be separated by treating the fixed cells with lyticase to digest the cell walls (Fig. E), indicating that the principal defect was in septum formation or cell separation and not in cytokinesis. Thus, it seems clear that cells can undergo cytokinesis and (with lesser and more variable efficiency) cell separation in the absence of Myo1p and hence of the contractile ring.
Figure 9 Phenotypic effects of MYO1 deletions. (A–E) Cells of wild-type strain YEF473 (A) and of myo1Δ/myo1Δ strains JMY1318 (B and C) and YEF1820 (D and E) were fixed during exponential growth in SC medium and examined by DIC (A–D (more ...)
In addition to the defect in cell separation, some of the myo1Δ cells were aberrantly large and/or multinucleate (Fig. , D, J, and K; cf. A, G, and H). Cell shape was also aberrant in some of the cells in various ways: some cells were rounder than wild-type, whereas others were more elongated. Some cells also had abnormally wide mother– bud necks (Fig. D). As with the growth defect, these phenotypes were considerably more severe for strain YEF1820 (e.g., 36% of the cells had two or more nuclei; n = 234) than for strain JMY1318, in which the large majority of the cells had a normal morphology. Although myo1Δ cells of either strain background completely lacked an actin ring (see above), the subsequent clustering of actin patches at the neck seemed to occur normally (Fig. , F and I), indicating that this process does not require the prior presence or contraction of the actomyosin ring.
Cytokinesis without F-actin
Ayscough et al. (1997)
reported that many of the cells in an asynchronous population could undergo cell separation in the presence of 200 μM LAT-A, suggesting that cytokinesis in S
does not require F-actin. We have confirmed this result. Using strain YEF1698 growing exponentially in SC medium, and scoring budded cells under conditions (sonication after fixation) that minimize the number of cells that have completed cytokinesis but not cell separation (Pringle and Mor, 1975
), we found that the proportion of budded cells decreased from 60 to 28% during a 4-h incubation in the presence of 200 μM LAT-A. Treatment of fixed cells from the population before LAT-A treatment with cell wall-digesting enzymes reduced the proportion of budded cells from 60 to 51%, implying that only 6% of the population had completed cytokinesis but not cell separation (recall that each budded cell produces two unbudded cells upon division); thus, most of the decrease in the percent budded cells during LAT-A treatment indeed involved the completion of both cytokinesis and cell separation. We also used time-lapse microscopy to examine possible cell separation in LAT-A–treated cells. As described above, untreated cells developed a visible septum shortly after contraction of the Myo1p ring, and cell separation was observed ~10 min later. In contrast, we observed neither septum formation nor cell separation during the 30 min after the disappearance of the Myo1p ring, in the LAT-A–treated cells. In one case, a cell that was before anaphase when first observed was seen to form a septum and undergo cell separation ~3 h after the beginning of LAT-A treatment. Thus, it appears that cytokinesis and/or septum formation is delayed or partially defective in the absence of F-actin.