Septins are a family of GTP-binding proteins whose heterooligomeric complex is the basic structural element of the septin filaments found in many eukaryotic organisms. In budding yeast, septins are mainly confined at the mother–daughter junction and are required for cell morphogenesis and division. Septins undergo assembly and disassembly in accordance with the progression of the cell cycle. In this report, we identified the yeast protein Syp1p as a new regulator of septin dynamics. Syp1p colocalizes with septins throughout most of the cell cycle. Syp1p interacts with the septin subunit Cdc10p and can be precipitated by Cdc10p and Cdc12p. In the syp1 mutant, both formation of a complete septin ring at the incipient bud site and disassembly of the septin ring in later stages of cell division are significantly delayed. In addition, overexpression of Syp1p causes marked acceleration of septin disassembly. The fluorescence recovery after photobleaching (FRAP) assay further showed that Syp1p promotes septin turnover in different cell cycle stages. These results suggest that Syp1p is involved in the regulation of cell cycle-dependent dynamics of the septin cytoskeleton in yeast.

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SEPTINS were first identified from the yeast Saccharomyces cerevisiae as a set of cdc mutants defective in cytokinesis and were subsequently found to be present in many other eukaryotic species including human (HARTWELL 1971; LONGTINE et al. 1996; SPILIOTIS and NELSON 2006). Septin proteins polymerize into filaments and form ring-like structures found at specific cellular locations (FIELD and KELLOGG 1999; VERSELE et al. 2004; VRABIOIU and MITCHISON 2006; SIRAJUDDIN et al. 2007). Yeast cells express seven septins, five of which (Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Shs1p/Sep7p) are the structural components of the septin ring at the mother-bud junction during vegetative growth (VERSELE et al. 2004), and the other two (Spr3p and Spr28p) are engaged in sporulation (VERSELE and THORNER 2005). The septin ring serves primarily as a scaffold for recruitment of proteins that function in cytokinesis (BI 2001; DOBBELAERE and BARRAL 2004), morphogenesis checkpoint (BARRAL et al. 1999; LEW 2003), bud site selection (FLESCHER et al. 1993; ZAHNER et al. 1996), pheromone-induced morphogenesis (GIOT and KONOPKA 1997), and chitin deposition (DEMARINI et al. 1997; KOZUBOWSKI et al. 2003). In addition, the septin ring is also known to function as diffusion barriers to compartmentalize the plasma and ER membranes (BARRAL et al. 2000; TAKIZAWA et al. 2000; DOBBELAERE and BARRAL 2004; LUEDEKE et al. 2005). As the organization and dynamics of septin filaments are key to their functions, it is important to understand how assembly and disassembly of the septin cytoskeleton are regulated.

Yeast is a desired model system to study the organization and regulation of septins. Yeast septins localize to the incipient bud site shortly before bud emergence where they quickly organize into a ring. During bud growth, the ring expands into an hourglass-like collar spanning the mother-bud neck. At the onset of cytokinesis, the septin collar splits into two septin rings with one each at the mother and daughter sides. After completion of cell division, the old septins normally disassemble before the new structures form for the next cell cycle (CID et al. 2001; LIPPINCOTT et al. 2001; VERSELE and THORNER 2004; IWASE et al. 2006). The septin filament reorganization during the cell cycle correlates with the dynamic properties of septins as detected by the fluorescence recovery after photobleaching (FRAP) experiments (DOBBELAERE et al. 2003). At the early or late cell cycle stages, when septins are in a state of active turnover, the subunits of the septin filaments are replaceable. During the S, G2, and M phases, on the other hand, septin filaments are stable and the subunits are "frozen" (DOBBELAERE et al. 2003).

Various proteins have been identified to regulate septin organization at different cell cycle stages. The small GTPase Cdc42p and its guanine nucleotide exchange factor Cdc24p are required for assembly of the early septin ring at the bud site (GLADFELTER et al. 2002; CAVISTON et al. 2003; IWASE et al. 2006). The effectors of Cdc42p, the Cla4p and Ste20p kinases, also take part in this process (CVRCKOVA et al. 1995; WEISS et al. 2000; VERSELE and THORNER 2004). During bud growth, septin phosphorylation by the kinase Gin4p results in stabilization of septin organization (DOBBELAERE et al. 2003). After cytokinesis, phosphorylation of Cdc3p by Cdc28p promotes the disassembly of the old septin ring (TANG and REED 2002). Dephosphorylation of septins by PP2A is also involved in septin disassembly (MITCHELL and SPRAGUE 2001; DOBBELAERE et al. 2003). Apart from these findings, however, our knowledge of the mechanism that regulates septin dynamics during the cell cycle remains rather scarce.

The yeast protein Syp1p was originally identified as a multicopy suppressor of the pfy1 mutant (MARCOUX et al. 2000). Syp1p was also found capable of restoring the axial bud site selection in the arf3 mutant when overexpressed (LAMBERT et al. 2007). As both Pfy1p and Arf3p are required for the functions of actin cytoskeleton, it is plausible that Syp1p may be involved in actin cytoskeleton and cell polarity as well. In addition, limited studies so far also suggest a role for Syp1p in the function or regulation of septins. Syp1p was localized to the bud neck and its overexpression could induce elongated buds in a fraction of the cell population (MARCOUX et al. 2000), similarly to the proteins that are involved in the organization of septins (DEMARINI et al. 1997; LIPPINCOTT and LI 1998; KIKYO et al. 1999). In this report, we investigated the relationship of Syp1p with septins. We demonstrate that Syp1p can interact with septins and is required for proper septin dynamics at different cell cycle stages. Syp1p, therefore, appears to be a new regulator of septin dynamics in yeast.

Strains, plasmids, media, and general methods:

Yeast strains and plasmids used in this study are listed in Tables 1 and 2, respectively. Gene deletion in YMC515Q, YMC536 was created by integrating an Schizosaccharomyces pombe HIS5 selection cassette to replace the chromosomal locus. YMC517, YMC520, YMC521, YMC532, YMC533, and YMC534 were generated by integrating pCDC12-GFP-305, pGAL-HA-306, pGAL-SYP1-HA-306, pSYP1-HA-306, pSYP1-GFP-305, and pSYP1-CFP-306 into wild-type cells, respectively. The same strategy was used to integrate pGAL-SYP1-Myc-306, pCDC10-GFP-305, and pSYP1-CFP-306 into respective strains. Yeast cells were grown in standard yeast extract-peptone-dextrose (YEPD) or synthetic complete (SC) medium lacking appropriate amino acids for plasmid maintenance. In experiments requiring the expression of genes under the GAL1 promoter, raffinose instead of dextrose was used as the carbon source and galactose was later added for GAL1 induction. Hydroxyurea (HU) (Sigma) was added to a final concentration of 15 mg/ml where required. Preparation of yeast extracts and immunoblotting followed previous procedures (ZENG et al. 2001).

View this table: In this window In a new window   TABLE 1 Yeast strains used in this study

 

View this table: In this window In a new window   TABLE 2 Plasmid constructs used in this study

 

Two-hybrid assay and GST fusion protein binding:

For the yeast two-hybrid assay, test genes were cloned to the HA-tagged GAL4 activation domain of pGADT7 or the Myc-tagged DNA binding domain of pGBKT7 as described in Table 2. The plasmids were cotransformed into the strain SFY526 and the expression of each fusion protein was confirmed by Western blotting with anti-HA or anti-Myc antibodies. The β-galactosidase activities were measured as instructed by the manufacturer (CLONTECH). For GST fusion protein binding, yeast lysate containing Syp1p-HA was incubated with GST fusion protein-coupled beads in the lysate buffer for 2 hr at 4°. The pellets were precipitated and washed five times with the RIPA buffer before being eluted into the SDS–PAGE sample buffer.

Microscopy, live cell imaging, and FRAP:

Cells were observed with a Zeiss Axiovert 200M microscope equipped with a Coolsnap HQ camera (Roper Scientific, Tucson, AZ), unless specified otherwise. To visualize septin dynamics, the CS U22 Laser power supply and Cascade 512B camera were used. Yeast cells expressing GFP and/or CFP-tagged proteins were allowed to grow to the early log phase at 30°. Cells were harvested, resuspended in fresh media, and adhered to the surface of an agarose- (2%) coated glass slide, covered with a cover slip and sealed with vaseline. All of the imaging procedures were kept within a closed chamber at 30°. Images were acquired at the intervals of 1 min for Cdc12p-GFP and 2 min for Syp1p-GFP with motorized GFP filter. At each time point, 7 images (at a speed of 100 ms/image for septins or 400 ms for Syp1p) were acquired at 0.5 µm increments, deconvolved, and reconstructed into 3D images.

The FRAP assays were performed using a Zeiss LSM 510 confocal microscope at 25°. Cells of overnight cultures in YPD containing Cdc12-GFP were harvested and resuspended in synthetic complete medium and spread on a 2% agarose pad. Half of the septin ring was bleached with 20 iterations of 70–80% laser intensity at 488 nm. Excitation for image acquisition ( = 488 nm) was set at 4–5% of the maximal laser intensity. Pictures were taken every 30 sec for a total of 10 min. Fluorescence intensity was analyzed using Metamorph Version 4.0 software and plotted against time. Control cells with small- or medium-sized buds were used to correct for general bleaching.

Septin disorganization as a result of Syp1p overexpression:

First, we were interested in finding out whether the elongated bud phenotype induced by Syp1p overproduction, as reported previously (MARCOUX et al. 2000), could be attributed to septin abnormalities. We repeated this experiment with the strain that contained GAL1-SYP1-HA and Cdc12-GFP. After 7 hr of galactose induction at 30°, elongated buds became evident in some populations of the cells (Figure 1A, arrowheads). Quantitative analysis showed that 46% of mononuclear (n > 100), excluding the ones with very small buds, and 23% of binuclear budded cells (n > 100) had elongated buds. It was also obvious that many Syp1p overexpressing cells exhibited various septin defects (Figure 1A, arrows). Some cells had deformed septins at the bud neck (Figure 1A, arrows 1–3); some had their septins diffused to the cell membrane (Figure 1A, arrows 4–7) and some had little or no septin structures visible. Overall, 34% of mononuclear (n > 100) and 57% of binuclear budded cells (n > 100) displayed abnormal septin structures. In comparison, similar septin abnormalities were rarely found in the vector-transformed control cells. Longer (overnight) induction of Syp1p overexpression caused more severe defects in septin organization, which could be better visualized by confocal microscopy. As shown in Figure 1B, the majority of these cells displayed aberrant septin structures including, most prominently, diffusions to the cell membrane. The number of the cells with elongated buds was also significantly increased (to >70% of the budded cell population). This experiment indicates that Syp1p overexpression can disrupt the normal septin organization and the septin defects may be the cause of the elongated bud phenotype exhibited by some of these cells. Similar results were also obtained using Cdc10-GFP as a septin filament marker (data not shown, but see below).

View larger version (67K): In this window In a new window Download PPT slide   FIGURE 1.— Phenotypes caused by Syp1p overexpression observed under an inverted microscope. (A) Wild-type cell containing CDC12-GFP (YMC517) was transformed with Gal-SYP1-HA or the vector. The resulting strains, YMC518 (vector) and YMC519 (Gal-SYP1-HA), were cultured in raffinose at 30° to log phase followed by the addition of galactose to 2% to induce Syp1p expression. After 7 hr, samples were sonicated and prepared for microscopy. The elongated buds are marked by arrowheads and disorganized septin structures by arrows. The quantitative data of cell morphology and septin organization in the budded cells are shown on the right. (B). The same strains as described in A were subjected to overnight induction of Syp1p overexpression followed by examination under confocal microscope (Zeiss LSM 510). (C) Left: YMC520 (W303::vector), YMC521 (W303::Gal-SYP1-HA), YMC522 (swe1::vector), YMC523 (swe1::Gal-SYP1-HA) were induced for Syp1p overexpression for 7 hr as stated above. Right: The swe1 strain integrated with Gal-SYP1 transformed with vector (YMC524) or SWE1 (YMC525) were cultured and induced for Syp1p overexpression for 7 hr and examined similarly as stated above. Bars, 5 µm.

 
It has been reported that septin defects induce bud elongation through the Swe1p-dependent cell cycle delay (BARRAL et al. 1999; LONGTINE et al. 2000; LEW 2003). To investigate whether the elongated-bud phenotype in the Syp1p overexpression cells was dependent on Swe1p, the same experiment was carried out in the swe1 mutant. As shown in Figure 1C, deletion of SWE1 effectively suppressed the elongated-bud phenotype (Figure 1C), and reintroducing the SWE1 gene back into the mutant restored the phenotype (Figure 1C). This result further supports the notion that the function of Syp1p in vivo may be related to septin organization.

Cells experiencing prolonged overexpression of Syp1p also exhibited division defects. When overnight cultures in galactose were diluted into fresh galactose medium and allowed to continue the Syp1p expression for another 7 hr, 40% of cells (n = 300) became multibudded (Figure 2A). Accordingly, the septin organization was also abnormal in these multibudded cells (Figure 2A, arrows).

View larger version (81K): In this window In a new window Download PPT slide   FIGURE 2.— Additional septin abnormalities caused by Syp1p overexpression. (A) Wild-type cell containing CDC10-GFP was integrated with vector or Gal-SYP1-HA at the ura3 locus, and the respective strains (YMC526 and YMC527) were cultured overnight at 30° in galactose medium followed by dilution into fresh galactose medium and incubated for another 7 hr. The arrowhead marks a multibudded cell. The arrows show the septin defect in the multibudded cell. The quantitative data of the multibudded cells are shown in graphs on the right. (B) The strains described in A were cultured at 30° to log phase and induced by galactose for 3 hr followed by the addition of 2 µg/ml -factor for 2 hr. The arrowheads show the septin defect in the cell overexpressed Syp1p. The quantitative data of septin disorganization are shown in graphs in the right. (C) Left: The septin mutant cdc10-1 (YEF473-1619) containing CDC12-GFP was integrated with vector or Gal-SYP1-HA to generate YMC528 and YMC529, respectively. The strains were cultured at 25° to log phase and induced with galactose for 6 hr. The arrows indicate the elongated bud cells and arrowheads show the septin defect in the elongated bud cells. Right: YMC526 (W303::vector), YMC527 (W303::Gal-SYP1), YMC528, and YMC529 were patched on a plate and allowed to grow at 25° for 2 days and followed by replica plating onto a fresh plate and incubated at 31° for 2 days. Bars, 5 µm.

 
In addition to its role in vegetative growth, the septin cytoskeleton has also been reported to take part in the mating process (GIOT and KONOPKA 1997). After mating pheromone treatment, the yeast cells form a projection termed "shmoo," with the septins rearranged into arrays along the projection axis (LONGTINE et al. 1998). To examine whether overexpression of Syp1p would affect septin organization during shmoo formation, the cells were induced by galactose for 3 hr before the addition of -factor into the medium. After another 2 hr of incubation, remarkable septin organization defects were observed in these cells. The percentage of cells with typical fibrous septin structures was decreased to 18% from 57% in the control cells (n = 200, Figure 2B). The majority of the abnormal septin structures were similar to those found in the Syp1p overexpression cells without -factor treatment shown in Figure 1A, except that more pronounced septin aggregations were evident in this case (Figure 2B, arrowheads).

Furthermore, we also observed synthetic effects between septin mutants and Syp1p overexpression. Syp1p overexpression caused extraordinarily long buds in the cdc10-1 mutant at 25° (Figure 2C, arrows), accompanied by severe septin disorganizations. There were no clear septin rings at the bud neck. Instead, the septins were present as clumps (Figure 2C, arrowhead). At 31°, which was a permissive temperature for the cdc10-1 mutant, Syp1p overexpression caused cell death (Figure 2C, right). Similar synthetic effects were also observed between Syp1p overexpression and another septin mutant cdc3 (data not shown). Taken together, these findings confirm that overexpression of Syp1p can lead to severe defects in septin organization and suggest a role for Syp1p in the regulation of septin organization in yeast.

Abnormal septin structures in HU-arrested syp1 cells:

As reported previously (MARCOUX et al. 2000), deletion of the SYP1 gene generated no obvious defects in cell growth and actin organization (data not shown). The syp1 cells also appeared to have a normal septin cytoskeleton under regular culture conditions. We noticed, however, that the syp1 cells arrested with the DNA synthesis inhibitor HU displayed an apparently different septin morphology from that of wild type. Their septin rings at the neck generally had a larger diameter (Figure 3). Upon careful measurement, the diameter of the septin ring at the mother–daughter neck of the syp1 cells was 1.90 ± 0.26 µm, compared with 1.62 ± 0.23 µm in the parental wild-type cells (n > 100 for each strain, Figure 3). This suggests that the organization of the septin ring in the HU-arrested syp1 mutant is somewhat abnormal.

View larger version (45K): In this window In a new window Download PPT slide   FIGURE 3.— Septin abnormality of the syp1 cells upon HU treatment. YMC517 (W303::CDC12-GFP) and YMC516Q (syp1::CDC12-GFP) were cultured to log phase and treated with HU for 3 hr. Bars, 5 µm.

 

Association of Syp1p with septins:

Although Syp1p has been reported to be localized to the mother–daughter junction (MARCOUX et al. 2000), its localization pattern and that of septins have not been directly compared. We therefore examined the localization of Syp1p-CFP and Cdc12p-YFP in live cells. The two markers were found to be colocalized with each other in the cells of different cell cycle stages (Figure 4A). For example, both proteins localized to the incipient bud site in unbudded cells (Figure 4A, arrow 1), stayed at the mother-bud neck in small- (Figure 4A, arrow 2) and large- (Figure 4A, arrow 3) budded cells, and remained faintly at the division sites of mother and daughter cells after cell separation (Figure 4A, arrow 4). These results suggest that the cellular localization of Syp1p closely coincides with that of septins.

View larger version (51K): In this window In a new window Download PPT slide   FIGURE 4.— Colocalization and interaction between Syp1p and septins. (A) W303 integrated with SYP1-CFP and CDC12-YFP (YMC530) was examined for Syp1p-CFP and Cdc12p-YFP localizations. The arrows show the positions of the proteins at different cell cycle stages. Bars, 5 µm. (B) YMC517 (WT::CDC12-GFP), YMC531 (cdc10-1::CDC12-GFP), YMC534 (WT::SYP1-CFP), and YMC535 (cdc10-1::SYP1-CFP) were cultured at 25° to log phase and one-half of the culture was shifted to 37° and the other half remained at 25°. After 5 hr, the cells were collected and fixed for visualization of Cdc12p-GFP and Syp1p-CFP. Bars, 5 µm. (C) Two-hybrid interaction between Syp1p and septins shown as the β-galactosidase activities. (D) Different septin components were fused to GST tag and expressed and purified from bacteria. The yeast lysate was prepared from the cell integrated with SYP1-HA (YMC532). The GST fusions were bead immobilized and incubated with yeast lysate. The precipitates were separated by gel electrophoresis, transferred to the membrane, and immunoblotted with anti-HA antibody to detect Syp1p (top). The membrane was then stained with Coomassie blue to detect GST and GST-septin fusion proteins (bottom). The arrows indicate different GST-septin fusion proteins.

 
As shown in Figure 3, Syp1p was not required for septins to localize to the neck region. The cellular localization of Syp1p, however, was found to be strictly dependent on septins. At a permissive temperature, the cdc10-1 mutant grew well with normal cell morphology. Under this condition, both Syp1p-CFP and Cdc12p-GFP displayed normal patterns of localization identical to the wild-type strain (Figure 4B). At the nonpermissive temperature of 37°, on the other hand, septins became completely diffuse over the cell (Figure 4B), whereas Syp1p similarly lost the neck localization and was diffuse as well (Figure 4B).

These results led us to investigate the possibility that Syp1p may physically associate with septins in vivo. We first used the two-hybrid assay to examine the interactions of Syp1p with different septin subunits. Indeed, Syp1p demonstrated a clear binding activity with Cdc10p (Figure 4C). To further ascertain the interaction, we attempted to precipitate Syp1p-HA from the yeast cell lysates using GST-septin fusion proteins. As shown in Figure 4D, Cdc10p was able to pull down Syp1p readily (Figure 4D, lane 4). Cdc12p could also pull down Syp1p in this assay (Figure 4D, lane 6). Longer exposure of the Western membrane revealed that a small amount of Syp1p was also present in the binding reactions of Cdc3p and Cdc11p, but not in that of the GST control (data not shown, but faintly visible in Figure 4D). These results suggest that Syp1p may bind to the septin filaments at a region with Cdc10p as a major interaction partner.

Localization of Syp1p in live cells:

To better understand the function of Syp1p, we next followed the localization pattern of Syp1p-GFP during the cell cycle in live cells using time-lapse fluorescent microscopy. In the early cell cycle stage, Syp1p-GFP appeared at the incipient bud site 8 min before bud emergence (Figure 5A, arrows). At first, it appeared as a patch on the cortex and then formed a ring, which remained at the base of the bud after bud emergence (Figure 5, A and B, arrowheads). During the early stage of bud growth, Syp1p-GFP stayed on as a ring at the mother-bud neck (Figure 5B). As the bud grew bigger, some portions of Syp1p-GFP started to appear on the cortex of the bud, while the signals at the bud neck were fading away until they completely disappeared (Figure 5C, frame 8, arrow). About 16 min later, some Syp1p-GFP signals began to congregate to the bud neck (Figure 5C, frame 12, arrowhead). This pattern remained until the completion of cytokinesis (Figure 5C, frame 15). After cell division, Syp1p-GFP reappeared at the incipient bud site in both mother and daughter cells as the next cell cycle initiated (Figure 5C, frame 16). The localization of Syp1p in live cells, therefore, appears to correlate very well with that of septins.

View larger version (127K): In this window In a new window Download PPT slide   FIGURE 5.— Dynamic localization of Syp1p-GFP during the cell cycle. (A and B) Time-lapse fluorescence images of Syp1p-GFP during (A) and after (B) bud emergence. The images were taken at an interval of 2 min. The strain was YMC533. The arrows in A show the first appearance of Syp1p-GFP at the incipient bud site. The arrowheads indicate the base of the buds. (C) Time-lapse fluorescence images of Syp1p-GFP comprising the late cell cycle stages and the early stages of the next cycle. The images were taken at an interval of 4 min. YMC533 was prepared as described above. The arrow indicates the disappearance of Syp1p-GFP from the neck. The arrowhead shows the reappearance of Syp1p-GFP. Bars, 5 µm.

 

The effects of the syp1 mutation on septin dynamics:

To investigate the possible roles of Syp1p in the regulation of septin dynamics, wild-type and the syp1 mutant cells carrying CDC12-GFP were examined with time-lapse microscopy. As shown in Figure 6A, Cdc12p-GFP first appeared in both the wild-type and the syp1 mutant cells as a hazy, irregularly shaped patch at the incipient bud site (Figure 6A, arrows), which took 4 min in the wild type to transform into a complete septin ring (Figure 6A, top, arrowhead). On the other hand, the same process took nearly twice as long in the syp1 cell (Figure 6A, bottom, arrowhead). The average time needed for formation of a complete ring in syp1 cells was 8 ± 2 min (n = 21), compared with only 5 ± 1 min in the wild-type cells (n = 33). After the septin ring formation, the ring stayed at the mother-bud neck until it was disassembled. We calculated the time from the point of ring formation to the point when the septin ring started to decrease in intensity (start of septin disassembly) and found it to be 75 min in both wild-type and the syp1 cells (n > 10 for each strain). We subsequently defined the time from the start of septin disassembly to septin disappearance in either the mother or the daughter side of the neck as the septin disassembly period. It was found that the septin disassembly period at the daughter side was significantly longer in the syp1 cells than that in the wild-type cells. In the wild-type cell, the duration of septin disassembly at the mother side was 20 min (Figure 6B, top, frames 1–10, arrowhead), while it was 28 min at the daughter side (Figure 6B, top, frames 1–14, arrowhead). In the syp1 cell, the mother side septin disassembly took about 22 min, close to that of wild type (Figure 6B, bottom, frames 1–11, arrowhead). However, the disassembly period in the daughter side was measured to be 36 min (Figure 6B, bottom, frames 1–18, arrowhead). The quantitative data compiled from 40 wild-type and 60 syp1 cells led to the conclusion that the average time required for septin disassembly at the daughter side of the cell is 10 min longer in syp1 cells (42 ± 13 min) than in wild type (32 ± 12 min). The mutant, nevertheless, apparently had a similar time frame as the wild type for disassembly of the septin ring at the mother side of the neck, which was usually much fainter in the first place.

View larger version (70K): In this window In a new window Download PPT slide   FIGURE 6.— Abnormal septin dynamics in the syp1 cells and the cells overexpressing Syp1p. Live cells of YMC517 (WT) and YMC516Q (syp1) containing CDC12-GFP were observed for septin dynamics. (A) The images were taken at an interval of 1 min. The arrows indicate the nascent septin structures appearing at the incipient bud sites. The arrowheads mark the point when a complete septin ring was formed. (B) The images were taken at an interval of 2 min. The arrowheads in frame 2 mark the point when the septin ring started to decrease in intensity. The arrowheads in frames 10 and 14 of the wild-type cell, and in frames 11 and 18 of the mutant, mark the point when the old septins disappeared in mother and daughter cells, respectively. (C) The images were taken at an interval of 1 min. YMC518 (vector) and YMC519 (Gal-SYP1) containing CDC12-GFP were induced for Syp1p expression by galactose for 3 hr. The majority of the cells still maintain normal septin morphology after 3 hr of Syp1p overexpression. The arrowheads indicate the point when septin rings begin to disassemble in the vector-containing (top) and the GAL-SYP1-containing (bottom) cells. Two representative GAL-SYP1-containing cells were shown: normal looking (a) and with an elongated bud (b). Bars, 5 µm.

 
The above observation suggests that Syp1p may be required for septin disassembly in the late stage of the cell cycle. Were this the case, one could expect to see accelerated septin disassembly in the cells overexpressing Syp1p. To put this possibility to a test, we followed the Cdc12-GFP marker in live cells that contained pGal-SYP1. Wild-type cells experiencing galactose shift routinely have a longer time frame of septin disassembly than in glucose. Whether this is due to a cell cycle response to galactose pulse or to other metabolic effects on the septin dynamics is not known. After 3 hr in galactose, the septin ring in the vector control cell gradually decreased in intensity over a long time (>30 min) before it completely disappeared (Figure 6C, top). Under the same condition, however, the septins in the cell overexpressing Syp1p disassembled very rapidly (Figure 6C, bottom). The rings disappeared as fast as just a few minutes, regardless of whether the cell had a normal or an elongated bud (Figure 6C). Statistical data indicated that the average duration of disassembly was 45 min in the control cells (n = 12), and 13 min in Syp1p overexpression cells (n = 20). These results are consistent with the observation of the delayed septin disassembly in the syp1 mutant and further support the conclusion that Syp1p functions in the disassembly of septin structures in late cell cycle stages.

Examination of septin dynamics using FRAP:

Thus far, we have identified two functions of Syp1p in the regulation of septin dynamics: the one related to the ring formation at the new budding site at the beginning of the cell cycle and the other the promotion of septin disassembly in the later stages of the cell cycle. The role of Syp1p in septin dynamics was further confirmed using the FRAP assay. In these experiments, half of the fluorescently labeled septin ring was first bleached with a laser beam. The fluorescence recovery in the bleached area, brought about by exchange of septin subunits from outside of the photobleached area, was then monitored over time. This analysis was carried out with unbudded cells and cells in late cell cycle stages, both of which are known to display a certain rate of septin turnover (DOBBELAERE et al. 2003). As shown in Figure 7A, unbudded wild-type cells with a newly formed septin ring showed active septin turnover, as has been previously reported (CAVISTON et al. 2003; DOBBELAERE et al. 2003). Following photobleaching, the GFP-fluorescence intensity in these cells recuperated from 40% of the original value at the time of 0 sec to the recovery plateau of 65% in 120 sec, with a half-time of 44 ± 15 sec (n = 14). The syp1 cells, on the other hand, recovered across the same range in 180 sec with a half-time of 69 ± 39 sec (n = 13), significantly slower than the wild-type cells. This result indicates that the turnover of the septin ring in the early cell cycle is delayed in the syp1 mutant.

View larger version (38K): In this window In a new window Download PPT slide   FIGURE 7.— FRAP studies of septin dynamics in syp1 and Syp1p overexpression cells. CDC12-GFP was locus integrated into the strains of YMC517 (wild type), YMC516Q (syp1), YMC518 (vector), and YMC519 (Gal-SYP1), respectively. The small box in the first frame of each section indicates the bleached region. A representative series of images for a half-bleached ring are shown along with a statistical graph to its right. Each value on the y-axis in the graphs represents the percentage of the fluorescence intensity of the septin ring in the bleached region to the fluorescence intensity of the same region before bleaching. (A) Unbudded cell. (B) Large-budded cells with a split ring. (C) Cell with a medium-sized bud. Bars, 5 µm.

 
Similarly, the syp1 deletion also resulted in a slower rate of fluorescence recovery in late cell cycle stages. As shown in Figure 7B, the average half-time of fluorescence recovery of photobleached split rings at the mother–daughter junction in syp1 cells was 54 ± 21 sec (n = 17), 16 sec longer than that of wild-type cells (38 ± 13 sec, n = 14). This suggests that Syp1p is also required for the efficient septin turnover in the late cell cycle.

In contrast to the cells of early and late cell cycle stages, the cells of mid-cell cycle stage, i.e., those with small- or medium-sized buds, do not undergo active septin turnover (DOBBELAERE et al. 2003). As a result, these cells essentially failed to recover any GFP fluorescence after photobleaching (Figure 7C, top, n = 6). Remarkably, overexpression of Syp1p caused significant changes to septin dynamics in these cells. Recovery of fluorescence was clearly observed (Figure 7C, bottom, n = 6). This result further supports the role of Syp1p as a promoter of septin disassembly.

Genetic interaction between SYP1 and RTS1:

Previously, the regulatory subunit of the phosphatase PP2A, Rts1p, was shown to be required for septin disassembly in the late cell cycle (DOBBELAERE et al. 2003). In rts1 cells, the split septin rings failed to disassemble properly (DOBBELAERE et al. 2003). It is possible, therefore, that Syp1p and Rts1p may share an overlapping function in the regulation of septin disassembly. In support of this hypothesis, we found that deletion of SYP1 rendered rts1 cells completely inviable at 34°, a semi-permissive temperature for the single mutant (Figure 8A, top), and conversely, overexpression of Syp1p significantly improved the viability of rts1 cells at this temperature (Figure 8A, bottom). Consistent with these findings, the syp1rts1 double mutant also displayed a marked increase in the populations of cells with either elongated buds or defective cytokinesis (Figure 8B).

View larger version (40K): In this window In a new window Download PPT slide   FIGURE 8.— Genetic interactions between SYP1 and RTS1. (A) The synthetic lethality between syp1 and rts1 and improved viability of rts1 by Syp1p overexpression. Top: Wild-type, YMC515Q (syp1), YMC536 (rts1), and YMC537 (syp1rts1) cells were cultured to log phase at 25° and spotted on a YPD plate after serial dilutions and incubated at the indicated temperatures for 2 days. Bottom: The same experiment was performed with the cells of indicated genotypes on a dropout plate. (B) Synthetic effect of syp1 and rts1 on cell morphology. Cells of indicated genotypes were cultured to log phase at 25° and shifted to 37° for 4 hr. The quantitative data of cell morphology in the budded cell population are shown in the graph below. Bars, 5 µm.

 

Evidence for Syp1p functioning in septin organization:

In this report, we identified Syp1p as a novel septin-associated protein that functions in the regulation of septin organization in yeast. It colocalizes with septin filaments throughout most of the cell cycle and physically interacts with the septin subunit Cdc10p in vitro. The finding that Syp1p can be precipitated from cell extracts by Cdc10p, Cdc12p, and weakly so by other septins, suggests that Syp1p associates with the septin complex in vivo. Consistent with the physical interaction between Syp1p and the septin complex, the localization of Syp1p is dependent on the integrity of septins at the neck. Both loss of function and overexpression of Syp1p affect the septin assembly and disassembly at specific cell cycle stages. These results strongly suggest a functional relationship between Syp1p and septin organization.

Regulation of septin dynamics by Syp1p:

The role of Syp1p in regulating the assembly and disassembly of septins is also well supported by its dynamic cellular localizations. The dynamic behavior of Syp1p and its persistent colocalization with septins throughout the cell cycle, except for a brief period at the late large-budded stage, suggest that Syp1p may be required for the normal septin organization at different cell cycle stages. Indeed, we have found that in the syp1 cells, the septin dynamics are altered at least at two stages of the cell cycle. Early in the cell cycle of the wild-type cells, septins first appear at the incipient bud site as cloudy and irregular structures, as has also been observed recently by IWASE et al. (2006). They then transform into a jagged ring, which further develops into a complete ring. The whole process takes 4–5 min. However, in the syp1 cells, the jagged ring stays for a longer time and the process of forming a complete ring lasts up to twice as long as in the wild type. In the absence of Syp1p, therefore, the septin ring formation at the beginning of the cell cycle is delayed.

Another aspect of septin dynamics that is altered in the syp1 cells is the septin disassembly at the daughter side of the neck in a late stage of the cell cycle. The syp1 cells consistently show a delay in this process compared with the wild type. The disassembly at the mother side of the neck appears to be unaffected. This observation, however, is complicated by the fact that the distribution of septins at the two sides of the neck is largely uneven in this strain background. Nevertheless, the finding that the septin filaments stay significantly longer in syp1 cells during the later stage of the cell cycle indicates that Syp1p is required for disassembly of the old septins. This notion is also supported by the result of the Syp1p overexpression experiment, in which the septin ring is found to be disassembled at a rate several times faster than in the vector control cells. The remarkably accelerated septin disassembly resulting from Syp1p overexpression may also explain the lethality caused by Syp1p overexpression to the septin mutants. Furthermore, the FRAP assay, which measures the rate of turnover of septin filaments in the cell, further confirmed the role of Syp1p in the dynamics of septin. Turnover of septins in the syp1 cells showed significant delay in both early and late cell cycle stages. The slow turnover of septins in the syp1 mutant may be the reason for the delayed septin ring formation in the early cell cycle stage and septin disassembly in the late cell cycle stage.

The possible functions of Syp1p in regulation of septin dynamics in yeast:

The synthetic lethality between mutants defective in Syp1p and Rts1p suggests that these two proteins share certain essential functions in vivo. Rts1p is a regulatory subunit of the phosphatase PP2A, which has been shown to be required for dephosphorylation of Shs1p in the process of septin disassembly (SHU et al. 1997; DOBBELAERE et al. 2003). Interestingly, the cellular localization of the PP2A complex is highly similar to that of Syp1p (GENTRY and HALLBERG 2002). Whether Syp1p acts as another PP2A regulatory subunit remains to be investigated.

Syp1p could also affect septin organization through its connection with actin cytoskeleton. In view of the previous report that Syp1p could suppress the pfy1 mutation when overexpressed (MARCOUX et al. 2000), we have conducted a number of experiments to examine the possible functions of Syp1p in relation to the actin cytoskeleton. We found no significant defects in the organization or the function of actin cytoskeleton in syp1 cells. The syp1 mutant contained normal actin cytoskeleton and was not impaired in endocytosis (our unpublished results). Nevertheless, we found that the localization of Syp1p to the budding site and the neck was abolished by treatment of the actin filament toxin Latrunculin A or by a mutation in the actin gene (our unpublished results). Syp1p, therefore, is dependent on actin cytoskeleton to establish an intimate colocalization with septin. How actin cytoskeleton affects the localization of Syp1p has not been explored further. It is worth noting that the yeast formins Bni1p and Bnr1p, which nucleate the assembly of actin cables, have both been implicated in septin dynamics as well (KIKYO et al. 1999; KADOTA et al. 2004). Bni1p is required for formation of the early septin ring (KADOTA et al. 2004) and overexpression of Bnr1p resulted in bud elongation with abnormal septin structures (KIKYO et al. 1999), similarly to what we have observed in syp1 and Syp1p overexpression cells. One could speculate, therefore, that Syp1p may cooperate with formins to regulate septin assembly and disassembly.

To conclude, we have demonstrated the functional relationship between Syp1p and septin organization in this report. Syp1p physically interacts with septin filaments and is involved in the processes of septin ring formation in the early stage of the cell cycle. Syp1p is also required for efficient septin turnover at multiple stages of the cell cycle. Further study of the role of Syp1p and its functional relationship with other septin regulators will help us better understand the mechanism that regulates the dynamics of the septin cytoskeleton during the cell cycle.

We are grateful to Uttam Surana for providing the swe1 strain and to Alan Munn for the cdc10-1 mutant. Desmond Dorairajoo and Jun Wang are thanked for the work with microscopy and other general technical assistance. We also thank the members of the M.C. laboratory for valuable discussions. This work was supported by the Agency for Science, Technology and Research of Singapore. M.C. holds an adjunct faculty appointment from the Department of Biochemistry, Faculty of Medicine, National University of Singapore.

BARRAL, Y., M. PARRA, S. BIDLINGMAIER and M. SNYDER, 1999 Nim1-related kinases coordinate cell cycle progression with the organization of the peripheral cytoskeleton in yeast. Genes Dev. 13: 176–187.[Abstract/Free Full Text]

BARRAL, Y., V. MERMALL, M. S. MOOSEKER and M. SNYDER, 2000 Compartmentalization of the cell cortex by septins is required for maintenance of cell polarity in yeast. Mol. Cell 5: 841–851.[CrossRef][Medline]

BI, E., 2001 Cytokinesis in budding yeast: the relationship between actomyosin ring function and septum formation. Cell Struct. Funct. 26: 529–537.[CrossRef][Medline]

CAVISTON, J. P., M. LONGTINE, J. R. PRINGLE and E. BI, 2003 The role of Cdc42p GTPase-activating proteins in assembly of the septin ring in yeast. Mol. Biol. Cell 14: 4051–4066.[Abstract/Free Full Text]

CID, V. J., L. ADAMIKOVA, M. SANCHEZ, M. MOLINA and C. NOMBELA, 2001 Cell cycle control of septin ring dynamics in the budding yeast. Microbiology 147: 1437–1450.[Abstract/Free Full Text]

CVRCKOVA, F., C. DE VIRGILIO, E. MANSER, J. R. PRINGLE and K. NASMYTH, 1995 Ste20-like protein kinases are required for normal localization of cell growth and for cytokinesis in budding yeast. Genes Dev. 9: 1817–1830.[Abstract/Free Full Text]

DEMARINI, D. J., A. E. ADAMS, H. FARES, C. DE VIRGILIO, G. VALLE et al., 1997 A septin-based hierarchy of proteins required for localized deposition of chitin in the Saccharomyces cerevisiae cell wall. J. Cell Biol. 139: 75–93.[Abstract/Free Full Text]

DOBBELAERE, J., and Y. BARRAL, 2004 Spatial coordination of cytokinetic events by compartmentalization of the cell cortex. Science 305: 393–396.[Abstract/Free Full Text]

DOBBELAERE, J., M. S. GENTRY, R. L. HALLBERG and Y. BARRAL, 2003 Phosphorylation-dependent regulation of septin dynamics during the cell cycle. Dev. Cell 4: 345–357.[CrossRef][Medline]

FIELD, C. M., and D. KELLOGG, 1999 Septins: Cytoskeletal polymers or signalling GTPases? Trends Cell Biol. 9: 387–394.[CrossRef][Medline]

FLESCHER, E. G., K. MADDEN and M. SNYDER, 1993 Components required for cytokinesis are important for bud site selection in yeast. J. Cell Biol. 122: 373–386.[Abstract/Free Full Text]

GENTRY, M. S., and R. L. HALLBERG, 2002 Localization of Saccharomyces cerevisiae protein phosphatase 2A subunits throughout mitotic cell cycle. Mol. Biol. Cell 13: 3477–3492.[Abstract/Free Full Text]

GIOT, L., and J. B. KONOPKA, 1997 Functional analysis of the interaction between Afr1p and the Cdc12p septin, two proteins involved in pheromone-induced morphogenesis. Mol. Biol. Cell 8: 987–998.[Abstract]

GLADFELTER, A. S., I. BOSE, T. R. ZYLA, E. S. BARDES and D. J. LEW, 2002 Septin ring assembly involves cycles of GTP loading and hydrolysis by Cdc42p. J. Cell Biol. 156: 315–326.[Abstract/Free Full Text]

HARTWELL, L. H., 1971 Genetic control of the cell division cycle in yeast. IV. Genes controlling bud emergence and cytokinesis. Exp. Cell Res. 69: 265–276.[CrossRef][Medline]

IWASE, M., J. LUO, S. NAGARAJ, M. LONGTINE, H. B. KIM et al., 2006 Role of a Cdc42p effector pathway in recruitment of the yeast septins to the presumptive bud site. Mol. Biol. Cell 17: 1110–1125.[Abstract/Free Full Text]

KADOTA, J., T. YAMAMOTO, S. YOSHIUCHI, E. BI and K. TANAKA, 2004 Septin ring assembly requires concerted action of polarisome components, a PAK kinase Cla4p, and the actin cytoskeleton in Saccharomyces cerevisiae. Mol. Biol. Cell 15: 5329–5345.[Abstract/Free Full Text]

KIKYO, M., K. TANAKA, T. KAMEI, K. OZAKI, T. FUJIWARA et al., 1999 An FH domain-containing Bnr1p is a multifunctional protein interacting with a variety of cytoskeletal proteins in Saccharomyces cerevisiae. Oncogene 18: 7046–7054.[CrossRef][Medline]

KOZUBOWSKI, L., H. PANEK, A. ROSENTHAL, A. BLOECHER, D. J. DEMARINI et al., 2003 A Bni4-Glc7 phosphatase complex that recruits chitin synthase to the site of bud emergence. Mol. Biol. Cell 14: 26–39.[Abstract/Free Full Text]

LAMBERT, A. A., M. P. PERRON, E. LAVOIE and D. PALLOTTA, 2007 The Saccharomyces cerevisiae Arf3 protein is involved in actin cable and cortical patch formation. FEMS Yeast Res. 7: 782–795.[CrossRef][Medline]

LEW, D. J., 2003 The morphogenesis checkpoint: how yeast cells watch their figures. Curr. Opin. Cell Biol. 15: 648–653.[CrossRef][Medline]

LIPPINCOTT, J., and R. LI, 1998 Dual function of Cyk2, a cdc15/PSTPIP family protein, in regulating actomyosin ring dynamics and septin distribution. J. Cell Biol. 143: 1947–1960.[Abstract/Free Full Text]

LIPPINCOTT, J., K. B. SHANNON, W. SHOU, R. J. DESHAIES and R. LI, 2001 The Tem1 small GTPase controls actomyosin and septin dynamics during cytokinesis. J. Cell Sci. 114: 1379–1386.[Abstract]

LONGTINE, M. S., D. J. DEMARINI, M. L. VALENCIK, O. S. AL-AWAR, H. FARES et al., 1996 The septins: roles in cytokinesis and other processes. Curr. Opin. Cell Biol. 8: 106–119.[CrossRef][Medline]

LONGTINE, M. S., H. FARES and J. R. PRINGLE, 1998 Role of the yeast Gin4p protein kinase in septin assembly and the relationship between septin assembly and septin function. J. Cell Biol. 143: 719–736.[Abstract/Free Full Text]

LONGTINE, M. S., C. L. THEESFELD, J. N. MCMILLAN, E. WEAVER, J. R. PRINGLE et al., 2000 Septin-dependent assembly of a cell cycle-regulatory module in Saccharomyces cerevisiae. Mol. Cell. Biol. 20: 4049–4061.[Abstract/Free Full Text]

LUEDEKE, C., S. B. FREI, I. SBALZARINI, H. SCHWARZ, A. SPANG et al., 2005 Septin-dependent compartmentalization of the endoplasmic reticulum during yeast polarized growth. J. Cell Biol. 169: 897–908.[Abstract/Free Full Text]

MARCOUX, N., S. CLOUTIER, E. ZAKRZEWSKA, P. M. CHAREST, Y. BOURBONNAIS et al., 2000 Suppression of the profilin-deficient phenotype by the RHO2 signaling pathway in Saccharomyces cerevisiae. Genetics 156: 579–592.[Abstract/Free Full Text]

MITCHELL, D. A., and G. F. SPRAGUE, JR., 2001 The phosphotyrosyl phosphatase activator, Ncs1p (Rrd1p), functions with Cla4p to regulate the G(2)/M transition in Saccharomyces cerevisiae. Mol. Cell. Biol. 21: 488–500.[Abstract/Free Full Text]

SHU, Y., H. YANG, E. HALLBERG and R. HALLBERG, 1997 Molecular genetic analysis of Rts1p, a B' regulatory subunit of Saccharomyces cerevisiae protein phosphatase 2A. Mol. Cell. Biol. 17: 3242–3253.[Abstract/Free Full Text]

SIRAJUDDIN, M., M. FARKASOVSKY, F. HAUER, D. KUHLMANN, I. G. MACARA et al., 2007 Structural insight into filament formation by mammalian septins. Nature 449: 311–315.[CrossRef][Medline]

SPILIOTIS, E. T., and W. J. NELSON, 2006 Here come the septins: novel polymers that coordinate intracellular functions and organization. J. Cell Sci. 119: 4–10.[Abstract/Free Full Text]

TAKIZAWA, P. A., J. L. DERISI, J. E. WILHELM and R. D. VALE, 2000 Plasma membrane compartmentalization in yeast by messenger RNA transport and a septin diffusion barrier. Science 290: 341–344.[Abstract/Free Full Text]

TANG, C. S., and S. I. REED, 2002 Phosphorylation of the septin cdc3 in g1 by the cdc28 kinase is essential for efficient septin ring disassembly. Cell Cycle 1: 42–49.[Medline]

VERSELE, M., and J. THORNER, 2004 Septin collar formation in budding yeast requires GTP binding and direct phosphorylation by the PAK, Cla4. J. Cell Biol. 164: 701–715.[Abstract/Free Full Text]

VERSELE, M., B. GULLBRAND, M. J. SHULEWITZ, V. J. CID, S. BAHMANYAR et al., 2004 Protein-protein interactions governing septin heteropentamer assembly and septin filament organization in Saccharomyces cerevisiae. Mol. Biol. Cell 15: 4568–4583.[Abstract/Free Full Text]

VERSELE, M., and J. THORNER, 2005 Some assembly required: yeast septins provide the instruction manual. Trends Cell Biol. 15: 414–424.[CrossRef][Medline]

VRABIOIU, A. M., and T. J. MITCHISON, 2006 Structural insights into yeast septin organization from polarized fluorescence microscopy. Nature 443: 466–469.[CrossRef][Medline]

WEISS, E. L., A. C. BISHOP, K. M. SHOKAT and D. G. DRUBIN, 2000 Chemical genetic analysis of the budding-yeast p21-activated kinase Cla4p. Nat. Cell Biol. 2: 677–685.[CrossRef][Medline]

ZAHNER, J. E., H. A. HARKINS and J. R. PRINGLE, 1996 Genetic analysis of the bipolar pattern of bud site selection in the yeast Saccharomyces cerevisiae. Mol. Cell. Biol. 16: 1857–1870.[Abstract/Free Full Text]

ZENG, G., X. YU and M. CAI, 2001 Regulation of yeast actin cytoskeleton-regulatory complex Pan1p/Sla1p/End3p by serine/threonine kinase Prk1p. Mol. Biol. Cell 12: 3759–3772.[Abstract/Free Full Text]

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