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Article |
Address correspondence to Michael Snyder, Department of Molecular, Cellular, and Developmental Biology, Yale University, P.O. Box 208103, New Haven, CT 06520-8103. Tel.: (203) 432-6139. Fax: (203) 432-3597. email: michael.snyder{at}yale.edu
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Abstract |
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Key Words: polarized growth; polarisome; Bni1; Cdc42; Fus1
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Introduction |
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The budding yeast Saccharomyces cerevisiae initiates polarized growth from the cell surface at several stages of its life cycle and is a useful organism for understanding the regulation of polarized growth events. During vegetative growth, the G1 Cdk complex (Cln-Cdc28) promotes bud emergence (Lew and Reed, 1993). Growth is initially restricted to the bud tip (apical growth) until rising activity of mitotic Cdk complexes (Clb-Cdc28) induces growth throughout the bud surface (Lew and Reed, 1993). During mating, haploid cells respond to pheromone produced by cells of the opposite mating type and form specialized structures called mating projections that are important for cellcell recognition. Growth is restricted to the projection tip and oriented along a pheromone gradient, allowing cells to seek out mates and then fuse with them (Jackson and Hartwell, 1990; Segall, 1993).
In the presence of a high concentration of mating pheromone, cells initiate and terminate projection growth in regular cycles (Bucking-Throm et al., 1973). This phenomenon is thought to enable cells to locate nearby partners and fuse (see Discussion). A similar phenomenon also occurs in budding cells; when the activity of the mitotic Clb-Cdc28 kinase is shut off, cells arrest and periodically form buds that grow to a defined length (Goebl et al., 1988; Schwob et al., 1994; Mathias et al., 1996; Chun and Goebl, 1997). Interestingly, these periodic initiations of polarized growth occur independently of the major budding yeast cell cycleregulating Cdk, Cdc28 (Haase and Reed, 1999). The biochemical nature of the oscillator that triggers the periodic initiation and termination of polarized growth in these cases is unknown.
During both budding and mating projection formation, polarization of the actin cytoskeleton drives the initiation of polarized growth sites (Madden and Snyder, 1998; Pruyne and Bretscher, 2000). Local activation of the essential rho-related GTPase Cdc42 is critical for the initiation of actin polarization. In the absence of Cdc42 activity, cells continue to grow isotropically, becoming large and round. Cellular Cdc42 dynamically cycles between the inactive GDP-bound form and the active GTP-bound form. The cycling of Cdc42 is regulated by the essential guanine nucleotide exchange factor (GEF) Cdc24 (Zheng et al., 1994) and the GTPase-activating proteins (GAPs) Bem3, Rga1, and Rga2 (Smith et al., 2002). Cdc42 is believed to signal through multiple effectors that preferentially interact with GTP-bound Cdc42. These include the PAK-like kinases Ste20 and Cla4, two related proteins, Gic1 and Gic2, and the formin homologue Bni1 (Johnson, 1999).
Bni1 localizes to sites of polarized growth (Evangelista et al., 1997; Fujiwara et al., 1998) and is important for the assembly of actin cables (Evangelista et al., 2002; Sagot et al., 2002), which are thought to direct organelle segregation and the polarized delivery of secretory vesicles and specific mRNAs to the cell surface (Bretscher, 2003). bni1 cells have defects in bud emergence, mating projection formation, and diploid bud site selection (Zahner et al., 1996; Evangelista et al., 1997; Ozaki-Kuroda et al., 2001). Bni1 has a homologue in yeast, Bnr1, and the simultaneous disruption of Bni1 and Bnr1 prevents cells from establishing polarity (Evangelista et al., 2002). The efficient activation and localization of Bni1 requires Spa2 and Pea2 (Fujiwara et al., 1998; Ozaki-Kuroda et al., 2001; Sagot et al., 2002). Spa2 and Pea2 localize to sites of polarized growth, and spa2
and pea2
cells have polarized growth defects that are similar to those observed in bni1
cells (Snyder, 1989; Gehrung and Snyder, 1990; Snyder et al., 1991; Chenevert et al., 1994; Valtz and Herskowitz, 1996). Multiple physical interactions have been detected between Bni1, Spa2, Pea2, and Bud6, and they have been proposed to act together as a complex termed the polarisome (Evangelista et al., 1997; Fujiwara et al., 1998; Sheu et al., 1998).
In this report, we have investigated the mechanisms that regulate mating projection formation and termination and the frequency of these events in haploid yeast cells treated with a high concentration of pheromone. Our results suggest that the Cdc42 regulator Bem3 and the polarisome components Spa2 and Pea2 act upstream of Bni1 to regulate the timing of mating projection formation. Surprisingly, we found that polarisome components as well as the cell fusion proteins Fus1 and Fus2 are important for termination of projection growth and delocalization of actin. Moreover, Fus1 does not regulate the timing of mating projection formation, suggesting that initiation and termination of growth are regulated by partially separate pathways. Our results define the first molecular regulators controlling the timing of periodic mating projection formation and growth termination in budding yeast and provide insight into the mechanisms of dynamic regulation of polarized growth in eukaryotes.
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Results |
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Our time-lapse observations of pheromone-treated wild-type cells revealed three features not previously reported. First, visible growth of existing projections stopped at approximately the same time or slightly after the emergence of a new projection (Fig. 1, B and C). Growth of the first projection terminated an average of 11.0 ± 7.4 min after the initiation of the second projection (n = 11). Second, projections that ceased growth were never observed to resume growth (Fig. 1, B and C). Third, projections grew to approximately the same length (within 20%) before terminating growth.
To more precisely investigate the correlation between the termination of previous projection growth sites and the formation of new projections, we treated wild-type cells with -factor for 6 h and then examined the actin cytoskeleton; polarized actin patches accumulate at active growth sites and are visible before projection growth (Gehrung and Snyder, 1990). After a 6-h incubation with
-factor, cells had an average of 2.96 ± 0.10 mating projections, and all cells had at least two projections. 90.6 ± 0.6% of cells had exactly one site of polarized actin, and 9.4 ± 0.6% had two sites of polarized actin (Fig. 2 A). In these latter cases, the second site of actin polarization was usually present as a broad region on the cell surface that was not associated with a visible projection (Fig. 2 A, arrow), suggesting that these sites represented presumptive projection sites or the earliest stages of projection growth. Interestingly, cells without polarized actin sites were not observed. Taken together, our results suggest that old projections terminate growth at approximately the same time or almost immediately following the initiation of new projections.
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Deletion of genes encoding the polarisome components Spa2, Pea2, or Bni1 decreases the frequency of mating projection formation
We next wished to identify molecular components important for controlling the timing of projection formation. cdc34-2 strains grown at the restrictive temperature arrest with low mitotic Cdk activity and form multiple buds with a regular periodicity (Goebl et al., 1988). Preliminary observations previously indicated that spa2 cdc34-2 cells do not initiate a second bud (Bidlingmaier and Snyder, 2002), suggesting that Spa2 may be a component of the unknown oscillatory mechanism that generates periodic initiation of polarized growth in cdc34-2 and pheromone-treated cells. Spa2 is a component of the "polarisome," a multiprotein complex that is important for polarized growth (Sheu et al., 1998). To determine if Spa2 is important for periodic mating projection formation, we treated spa2 cells with
-factor and measured the timing of projection emergence (Fig. 3 A). The formation of the first projection is slightly delayed in spa2
cells. The time at which 50% of spa2
cells had formed one projection was 85 min, as compared with 67 min for wild-type cells (Fig. 3 A). The emergence of second projections in spa2
cells was severely delayed relative to wild-type cells. After 375 min of
-factor treatment, only 40% of spa2
cells had formed a second projection (Fig. 3 A). In contrast, 50% of wild-type cells had formed a second projection by 210 min (Fig. 1 A). spa2
cells had an average initiation period of 201 ± 13 min (compared with 119 ± 4 for wild-type cells). Projection morphology and growth rate were also affected in spa2
cells. The projections formed by spa2
cells were on average 60% wider than wild-type projections (2.4 ± 0.2 vs. 1.5 ± 0.1 µm, respectively), and the average total extension rate for spa2
projections was almost three times greater than the wild-type extension rate (42.7 ± 2.7 vs. 16.1 ± 0.8 nm/min, respectively).
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Other known polarisome components include Pea2 and Bud6, whose molecular functions are unknown, and the formin homologue Bni1, which has recently been shown to promote the assembly of actin filaments (Evangelista et al., 2002; Sagot et al., 2002; Pring et al., 2003). To determine if Pea2, Bud6, or Bni1 are important for the frequency of periodic mating projection initiation, we treated pea2, bud6
, and bni1
cells with
-factor for 6 h and counted the total number of projections formed to determine the initiation period. Examination of intermediate time points confirmed that projections emerged sequentially in all cases (unpublished data). Similar to spa2
cells, pea2
and bni1
cells had initiation periods that are significantly longer than the wild-type initiation period (180 ± 7 and 213 ± 9 min, respectively, vs. 119± 4 for wild type; Fig. 4 A) and formed projections that are wider than wild-type projections (Fig. 4 B). Deletion of BUD6 did not affect the frequency of mating projection formation or the morphology of the projections (unpublished data). We also analyzed the rate of projection growth in pea2
and bni1
cells. Similar to spa2
cells, the average total projection growth rate for pea2
cells was almost three times the rate of wild-type projection growth (47.3 ± 4.6 nm/min). The average total growth rate was lower for bni1
projections (27.6 ± 0.8 nm/min) but still greater than the wild-type total growth rate. Thus, similar to spa2
cells, pea2
and bni1
cells initiate mating projections less frequently than wild-type cells and form wider, faster growing projections.
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To investigate the growth termination phenotype further, we analyzed the actin cytoskeleton in pheromone-treated spa2, pea2
, and bni1
cells. After a 6-h incubation with
-factor, >60% of spa2
, pea2
, and bni1
cells with two visible projections had actin polarized in the tips of both projections, indicating that they were growing simultaneously (Fig. 5, A and B). spa2
and pea2
projections also had actin cables; these were absent or difficult to detect in bni1
projections (Fig. 5 A). Thus, in addition to their importance in periodic mating projection initiation, Spa2, Pea2, and Bni1 are important for actin delocalization and growth termination.
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Regulators of Cdc42 activity control the frequency of projection initiation in a Bni1-dependent manner
As Bni1 is a downstream effector of the small GTPase Cdc42, we wished to determine if Cdc42 is important for periodic mating projection initiation. We attempted to determine if Cdc42 activity regulates the periodicity of mating projection formation by analyzing cells containing the hyperactive cdc42G60D allele, which causes cells to produce multiple buds that grow simultaneously (Caviston et al., 2002). However, the analysis was complicated by the fact that although cells arrested, most lysed after a 5-h -factor treatment, and multibudded cells (
50% of population) did not appear to undergo a morphological response to pheromone (unpublished data).
cdc42G60D cells have dramatically elevated levels of GTP-bound Cdc42 (Caviston et al., 2002); we therefore determined if more subtle changes in Cdc42 activity would affect the timing of mating projection initiation. Cdc42 is positively regulated by the GEF Cdc24 (Zheng et al., 1994) and negatively regulated by the GAPs Bem3, Rga1, and Rga2 (Smith et al., 2002). We analyzed projection formation in pheromone-treated cdc24-2, bem3, rga1
, and rga2
cells. At the permissive temperature of 25°C, the projection initiation period of pheromone-treated cdc24-2 cells was similar to wild-type cells (unpublished data). However, at the semipermissive temperature of 30°C, cdc24-2 cells had a longer projection initiation period than wild-type cells (158 ± 9 vs. 119 ± 4 min, respectively; Fig. 6 A). In contrast, bem3
cells had a shorter projection initiation period than wild-type cells (101 ± 4 min; Fig. 6 A). The differences between the projection initiation periods of cdc24-2 and bem3
cells and wild-type cells were statistically significant (P < 0.001 in both cases). The morphological response to pheromone was unaffected in rga1
and rga2
cells (unpublished data). Notably, >90% of cdc24-2 and bem3
cells had only one polarized actin site, indicating that growth termination was not affected in these mutants. The average total projection growth rate in bem3
and cdc24-2 cells (16.3 ± 1.3 and 15.0 ± 1.1 nm/min, respectively) was also similar to wild type, indicating that the timing alterations were not due to general effects on cell growth. Thus, Bem3 and Cdc24 regulate the frequency of periodic mating projection initiation.
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Fus1 is important for the termination of projection growth but not the frequency of projection initiation
A screen for genes required for mating in chemotropism-defective cells identified a small set of genes that includes SPA2, PEA2, BNI1, FUS1, and FUS2 (Dorer et al., 1997). Transcription of both FUS1 and FUS2 is dramatically up-regulated in response to pheromone (Trueheart et al., 1987), and fus1 and fus2
mutants have cell fusion defects that are similar to those observed in spa2
mutants (Gammie et al., 1998). These similarities led us to investigate the effect of deleting FUS1 and FUS2 on the timing of mating projection initiation and termination in pheromone-treated cells. We incubated fus1
and fus2
cells with
-factor for 6 h and analyzed projection formation and actin distribution. The projection initiation period in fus1
cells was not significantly different from wild-type cells (124 ± 5 min) (Fig. 7 A). However, growth termination was severely delayed; fus1
projections grew to greater lengths than wild-type projections, and 68% of cells had more than one site of polarized actin (Fig. 7, B and C). Thus, Fus1 is important for growth termination and delocalization of actin patches. In fus2
cells, the initiation period was slightly increased (145 ± 4 min) (Fig. 7 A), although much less than in polarisome mutants. Growth termination was also delayed in fus2
cells; 64% of cells had more than one site of polarized actin, and the projections grew to greater lengths than wild-type projections (Fig. 7, B and C). These results indicate that Fus1 and Fus2 are important for actin delocalization and the termination of projection growth. The observation that the frequency of mating projection initiation is not affected in fus1
cells further supports the idea that the frequency of projection initiation and termination is regulated by partially separate pathways.
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To determine if Pho85, Slt2, and Cla4 are important for periodic mating projection initiation or mating projection growth termination, we analyzed -factortreated pho85
, slt2
, and cla4
cells. For slt2
cells, the experiments were performed in the presence of 1 M sorbitol to prevent cell lysis. In each mutant strain, the frequency of mating projection formation was unaffected (unpublished data). The termination of growth (as determined by projection size) and the delocalization of actin patches was also unaffected (unpublished data). Thus, Pho85, Slt2, and Cla4 do not appear to be important for periodic mating projection initiation or mating projection growth termination.
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Discussion |
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The cell fusion proteins Fus1 and Fus2 are important for projection growth termination
We have shown that growth termination and actin delocalization are also delayed in fus1 and fus2
cells. The expression of FUS1 and FUS2 is dramatically induced in response to pheromone, and Fus1 and Fus2 localize to the tips of mating projections and are important for the degradation of cell wall in the cell-fusion zone (McCaffrey et al., 1987; Trueheart et al., 1987; Elion et al., 1995; Gammie et al., 1998). Significantly, the frequency of periodic mating projection initiation is unaffected in fus1
cells. Pheromone induction of FUS1 expression is normal in polarisome mutants (Sheu et al., 1998; Roberts et al., 2000), and overexpression of FUS1 using a high-copy plasmid did not suppress the growth termination defects of polarisome mutants, suggesting that reduced expression of FUS1 is not the cause of growth termination defects in polarisome mutants. Fus1 is a transmembrane protein with an intracellular SH3 domain (Trueheart et al., 1987; Trueheart and Fink, 1989) and interacts with actin in a two-hybrid assay (Uetz et al., 2000). It is possible that Fus1 concentrated in projection tips acts to locally destabilize the actin cytoskeleton, leading to growth termination.
Cdc42 may control the frequency of projection initiation by regulating the activity of Bni1
We have shown that the Cdc42 GAP, Bem3, and the Cdc42 GEF, Cdc24, regulate the frequency of periodic mating projection. Pheromone-treated cdc24-2 cells have increased projection initiation periods relative to wild-type cells at the semipermissive temperature of 30°C, while bem3 cells have a decreased initiation period. These results suggest that Cdc42-GTP levels may regulate projection initiation frequency. Deletion of BEM3 suppresses the projection initiation frequency defects of spa2
and pea2
cells, but not bni1
cells. It is likely that deletion of BEM3 results in higher levels of GTP-bound Cdc42, which in turn binds and activates Bni1 (Fig. 8). Thus, increased Bni1 activity caused by deletion of BEM3 may be sufficient to compensate for the loss of Spa2 or Pea2 function.
Bni1 is probably not the only Cdc42-GTP effector important for projection initiation, as bni1 cells still form projections, albeit at reduced frequency. Presumably additional effectors also exist; candidates include Bnr1, a homologue of Bni1, and Arp2/3 (Imamura et al., 1997; Lechler et al., 2001).
Interestingly, Cdc42 activity does not appear to affect projection growth termination; cdc24-2 cells incubated with pheromone at the semirestrictive temperature form normal length projections and depolarize actin, and deletion of BEM3 does not suppress the projection growth termination and actin delocalization defects of spa2 and pea2
cells. Thus, it is likely that the frequency of projection initiation and termination is regulated by partially separate pathways. Consistent with this, Fus1 is important for projection growth termination but not initiation frequency (Fig. 8).
Model for periodic mating projection initiation
Our results indicate that the polarisome and Cdc42 regulators control the frequency of periodic mating projection initiation. We propose a model in which periodic mating projection initiation is driven by oscillating levels of Cdc42-GTP in the cell periphery. Initially, a low level of Cdc42-GTP is evenly spread throughout the plasma membrane. Exposure to pheromone stimulates the export of Cdc24 from the nucleus to activated receptors at the cell surface. The GEF activity of Cdc24 causes Cdc42-GTP levels to rise. In the absence of a pheromone gradient, Cdc42-GTP levels initially rise evenly throughout the plasma membrane. It has recently been reported that a sufficient concentration of Cdc42-GTP at the cell surface can promote actomyosin-dependent spontaneous generation of polarity (Wedlich-Soldner et al., 2003). The authors propose that a critical local concentration of Cdc42-GTP on the plasma membrane can produce a positive feedback loop in which Cdc42-GTPinduced actin polymerization increases the probability of further Cdc42 accumulation to that site (Wedlich-Soldner et al., 2003). Our results are consistent with this model and further suggest that Bni1, facilitated by Spa2 and Pea2, is an important downstream effector of Cdc42-GTP that promotes the initiation of mating projections. Thus, once a critical local threshold of Cdc42-GTP is reached, an actin-dependent positive feedback is established, leading to polarization of secretion and clustering of receptors and growth machinery at the projection tip. As a result of this polarization, we propose that a critical rate-limiting factor, or factors, is depleted from the remaining plasma membrane. Potential candidates for these factors include Cdc42 and pheromone receptor. As a result of the depletion of critical factors, the concentration of Cdc42-GTP in the remaining plasma membrane drops below the critical threshold for initiation. In the continuing presence of pheromone, Cdc42-GTP levels rise throughout the cell periphery until the critical threshold concentration is reached again, resulting in the initiation of the next mating projection. We attempted to detect oscillations in the level of Cdc42-GTP in cells undergoing periodic mating projection formation; however, our assays were not able to reliably detect Cdc42-GTP in pheromone-treated cells. Methods for detecting the quantity and location of Cdc42-GTP in individual cells may be required to detect Cdc42-GTP oscillations.
This model provides a framework for understanding many of our observations. Mutants that affect Cdc42-GTP levels (such as bem3 and cdc24-2) would change the time it takes cells to reach the critical threshold for initiation. As spa2
and pea2
mutants are expected to have reduced Bni1 activity, and thus require higher local concentrations of Cdc42-GTP for initiation, the refractory period between projection initiations will be increased in these mutants.
Relationship between initiation and termination of projection growth
During the cell cycle, the initiation of many events is dependent upon the completion of a previous event. In the case of periodic projection formation, it is possible that the initiation of a new projection is delayed until the previous projection has terminated growth. Our time-lapse observations and actin staining of wild-type cells treated with high concentration of pheromone indicate that termination of projection growth occurs almost immediately (10 min) following the initiation of new projections. Additionally, after the emergence of the first projection, cells without polarized actin are not observed. Thus, it appears that old projections do not completely terminate growth before the initiation of the next projection. It is possible that growth termination begins before the initiation of the next projection but is not completed until after the emergence of the next projection. However, in polarisome, fus1
, and fus2
mutants, previous projections continue to grow well after new projections have initiated. Thus, the termination of old projections does not appear to be a strict prerequisite for the initiation of new projections.
Our results suggest that projection initiation and termination are regulated by partially separate pathways and that the termination of the previous projection is not required for the initiation of the next projection. Nevertheless, there appears to be a relationship between projection termination and initiation. Lat-Ainduced termination of projection growth promotes the initiation of new projections in both wild-type and spa2 cells (Fig. 8). Perhaps the disassembly of projection growth sites releases an essential factor, or factors, that is rate limiting for the initiation of new projections. Why do the timing of initiation and termination appear to be tightly linked in wild-type cells? Perhaps the frequency of termination may be naturally close to the frequency of initiation in wild-type cells. If growth termination begins before the initiation of the next projection, the accompanying release of critical factors may promote the initiation of a new projection.
Biological significance of periodic mating projection initiation and termination
The ability of cells to orient projection growth along a pheromone gradient (chemotropism) allows mating cells to efficiently seek out a partner and position projection tips in preparation for cell fusion (Madden and Snyder, 1992; Erdman et al., 1998). In the presence of a high isotropic concentration of pheromone, cells execute a response termed default mating (Dorer et al., 1997). Interestingly, Spa2, Pea2, Bni1, Fus2, and Fus1 are important for default mating (Dorer et al., 1997). It has been suggested that default mating places a more stringent requirement on cell fusion than chemotropic mating (Dorer et al., 1997).
We propose that in the absence of a pheromone gradient to direct projection growth toward a nearby partner, periodic mating projection initiation may allow cells to probe in different directions for a partner. In spa2, pea2
, and bni1
mutants, this probing may be less efficient because projections initiate less frequently and continue to grow in the same direction for long periods of time. Thus, periodic mating projection formation cycles may be an important biological response that facilitates efficient mating in the presence of a saturating concentration of pheromone.
Interestingly, our results indicate that spa2, pea2
, bni1
, fus1
, and fus2
mutants share another common defect in projection growth termination. New cell wall must be continuously synthesized at the tips of growing projections to maintain cell integrity. However, cell wall material between mating cells must be removed before fusion can occur. Thus, delayed projection growth termination may contribute to the cell fusion defects observed in these mutants. In addition, it is also possible that the presence of more than one actively growing projection interferes with the process of mating.
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Materials and methods |
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Time-lapse analysis of living -factortreated cells
To make YPAD/agarose pads, YPAD + 2% agarose was melted and 5 µg/ml -factor was added. Several drops were placed on a microscope slide and covered with a coverslip. Cells were grown to log phase in YPAD, and
-factor was added at a concentration of 5 µg/ml. The coverslips were carefully removed, a drop of cells was added directly to the YPAD/agarose pad, and the cells were recovered with a coverslip. Images of the same field of cells were taken at 10-min intervals during the course of the experiments. Cells grown on the pads remained viable for at least 8 h. The timing of the emergence of first and second projections for multiple cells was determined from the time-lapse images. Projection growth (first and second) was measured for each 10-min interval, and a 30-min moving average was calculated and used to plot the growth rate as a function of time. An average over the entire period of growth for multiple cells was used to calculate the average growth rates of first projections (before and after second projection formation) and second projections. Standard deviations for the timing of projection formation and growth rates are indicated as ±.
Lat-A treatment of -factortreated cells
Exponentially growing cells were treated with 5 µg/ml -factor until >85% had formed one mating projection. Cells were pelleted and resuspended in 50 µl YPAD + 5 µg/ml
-factor. Lat-A (Molecular Probes) was added from a 10 mM DMSO stock to a final concentration of 200 µM. After 5 min, the cells were washed three times with YPAD + 5 µg/ml
-factor and returned to growth in YPAD + 5 µg/ml
-factor. Actin staining confirmed that the actin cytoskeleton had been disrupted in >95% of the cells. At the indicated times following Lat-A treatment, the cells were fixed, and the number of mating projections was analyzed. At least 100 cells were analyzed for each experiment; the average of two experiments was calculated and standard deviations are presented as ±.
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Acknowledgments |
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This research was supported by National Institutes of Health (NIH) grant GM36494 to M. Snyder. S. Bidlingmaier was supported by an NIH training grant.
Submitted: 10 July 2003
Accepted: 4 December 2003
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