Hsl1p, a Swe1p inhibitor, is degraded via the anaphase-promoting complex

Ubiquitination and subsequent degradation of critical cell cycle regulators is a key mechanism exploited by the cell to ensure an irreversible progression of cell cycle events. The anaphase-promoting complex (APC) is a ubiquitin ligase that targets proteins for degradation by the 26S proteasome. Here we identify the Hsl1p protein kinase as an APC substrate that interacts with Cdc20p and Cdh1p, proteins that mediate APC ubiquitination of protein substrates. Hsl1p is absent in G(1), accumulates as cells begin to bud, and disappears in late mitosis. Hsl1p is stabilized by mutations in CDH1 and CDC23, both of which result in compromised APC activity. Unlike Hsl1p, Gin4p and Kcc4p, protein kinases that have sequence homology to Hsl1p, were stable in G(1)-arrested cells containing active APC. Mutation of a destruction box motif within Hsl1p (Hsl1p(db-mut)) stabilized Hsl1p. Interestingly, this mutation also disrupted the Hsl1p-Cdc20p interaction and reduced the association between Hsl1p and Cdh1p in coimmunoprecipitation studies. These findings suggest that the destruction box motif is required for Cdc20p and, to a lesser extent, for Cdh1p to target Hsl1p to the APC for ubiquitination. Hsl1p has been previously shown to inhibit Swe1p, a protein kinase that negatively regulates the cyclin-dependent kinase Cdc28p, by promoting Swe1p degradation via SCF(Met30) in a bud morphogenesis checkpoint. Results of the present work indicate that Hsl1p is degraded in an APC-dependent manner and suggest a link between the SCF (Skp1-cullin-F box) and APC-proteolytic systems that may help to coordinate the proper progression of cell cycle events.     

Molecular Dissection of the Checkpoint Kinase Hsl1p

Cell shape can influence cell behavior. In Saccharomyces cerevisiae, bud emergence can influence cell cycle progression via the morphogenesis checkpoint. This surveillance pathway ensures that mitosis always follows bud formation by linking degradation of the mitosis-inhibitory kinase Swe1p (Wee1) to successful bud emergence. A crucial component of this pathway is the checkpoint kinase Hsl1p, which is activated upon bud emergence and promotes Swe1p degradation. We have dissected the large nonkinase domain of Hsl1p by using evolutionary conservation as a guide, identifying regions important for Hsl1p localization, function, and regulation. An autoinhibitory motif restrains Hsl1p activity when it is not properly localized to the mother-bud neck. Hsl1p lacking this motif is active as a kinase regardless of the assembly state of cytoskeletal septin filaments. However, the active but delocalized Hsl1p cannot promote Swe1p down-regulation, indicating that localization is required for Hsl1p function as well as Hsl1p activation. We also show that the septin-mediated Hsl1p regulation via the novel motif operates in parallel to a previously identified Hsl1p activation pathway involving phosphorylation of the Hsl1p kinase domain. We suggest that Hsl1p responds to alterations in septin organization, which themselves occur in response to the local geometry of the cell cortex.     

DNA replication checkpoint control of Wee1 stability by vertebrate Hsl7

G2/M checkpoints prevent mitotic entry upon DNA damage or replication inhibition by targeting the Cdc2 regulators Cdc25 and Wee1. Although Wee1 protein stability is regulated by DNA-responsive checkpoints, the vertebrate pathways controlling Wee1 degradation have not been elucidated. In budding yeast, stability of the Wee1 homologue, Swe1, is controlled by a regulatory module consisting of the proteins Hsl1 and Hsl7 (histone synthetic lethal 1 and 7), which are targeted by the morphogenesis checkpoint to prevent Swe1 degradation when budding is inhibited. We report here the identification of Xenopus Hsl7 as a positive regulator of mitosis that is controlled, instead, by an entirely distinct checkpoint, the DNA replication checkpoint. Although inhibiting Hsl7 delayed mitosis, Hsl7 overexpression overrode the replication checkpoint, accelerating Wee1 destruction. Replication checkpoint activation disrupted Hsl7-Wee1 interactions, but binding was restored by active polo-like kinase. These data establish Hsl7 as a component of the replication checkpoint and reveal that similar cell cycle control modules can be co-opted for use by distinct checkpoints in different organisms.     

Feedback control of Swe1p degradation in the yeast morphogenesis checkpoint

Saccharomyces cerevisiae cells exposed to a variety of physiological stresses transiently delay bud emergence or bud growth. To maintain coordination between bud formation and the cell cycle in such circumstances, the morphogenesis checkpoint delays nuclear division via the mitosis-inhibitory Wee1-family kinase, Swe1p. Swe1p is degraded during G2 in unstressed cells but is stabilized and accumulates following stress. Degradation of Swe1p is preceded by its recruitment to the septin scaffold at the mother-bud neck, mediated by the Swe1p-binding protein Hsl7p. Following osmotic shock or actin depolymerization, Swe1p is stabilized, and previous studies suggested that this was because Hsl7p was no longer recruited to the septin scaffold following stress. However, we now show that Hsl7p is in fact recruited to the septin scaffold in stressed cells. Using a cyclin-dependent kinase (CDK) mutant that is immune to checkpoint-mediated inhibition, we show that Swe1p stabilization following stress is an indirect effect of CDK inhibition. These findings demonstrate the physiological importance of a positive-feedback loop in which Swe1p activity inhibits the CDK, which then ceases to target Swe1p for degradation. They also highlight the difficulty in disentangling direct checkpoint pathways from the effects of positive-feedback loops active at the G2/M transition.     

Determinants of Swe1p degradation in Saccharomyces cerevisiae

Swe1p, the sole Wee1-family kinase in Saccharomyces cerevisiae, is synthesized during late G1 and is then degraded as cells proceed through the cell cycle. However, Swe1p degradation is halted by the morphogenesis checkpoint, which responds to insults that perturb bud formation. The Swe1p stabilization promotes cell cycle arrest through Swe1p-mediated inhibitory phosphorylation of Cdc28p until the cells can recover from the perturbation and resume bud formation. Swe1p degradation involves the relocalization of Swe1p from the nucleus to the mother-bud neck, and neck targeting requires the Swe1p-interacting protein Hsl7p. In addition, Swe1p degradation is stimulated by its substrate, cyclin/Cdc28p, and Swe1p is thought to be a target of the ubiquitin ligase SCF(Met30) acting with the ubiquitin-conjugating enzyme Cdc34p. The basis for regulation of Swe1p degradation by the morphogenesis checkpoint remains unclear, and in order to elucidate that regulation we have dissected the Swe1p degradation pathway in more detail, yielding several novel findings. First, we show here that Met30p (and by implication SCF(Met30)) is not, in fact, required for Swe1p degradation. Second, cyclin/Cdc28p does not influence Swe1p neck targeting, but can directly phosphorylate Swe1p, suggesting that it acts downstream of neck targeting in the Swe1p degradation pathway. Third, a screen for functional but nondegradable mutants of SWE1 identified two small regions of Swe1p that are key to its degradation. One of these regions mediates interaction of Swe1p with Hsl7p, showing that the Swe1p-Hsl7p interaction is critical for Swe1p neck targeting and degradation. The other region did not appear to affect interactions with known Swe1p regulators, suggesting that other as-yet-unknown regulators exist.     

The checkpoint kinase Hsl1p is activated by Elm1p-dependent phosphorylation

Saccharomyces cerevisiae cells growing in the outdoor environment must adapt to sudden changes in temperature and other variables. Many such changes trigger stress responses that delay bud emergence until the cells can adapt. In such circumstances, the morphogenesis checkpoint delays mitosis until a bud has been formed. Mitotic delay is due to the Wee1 family mitotic inhibitor Swe1p, whose degradation is linked to bud emergence by the checkpoint kinase Hsl1p. Hsl1p is concentrated at the mother-bud neck through association with septin filaments, and it was reported that Hsl1p activation involved relief of autoinhibition in response to septin interaction. Here we challenge the previous identification of an autoinhibitory domain and show instead that Hsl1p activation involves the phosphorylation of threonine 273, promoted by the septin-associated kinase Elm1p. We identified elm1 mutants in a screen for defects in Swe1p degradation and show that a phosphomimic T273E mutation in HSL1 bypasses the need for Elm1p in this pathway.     

Septin-dependent assembly of a cell cycle-regulatory module in Saccharomyces cerevisiae

Saccharomyces cerevisiae septin mutants have pleiotropic defects, which include the formation of abnormally elongated buds. This bud morphology results at least in part from a cell cycle delay imposed by the Cdc28p-inhibitory kinase Swe1p. Mutations in three other genes (GIN4, encoding a kinase related to the Schizosaccharomyces pombe mitotic inducer Nim1p; CLA4, encoding a p21-activated kinase; and NAP1, encoding a Clb2p-interacting protein) also produce perturbations of septin organization associated with an Swe1p-dependent cell cycle delay. The effects of gin4, cla4, and nap1 mutations are additive, indicating that these proteins promote normal septin organization through pathways that are at least partially independent. In contrast, mutations affecting the other two Nim1p-related kinases in S. cerevisiae, Hsl1p and Kcc4p, produce no detectable effect on septin organization. However, deletion of HSL1, but not of KCC4, did produce a cell cycle delay under some conditions; this delay appears to reflect a direct role of Hsl1p in the regulation of Swe1p. As shown previously, Swe1p plays a central role in the morphogenesis checkpoint that delays the cell cycle in response to defects in bud formation. Swe1p is localized to the nucleus and to the daughter side of the mother bud neck prior to its degradation in G(2)/M phase. Both the neck localization of Swe1p and its degradation require Hsl1p and its binding partner Hsl7p, both of which colocalize with Swe1p at the daughter side of the neck. This localization is lost in mutants with perturbed septin organization, suggesting that the release of Hsl1p and Hsl7p from the neck may reduce their ability to inactivate Swe1p and thus contribute to the G(2) delay observed in such mutants. In contrast, treatments that perturb actin organization have little effect on Hsl1p and Hsl7p localization, suggesting that such treatments must stabilize Swe1p by another mechanism. The apparent dependence of Swe1p degradation on localization of the Hsl1p-Hsl7p-Swe1p module to a site that exists only in budded cells may constitute a mechanism for deactivating the morphogenesis checkpoint when it is no longer needed (i.e., after a bud has formed).     

Phosphorylation of Hsl1 by Hog1 leads to a G2 arrest essential for cell survival at high osmolarity

Control of cell cycle progression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. Exposure of yeast to osmostress leads to activation of the Hog1 SAPK, which controls cell cycle at G1 by the targeting of Sic1. Here, we show that survival to osmostress also requires regulation of G2 progression. Activated Hog1 interacts and directly phosphorylates a residue within the Hsl7-docking site of the Hsl1 checkpoint kinase, which results in delocalization of Hsl7 from the septin ring and leads to Swe1 accumulation. Upon Hog1 activation, cells containing a nonphosphorylatable Hsl1 by Hog1 are unable to promote Hsl7 delocalization, fail to arrest at G2 and become sensitive to osmostress. Together, we present a novel mechanism that regulates the Hsl1-Hsl7 complex to integrate stress signals to mediate cell cycle arrest and, demonstrate that a single MAPK coordinately modulates different cell cycle checkpoints to improve cell survival upon stress.     

Dynamic Localization of the Swe1 Regulator Hsl7 During the Saccharomyces cerevisiae Cell Cycle

In Saccharomyces cerevisiae, entry into mitosis requires activation of the cyclin-dependent kinase Cdc28 in its cyclin B (Clb)-associated form. Clb-bound Cdc28 is susceptible to inhibitory tyrosine phosphorylation by Swe1 protein kinase. Swe1 is itself negatively regulated by Hsl1, a Nim1-related protein kinase, and by Hsl7, a presumptive protein-arginine methyltransferase. In vivo all three proteins localize to the bud neck in a septin-dependent manner, consistent with our previous proposal that formation of Hsl1-Hsl7-Swe1 complexes constitutes a checkpoint that monitors septin assembly. We show here that Hsl7 is phosphorylated by Hsl1 in immune-complex kinase assays and can physically associate in vitro with either Hsl1 or Swe1 in the absence of any other yeast proteins. With the use of both the two-hybrid method and in vitro binding assays, we found that Hsl7 contains distinct binding sites for Hsl1 and Swe1. A differential interaction trap approach was used to isolate four single-site substitution mutations in Hsl7, which cluster within a discrete region of its N-terminal domain, that are specifically defective in binding Hsl1. When expressed in hsl7Delta cells, each of these Hsl7 point mutants is unable to localize at the bud neck and cannot mediate down-regulation of Swe1, but retains other functions of Hsl7, including oligomerization and association with Swe1. GFP-fusions of these Hsl1-binding defective Hsl7 proteins localize as a bright perinuclear dot, but never localize to the bud neck; likewise, in hsl1Delta cells, a GFP-fusion to wild-type Hsl7 or native Hsl7 localizes to this dot. Cell synchronization studies showed that, normally, Hsl7 localizes to the dot, but only in cells in the G1 phase of the cell cycle. Immunofluorescence analysis and immunoelectron microscopy established that the dot corresponds to the outer plaque of the spindle pole body (SPB). These data demonstrate that association between Hsl1 and Hsl7 at the bud neck is required to alleviate Swe1-imposed G2-M delay. Hsl7 localization at the SPB during G1 may play some additional role in fine-tuning the coordination between nuclear and cortical events before mitosis.     

Mathematical model of the morphogenesis checkpoint in budding yeast

The morphogenesis checkpoint in budding yeast delays progression through the cell cycle in response to stimuli that prevent bud formation. Central to the checkpoint mechanism is Swe1 kinase: normally inactive, its activation halts cell cycle progression in G2. We propose a molecular network for Swe1 control, based on published observations of budding yeast and analogous control signals in fission yeast. The proposed Swe1 network is merged with a model of cyclin-dependent kinase regulation, converted into a set of differential equations and studied by numerical simulation. The simulations accurately reproduce the phenotypes of a dozen checkpoint mutants. Among other predictions, the model attributes a new role to Hsl1, a kinase known to play a role in Swe1 degradation: Hsl1 must also be indirectly responsible for potent inhibition of Swe1 activity. The model supports the idea that the morphogenesis checkpoint, like other checkpoints, raises the cell size threshold for progression from one phase of the cell cycle to the next.     

Control of Swe1p degradation by the morphogenesis checkpoint.

In the budding yeast Saccharomyces cerevisiae, a cell cycle checkpoint coordinates mitosis with bud formation. Perturbations that transiently depolarize the actin cytoskeleton cause delays in bud formation, and a 'morphogenesis checkpoint' detects the actin perturbation and imposes a G2 delay through inhibition of the cyclin-dependent kinase, Cdc28p. The tyrosine kinase Swe1p, homologous to wee1 in fission yeast, is required for the checkpoint-mediated G2 delay. In this report, we show that Swe1p stability is regulated both during the normal cell cycle and in response to the checkpoint. Swe1p is stable during G1 and accumulates to a peak at the end of S phase or in early G2, when it becomes unstable and is degraded rapidly. Destabilization of Swe1p in G2 and M phase depends on the activity of Cdc28p in complexes with B-type cyclins. Several different perturbations of actin organization all prevent Swe1p degradation, leading to the persistence or further accumulation of Swe1p, and cell cycle delay in G2.     

Phosphorylation-Independent Inhibition of Cdc28p by the Tyrosine Kinase Swe1p in the Morphogenesis Checkpoint

The morphogenesis checkpoint in budding yeast delays cell cycle progression in G(2) when the actin cytoskeleton is perturbed, providing time for cells to complete bud formation prior to mitosis. Checkpoint-induced G(2) arrest involves the inhibition of the master cell cycle regulatory cyclin-dependent kinase, Cdc28p, by the Wee1 family kinase Swe1p. Results of experiments using a nonphosphorylatable CDC28(Y19F) allele suggested that the checkpoint stimulated two inhibitory pathways, one that promoted phosphorylation at tyrosine 19 (Y19) and a poorly characterized second pathway that did not require Cdc28p Y19 phosphorylation. We present the results from a genetic screen for checkpoint-defective mutants that led to the repeated isolation of the dominant CDC28(E12K) allele that is resistant to Swe1p-mediated inhibition. Comparison of this allele with the nonphosphorylatable CDC28(Y19F) allele suggested that Swe1p is still able to inhibit CDC28(Y19F) in a phosphorylation-independent manner and that both the Y19 phosphorylation-dependent and -independent checkpoint pathways in fact reflect Swe1p inhibition of Cdc28p. Remarkably, we found that a Swe1p mutant lacking catalytic activity could significantly delay the cell cycle in vivo during a physiological checkpoint response, even when expressed at single copy. The finding that a Wee1 family kinase expressed at physiological levels can inhibit a nonphosphorylatable cyclin-dependent kinase has broad implications for many checkpoint studies using such mutants in other organisms.     

Hsl7 Localizes to a Septin Ring and Serves as an Adapter in a Regulatory Pathway That Relieves Tyrosine Phosphorylation of Cdc28 Protein Kinase in Saccharomyces cerevisiae

Successful mitosis requires faithful DNA replication, spindle assembly, chromosome segregation, and cell division. In the budding yeast Saccharomyces cerevisiae, the G(2)-to-M transition requires activation of Clb-bound forms of the protein kinase, Cdc28. These complexes are held in an inactive state via phosphorylation of Tyr19 in the ATP-binding loop of Cdc28 by the Swe1 protein kinase. The HSL1 and HSL7 gene products act as negative regulators of Swe1. Hsl1 is a large (1,518-residue) protein kinase with an N-terminal catalytic domain and a very long C-terminal extension. Hsl1 localizes to the incipient site of cytokinesis in the bud neck in a septin-dependent manner; however, the function of Hsl7 was not previously known. Using both indirect immunofluorescence with anti-Hsl7 antibodies and a fusion of Hsl7 to green fluorescent protein, we found that Hsl7 also localizes to the bud neck, congruent with the septin ring that faces the daughter cell. Both Swe1 and a segment of the C terminus of Hsl1 (which has no sequence counterpart in two Hsl1-related protein kinases, Gin4 and Kcc4) were identified as gene products that interact with Hsl7 in a two-hybrid screen of a random S. cerevisiae cDNA library. Hsl7 plus Swe1 and Hsl7 plus Hsl1 can be coimmunoprecipitated from extracts of cells overexpressing these proteins, confirming that Hsl7 physically associates with both partners. Also consistent with the two-hybrid results, Hsl7 coimmunoprecipitates with full-length Hsl1 less efficiently than with a C-terminal fragment of Hsl1. Moreover, Hsl7 does not localize to the bud neck in an hsl1Delta mutant, whereas Hsl1 is localized normally in an hsl7Delta mutant. Phosphorylation and ubiquitinylation of Swe1, preludes to its destruction, are severely reduced in cells lacking either Hsl1 or Hsl7 (or both), as judged by an electrophoretic mobility shift assay. Collectively, these data suggest that formation of the septin rings provides sites for docking Hsl1, exposing its C terminus and thereby permitting recruitment of Hsl7. Hsl7, in turn, presents its cargo of bound Swe1, allowing phosphorylation by Hsl1. Thus, Hsl1 and Hsl7 promote proper timing of cell cycle progression by coupling septin ring assembly to alleviation of Swe1-dependent inhibition of Cdc28. Furthermore, like septins and Hsl1, homologs of Hsl7 are found in fission yeast, flies, worms, and humans, suggesting that its function in this control mechanism may be conserved in all eukaryotes.     

A role for the Pkc1p/Mpk1p kinase cascade in the morphogenesis checkpoint

In many cells the timing of entry into mitosis is controlled by the balance between the activity of inhibitory Wee1-related kinases (Swe1p in budding yeast) and the opposing effect of Cdc25-related phosphatases (Mih1p in budding yeast) that act on the cyclin-dependent kinase Cdc2 (Cdc28p in budding yeast). Wee1 and Cdc25 are key elements in the G2 arrest mediated by diverse checkpoint controls. In budding yeast, a 'morphogenesis checkpoint' that involves Swe1p and Mih1p delays mitotic activation of Cdc28p. Many environmental stresses (such as shifts in temperature or osmolarity) provoke transient depolarization of the actin cytoskeleton, during which bud construction is delayed while cells adapt to environmental conditions. During this delay, the morphogenesis checkpoint halts the cell cycle in G2 phase until actin can repolarize and complete bud construction, thus preventing the generation of binucleate cells. A similar G2 delay can be triggered by mutations or drugs that specifically impair actin organization, indicating that it is probably actin disorganization itself, rather than specific environmental stresses, that triggers the delay. The G2 delay involves stabilization of Swe1p in response to various actin perturbations, although this alone is insufficient to produce a long G2 delay.     

Budding yeast Dma1 and Dma2 participate in regulation of Swe1 levels and localization

Timely down-regulation of the evolutionarily conserved protein kinase Swe1 plays an important role in cell cycle control, as Swe1 can block nuclear division through inhibitory phosphorylation of the catalytic subunit of cyclin-dependent kinase. In particular, Swe1 degradation is important for budding yeast cell survival in case of DNA replication stress, whereas it is inhibited by the morphogenesis checkpoint in response to alterations in actin cytoskeleton or septin structure. We show that the lack of the Dma1 and Dma2 ubiquitin ligases, which moderately affects Swe1 localization and degradation during an unperturbed cell cycle with no apparent phenotypic effects, is toxic for cells that are partially defective in Swe1 down-regulation. Moreover, Swe1 is stabilized, restrained at the bud neck, and hyperphosphorylated in dma1Δ dma2Δ cells subjected to DNA replication stress, indicating that the mechanism stabilizing Swe1 under these conditions is different from the one triggered by the morphogenesis checkpoint. Finally, the Dma proteins are required for proper Swe1 ubiquitylation. Taken together, the data highlight a previously unknown role of these proteins in the complex regulation of Swe1 and suggest that they might contribute to control, directly or indirectly, Swe1 ubiquitylation.     

Hsl7 is a substrate-specific type II protein arginine methyltransferase in yeast

The Saccharomyces cerevisiae protein Hsl7 is a regulator of the Swe1 protein kinase in cell cycle checkpoint control. Hsl7 has been previously described as a type III protein arginine methyltransferase, catalyzing the formation of ω-monomethylarginine residues on non-physiological substrates. However, we show here that Hsl7 can also display type II activity, generating symmetric dimethylarginine residues on calf thymus histone H2A. Symmetric dimethylation is only observed when enzyme and the methyl-accepting substrate were incubated for extended times. We confirmed the Hsl7-dependent formation of symmetric dimethylarginine by amino acid analysis and thin layer chromatography with wild-type and mutant recombinant enzymes expressed from both bacteria and yeast. This result is significant because no type II activity has been previously demonstrated in S. cerevisiae. We also show that Hsl7 has little or no activity on GST-GAR, a commonly used substrate for protein arginine methyltransferases, and only minimal activity on myelin basic protein. This enzyme thus may only recognize only a small subset of potential substrate proteins in yeast, in contrast to the situation with Rmt1, the major type I methyltransferase.     

A monitor for bud emergence in the yeast morphogenesis checkpoint

Cell cycle transitions are subject to regulation by both external signals and internal checkpoints that monitor satisfactory progression of key cell cycle events. In budding yeast, the morphogenesis checkpoint arrests the cell cycle in response to perturbations that affect the actin cytoskeleton and bud formation. Herein, we identify a step in this checkpoint pathway that seems to be directly responsive to bud emergence. Activation of the kinase Hsl1p is dependent upon its recruitment to a cortical domain organized by the septins, a family of conserved filament-forming proteins. Under conditions that delayed or blocked bud emergence, Hsl1p recruitment to the septin cortex still took place, but hyperphosphorylation of Hsl1p and recruitment of the Hsl1p-binding protein Hsl7p to the septin cortex only occurred after bud emergence. At this time, the septin cortex spread to form a collar between mother and bud, and Hsl1p and Hsl7p were restricted to the bud side of the septin collar. We discuss models for translating cellular geometry (in this case, the emergence of a bud) into biochemical signals regulating cell proliferation.     

Eavesdropping on the cytoskeleton: progress and controversy in the yeast morphogenesis checkpoint

The morphogenesis checkpoint provides a link between bud formation and mitosis in yeast. In this pathway, insults affecting the actin or septin cytoskeleton trigger a cell cycle arrest, mediated by the Wee1 homolog Swe1p, which catalyzes the inhibitory phosphorylation of the mitosis-promoting cyclin-dependent kinase (CDK) on a conserved tyrosine residue. Analyses of Swe1p phosphorylation have mapped 61 sites targeted by CDKs and Polo-related kinases, which control both Swe1p activity and Swe1p degradation. Although the sites themselves are not evolutionarily conserved, the control of Swe1p degradation exhibits many conserved features, and is linked to DNA-responsive checkpoints in vertebrate cells. At the 'sensing' end of the checkpoint, recent work has begun to shed light on how septins are organized and how they impact Swe1p regulators. However, the means by which Swe1p responds to actin perturbations once a bud has formed remains controversial.