Bromodeoxyuridine labeling and flow cytometric identification of replicating Saccharomyces cerevisiae cells: lengths of cell cycle phases and population variability at specific cell cycle positions.

An immunofluorescent staining procedure has been developed to identify, with flow cytometry, replicating cells of Saccharomyces cerevisiae after incorporation of bromodeoxyuridine (BrdUrd) into the DNA. Incorporation of BrdUrd is made possible by using yeast strains with a cloned thymidine kinase gene from the herpes simplex virus. An exposure time of 4 min to BrdUrd results in detectable labeling of the DNA. The BrdUrd/DNA double staining procedure has been optimized and the flow cytometry measurements yield histograms comparable to data typically obtained for mammalian cells. On the basis of the accurate assessment of cell fractions in individual cell cycle phases of the asynchronously growing cell population, the average duration of the cell cycle phases has been evaluated. For a population doubling time of 100 min it was found that cells spend in average 41 min in the replicating phase and 24 min in the G2+M cell cycle period. Assuming that mother cells immediately reenter the S phase after cell division, daughter cells spend 65 min in the G1 cell cycle phase. Together with the single cell fluorescence parameters, the forward-angle light scattering intensity (FALS) has been determined as an indicator of cell size. Comparing different temporal positions within the cell cycle, the determined FALS distributions show the lowest variability at the beginning of the S phase. The developed procedure in combination with multiparameter flow cytometry should be useful for studying the kinetics and regulation of the budding yeast cell cycle.     

Symmetric cell division in pseudohyphae of the yeast Saccharomyces cerevisiae.

Laboratory strains of Saccharomyces cerevisiae are dimorphic; in response to nitrogen starvation they switch from a yeast form (YF) to a filamentous pseudohyphal (PH) form. Time-lapse video microscopy of dividing cells reveals that YF and PH cells differ in their cell cycles and budding polarity. The YF cell cycle is controlled at the G1/S transition by the cell-size checkpoint Start. YF cells divide asymmetrically, producing small daughters from full-sized mothers. As a result, mothers and daughters bud asynchronously. Mothers bud immediately but daughters grow in G1 until they achieve a critical cell size. By contrast, PH cells divide symmetrically, restricting mitosis until the bud grows to the size of the mother. Thus, mother and daughter bud synchronously in the next cycle, without a G1 delay before Start. YF and PH cells also exhibit distinct bud-site selection patterns. YF cells are bipolar, producing their second and subsequent buds at either pole. PH cells are unipolar, producing their second and subsequent buds only from the end opposite the junction with their mother. We propose that in PH cells a G2 cell-size checkpoint delays mitosis until bud size reaches that of the mother cell. We conclude that yeast and PH forms are distinct cell types each with a unique cell cycle, budding pattern, and cell shape.     

Multiple methods of visualizing the yeast vacuole permit evaluation of its morphology and inheritance during the cell cycle

The vacuole of the yeast Saccharomyces cerevisiae was visualized with three unrelated fluorescent dyes: FITC-dextran, quinacrine, and an endogenous fluorophore produced in ade2 yeast. FITC-dextran, which enters cells by endocytosis, had been previously developed as a vital stain for yeast vacuoles. Quinacrine, which diffuses across membranes and accumulates in acidic compartments in mammalian cells, can also be used as a marker for yeast vacuoles. ade2 yeast accumulate an endogenous fluorophore in their vacuoles. Using these stains, yeast were examined for vacuole morphology throughout the cell division cycle. In both the parent cell and the bud, a single vacuole was the most common morphology at every stage. Two or more vacuoles could also be found in the mother cell or in the bud; however, this morphology was not correlated with any stage of the cell division cycle. Even small buds (in early S phase) often contained a small vacuole. By the time the bud was half the diameter of the mother cell, it almost always bore a vacuole. This picture of vacuole division and segregation differs from what is seen with synchronized cultures. In ade2 yeast, the bud usually inherits a substantial portion of its vacuole contents from the mother cell. We propose that vacuolar segregation is accomplished by vesicular traffic between the parent cell and the bud.     

A retention mechanism for distribution of mitochondria during cell division in budding yeast.

Mitochondria are indispensable for normal eukaryotic cell function. As they cannot be synthesized de novo and are self-replicating, mitochondria must be transferred from mother to daughter cells. Studies in the budding yeast Saccharomyces cerevisiae indicate that mitochondria enter the bud immediately after bud emergence, interact with the actin cytoskeleton for linear, polarized movement of mitochondria from mother to bud, but are equally distributed among mother and daughter cells [1] [2] [3]. It is not clear how the mother cell maintains its own supply of mitochondria. Here, we found that mother cells retain mitochondria by immobilization of some mitochondria in the 'retention zone', the base of the mother cell distal to the bud. Retention requires the actin cytoskeleton as mitochondria colocalized with actin cables in the retention zone, and mutations that perturb actin dynamics or actin-mitochondrial interactions produced retention defects. Our results support the model that equal distribution of mitochondria during cell division is a consequence of two actin-dependent processes: movement of some mitochondria into the daughter bud and immobilization of others in the mother cell.     

Structural rearrangements of tubulin and actin during the cell cycle of the yeast Saccharomyces

The distribution of actin and tubulin during the cell cycle of the budding yeast Saccharomyces was mapped by immunofluorescence using fixed cells from which the walls had been removed by digestion. The intranuclear mitotic spindle was shown clearly by staining with a monoclonal antitubulin; the presence of extensive bundles of cytoplasmic microtubules is reported. In cells containing short spindles still entirely within the mother cells, one of the bundles of cytoplasmic microtubules nearly always extended to (or into) the bud. Two independent reagents (anti-yeast actin and fluorescent phalloidin) revealed an unusual distribution of actin: it was present as a set of cortical dots or patches and also as distinct fibers that were presumably bundles of actin filaments. Double labeling showed that at no stage in the cell cycle do the distributions of actin and tubulin coincide for any significant length, and, in particular, that the mitotic spindle did not stain detectably for actin. However, both microtubule and actin staining patterns change in a characteristic way during the cell cycle. In particular, the actin dots clustered in rings about the bases of very small buds and at the sites on unbudded cells at which bud emergence was apparently imminent. Later in the budding cycle, the actin dots were present largely in the buds and, in many strains, primarily at the tips of these buds. At about the time of cytokinesis the actin dots clustered in the neck region between the separating cells. These aspects of actin distribution suggest that it may have a role in the localized deposition of new cell wall material.     

Volume growth of daughter and parent cells during the cell cycle of Saccharomyces cerevisiae a/alpha as determined by image cytometry.

The pattern of volume growth of Saccharomyces cerevisiae a/alpha was determined by image cytometry for daughter cells and consecutive cycles of parent cells. An image analysis program was specially developed to measure separately the volume of bud and mother cell parts and to quantify the number of bud scars on each parent cell. All volumetric data and cell attributes (budding state, number of scars) were stored in such a way that separate volume distributions of cells or cell parts with any combination of properties--for instance, buds present on mothers with two scars or cells without scars (i.e., daughter cells) and without buds--could be obtained. By a new method called intersection analysis, the average volumes of daughter and parent cells at birth and at division could be determined for a steady-state population. These volumes compared well with those directly measured from cells synchronized by centrifugal elutriation. During synchronous growth of daughter cells, the pattern of volume increase appeared to be largely exponential. However, after bud emergence, larger volumes than those predicted by a continuous exponential increase were obtained, which confirms the reported decrease in buoyant density. The cycle times calculated from the steady-state population by applying the age distribution equation deviated from those directly obtained from the synchronized culture, probably because of inadequate scoring of bud scars. Therefore, for the construction of a volume-time diagram, we used volume measurements obtained from the steady-state population and cycle times obtained from the synchronized population. The diagram shows that after bud emergence, mother cell parts continue to grow at a smaller rate, increasing about 10% in volume during the budding period. Second-generation daughter cells, ie., cells born from parents left with two scars, were significantly smaller than first-generation daughter cells. Second- and third-generation parent cells showed a decreased volume growth rate and a shorter budding period than that of daughter cells.     

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.     

Astral microtubules are not required for anaphase B in Saccharomyces cerevisiae

tub2-401 is a cold-sensitive allele of TUB2, the sole gene encoding beta-tubulin in the yeast, Saccharomyces cerevisiae. At 18 degrees C, tub2-401 cells are able to assemble spindle microtubules but lack astral microtubules. Under these conditions, movement of the spindle to the bud neck is blocked. However, spindle elongation and chromosome separation are unimpeded and occur entirely within the mother cell. Subsequent cytokinesis produces one cell with two nuclei and one cell without a nucleus. The anucleate daughter can not bud. The binucleate daughter proceeds through another cell cycle to produce a cell with four nuclei and another anucleate cell. With additional time in the cold, the number of nuclei in the nucleated cells continues to increase and the percentage of anucleate cells in the population rises. The results indicate that astral microtubules are needed to position the spindle in the bud neck but are not required for spindle elongation at anaphase B. In addition, cell cycle progression does not depend on the location or orientation of the spindle.     

Buoyant density variation during the cell cycle of Saccharomyces cerevisiae

Cell buoyant densities of the budding yeast Saccharomyces cerevisiae were determined for rapidly growing asynchronous and synchronous cultures by equilibrium sedimentation in Percoll gradients. The average cell density in exponentially growing cultures was 1.1126 g/ml, with a range of density variation of 0.010 g/ml. Densities were highest for cells with buds about one-fourth the diameter of their mother cells and lowest when bud diameters were about the same as their mother cells. In synchronous cultures inoculated from the least-dense cells, there was no observable perturbation of cell growth: cell numbers increased without lag, and the doubling time (66 min) was the same as that for the parent culture. Starting from a low value at the beginning of the cycle, cell buoyant density oscillated between a maximum density near midcycle (0.4 generations) and a minimum near the end of the cycle (0.9 generations). The pattern of cyclic variation of buoyant density was quantitatively determined from density measurements for five cell classes, which were categorized by bud diameter. The observed variation in buoyant density during the cell cycle of S. cerevisiae contrasts sharply with the constancy in buoyant density observed for cells of Escherichia coli, Chinese hamster cells, and three murine cell lines.     

Artificial tethering to nuclear pores promotes partitioning of extrachromosomal DNA during yeast asymmetric cell division

SUMMARY: Asymmetric cell division in unicellular organisms enables sequestration of senescence factors to specific subpopulations. Accumulation of autonomously replicating sequence (ARS) plasmids, which frequently emerge from recombination within the highly repetitive ribosomal DNA locus, is linked to limited replicative life span of Saccharomyces cerevisiae cells [1]. During budding yeast cell division, ARS plasmids are retained in the ageing mother cell, such that only 1 out of 10 plasmids enters the rejuvenated bud [2]. Binding of ARS plasmids to nuclear structures retained in the mother cell was speculated to explain asymmetric plasmid segregation [2]. Association with nuclear pore complexes (NPCs) was proposed to underlie retention of ARS plasmids in the mother cell [3]. However, the role of NPCs in segregation of ARS plasmids is unclear, as NPCs are partitioned between mother and bud nuclei during mitosis [4,5]. Here we analyzed how segregation of ARS plasmids is influenced by their interaction with NPCs. We found that artificial tethering to NPCs promotes transport of ARS plasmids into the bud. Moreover, our experiments provide support for the notion that interaction with ARS plasmids does not affect movement of NPCs into the bud. We conclude that binding to NPCs cannot by itself contribute to asymmetric segregation of ARS plasmids.     

Studies concerning the temporal and genetic control of cell polarity in Saccharomyces cerevisiae

The establishment of cell polarity was examined in the budding yeast, S. cerevisiae. The distribution of a polarized protein, the SPA2 protein, was followed throughout the yeast cell cycle using synchronized cells and cdc mutants. The SPA2 protein localizes to a patch at the presumptive bud site of G1 cells. Later it concentrates at the bud tip in budded cells. At cytokinesis, the SPA2 protein is at the neck between the mother and daughter cells. Analysis of unbudded haploid cells has suggested a series of events that occurs during G1. The SPA2 patch is established very early in G1, while the spindle pole body residues on the distal side of the nucleus. Later, microtubules emanating from the spindle pole body intersect the SPA2 crescent, and the nucleus probably rotates towards the SPA2 patch. By middle G1, most cells contain the SPB on the side of the nucleus proximal to the SPA2 patch, and a long extranuclear microtubule bundle intersects this patch. We suggest that a microtubule capture site exists in the SPA2 staining region that stabilizes the long microtubule bundle; this capture site may be responsible for rotation of the nucleus. Cells containing a polarized distribution of the SPA2 protein also possess a polarized distribution of actin spots in the same region, although the actin staining is much more diffuse. Moreover, cdc4 mutants, which form multiple buds at the restrictive temperature, exhibit simultaneous staining of the SPA2 protein and actin spots in a subset of the bud tips. spa2 mutants contain a polarized distribution of actin spots, and act1-1 and act1-2 mutants often contain a polarized distribution of the SPA2 protein suggesting that the SPA2 protein is not required for localization of the actin spots and the actin spots are not required for localization of the SPA2 protein. cdc24 mutants, which fail to form buds at the restrictive temperature, fail to exhibit polarized localization of the SPA2 protein and actin spots, indicating that the CDC24 protein is directly or indirectly responsible for controlling the polarity of these proteins. Based on the cell cycle distribution of the SPA2 protein, a "cytokinesis tag" model is proposed to explain the mechanism of the non-random positioning of bud sites in haploid yeast cells.     

Morphologically-structured models of growing budding yeast populations

It has been well recognized that many key aspects of cell cycle regulation are encoded into the size distributions of growing budding yeast populations due to the tight coupling between cell growth and cell division present in this organism. Several attempts have been made to model the cell size distribution of growing yeast populations in order to obtain insight on the underlying control mechanisms, but most were based on the age structure of asymmetrically dividing populations. Here we propose a new framework that couples a morphologically-structured representation of the population with population balance theory to formulate a dynamic model for the size distribution of growing yeast populations. An advantage of the presented framework is that it allows derivation of simpler models that are directly identifiable from experiments. We show how such models can be derived from the general framework and demonstrate their utility in analyzing yeast population data. Finally, by employing a recently proposed numerical scheme, we proceed to integrate numerically the full distributed model to provide predictions of dynamics of the cell size structure of growing yeast populations.     

Calmodulin concentrates at regions of cell growth in Saccharomyces cerevisiae

Calmodulin was localized in Saccharomyces cerevisiae by indirect immunofluorescence using affinity-purified polyclonal antibodies. Calmodulin displays an asymmetric distribution that changes during the cell cycle. In unbudded cells, calmodulin concentrates at the presumptive site of bud formation approximately 10 min before bud emergence. In small budded cells, calmodulin accumulates throughout the bud. As the bud grows, calmodulin concentrates at the tip, then disperses, and finally concentrates in the neck region before cytokinesis. An identical staining pattern is observed when wild-type calmodulin is replaced with mutant forms of calmodulin impaired in binding Ca2+. Thus, the localization of calmodulin does not depend on its ability to bind Ca2+ with a high affinity. Double labeling of yeast cells with affinity-purified anti-calmodulin antibody and rhodamine-conjugated phalloidin indicates that calmodulin and actin concentrate in overlapping regions during the cell cycle. Furthermore, disrupting calmodulin function using a temperature-sensitive calmodulin mutant delocalizes actin, and act1-4 mutants contain a random calmodulin distribution. Thus, calmodulin and actin distributions are interdependent. Finally, calmodulin localizes to the shmoo tip in cells treated with alpha-factor. This distribution, at sites of cell growth, implicates calmodulin in polarized cell growth in yeast.     

PAK-family kinases regulate cell and actin polarization throughout the cell cycle of Saccharomyces cerevisiae

During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and cell surface growth are polarized, mediating bud emergence, bud growth, and cytokinesis. We have determined whether p21-activated kinase (PAK)-family kinases regulate cell and actin polarization at one or several points during the yeast cell cycle. Inactivation of the PAK homologues Ste20 and Cla4 at various points in the cell cycle resulted in loss of cell and actin cytoskeletal polarity, but not in depolymerization of F-actin. Loss of PAK function in G1 depolarized the cortical actin cytoskeleton and blocked bud emergence, but allowed isotropic growth and led to defects in septin assembly, indicating that PAKs are effectors of the Rho-guanosine triphosphatase Cdc42. PAK inactivation in S/G2 resulted in depolarized growth of the mother and bud and a loss of actin polarity. Loss of PAK function in mitosis caused a defect in cytokinesis and a failure to polarize the cortical actin cytoskeleton to the mother-bud neck. Cla4-green fluorescent protein localized to sites where the cortical actin cytoskeleton and cell surface growth are polarized, independently of an intact actin cytoskeleton. Thus, PAK family kinases are primary regulators of cell and actin cytoskeletal polarity throughout most or all of the yeast cell cycle. PAK-family kinases in higher organisms may have similar functions.     

Analysis of protein distribution in budding yeast

Flow cytometry is a fast and sensitive method that allows monitoring of different cellular parameters on large samples of a population. Protein distributons give relevant information on growth dynamics, since they are related to the age distribution and depend on the law of growth of the population and the law of protein accumulation during the cell cycle. We analyzed protein distributions to evaluate alternative growth models for the budding yeast Saccharomyces cerevisiae and to monitor the changes in population dynamics that result from environmental modifications; such an analysis could potentially give parameters useful in the control of biotechnological processes. Theoretical protein distributions (taking into account the unequal division of yeast cells and the exponential law of protein accumulation during a cell cycle) quantitatively fit experimental distributions, once appropriate variability sources are introduced. Best fits are obtained when the protein threshold required for bud emergence increases at each new generation of parent cells.     

Synchronous cell growth occurs upon synchronizing the two regulatory steps of the Saccharomyces cerevisiae cell cycle

There are two known asynchronous steps in the budding yeast Saccharomyces cerevisiae cell cycle, where an asynchronous step is one which is completed in different lengths of time by different cells in an isogenic population. It is shown here that elimination of the asynchrony due to cell size by preincubation of cells with the mating pheromone alpha-factor, and decreasing the asynchrony in the cdc28 'start' step by lowering the pH, yields highly synchronous cell growth measured as the time period between the emergence of buds. In one experiment, cell budding for 92% of cells occurred within a 12-min period for at least two generations. Under identical conditions, cell number increase is not as synchronous as bud emergence indicating that there is a third asynchronous step, which is concluded to be at cell separation. These results are consistent with there being two--and only two--asynchronous steps in the cell cycle, measured from bud emergence to bud emergence. Surprisingly, these two steps are also the two major regulatory steps of the cell cycle. It is concluded that asynchrony may be a general feature of cell cycle regulatory steps. The asynchrony in the completion of the cdc28 'start' step which occurs in the first cell cycle after alpha-factor washout is shown here to be almost or entirely eliminated for the second passage through this step after alpha-factor washout. The 'true' time between the onset of budding and the point where 50% of cells have budded (called t50BE) is 17 and less than or equal to 2 min for the first and second budding, respectively, after alpha-factor washout. The cell cycle models requiring a transition probability, or asynchrony, at 'start' for every cell cycle are therefore incorrect.     

Changes in activities of several enzymes involved in carbohydrate metabolism during the cell cycle of Saccharomyces cerevisiae.

Activity changes of a number of enzymes involved in carbohydrate metabolism were determined in cell extracts of fractionated exponential-phase populations of Saccharomyces cerevisiae grown under excess glucose. Cell-size fractionation was achieved by an improved centrifugal elutriation procedure. Evidence that the yeast populations had been fractionated according to age in the cell cycle was obtained by examining the various cell fractions for their volume distribution and their microscopic appearance and by flow cytometric analysis of the distribution patterns of cellular DNA and protein contents. Trehalase, hexokinase, pyruvate kinase, phosphofructokinase 1, and fructose-1,6-diphosphatase showed changes in specific activities throughout the cell cycle, whereas the specific activities of alcohol dehydrogenase and glucose-6-phosphate dehydrogenase remained constant. The basal trehalase activity increased substantially (about 20-fold) with bud emergence and decreased again in binucleated cells. However, when the enzyme was activated by pretreatment of the cell extracts with cyclic AMP-dependent protein kinase, no significant fluctuations in activity were seen. These observations strongly favor posttranslational modification through phosphorylation-dephosphorylation as the mechanism underlying the periodic changes in trehalase activity during the cell cycle. As observed for trehalase, the specific activities of hexokinase and phosphofructokinase 1 rose from the beginning of bud formation onward, finally leading to more than eightfold higher values at the end of the S phase. Subsequently, the enzyme activities dropped markedly at later stages of the cycle. Pyruvate kinase activity was relatively low during the G1 phase and the S phase, but increased dramatically (more than 50-fold) during G2. In contrast to the three glycolytic enzymes investigated, the highest specific activity of the gluconeogenic enzyme fructose-1, 6-diphosphatase 1 was found in fractions enriched in either unbudded cells with a single nucleus or binucleated cells. The observed changes in enzyme activities most likely underlie pronounced alterations in carbohydrate metabolism during the cell cycle.     

A bimolecular mechanism for the cell size control of the cell cycle

A molecular model for the control of cell size has been developed. It is based on two molecules, one (I) acts as an inhibitor of the entrance into S phase, and it is synthetised just after cell separation in a fixed amount per nucleus. The other (A) is an activator of the S phase, and it is synthetised at a ratio proportional to the overall protein accumulation. The activator reacts stoichiometrically with (I), and after all the (I) molecules have been titrated, (A) begins to accumulate. When it reaches a threshold value, it triggers the onset of DNA replication. This model was tested by simulation and when applied to the case of unequal division explains a number of features of an exponentially growing yeast cell population: (a) the lengths of TP (cycle time of parent cells) and TD (cycle time of daughter cells) verify the condition exp(- KTP ) + exp(- KTD ) = 1; (b) the changes of the average cell size of populations at different growth rates; (c) the frequency of parents and daughters at various growth rates; (d) the increase of cell size at bud initiation for cells of increasing genealogical age; (e) the existence of a TP - TB period (difference between the cycle time of parents and the length of budded phase) that depends linearly upon the doubling time of the population.     

Localised axial progenitor cell populations in the avian tail bud are not committed to a posterior Hox identity

The outgrowth of the vertebrate tail is thought to involve the proliferation of regionalised stem/progenitor cell populations formed during gastrulation. To follow these populations over extended periods, we used cells from GFP-positive transgenic chick embryos as a source for donor tissue in grafting experiments. We determined that resident progenitor cell populations are localised in the chicken tail bud. One population, which is located in the chordoneural hinge (CNH), contributes descendants to the paraxial mesoderm, notochord and neural tube, and is serially transplantable between embryos. A second population of mesodermal progenitor cells is located in a separate dorsoposterior region of the tail bud, and a corresponding population is present in the mouse tail bud. Using heterotopic transplantations, we show that the fate of CNH cells depends on their environment within the tail bud. Furthermore, we show that the anteroposterior identity of tail bud progenitor cells can be reset by heterochronic transplantation to the node region of gastrula-stage chicken embryos.     

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.