CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae

Budding in the yeast Saccharomyces cerevisiae involves a polarized deposition of new cell surface material that is associated with a highly asymmetric disposition of the actin cytoskeleton. Mutants defective in gene CDC24, which are unable to bud or establish cell polarity, have been of great interest with regard to both the mechanisms of cellular morphogenesis and the mechanisms that coordinate cell-cycle events. To gain further insights into these problems, we sought additional mutants with defects in budding. We report here that temperature-sensitive mutants defective in genes CDC42 and CDC43, like cdc24 mutants, fail to bud but continue growth at restrictive temperature, and thus arrest as large unbudded cells. Nearly all of the arrested cells appear to begin nuclear cycles (as judged by the occurrence of DNA replication and the formation and elongation of mitotic spindles), and many go on to complete nuclear division, supporting the hypothesis that the events associated with budding and those of the nuclear cycle represent two independent pathways within the cell cycle. The arrested mutant cells display delocalized cell- surface deposition associated with a loss of asymmetry of the actin cytoskeleton. CDC42 maps distal to the rDNA on chromosome XII and CDC43 maps near lys5 on chromosome VII.     

Electron microscopic studies onNosema mesnili Paillot (Microsporidia: Nosematidae) infecting the Malpighian tubules ofPieris canidia larva

Summary An entire coding region of theCDC24/CLS4 gene and its truncated derivatives were overexpressed in yeast cells under the control of theGAL1 promoter. Western blotting analysis of the yeast cell lysates showed that the CDC24/CLS4 protein (Cdc24p) was induced to reach its maximum level after 9 h incubation of the cells in galactose medium. Overexpression of Cdc24p within the cells caused the morphological change, accumulating large spherical unbudded cells which exhibited actin cytoskeleton disturbed, chitin delocalized on the cell surface, and cell viability decreased. Multiple nuclei were observed in these cells, indicating that only budding cycle but not nuclear division cycle is blocked by the overproduction of Cdc24p. In order to identify the region of Cdc24p responsible for the growth inhibition, several truncatedCDC24 genes were expressed. Surprisingly, overexpression of fragments either containing the C-terminal 76 amino acid residues or deleting the same region inhibited cellular growth. This suggests that Cdc24p contains multiple functional domains for its tasks, likely cooperating signals of bud positioning and bud timing.     

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.     

The SPA2 protein of yeast localizes to sites of cell growth

A yeast gene, SPA2, was isolated with human anti-spindle pole autoantibodies. The SPA2 gene was fused to the Escherichia coli trpE gene, and polyclonal antibodies were prepared to the fusion protein. Immunofluorescence experiments indicate that the SPA2 gene product has a sharply polarized distribution in yeast cells. In budded cells the SPA2 protein is present at the tip of the bud; in unbudded cells, it is localized to one edge of the cell. When a-cells are induced to form schmoos with alpha-factor, the SPA2 protein is found at the tip of the schmoo. These areas of SPA2 localization correspond to cellular sites expected to be involved in bud formation and/or cell growth. The SPA2 antigen is present in a-cells, alpha-cells, and a/alpha-diploid cells, but is absent in mutant cells in which the SPA2 gene has been disrupted. spa2 mutant cells are viable, but display defects in the direction and control of cell growth. Compared to wild-type cells, spa2 mutant cells have slightly altered budding patterns. Entry into stationary phase is impaired for spa2 mutants, and mutants with one particular allele, spa2-7, form multiple buds under nutrient-limiting conditions. Thus, SPA2 is a newly identified yeast gene that is involved in the direction and control of cell division, and whose gene product localizes to the site of cell growth.     

Cell size modulation by CDC25 and RAS2 genes in Saccharomyces cerevisiae.

A detailed kinetic analysis of the cell cycle of cdc25-1, RAS2Val-19, or cdc25-1/RAS2Val-19 mutants during exponential growth is presented. At the permissive temperature (24 degrees C), cdc25-1 cells show a longer G1/unbudded phase of the cell cycle and have a smaller critical cell size required for budding without changing the growth rate in comparison to an isogenic wild type. The RAS2Val-19 mutation efficiently suppresses the ts growth defect of the cdc25-1 mutant at 36 degrees C and the increase of G1 phase at 24 degrees C. Moreover, it causes a marked increase of the critical cell mass required to enter into a new cell division cycle compared with that of the wild type. Since the critical cell mass is physiologically modulated by nutritional conditions, we have also studied the behavior of these mutants in different media. The increase in cell size caused by the RAS2Val-19 mutation is evident in all tested growth conditions, while the effect of cdc25-1 is apparently more pronounced in rich culture media. CDC25 and RAS2 gene products have been showed to control cell growth by regulating the cyclic AMP metabolic pathway. Experimental evidence reported herein suggests that the modulation of the critical cell size by CDC25 and RAS2 may involve adenylate cyclase.     

Septin function in Candida albicans morphogenesis

The septin proteins function in the formation of septa, mating projections, and spores in Saccharomyces cerevisiae, as well as in cell division and other processes in animal cells. Candida albicans septins were examined in this study for their roles in morphogenesis of this multimorphic, opportunistically pathogenic fungus, which can range from round budding yeast to elongated hyphae. C. albicans green fluorescent protein labeled septin proteins localized to a tight ring at the bud and pseudohyphae necks and as a more diffuse array in emerging germ tubes of hyphae. Deletion analysis demonstrated that the C. albicans homologs of the S. cerevisiae CDC3 and CDC12 septins are essential for viability. In contrast, the C. albicans cdc10Delta and cdc11Delta mutants were viable but displayed conditional defects in cytokinesis, localization of cell wall chitin, and bud morphology. The mutant phenotypes were not identical, however, indicating that these septins carry out distinct functions. The viable septin mutants could be stimulated to undergo hyphal morphogenesis but formed hyphae with abnormal curvature, and they differed from wild type in the selection of sites for subsequent rounds of hyphal formation. The cdc11Delta mutants were also defective for invasive growth when embedded in agar. These results further extend the known roles of the septins by demonstrating that they are essential for the proper morphogenesis of C. albicans during both budding and filamentous growth.     

Cellular morphogenesis in the Saccharomyces cerevisiae cell cycle: localization of the CDC3 gene product and the timing of events at the budding site

Budding cells of the yeast Saccharomyces cerevisiae possess a ring of 10-nm-diameter filaments, of unknown biochemical nature, that lies just inside the plasma membrane in the neck connecting the mother cell to its bud. Electron microscopic observations suggest that these filaments assemble at the budding site coincident with bud emergence and disassemble shortly before cytokinesis (Byers, B. and L. Goetsch. 1976. J. Cell Biol. 69:717-721). Mutants defective in any of four genes (CDC3, CDC10, CDC11, or CDC12) lack these filaments and display a pleiotropic phenotype that involves abnormal bud growth and an inability to complete cytokinesis. We showed previously by immunofluorescence that the CDC12 gene product is probably a constituent of the ring of 10-nm filaments (Haarer, B. and J. Pringle. 1987. Mol. Cell. Biol. 7:3678-3687). We now report the use of fusion proteins to generate polyclonal antibodies specific for the CDC3 gene product. In immunofluorescence experiments, these antibodies decorated the neck regions of wild-type and mutant cells in patterns suggesting that the CDC3 gene product is also a constituent of the ring of 10-nm filaments. We also used the CDC3-specific and CDC12-specific antibodies to investigate the timing of localization of these proteins to the budding site. The results suggest that the CDC3 protein is organized into a ring at the budding site well before bud emergence and remains so organized for some time after cytokinesis. The CDC12 product appears to behave similarly, but may arrive at the budding site closer to the time of bud emergence, and disappear from that site more quickly after cytokinesis, than does the CDC3 product. Examination of mating cells and cells responding to purified mating pheromone revealed novel arrangements of the CDC3 and CDC12 products in the regions of cell wall reorganization. Both proteins were present in normal-looking ring structures at the bases of the first zygotic buds.     

Unequal division in Saccharomyces cerevisiae and its implications for the control of cell division

The budding yeast, Saccharomyces cerevisiae, was grown exponentially at different rates in the presence of growth rate-limiting concentrations of a protein synthesis inhibitor, cycloheximide. The volumes of the parent cell and the bud were determined as were the intervals of the cell cycle devoted to the unbudded and budded periods. We found that S. cerevisiae cells divide unequally. The daughter cell (the cell produced at division by the bud of the previous cycle) is smaller and has a longer subsequent cell cycle than the parent cell which produced it. During the budded period most of the volume increase occurs in the bud and very little in the parent cell, while during the unbudded period both the daughter and the parent cell increase significantly in volume. The length of the budded interval of the cell cycle varies little as a function of population doubling time; the unbudded interval of the parent cell varies moderately; and the unbudded interval for the daughter cell varies greatly (in the latter case an increase of 100 min in population doubling time results in an increase of 124 min in the daughter cell's unbudded interval). All of the increase in the unbudded period occurs in that interval of G1 that precedes the point of cell cycle arrest by the S. cerevisiae alpha-mating factor. These results are qualitatively consistent with and support the model for the coordination of growth and division (Johnston, G. C., J. R. Pringle, and L. H. Hartwell. 1977. Exp. Cell. Res. 105:79-98.) This model states that growth and not the events of the DNA division cycle are rate limiting for cellular proliferation and that the attainment of a critical cell size is a necessary prerequisite for the "start" event in the DNA-division cycle, the event that requires the cdc 28 gene product, is inhibited by mating factor and results in duplication of the spindle pole body.     

Yeast calmodulin localizes to sites of cell growth

Summary Intracellular localization of calmodulin was examined in the budding yeast,Saccharomyces cerevisiae. Distribution of calmodulin changes in a characteristic way during the cell cycle. Calmodulin localizes to a patch at the presumptive bud site of unbudded cells. It concentrates at the bud tip in small-budded cells, and later it diffuses throughout the entire bud. At cytokinesis, calmodulin is largely at the neck between the mother and daughter cells. Double staining experiments have shown that the location of some polarized actin dots is coincident with that of calmodulin dots. Polarized localization of actin dots is observed in cells depleted of calmodulin, suggesting that calmodulin is not required for localization of the actin dots. Thecdc24 mutant that has a defect in bud assembly at the restrictive temperature fails to exhibit polarized localization of calmodulin, indicating that theCDC24 gene product is responsible for controlling the polarity of calmodulin.     

Unique morphogenesis in Saccharomyces cerevisiae strain GS1731

During the lag and early exponential phase of growth, 50–60% of budded cells of Saccharomyces cerevisiae strain GS1731 were multiply budded. During subsequent culture growth, the frequency of multiply budded cells decreased until by stationary phase multiply budded cells were rare. Data from renewed growth of a culture after hydroxyurea treatment indicated that GS1731 mother cells could assemble up to three pre-bud sites and begin bud growth and development in each. Light and scanning electron microscopy showed two or three very small buds emerging simultaneously on a mother cell and either reaching full size at the same time or enlarging sequentially. Immunofluorescence studies revealed that these multiply budded cells had multiple bundles of cytoplasmic microtubules. DAPI staining of nuclei revealed that some of the unbudded mother cells were multinucleate and completed cytokinesis giving rise to normal daughter cells.     

Effects of temperature on the yeast cell cycle analyzed by flow cytometry

The effects of temperature (in the range 15-36 degrees C) on growth and the nuclear and budding cycle have been studied in populations of the yeast Saccharomyces cerevisiae exponentially growing in batch on yeast nitrogen base (YNB) glucose medium. The maximal rate of exponential growth is achieved at 30 degrees C, and a transition point is apparent at about 20 degrees C. At all tested temperatures DNA replication begins when cells are still unbudded and both the budded period and the postreplicative period have the same temperature dependence. A temperature compensatory mechanism seems to operate in S phase, during which duration remains relatively constant, in the range 21-36 degrees C, while duration of G2+ M phases shows a much more pronounced temperature dependence. The results are discussed in terms of a cell-cycle model for budding yeast.     

Yeast myosin heavy chain mutant: maintenance of the cell type specific budding pattern and the normal deposition of chitin and cell wall components requires an intact myosin heavy chain gene

Recent studies with myosin heavy chain mutants in the slime mold Dictyostelium discoideum and the yeast Saccharomyces cerevisiae indicate that the myosin heavy chain gene is not essential for cell survival under laboratory growth conditions. However, cells lacking a normal myosin heavy chain gene demonstrate substantial alterations in growth and cell division. In this study, we report that a disruption mutant in the rod portion of the yeast myosin heavy chain gene, MYO1, produces abnormal chitin distribution and cell wall organization at the mother-bud neck in a high proportion of dividing cells. It is suggested that this phenotype is the cause of the cell division defect and the osmotic sensitivity of yeast MYO1 mutants. In the absence of a normal MYO1 polypeptide, yeast cells alter their cell type specific budding pattern. It is concluded that an intact myosin heavy chain gene is required to maintain the cell type specific budding pattern and the correct localization and deposition of chitin and cell wall components during cell growth and division.     

Characterization of the CDC10 product and the timing of events of the budding site of Saccharomyces cerevisiae

Budding cells of the yeast Saccharomyces cerevisiae possess a ring of septin filaments of unknown biochemical nature that lies under the inner surface of the plasma membrane in the neck that connects the mother cell to its bud. Mutants, defective in any of the four genes (CDC3, CDC10, CDC11, CDC12), lack these septin filaments and display a pleiotropic phenotype that involves abnormal bud growth and an inability to complete cytokinesis. The cloned CDC10 was fused to bacterial genes to generate antibodies specific for the CDC10 product was a constituent of the septin filaments. Cdc10p-specific antibodies for septin staining and actin-specific rhodamine-phalloidine were used to investigate the timing of the localization of septin and actin at the budding site using the immunofluorescence microscopic technique. In wild-type cells, the timing of the appearance and disappearance of these proteins was indistinguishable. In addition, the cdc10 mutant did not prevent actin localization at the budding site. The mutant that was blocked in the actin function also did not prevent the septin localization of the Cdc10p. This result may suggest an organizational independence between these proteins in the bud formation. Finally, the localization of septin and actin in the cdc24 mutant cell was examined. It was found that the CDC24 function was necessary for the organization of septin and actin at the budding site.     

Growth and the inducibility of mycelium formation in Candida albicans: a single-cell analysis using a perfusion chamber

In Candida albicans, cells actively growing in the budding form cannot be immediately induced to form a mycelium until they enter stationary phase. However, if exponential phase cells are starved for a minimum of 10 to 20 min, they are inducible. Using a video-monitored perfusion chamber, we found that starved cells were able to form mycelia regardless of their position in the budding cycle. When starved exponential cells were released into fresh nutrient medium at high temperature and pH, conditions conducive to mycelium formation, unbudded cells evaginated after an average lag period of 75 min and then grew exclusively in the mycelial form. Depending upon the volume, or maturity, of the bud, budded cells entered two different avenues of outgrowth leading to mycelium formation. If the daughter bud was small, growth resumed by apical elongation of the bud, leading to a 'shmoo' shape which tapered into an apical mycelium. If the daughter bud was large, the cell underwent a sequence of evaginations: first, the mother cell evaginated after an average period of 75 min; then the daughter bud evaginated 40 min later. Both evaginations then grew in the mycelial form. In this latter sequence, the evagination on the mother cell was positioned non-randomly, occurring in the majority of cells adjacent to the bud. All buds undergoing evagination contained a nucleus, but roughly 20% of buds undergoing apical elongation did not.     

Septins,under Cla4p regulation,and the chitin ring are required for neck integrity in budding yeast

CLA4, encoding a protein kinase of the PAK type, and CDC11, encoding a septin, were isolated in a screen for synthetic lethality with CHS3, which encodes the chitin synthase III catalytic moiety. Although Ste20p shares some essential function with Cla4p, it did not show synthetic lethality with Chs3p. cla4 and cdc11 mutants exhibited similar morphological and septin localization defects, including aberrant and ectopic septa. Myo1p, which requires septins for localization, formed abnormally wide rings in cla4 mutants. In cultures started with unbudded cells, an inhibitor of Chs3p activity, nikkomycin Z, aggravated the abnormalities of cla4 and cdc11 mutants and gave rise to enlarged necks at the mother-bud junction, leading to cell death. It is concluded that Cla4p is required for the correct localization and/or assembly of the septin ring and that both the septin ring and the Chs3p-requiring chitin ring at the mother-bud neck cooperate in maintaining the neck constricted throughout the cell cycle, a vital function in budding yeast.     

The JNM1 gene in the yeast Saccharomyces cerevisiae is required for nuclear migration and spindle orientation during the mitotic cell cycle

JNM1, a novel gene on chromosome XIII in the yeast Saccharomyces cerevisiae, is required for proper nuclear migration. jnm1 null mutants have a temperature-dependent defect in nuclear migration and an accompanying alteration in astral microtubules. At 30 degrees C, a significant proportion of the mitotic spindles is not properly located at the neck between the mother cell and the bud. This defect is more severe at low temperature. At 11 degrees C, 60% of the cells accumulate with large buds, most of which have two DAPI staining regions in the mother cell. Although mitosis is delayed and nuclear migration is defective in jnm1 mutant, we rarely observe more than two nuclei in a cell, nor do we frequently observe anuclear cells. No loss of viability is observed at 11 degrees C and cells continue to grow exponentially with increased doubling time. At low temperature the large budded cells of jnm1 mutants exhibit extremely long astral microtubules that often wind around the periphery of the cell. jnm1 mutants are not defective in chromosome segregation during mitosis, as assayed by the rate of chromosome loss, or nuclear migration during conjugation, as assayed by the rate of mating and cytoduction. The phenotype of a jnm1 mutant is strikingly similar to that for mutants in the dynein heavy chain gene (Eshel, D., L. A. Urrestarazu, S. Vissers, J.-C. Jauniaux, J. C. van Vliet-Reedijk, R. J. Plants, and I. R. Gibbons. 1993. Proc. Natl. Acad. Sci. USA. 90:11172-11176; Li, Y. Y., E. Yeh, T. Hays, and K. Bloom. 1993. Proc. Natl. Acad. Sci. USA. 90:10096-10100). The JNM1 gene product is predicted to encode a 44-kD protein containing three coiled coil domains. A JNM1:lacZ gene fusion is able to complement the cold sensitivity and microtubule phenotype of a jnm1 deletion strain. This hybrid protein localizes to a single spot in the cell, most often near the spindle pole body in unbudded cells and in the bud in large budded cells. Together these results point to a specific role for Jnm1p in spindle migration, possibly as a subunit or accessory protein for yeast dynein.     

Aip3p/Bud6p, a yeast actin-interacting protein that is involved in morphogenesis and the selection of bipolar budding sites.

A search for Saccharomyces cerevisiae proteins that interact with actin in the two-hybrid system and a screen for mutants that affect the bipolar budding pattern identified the same gene, AIP3/BUD6. This gene is not essential for mitotic growth but is necessary for normal morphogenesis. MATa/alpha daughter cells lacking Aip3p place their first buds normally at their distal poles but choose random sites for budding in subsequent cell cycles. This suggests that actin and associated proteins are involved in placing the bipolar positional marker at the division site but not at the distal tip of the daughter cell. In addition, although aip3 mutant cells are not obviously defective in the initial polarization of the cytoskeleton at the time of bud emergence, they appear to lose cytoskeletal polarity as the bud enlarges, resulting in the formation of cells that are larger and rounder than normal. aip3 mutant cells also show inefficient nuclear migration and nuclear division, defects in the organization of the secretory system, and abnormal septation, all defects that presumably reflect the involvement of Aip3p in the organization and/or function of the actin cytoskeleton. The sequence of Aip3p is novel but contains a predicted coiled-coil domain near its C terminus that may mediate the observed homo-oligomerization of the protein. Aip3p shows a distinctive localization pattern that correlates well with its likely sites of action: it appears at the presumptive bud site prior to bud emergence, remains near the tips of small bund, and forms a ring (or pair of rings) in the mother-bud neck that is detectable early in the cell cycle but becomes more prominent prior to cytokinesis. Surprisingly, the localization of Aip3p does not appear to require either polarized actin or the septin proteins of the neck filaments.     

Structural heterogeneity in populations of the budding yeast Saccharomyces cerevisiae.

Bud scar analysis integrated with mathematical analysis of DNA and protein distributions obtained by flow microfluorometry have been used to analyze the cell cycle of the budding yeast Saccharomyces cerevisiae. In populations of this yeast growing exponentially in batch at 30 degrees C on different carbon and nitrogen sources with duplication times between 75 and 314 min, the budded period is always shorter (approximately 5 to 10 min) than the sum of the S + G2 + M + G1* phases (determined by the Fried analysis of DNA distributions), and parent cells always show a prereplicative unbudded period. The analysis of protein distributions obtained by flow microfluorometry indicates that the protein level per cell required for bud emergence increases at each new generation of parent cells, as observed previously for cell volume. A wide heterogeneity of cell populations derives from this pattern of budding, since older (and less frequent) parent cells have shorter generation times and produce larger (and with shorter cycle times) daughter cells. A possible molecular mechanism for the observed increase with genealogical age of the critical protein level required for bud emergence is discussed.     

The relationship between yeast cell size and cell division in Candida albicans

The mean size and percentage of budded and unbudded cells of Candida albicans grown in batch culture over a wide range of doubling times have been measured. Cell volume decreased with increased doubling time and a nonlinear approach to an asymptotic minimum was observed. When cells were separated by age according to bud scars, each age showed a similar decrease. During each cell division cycle, size increased slowly during both budded and unbudded periods so that each generation was significantly larger than the preceding. There was no difference in size between the parent portion of budded cells and unbudded cells of the same age. Time-lapse photomicroscopy of cells growing on solid medium showed that cells divide asymmetrically with larger parents having a shorter subsequent cycle time than the smaller daughter, although the time utilized for bud formation was similar. When cells were shifted from a medium supporting a low growth rate and small size to a medium supporting a faster growth rate and larger size, both budded and unbudded cells increased significantly in size. As the doubling time increased, both the budded and unbudded portions of parental and daughter cycles increased.     

Relationship of actin and tubulin distribution to bud growth in wild-type and morphogenetic-mutant Saccharomyces cerevisiae

The distribution of actin in wild-type cells and in morphogenetic mutants of the budding yeast Saccharomyces cerevisiae was explored by staining cells with fluorochrome-labeled phallotoxins after fixing and permeabilizing the cells by several methods. The actin appeared to be localized in a set of cortical spots or patches, as well as in a network of cytoplasmic fibers. Bundles of filaments that may possibly correspond to the fibers visualized by fluorescence were observed with the electron microscope. The putative actin spots were concentrated in small and medium-sized buds and at what were apparently the sites of incipient bud formation on unbudded cells, whereas the putative actin fibers were generally oriented along the long axes of the mother-bud pairs. In several morphogenetic mutants that form multiple, abnormally elongated buds, the actin patches were conspicuously clustered at the tips of most buds, and actin fibers were clearly oriented along the long axes of the buds. There was a strong correlation between the occurrence of active growth at particular bud tips and clustering of actin spots at those same tips. Near the end of the cell cycle in wild- type cells, actin appeared to concentrate (as a cluster of spots or a band) in the neck region connecting the mother cell to its bud. Observations made using indirect immunofluorescence with a monoclonal anti-yeast-tubulin antibody on the morphogenetic mutant cdc4 (which forms multiple, abnormally elongated buds while the nuclear cycle is arrested) revealed the surprising occurrence of multiple bundles of cytoplasmic microtubules emanating from the one duplicated spindle-pole body per cell. It seems that most or all of the buds contain one or more of these bundles of microtubules, which often can be seen to extend to the very tips of the buds. These observations are consistent with the hypotheses that actin, tubulin, or both may be involved in the polarization of growth and localization of cell-wall deposition that occurs during the yeast cell cycle.