Analysis of budding patterns

[7]

ANALYSIS OF BUDDINGPATrERNS

131

OD42o is absorbance by o-nitrophenol (+light scattering); OD6oo is the optical density of the culture at the time of harvesting. OD55o is the light scattering by cell debris; volume assayed is the volume of culture used in the assay in ml; time is reaction time in min. Acknowledgments I thankMatthiasRottmannand SonjaDieterfor providingthe figuresand workingon the protocols described in this article, and HenrikeLotz and Kai Sohn for commentson the manuscript. This work was supportedin part by Grant#03121805 from the BMBF and Ru608/2-1 fromthe DFG.

[7] A n a l y s i s

of Budding

Patterns

By MATTHEWLORD, TRACY CHEN, ATSUSHI FUJITA, and JOHN CHANT

Introduction It has long been known that Saccharomyces can bud and divide in spatially ordered patterns.1 These patterns, determined by cell type and environmental conditions, serve as a paradigm for regulated morphogenetic differentiation. In the past decade or so, efforts to understand the molecular mechanisms underlying the production and regulation of these patterns have intensified. This chapter describes the most commonly employed methods for analyzing budding pattern, bud scar staining, the ace2 colony assay, the use of pseudohyphal growth, and the microcolony method. Each has its own utility. The method chosen depends on the goal of one's experiment. Vegetatively growing yeast can produce two patterns of bud-site selection (Fig. 1A). Haploids (a or a cells) exhibit the axial pattern. Diploids (a/a cells) exhibit the bipolar pattern. Budding pattern is determined by cell type rather than ploidy. For example, a/a or or/or diploids bud in the axial pattern. In the axial pattern, ceils bud immediately adjacent to the previous site of division between mother and daughter (Fig. 1A). 1-3 In the bipolar pattern, cells bud from the poles of their ellipsoidal shapes, t-3 Diploid cells choose poles in no particular order, although biases do exist. The strongest bias is that newborn daughters bud from the pole furthest from their mothers (the distal pole) (Fig. 1A). 3

1D. Freifelder, J. Bacteriol. 80, 567 (1960). 2 j. B. Hicks,J. N. Strathern, and I. Herskowitz,Genetics 85, 373 (1977). 3j. Chant and J. R. Pringle, J. Cell Biol. 129, 751 (1995).

METHODS IN ENZYMOLOGY, VOL. 350

Copyright 2002, Elsevier Science (USA). All rights reserved. 0076-6879/02 $35.00

132

BASIC TECHNIQUES

A

Axial

[7]

Bipolar

or

Random (mutant)

B

Axial

©._,... ©...,,,. @ Bipolar

©...,,,. ©

Unipolar (Pseudohyphal)

nitrogen starvation Random (mutant)

(D-

G

FIG. 1. Budding patterns in yeast. (A) Budding pattern of yeast at the two-cell/two-bud stage: the axial, the bipolar, and the random budding patterns. (B) Budding patterns as represented by the arrangement of division scars on the cell surface. Small rings represent bud scars. The larger ring, depicted at the left pole of each cell, represents the birth scar. T h e m o s t c o m m o n l y e m p l o y e d m e t h o d for analyzing budding pattern is the observation o f patterns o f division scars on the cell surface (Fig. 1B). 3 E v e r y cell is born with a birth scar m a r k i n g the f o r m e r site o f attachment to its mother. E a c h t i m e a cell c o m p l e t e s a round o f budding, a bud scar remains on the cell surface. B e c a u s e yeast possess a thick cell wall, these scars are p e r m a n e n t and serve as a

[7]

ANALYSIS OF BUDDING PATTERNS

133

historical record of a cell's budding pattern over many generations. These scars can also be used to assess a mother cell's age. 4 In the axial pattern, in which a cell buds adjacent to its most recent division site, bud scars form a connected chain starting at the birth scar (Fig. 1B). The path of this chain is essentially a random walk, except that cells cannot form buds on top of preexisting bud scars. A cluster, a line, or some intermediate pattern of scars can result. In the bipolar pattern, cells develop clusters of scars at their poles (Fig. 1B). The number of scars at each pole is variable because there is no set order for using one pole or the other. For a comprehensive description of these patterns, see Chant and Pringle. 3 Much of the work surrounding the problem of budding pattern involves the use of mutants exhibiting specific alterations. Mutations in several genes (RSR1/BUD1, BUD2, or BUD5)5-7 alter the axial and bipolar patterns to an undirected pattern that approaches random selection of sites (Figs. 1A,B). Mutations in other genes (BUD3, BUD4, BUDIO/AXL2/SR04, and AXL1) 6,8-1° convert haploids from budding in the axial pattern to budding in the bipolar pattern but have little to no effect on the bipolar pattern of diploids. Correspondingly, there are a number of mutations which affect the bipolar pattern without affecting the axial pattern. Mutations in BUD8 and BUD9 convert the bipolar pattern to unipolar patterns.ll BUD8 mutants choose buds exclusively from the birth scar pole. BUD9 mutants choose buds from the distal pole only (Fig. 1B). Mutations in RAX1 and RAX2 convert the bipolar pattern to a mixture of random and axial, t2 A very large number of mutations simply randomize the bipolar pattern without affecting the axial pattern (BUD7, BNI1, SPA2, PEA2, and others). 11,13-15 Calcofluor Staining of Bud Scars Calcofluor staining is the most accurate way to analyze a cell's budding pattern in a detailed and quantitative manner. Calcofluor is a fluorescent dye that stains

4 M. Hayashibe and S. Katohda, J. Gen. Appl. Microbiol. 19, 23 (1973). 5 A. Bender and J. R. Pringle, Proc. Natl. Acad. Sci. U.S.A. 86, 9976 (1989). 6 j. Chant and I. Herskowitz, Cell 65, 1203 (1991). 7 j. Chant, K. Corrado, J. R. Pringle, and I. Herskowitz, Cell 65, 1213 (1991). 8 A. Halme, M. Michelitch, E. L. Mitchell, and J. Chant, Curr. Biol. 6, 570 (1996). 9 T. Roemer, K. Madden, J. Chang, and M. Snyder, Genes Dev. 10, 777 (1996). ~0 A. Fujita, C. Oka, Y. Arikawa, T. Katagai, A. Tonouchi, S. Kuhara, and Y. Misumi, Nature 372, 567 (1994). Jl j. E. Zahner, H. A. Harkins, andJ. R. Pringle, Mol. Cell. Biol. 16, 1857 (1996). 12 T. Chen, T. Hiroko, A. Chaudhuri, E Inose, M. Lord, S. Tanaka, J. Chant, and A. Fujita, Science 290, 1975 (2000). 13 N. Valtz and I. Herskowitz, J. Cell Biol. 135, 725 (1996). 14 M. Snyder, J. Cell Biol. 108, 1419 (1989). 15 S. Yang, K. R. Ayscough, and D. G. Drubin, J. Cell Biol. 136, 111 (1997).

134

BASIC TECHNIQUES

Axial

B 1~3 ,.) id r ~

s:

[7]

I~

e,,

J

FIG.2. Bud scar staining with Calcofluor.Upper left: The axial budding pattern of haploids. Upper right: The bipolar budding pattern of diploids. Lower left: The random budding pattern in bud5A haploids. Lower right: The unipolar budding pattern in bud9AIbud9A diploids. Examples of birth scars on cells are indicated by arrowheads. c h i t n - f i c h structures o f the Saccharomyces cerevisiae cell wall. 16 Examples o f budding patterns seen from Calcofluor-stained cell samples are shown in Fig. 2. Bud scars stain brightly, while birth scars are considerably more subtle (as indicated by arrowheads in Fig. 2). Standard M e t h o d 1. Grow a logarithmic culture overnight to an OD600 of approximately 0.2-1. Typically we dilute a saturated culture 5000-fold (1/zl in 5 ml) and grow overnight. 16j. R. Pringle, Methods Enzymol. 194, 732 (1991).

[7]

ANALYSIS OF BUDDINGPATTERNS

135

2. Fix the cells by adding formaldehyde to the medium at a final concentration of 3.7%. Incubate the culture for a further 45-60 min at 30 ° or room temperature. (Formaldehyde is toxic. Please observe the recommended precautions.) 3. Pellet cells and wash twice with water (1 ml per wash). 4. Pellet cells and resuspend in Calcofluor solution (1 mg/ml in water; a 10 mg/ml stock can be kept indefinitely at -20°). We resuspend cells derived from a 5-ml culture in 0.5 ml of the Calcofluor solution. 5. Incubate the sample at room temperature for 5 rain. 6. (Optional) Sonicate for 5 sec on a low setting with a microtip probe (e.g., setting 3 out of 10 on a Branson 450 Sonifier) to eliminate cell clumping, which varies from strain to strain. We strongly recommend this step whenever performing quantitation. 7. Pellet cells and wash with water. Repeat. Resuspend the final pellet in an appropriate volume of water (50-500 #1). At this stage samples can be stored for months at 4 ° . 8. Mount cells on a slide (typically 3-5 #1 with an 18 mm × 18mm coverslip) and squash cells between the slide and coverslip as follows: lay the slide (coverslip up) on a pad of paper towels and place a small pad of paper towels on top with an appropriate weight (a heavy biochemistry textbook is useful). Press for 5-20 min. Squashing immobilizes the cells and allows observation of the cells in one focal plane. (Note that some drying of the cell suspension occurs, resulting in parts of the sample being wet with other parts having dried out. Observe the cells in the remaining droplets of moisture.) 9. Observe stained cells by epifluorescence microscopy using a DAPI filter set. Photobleaching is rarely a problem when using a standard fluorescent microscope since Calcofluor is extremely bright.

Important Considerations (i) For accurate assessment of budding pattems, one must use cultures grown overnight in log phase since budding patterns, especially axial, lose fidelity when cells are in stationary phase. 3 If one wishes to observe cells with multiple scars, the cells should have been grown in logarithmic phase for the period during which the scars were produced. If a cell has eight scars, then this pattern was produced over roughly a 12-hour period in rich medium. (ii) One should be sure to use extremely clean slides and coverslips--a flesh batch. Dust and other small particles hinder efficient cell pressing between slide and coverslip. Dirt also photographs readily. (iii) Efficient flattening of cells between slide and coverslip is essential for observing the cell surface and photographing the cells. (iv) Strain background and growth conditions can affect budding pattern. It is essential to include control samples (wild-type cells from the relevant

136

BASIC TECHNIQUES

[71

strain background) to which one can clearly compare the phenotypes of the samples under study.

Quantitative Scoring Some degree of quantitation with controls is often necessary as first impressions from gazing through the microscope can be misleading. Investigators have generally devised their own schemes to score budding patterns as axial, bipolar, unipolar, or random. Most schemes are very similar. Here we outline our scoring methods. 12'17 We generally score cells with 1 bud scar and cells possessing 3 - 4 bud scars. We count 100-200 cells per sample. Quantitative scoring is essential if one wishes to distinguish subtle differences in budding pattern. First Bud Scar Counts. The position of the first bud scar in relation to the birth scar is scored as follows.

Proximal: If the first bud scar is immediately adjacent to the birth scar, we class this position as proximal. Cells budding axially place their first bud scar proximally 98-100% of the time. Some strains budding in the bipolar pattern place their first bud sites at the proximal pole to a limited degree (0-15%). Distal: If the first bud scar is positioned at the opposite pole from the birth scar we class this position as distal. Distal is typical of the bipolar budding pattern. Medial: If the first bud scar is neither adjacent to the birth scar nor within a bud scar's diameter of the distal pole, we class this position as medial. Medial is typical of the random budding pattern. When cultures are grown exponentially, cells budding in the axial or bipolar patterns essentially never bud in a medial position. Three to Four Bud Scar Counts. Three to four bud scar counts are scored as follows. Axial: If all three/four bud scars are connected in a chain with no spaces between scars and at least one scar is immediately adjacent to the birth scar, we class this cell as axial. Bipolar: If all three/four bud scars are positioned at the distal pole or distributed between the proximal and distal poles, we class this cell as bipolar. Bud scars at either pole need not be touching; however, they should be either adjacent to the birth scar, touching another bud scar, or within a bud scar's diameter from the distal pole. 17M. Lord, M. C. Yang, M. Mischke, and J. Chant, J. Cell BioL 151, 1501 (2000).

[7]

ANALYSIS OF BUDDING PATTERNS

137

Random: If any one or more of the three/four bud scars is situated in the cell's midsection, we class this cell as random. Fast Method This approach is recommended for preliminary checking of budding phenotypes. This method is not recommended for preparing cells for in-depth analysis, or for measurement of subtle budding phenotypes. Some cells will be in stationary phase, a point at which bud-site selection is not maintained with great efficiency. The axial pattern is particularly sensitive to stationary phase or slow growth. 3 1. With a toothpick scrape an appropriate amount of cells from a patch on a freshly grown overnight plate. Resuspend the cells in 0.5 ml of 1 mg/ml-Calcofluor solution. 2. Mount cells on a slide, press, and observe (as described in instructions 8 and 9 of the Standard Method). If speed is critical, cells can be squashed by briefly pressing cells between slide and coverslip using one's thumb.

Use of Calcofluor Staining in Combination with Other Analyses Certain dye-based methods for examining subcellular structure (e.g., DNA 18 or actin staining 19) can be performed in conjunction with the standard bud scar staining protocol. Covisualization of proteins is best performed using green fluorescent protein (GFP) fusions. Immunofluorescence is not especially compatible with bud scar staining, as permeating the cells for antibody access requires the removal of the cell wall, which results in loss of bud scars. This procedure relies on quick preparation of live samples and is particularly useful for GFP fusions. 1. From a freshly grown overnight patch of cells, thinly repatch some cells on a growth plate and incubate for a further 4 - 6 hr at 30 °. 2. Take a toothpick scraping of the new cell patch and resuspend in an appropriate small volume (30-100 #1) of 0.1 mg/ml Calcofluor solution. 3. Mount 3-5/zl of the cell suspension on a slide, cover with an 18 mm × 18 mm coverslip, and press briefly using the thumb. Immediately view cells by fluorescence microscopy using DAPI (bud scar staining) and GFP filters. Generally, GFP fusions should be observed first since GFP is more prone to photobleaching than Calcofluor. 18 j. R. Pringle, A. E. M. Adams, D. G. Drubin, and B. K. Haarer, Methods Enzymol. 194, 565 (1991 ). 19 A. E. M. Adams and J. R. Pringle, Methods Enzymol. 194, 729 (1991).

138

BASIC TECHNIQUES

Calcofluor

[7]

Bud4,GFP

FIG. 3. Covisualization of Calcottuor-stained bud scars and Bud4-GFP. 21 Left: Calcofluor staining of an axial budding strain. Right: Bud4-GFP.

Figure 3 depicts an example of this method: covisualization of Bud4-GFP (an axial landmark factor 2°) with bud scars. Colony Morphology-Based Analysis of Budding Patterns

ace2 A Colony Morphology-Based Analysis Use of colony morphology to determine the budding pattern of a cell is a facile method to screen a large population of isolates for alterations in budding pattern. The ace2A method can be used to distinguish cells budding in an axial manner from cells budding in a bipolar or random manner. ACE2 encodes a transcription factor that is involved in activation of CTS1, encoding a chitinase necessary for cell separation. 22,23 ace2A mother and daughter cells cannot separate from each other resulting in attached clumps of cells (Fig. 4A). ace2A haploid cells possessing an axial budding pattern form a fight cluster, whereas ace2A/ace2A diploid cells possessing a bipolar budding pattern form extended chains of cells. These defects in cell separation influence colony morphology (Fig. 4B). Colonies of ace2A cells budding in an axial manner are round with a lustrous surface, an appearance very similar to that of wild-type ACE2 colonies with which yeast biologists are familiar, ace2A cells exhibiting a bipolar pattern produce colonies with a notched outline and a rough surface, ace2 A 20 S. L. Sanders and 1. Herskowitz, J. CellBioL 134, 413 (1996). 21 M. Lord and J. Chant (unpublished observations). 22 G. Butler and D. J. Thiele, Mol. Cell. Biol. 11~ 476 (1991). 23 p. R. Dohrmann, G. Butler, K. Tamai, S. Dorland, J. R. Greene, D. 1. Thiele, and D. J. Stillman, Genes Dev. 6, 93 (1992).

[7]

ANALYSIS OF BUDDING PATTERNS

139

A

B a

a

a/ct

m

wild-type

ace2

a

ace2 bud5

ace21ace2

a

ace2 budlO

FIG. 4. The morphologies of ace2A cells and colonies. (A) Left: ace2A haploid cells exhibiting the axial budding pattern, right: ace2A/ace2A diploid cells exhibiting the bipolar budding pattern. Cells were stained with Calcofluor. (B) Colony morphologies of various strains: (l) A colony of wild-type haploid cells budding in an axial pattern; (2) a colony of ace2A haploid cells budding in an axial manner; (3) a colony of ace2A/ace2A diploid cells budding in a bipolar pattern; (4) a colony of ace2A bud5A haploid cells budding in a random pattern; (5) a colony of ace2 A budlOA haploid cells budding in a bipolar pattern.

140

BASICTECHNIQUES

[7]

bud5A haploids budding in a random fashion produce colonies with a slightly notched outline and a surface that is a little smoother than that of a colony budding in a bipolar manner (Fig. 4B). The bipolar-random difference is very subtle; consequently, use of ace2A colony morphology to distinguish bipolar from random budding is to be approached cautiously, ace2A budlOA haploids exhibiting the bipolar budding pattern show the same colony morphology as ace2A/ace2A diploids (Fig. 4B). Use of YPD plates is optimal for performing this assay. CSM plates can also be used. To screen large numbers of colonies, spread cells to yield 100-1000 colonies per plate. A stereoscopic microscope is helpful for the observation of colony morphology. Analysis of budding pattern by this method provides a powerful means by which to screen for mutants altered in bud-site selection. For example, mutations in AXL1, an axial-specific gene, were identified in a screen for nonaxial colonies, 10 and the bipolar-specific genes, RAX1 and RAX2, were discovered as partial axial revertants of an axllA strain. 12 It should be kept in mind that mutations unrelated to budding pattern can affect colony morphology. Direct observation of budding pattern by Calcofluor staining should be relied upon as a secondary screen. Pseudohyphal Colony Morphology-Based Analysis Diploid cells starved of nitrogen adopt a unipolar budding pattern in which buds emerge at the distal cell pole. 24 This unipolar pattern enables diploids to form filaments of elongated cells called pseudohyphae on appropriate media (SLAD). 24 Pseudohyphal colonies form a distinctive morphology: A highly fuzzy outline of filamentous growth surrounds the colony.24 Haploid mutants that bud in the bipolar pattern can also exhibit pseudohyphal-type colonies in appropriate strain backgrounds.2°'25 Diploids possessing a random pattern of budding are defective in pseudohyphal growth and form colonies which have bumpy outlines.24 Unlike the ace2 A assay, pseudohyphal colony morphology permits random budding colonies to be clearly distinguished from bipolar budding colonies. A n a l y s i s of M i c r o c o l o n y B u d d i n g P a t t e r n s This method utilizes direct microscopic observation of growing microcolonies on agar plates. Growing cells can be monitored over time and their budding patterns scored by analyzing the position at which two new buds emerge from a motherdaughter cell pair. 6 One sees patterns essentially as shown in Fig. IA.

C. J. Gimeno, E O. Ljungdahl, C. A. Styles, and G. R. Fink, Cell 68, 1077 (1992). 25 H.-U. M6sch and G. R. Fink, Genetics 145, 671 (1997).

[8]

USES AND ABUSES OF H O ENDONUCLEASE

1 41

Method 1. Grow up a logarithmic culture to an OD of approximately 0.2-1. 2. Take 5/zl of the cell culture and dilute it in 1 ml water. 3. Disrupt cell clumps by sonicating for 5 sec on a low setting with a microtip probe (e.g., setting 3 out of 10 on a Branson 450 Sonifier). 4. Plate a drop of the cell suspension on a YPD agar plate, allow to dry, and incubate at 30 ° for 2-3 hr. 5. View cells on a tetrad dissection microscope. 6. Score budding pattern at the two cell/two bud stage. Patterns should resemble those depicted in Fig. 1A. Considering the ease by which the budding pattern of a given cell can be scored quantitatively by staining with Calcofluor, this plate-assay method is not recommended to characterize the phenotype of a single strain. However, this method provides a way to screen through mutagenized cells to detect alterations in budding patterns. Indeed, the first four genes found to play a direct role in bud-site selection (RSR1/BUD1, BUD2, BUD3, BUD4)6 were found using this method of screening. Acknowledgments M. Lord was supported by a Human Frontiers Science Program Organization long-term fellowship. Work in J. Chant's laboratory is supported by the National Institutes of Health Grant GM 49782.

[81 U s e s a n d A b u s e s of HO E n d o n u c l e a s e By JAMES E. H A B E R Introduction The site-specific HO endonuclease is a member of a family of endonucleases implicated in the transposition of intron sequences.1 HO evolved to catalyze the homothallic switching of mating-type (MAT) genes in Saccharomyces cerevisiae, but over the past three decades, the uses to which HO has been put go much further than simply changing the mating type of a cell. HO-induced double-strand breaks (DSBs) have been used to study and define in detail several different mechanisms of homologous recombination in both mitotic and meiotic cells, as well as to l B. Dujon,

Gene82, 91 (1989).

METHODSIN ENZYMOLOGY,VOL.350

Copyright2002,ElsevierScience(USA). All fightsreserved. 0076-6879/02$35.00