206

Biochimica et Biophysica Acta, 1035 (1990) 206-213 Elsevier

BBAGEN 23345

Cell size specific binding of the fluorescent dye calcofluor to budding yeast Susan A. Moore Department of Chemistry & Biochemistry California State University, Fullerton, CA and Environmental Research Laboratory U.S. Environmental Protection Agency Athens, GA (U.S.A.) (Received 29 December 1989) (Revised manuscript received 23 March 1990)

Key words: Cell size; Fluorescent dye; Calcofluor; Budding yeast

The yeast Saccharomyces cerevisiae cell surface outside of the bud scars displayed an increasing fluorescence intensity with increasing cell size (volume), where fluorescence was due to irreversible binding of the fluorescent dye caicofluor. The increase in fluorescence intensity appeared to be due to an increase in the density of fluorescence per unit surface area of the cell. Exposure time measurements from a photomicroscope were used to quantitate fluorescence intensity on individual cells. The cell size dependent increase in fluorescence intensity was displayed by unbudded cells from stationary phase populations, and unbudded and parent cells from exponentially growing populations. Abnormally large cells generated during the arrest of cell division with a-factor or restrictive temperature for cdc3, 8, 13, 24, and 28 cell division cycle mutants, displayed significantly greater fluorescence intensity compared to the smaller cells generated during the arrest of division for cdc25, 33, and 35 mutant strains. Fluorescence intensity on newly emerging buds was broadly dependent on both the size of the bud, and the size of the parent cedis on which the buds were growing.

Introduction

A correlation of cell size a n d / o r shape with biological response has been established in several instances. Respiratory burst oscillations have been found to correlate with fluctuations in neutrophil shape detected by turbidometry, suggesting that the two responses are interrelated [1]. N A D P H oxidase becomes cytoskeletalassociated in response to chemotactic stimuli in neutrophils, and it appears that the cytoskeleton regulates the respiratory burst oxidase activity [1,2]. The cell cycle of amphibian eggs has been correlated with a cell surface contraction phenomenon [3,4]. In human blood platelets, a variety of stimuli, including treatment with thrombin, have been correlated with changes in cell size and shape [5]. In this report, we describe a correlation between cell size and Calcofluor White M2R dye binding to the cell surface of budding yeast. The increase in fluorescence is due to an increase in the density of fluorescence per unit surface area, suggesting that the

Correspondence (present address): S.A. Moore, Environmental Research Laboratory, U.S. Environmental Protection Agency, College Station Road, Athens, GA 30613, U.S.A.

composition of the cell surface may be correlated with cell size. Calcofluor White M2R (Scheme I and Ref. 6) is a stilbene-derived fluorescent dye used to stain plant cell walls and other cellulosic-containing structures for study with fluorescent light microscopy [7-10]. Calcofluor is absorbed onto a variety of polysaccharides containing contiguous fl-l,4-1inked D-glucopyranosyl units, such as fl-D-glucans, xyloglucans, substituted celluloses [9], mixed fl-1,3- and fl-l,4-D-glucans [10,11] and chitin [12]. Calcofluor binding appears to require a ribbon-like polysaccharide conformation involving repetitive flat hydrophobic surfaces [9-11]. Calcofluor binds to the hydrophobic pockets and also forms hydrogen bonds to the polysaccharide [13,14]. In yeast, calcofluor binds to the microfibrillar system of the cell wall [14]. Calcofluor appears to bind to chitin on the S. cerevisiae yeast cell

CALCOFLUOR

HN, ~ r - H N ~ C H ,~ - ~ ~ ~ I~.,~,. SO3Na $O3N=, N~T..N N(CH2CH2OH)2 N(CH2CH2OH)2

0304-4165/90/$03.50 © 1990 Elsevier Science Publishers B.V. (BiomedicalDivision)

Scheme I.

207 surface [12,15-17], but this binding may not be specific [14,18]. Staining of S. cerevisiae ceils with the fluorescent dye calcofluor is most intense at the ring which exists between the parent cell and bud [18,19]. After cell separation this ring becomes the intensely staining bud scar of the parent cells [14,19]. The cell surface outside of the ring and bud scars stains only very weakly after a pulse with calcofluor for exponentially growing cells (see figures in Ref. 19). Similar behavior can be seen in the binding of the fluorescent dye primuline (see Ref. 20) and gold-linked-wheat-agglutinin (see Ref. 21) to exponentially dividing S. cerevisiae yeast cells. Gold-linked-wheat-agglutinin appears to bind specifically to chitin on the S. cerevisiae yeast cell surface [14,22]. In contrast, extensive dye binding occurred over the entire cell surface of cells arrested at the CDC28 'start' step of cell division by the mating pheromone a-factor and pulsed with primuline [20], and of temperature-sensitive cdc24 mutant cells arrested at the cdc24 step of cell division and pulsed with calcofluor [19]. Similarly, gold-linked-wheat-agglutinin bound over the entire surface of division arrested cdc3, cdc4, cdc24, and cdc28 S. cerevisiae yeast cells [21]. It was concluded that generalized extrusion onto the yeast cell surface of chitin, which binds gold-linked-wheat-agglutinin, is a nonspecific effect of cell division arrest [21]. This is consistent with the extensive binding of the dyes calcofluor and primuline to the cell surface of division arrested cells [19,20]. The nature of this nonspecific event has not been established. It is concluded here that binding of calcofluor over the entire cell surface of S. cerevisiae yeast occurs as a function of cell size. The nonspecific effect of division arrest that produced extensive dye binding in the previous reports [19-21] appears to be the generation of abnormally large cells [23,36] that occurred upon the division arrest of the particular strains that were studied. Materials and Methods

Cell strains and materials All Saccharomyces cerevisiae yeast strains used were obtained from the Yeast Genetics Stock Center (University of California, Berkeley, CA) and were: X2180-1A M A T a SUC2 mal mel gal2 CUP1; 381G M A T a SUP4-3 ts cry1-1 his4-580a trpl a ade2-1 o try1 o lys2o; 104 cdc3-1ts; STX199-1D cdc8-1 ts M A T a adel trpl ural gal," 428 cdcl3-1t~; 182-6-3 cdc24-1 ts M A T a ural tyrl arg4 thr4 ade his trp gal," 321 cdc25-1ts; 185-3-4 cdc28-1 ts M A T a adel ade2 ural tyrl his7 lys2 gall leul; E-17 cdc33-1 ts M A T a adel ade2 gall his7 lys2 tyrl ural pet," BR214-4a cdc35-1 t~ M A T a adel arg4 his7 trpl ural. Genotypes are from Ref. 24. Unless a different genotype is designated, all cdc mutants were derived in parent strain A364A M A T a adel ade2 ural his7 lys2 tyrl gall [24].

Pure synthetic a-factor (Sigma Chemicals) was > 90% pure, based on thin-layer chromatography and amino acid analysis as previously described [25]. The Ks0 for cell division arrest in the %UB assay at 102 381G cells/ml was 2.4 + 0 . 3 . 1 0 -1° M a-factor, based on the molar absorption coefficient for a-factor of 1.4.10 4 at 280 nm in 0.01 M hydrochloric acid. This value of Ks0 is identical to that previously reported for highly purified natural a-factor [25]. Calcofluor White M2R (Purified) was obtained from Polysciences, Warrington, PA, and was used without further purification. This material showed at least three bands of UV lightabsorbing material upon thin-layer chromatography on silica gel plates in n-butanol/proprionic a c i d / w a t e r (2 : 1 : 1.2). At least some of these impurities are probably photoinduced isomers that are related to the fluorescence properties of calcofluor [26,27]. Primuline (Sigma Chemicals) and polyoxin-D (Streptomyces, Calbiochemicals) were used without further purification. Medium was YM1 + 2% glucose (pH 5.8) [25]. In most cases Yeast Nitrogen Base (YNB, Difco) was omitted from the medium because it was found, when old, to retard second bud emergence after the removal of a-factor. The omission of YNB had no effect on the binding behavior of calc0fluor and the generation time of X2180-1A wild-type cells. Formaldehyde was 37% reagent grade containing 10-15% methanol as a preservative (Fisher Scientific). This material was prefiltered prior to use. Millipore filters (0.45 /~m, Millipore, Bedford, MA) were used for all filtrations. Preparation of cells Exponentially growing (log phase) cells were prepared as previously described [25]. Stock cultures of stationary phase cells were prepared and stored at 4 ° C as previously described [25]. Stationary phase cells prepared in this way and stored for 1-5 weeks at 4 ° C were used for the calcofluor binding assays. Freshly made stationary phase cultures of X2180-1A were prepared by inoculating approx. 105 cells per rnl of stationary phase cells into medium (pH 5.8) and allowing the cells to regrow to stationary phase ( = 108 cells/ml). This required 3 - 4 days, after which time the culture showed more than 95% unbudded cells. The calcofluor binding properties of these cells were identical to those for stationary phase cultures of X2180-1A that had been stored at 4 ° C for 1-5 weeks. Preparation of calcofluor and primuline staining solutions For the quantitative analysis of fluorescence, considerable care was employed in the preparation of calcofluor staining solutions. Finely ground calcofluor at 20-50 mg was added to 50 ml medium followed by agitating at room temperature for 30 min. The majority of the calcofluor remained undissolved. The solution was then filtered twice through a 0.45/~m filter, centri-

208 fuged for 15 min at 1000 × g, and the supernatant collected. This yielded precipitate-free solutions of 1030 /~g/ml (10-30 /~M) calcofluor, consistent with the manufacturer's specifications (PolySciences Datasheet No. 265, April, 1982). The concentration of calcofluor in these solutions was determined spectrophotometrically at 350 nm using a molar absorption coefficient of 4.8 • 1 0 4 for calcofluor in pH 5.8 medium. The absorption coefficient was determined by dissolving 10 mg of calcofluor in 2.0 ml of 95% ethanol. The solution was centrifuged to remove the small amount of material that did not dissolve upon gentle heating. An aliquot of 20 /~1 of the supernatant was added to 3.0 ml medium (pH 5.8) and the absorbance spectrum was measured in a double-beam spectrophotometer against a blank containing only medium. The absorption coefficient of this material was 50 absorbance u n i t s / c m per (mg/ml) at 350 nm. The molar absorption coefficient was calculated from this value using the M r for the sodium salt of calcofluor of 961. The absorption coefficient determined here agrees within 10% with that reported by the manufacturer (Polysciences) for calcofluor in aqueous solution. During the assay for irreversible calcofluor binding, it is essential that undissolved calcofluor be removed from the staining solutions prior to incubation with cells. This is because, when present, solid calcofluor is collected on filters along with yeast cells. Upon resuspension, the calcofluor dissolves and restains the cells through reversible binding, producing spurious results in the assay for irreversible binding. The absence of solid particles of calcofluor in the staining solutions was verified under the fluorescence microscope. Calcofluorstaining solutions were made fresh daily and maintained at room temperature. To insure the absence of precipitate, calcofluor solutions were centrifuged a n d / o r filtered several times throughout an experiment. Primuline-staining solutions were prepared by dissolving primuline in distilled water at 10 mg/ml, followed by filtration through 0.45 ~m filters.

Irreversible binding of caleofluor to temperature-sensitive cdc strains, stationary phase and log phase cells Irreversible binding of calcofluor to cdc strains was performed as follows. Strains of edc mutants were grown at the permissive temperature of 2 5 ° C to approx. 1 0 6 log phase cells/ml, and placed at the restrictive temperature of 35.5 + 0.5°C for 4 - 6 h. After this time each cdc strain showed 80-100% of cells arrested with the morphology that is characteristic of the mutant strain, e.g., as unbudded cells for cdc24, edc25, cdc28, cdc33, cdc35, and as large budded cells for cdc3, cde8, and edcl3. Arrested cells of the cde3, ede8, edcl3, ede24, and cdc28 strains were abnormally large at the end of the arrest period as previously reported [23,36]. Cells were

induced to recover from arrest by shifting the cultures to the permissive temperature of 25 o C. At times immediately before and after the shift, aliquots were removed and brought to 2% formaldehyde. Calcofluor-staining solution (pH 5.8) was added to yield a final concentration of 10-20 /~M calcofluor. The aliquots were incubated for 1-10 min, filtered onto 0.45/~m filters, washed with 40 ml medium (pH 5.8) and resuspended in 0.5 ml medium containing 2% formaldehyde, or in medium lacking formaldehyde where indicated. Care was taken to avoid dehydration of the cells during filtration. Stationary and log phase cells were pulsed with calcofluor in an identical manner. The irreversible binding behavior of calcofluor to cells was unaltered by either sonication of cells, or fixation of cells with formaldehyde immediately before or after the treatment with calcofluor. Omission of formaldehyde did not diminish the cell size dependence of irreversible calcofluor binding (Fig. 2). Irreversible calcofluor binding was independent of the length of time of the pulse with calcofluor from 1-30 min, and the length of time between the calcofluor pulse and the assay for fluorescence up to 2-3 h. Nevertheless the assay for fluorescence was always performed immediately after the pulse with calcofluor in order to avoid spurious results that arise if a small amount of calcofluor precipitate is resuspended and solubilized with cells.

Quantitative determination of fluorescence intensity Fluorescence intensity was quantitated using an Olympus BHT fluorescence-photomicroscope equipped with an automatic exposure time measuring system (Olympus PM-10ADS). Exposure times were measured within a 1% spot of the photographic frame. The spot corresponded to a 5 or 2/~m radius circle using a 40 x and 100 x objective, respectively. The Olympus UV (excitation) - blue (emission) fluorescence cube along with 40 x fluorescence and phase contrast objectives were used to measure exposure times due to calcofluor fluorescence, except where otherwise indicated, as follows. With the fluorescence light blocked off, a cell was positioned within the 5 # m circle that is monitored by the automatic exposure time apparatus. When present, bud scars and rings were positioned outside of the circle, such that only the cell body lacking these structures was monitored. Movement of the bud scar or ring to various distances away from the perimeter of the circle changed the fluorescence (i.e., %flmax) of the cell body by < 10%, indicating that reflected fluorescence from these structures was not significant. With the visible light off, the fluorescence light was turned on and the exposure time of the cell was determined within 3-10 s without oil or water between the fluorescence objective and the cover slip (dry fluorescence) in a darkened room. The cell was moved and the back-

209 ground exposure time was determined with nothing in the circle. The average exposure time (exp. time) detected in this way for the background ranged from 11.5-12.5 min, and for cells ranged from 2 s to 12.5 min. In order to avoid the bleaching of cellular fluorescence, the UV light was kept off until just prior to exposure time measurements, and only one cell was assayed per microscope field. Cells were selected under phase contrast light for the measurement of fluorescence intensity. The exposure time decreased dramatically when fluorescent cells were positioned within, compared to just outside of the circle detected by the exposure meter, verifying that exposure times were based only on fluorescence within the area of the circle. Eqn. 1 was used to convert exposure time measurements into relative fluorescence intensity. This equation has the advantage that it converts the measured value (exposure time), which decreases as fluorescence intensity increases, into %flmax, which increases as fluorescence intensity increases. Eqn. 1 also has the advantage that it normalizes the data for the small variations in background fluorescence.

%flmax

exp. time(background) --exp. timeccen) exp. time(background) X 100%

(1)

When the exposure time (exp. time) of a cell approaches zero at extremely high fluorescence intensity, the value of %flmax approaches 100%. Therefore 100% represents the maximum difference between the background and cell fluorescence intensity that is detected by this procedure. Exposure time measurements with a 100 × objective were performed with immersion oil.

Measurement of cell volumes, cell concentration, and % unbudded cells Cell volumes were measured from phase contrast polaroid photographs using Polaroid 667 film (ASA 3200). The photo-eyepiece contained a sizing grid of 2.06 /tm per unit that was photographed along with cells. Cell volume was calculated from the photographs according to Eqn. 2 assuming cells are prolate elipsoids (28). V(/.tm 3) = ¢r/6 × l w 2

(2)

Measurement of % unbudded cells, cell concentrations, and the procedure for sonicating cells have been previously described [25]. Sonication had no effect on the fluorescence intensity (%flmax) and its quantitative dependence on cell size.

Qualitative determination of fluorescence intensity Qualitative determinations of fluorescence intensity were performed by assaying more than 200 cells by

visual inspection. Greater than 95% of cells showed the indicated irreversible calcofluor binding behavior. On occasion shrunken, dense yellow, dimly fluorescent cells were observed, suggesting that these cells had internalized calcofluor. Such cells were concluded to be dead, based on their shrunken, opaque appearance in phase contrast microscopy. They typically comprised less than 5% of the population. Similar extensive binding of gold-linked-wheat-agglutinin to dead cells has been observed [21]. The qualitative assay of fluorescence due to irreversible primuline binding was carried out in an identical fashion to that for calcofluor except a B-515W fluorescence filter set was used with a Nikon fluorescence photomicroscope. Results

Cell size dependent binding of calcofluor to stationary and log phase cells Stationary phase X2180-1A cells were incubated with calcofluor followed by filtration and washing to remove calcofluor. Cells retained dye to varying degrees after the washing procedure. Irreversible binding of calcofluor is defined as the binding of dye that remains, after extensive washing of cells. Fluorescence intensity was quantitated for individual cells by measuring exposure times in a fluorescence photomicroscope. Eqn. 1 was used to convert exposure times into percent of maximum fluorescence intensity (%flma×)" When stationary phase populations were assayed for the irreversible binding of calcofluor, larger cells showed a greater fluorescence intensity on the cell body outside of the bud scars, compared to smaller cells (Fig. 1, closed circles). This behaviour was not altered by the sonication of cells (see Materials and Methods). The cell size dependence of the fluorescence intensity that results from irreversible binding of calcofluor appears to be a general phenomenon for stationary phase S. cerevisiae yeast since the stationary phase populations of a number of strains displayed this dependence at least qualitatively including X2180-1A (Fig. 1), 381G, cdc3-1, cdcS-1, cdc13-1, cdc24-1, cdc25-1, cdc28-1, cdc33-1, and cdc35-1. Stationary phase cells that were assayed prior to the washing of cells showed a greater fluorescence intensity compared to cells that had been washed. A cell size dependent binding of calcofluor was observed under these conditions that detect reversible binding of calcofluor (Fig. 1, open circles). The fluorescence intensity due to the irreversible binding of calcofluor was found to vary in a cell size dependent fashion for log phase unbudded cells (Fig. 2, closed circles) and parent portions of budded cells (Fig. 3). Formaldehyde was normally added to cells prior to the pulse with calcofluor in order to prevent cell growth. The absence of formaldehyde produced a slight increase in the fluorescence intensity at any given cell size, but

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cell volume (#m 3) Fig. 1. Cell volume dependence of fluorescence intensity associated with calcofluor binding to unbudded stationary phase cells. Fluorescence intensity after a pulse with calcofluor (O) was determined as follows. X2180-1A stationary phase cells at 2.106 cells/ml containing 83% unbudded cells were incubated for 10 min in YM1 + 2% glucose (pH 5.8) medium containing 24 #M calcofluor and 1% formaldehyde. Cells were filtered onto a 0.45/tm Millipore filter, washed with 40 ml of medium and resuspended at 2. l0 T cells/ml in medium containing 1% formaldehyde. Fluorescence intensity (%fl max) was determined for the cell body of individual unbudded cells as described in Materials and Methods (Eqn. 1). When present, the fluorescent bud scars were positioned outside of the spot meter circle for the measurement of fluorescence intensity. Cell volumes were determined from phase contrast photographs that included a sizing grid (Eqn. 2). Fluorescence intensity during the continuous present of calcofluor ( o ) was measured by incubating X2180-1A stationary phase cells at 108 cells/ml with 24 #M calcofluor for 10 min. Cells were assayed over the next 60 rain for %flmax and cell volume.

Fig. 3. Cell volume depende£ce of the fluorescence intensity associated with calcofluor irreversibly bound to parent portions of log phase budded cells. Exponentially growing X2180-1A cells at 5-105 cells/ml in YMI +2% glucose (pH 5.8) medium were incubated with 12 #M calcofluor for 10 rain. Cells were filtered, washed, resuspended, and assayed for fluorescence intensity (%flma~) and cell volume of individual cells as described in the legend of Fig. 1. The fluorescent bud scars and ring between parent cells and buds were positioned outside of the spot meter circle for the measurement of parent cell fluorescence intensity.

fluorescence from the highly fluorescent parent-bud ring that was positioned immediately outside of the circle that was detected by the exposure meter. Despite this I O0

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cell volume (p.m3) Fig. 2. Cell volume dependence of the fluorescence intensity associated with calcofluor irreversibly bound to log phase unbudded cells. Exponentially growing X2180-1A cells at 106 cells/ml in YM1 + 2% glucose (pH 5.8) medium containing (O) or lacking ( o ) 1% formaldehyde were incubated with 16 FM calcofluor for 10 rnin. Cells were filtered onto a 0.45 # m Millipore filter, washed with 40 ml of medium, resuspended at 107 cells/ml in medium containing 1% formaldehyde, and assayed for fluorescence intensity (%fl max ) and cell volume.

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Fig. 4. Volume dependence of fluorescence intensity associated with calcofluor irreversibly bound to buds of abnormally large and log phase parent cells. X2180-1A log phase cells at 103 cells/ml in YM1 +2% glucose (pH 5.8) medium were arrested for 18.5 h with 42 nM o-factor, 23°C, to arrest cell division and produce abnormally large cells. Bud emergence was induced by filtering cells onto a 0.45 ttm Millipore filter, washing with 40 ml medium to remove o-factor, and resuspending cells in medium at 2-104 cells/ml, 23 ° C. At various times between 60 and 180 rain after the removal of a-factor, 1 ml aliquots were fixed with 1% formaldehyde and treated with 15 tLM calcofluor for 10 min. This was followed by filtration, washing with 40 ml medium, resuspension at 105 cells/ml in medium containing 1% formaldehyde, and measurement of fluorescence intensity (%flmax) and volume for the buds that had formed (I). The %flm,x and volume associated with the buds on parent cells from an exponentially growing population of X2180-1A cells (D) were measured as described in the legend of Fig. 3. The dashed line represents the cell volume dependence of fluorescence intensity associated with calcofluor that is irreversibly bound to stationary phase unbudded cells and was obtained from Fig. 1, closed circles.

211 error, which is reflected as an increased scatter in the data, a correlation of fluorescence intensity with bud size was observed (Fig. 4, open squares). In these studies, the fluorescence intensity of a bud (%flmax) was never observed to be greater than that for its attached parent cell.

Irreversible binding of calcofluor to division arrested cells that grow abnormally large during arrest X2180-1A cells were arrested at the 'start' step of cell division with a-factor for 3 h at 34 ° C. After this time cells were 95-100% unbudded [25,29]. The cell doubling time in the absence of a-factor was 84 min; the time between the 'start' step and cell separation was 57 min [30]; and the generation time upon recovery from a-factor arrest was 57 min [30]. Hence cells were arrested for two to three generation times, and had grown 3- to 4-fold larger in mass and volume compared to log phase cells [25,31]. Upon the removal of a-factor, the cells resumed bud emergence and cell division. Both before and after the removal of a-factor, these abnormally large cells showed extensive irreversible binding to calcofluor, with %flmax values in the 90-99% range similar to abnormally large cells in Figs. 1-3. Abnormally large cells that were generated in other ways showed similar behavior. Cell division was blocked at 3 6 ° C using temperature-sensitive cdc3-1, cdc8-1, cdc13-1, cdc24-1, and cdc28-1 mutant strains. Cell division was reinitiated by transfer to 25 ° C. During the block of cell division at 3 6 ° C the cells continued to grow in size [23,36] resulting in the production of abnormally large cells. Before and after release from the temperature block these abnormally large cells displayed intense fluorescence due to the irreversible binding of calcofluor over the entire cell surface with % f l m a x values in the 90-99% range (data not shown). Irreversible binding of calcofluor to division arrested cells that do not grow abnormally large during arrest Temperature-sensitive cdc25-1, cdc33-1, and cdc35-1 cells were arrested at the restrictive temperature of 36 ° C for 5 and 24 h. Cells were then shifted to 25 o C, which induced cell division after a short lag. During arrest, the cells did not grow significantly beyond the size of normal log phase cells [23,36]. Prior and subsequent to the temperature shift to 25 o C, these prearrested but normal-sized cells displayed relatively weak fluorescence intensity due to the irreversible binding of calcofluor. The %flmax values of individual cells (data not shown) were identical to those for log phase cells (Figs. 2 and 3) of comparable volume. Irreversible binding of calcofluor to new buds growing on abnormally large prearrested cells The buds that were formed on a-factor prearrested and abnormally large X2180-1A cells were observed to

grow surprisingly large before significant fluorescence was observed from the irreversible binding of calcofluor. Quantitation of the fluorescence intensity relative to the size of the bud is shown in Fig. 4. Although the data shows considerable scatter, fluorescence intensity is generally lower on buds growing on abnormally large cells (Fig. 4, closed circles) compared to buds of the same size growing on smaller log phase cells (Fig. 4, open squares), or compared to unbudded stationary phase cells of comparable size (Fig. 4, dashed line). Hence, new buds growing on abnormally large parent cells grow surprisingly large before a significant fluorescence is observed as a result of the irreversible binding of calcofluor. This behavior was observed for the second and third generations of bud formation after release from division arrest under the conditions of Fig. 4. Identical behavior was observed using the fluorescent dye primuline in place of calcofluor under otherwise identical conditions. Similar behavior was observed qualitatively for the new buds formed on abnormally large prearrested cells after release from division arrest by a shift to 25 ° C for prearrested and abnormally large cdc3-1, cdc8-1, cdc13-1, cdc24-1, and cdc28-1 mutant strains.

Calcofluor fluorescence per unit area of the cell surface An increase in the volume of a cell correlated with an increase in the intensity of fluorescence per unit surface area of the cell, where fluorescence is due to the irreversible binding of calcofluor. This was observed in several ways. First, this was obvious by visual inspection. For example, both small and large stationary phase cells were readily visible under phase contrast fight. Under fluorescence, however, small cells were invisible, whereas the 'equivalent area' on larger cells displayed clear and sometimes extensive fluorescence. Secondly, when stationary phase X2180-1A cells of 154, 108 and 54 g m 3 were measured, first as a whole cell and then as one-half of the same cell, there was a drop of 4, 6 and 9 %fl . . . . respectively, under conditions identical to Fig. 1, closed circles. The average drop was 8 + 4 %flm~x (+S.D.), and the data fit the equation: %flmax(half cell) = 1.17 × %flmax(whole cen)-19%, for 14 measurements at whole cell sizes ranging from 35 to 154 g m 3. The relatively small decrease in %flma~ for a halving of the fraction of a cell within the spot that is detected by the exposure meter, is due to the characteristics of the silicon blue diode light detector and the equations that calculate %limax and cell volume (Eqns. 1 and 2). In comparison to these results, when cells of 154, 108 and 54 g m 3 were compared to different, rather than the same cells of one-half size from a stationary phase population, there was a drop of 29, 28 and 20 %fl . . . . respectively (determined from Fig. 1, closed circles). The average drop among different cells for a 2-fold decrease in cell size was 26 + 9 %flmax between

212 35-140 # m 3. The data between 20 and 140 /,m 3 fit a straight line described by the equation: %flmax(size 0.5X) = 0.52 X %flmax(size 1X) -t- 8% (Fig. 1, closed circles). Comparison of the average change in %flmax in going from size 0.5X to 1X for the same cell (8 + 4%) as opposed to different cells (26 + 9%) supports the conclusion that an increase in cell size among different cells within a population is correlated with an increase in fluorescence intensity per unit surface area. The increase in %flmax with increasing cell size among different cells is too large to be accounted for solely by an increase in size with identical irreversible calcofluor binding per unit surface area. Thirdly, the %flmax was determined for stationary phase cells using a 100 × objective where cells above 30 btm3 occupied the entire circular area that is detected by the exposure meter. An increase in %flmax with increasing total cell volume was observed under these conditions, which detect changes in fluorescence per unit surface area (data not shown, the cell size dependence under these conditions was intermediate between the two curves shown in Fig. 1).

Irreversible binding of calcofluor to the cell body and projection tip during cell division arrest with a-factor The fluorescent dye primuline binds to a greater extent to the projection that is formed during the arrest of cell division with a-factor, compared to the remainder of the cell [20]. This result was confirmed and extended here using calcofluor. X2180-1A cells were arrested in 0.6 /*M a-factor in YM1 medium (pH 5.8) 24°C. After 2 h, only the developing projection displayed intense fluorescence after a pulse with calcofluor. After > 5 h the entire cell surface displayed extensive fluorescence after a pulse with the dye, and the projection was not distinguished from the remainder of the cell in its fluorescence intensity. The presence of polyoxin-D in the range of 20-200 /,g/rnl did not alter these results. Discussion

The calcofluor binding behavior described here represents that on the cell body outside of the bud scars or rings that connect parent cells and buds. Yeast cells outside of the bud scars readily bind calcofluor on their surface and display moderate to intense fluorescence during the continuous presence of the dye. After extensive washing of cells to remove calcofluor, some fluorescence remains which represents calcofluor that was irreversibly bound.

Cell size dependence of irreversible calcofluor binding The fluorescence intensity due to the irreversible binding of calcofluor is dependent on cell volume in S.

cerevisiae yeast. This is the case for stationary phase (Fig. 1) and exponentially growing (Figs. 2 and 3) cells. Abnormally large cells that are generated by the arrest of cell division using a-factor or restrictive temperature for cdc3, cdc8, cdcl3, cdc24, and cdc28 temperature-sensitive mutants showed high fluorescence intensity, whereas the much smaller log phase sized unbudded cells that were generated by the arrest of cell division with the cdc25, cdc33, and cdc35 strains showed much weaker fluorescence intensity due to the irreversible binding of calcofluor. Hence, intense fluorescence due to calcofluor binding is not a consequence of cell division arrest, but rather of the cell size achieved during the block. The increase in fluorescence intensity that is associated with increasing cell volume, reflects an increase in the fluorescence intensity per unit area of the cell surface. Such an effect can occur in several ways. An increase in cell volume may result in a greater density of calcofluor binding sites per unit area of the cell surface. For example, an increase in binding sites might be expected to occur as the cell wall thickens during cell growth. Alternatively, a structural change at the surface of the cell may occur such that fluorescence intensity, but not the total number of calcofluor binding sites, increases per unit area of the cell surface. The macromolecule on the surface of the cell to which calcofluor binds appears to be chitin [12,15-17]. However, there is some controversy over this [14,18]: e.g., Schizosaccharomyces pombe yeast cells bind calcofluor [32] but do not contain chitin [33,34]. The volume-dependent cell surface change reported here provides a marker of cell volume that is detectable within a small unit area of the cell surface. This observation may be technically useful. It also leads to the speculation that the cell could monitor its total volume by monitoring small sections of its surface for the degree of change that reflects cell volume. Previous reports have suggested that the cell may monitor its volume during growth. A sharply defined critical cell volume does not occur in the log phase cell cycle, because the variation in the cell volume at bud emergence is large, and identical to that at cell separation [28]. A critical size requirement occurs at or immediately prior to the genetically defined 'start' step of the cell cycle of S. cerevisiae yeast [35-40], although size refers to the overall macromolecular status of the cell and not to cell volume [28,39]. A dependence of the time of bud emergence on cell volume does occur for abnormally small stationary phase cells [36], that may reflect a critical cell volume requirement at the G o -+ G 1 transition. The variation in cell volume that is observed at the time of bud emergence for log phase cells [28], appears to entirely account for the variation in cell volume that is observed at the time of bud emergence for stationary phase cells [36], consistent with a critical cell volume requirement at the G o --* G 1 transition.

213 Hence, it appears that S. cerevisiae m a y measure a n d r e s p o n d to their cell v o l u m e d u r i n g their life cycle.

Calcofluor binding associated with new growth Based o n the fluorescence p a t t e r n s that resulted from calcofluor staining, it was f o u n d for S. pombe fission yeast that a cell must reach a critical length before the majority of growth occurs at the newly emerging end of the cell, rather t h a n at b o t h ends simultaneously. The critical length at which this new end take off ( N E T O ) occurs is strain a n d cell cycle d e p e n d e n t [32]. F o r the S. cerevisiae yeast m o n i t o r e d here, the fluorescence intensity that is associated with the irreversible b i n d i n g of calcofluor is smoothly d e p e n d e n t o n cell v o l u m e for all of the cell types studied, i n c l u d i n g newly emerging buds. A n a b r u p t increase in fluorescence intensity, associated with a critical cell volume, was n o t detected within the experimental error of the data. Interestingly the sensitivity of the cell size d e p e n d e n c e of fluorescence intensity for newly emerging b u d s (cf. the slopes of the lines in Fig. 4) appears to be inversely related to the volume of the p a r e n t cells o n which the b u d s were growing.

Acknowledgements This work was f u n d e d in part b y the B a n t i n g Research F o u n d a t i o n , T o r o n t o , C a n a d a , a n d grant No. DCB-8417345 from the N a t i o n a l Science F o u n d a t i o n , W a s h i n g t o n , D.C.

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