Vol. 137, No. 3
JOURNAL OF BACTERIOLOGY, Mar. 1979, p. 1456-1458 0021-9193/79/03-1456/03$02.00/0
Nuclear Division in Temperature-Sensitive Multicellular Mutants of Wangiella dermatitidis ROWENA L. ROBERTS, RUJU J. LO, AND PAUL J. SZANISZLO* Department of Microbiology, The University of Texas at Austin, Austin, Texas 78712 Received for publication 9 January 1979
Temperature-sensitive morphological mutants of Wangiella dermatitidis were stained with mithramycin, a fluorescent nuclear stain. Multicellular forms of the mutants each contained two or more nuclei, documenting that nuclear division accompanied the yeast-to-multicellular form conversion. Wangiella dermatitidis is a polymorphic zoopathogenic fungus. The vegetative.forms of this organism include yeastlike cells, multicellular forms, and moniliform and true hyphae (3, 6, 9). Multicellular forms are large, thick-walled, muriform units which resemble the tissue phase of a chromomycotic agent (1, 5). Multicellularity can be induced by culture of yeastlike cells under acidic conditions (9). Recently we reported the isolation of three temperature-sensitive (Ts) .multicellular (Mc) mutants of W. dermatitidis 8656 (7). These mutants, designated Mc 1, 2, and 3, grow in the yeastlike morphology at the permissive temperature (250C), but when cultured at near neutral pH and incubated at the restrictive temperature (370C) they express the multicellular phenotype. The parental strain grows in the yeastlike morphology at both 25 and 370C. Yeastlike cells of Mc 2 and 3 incubated at 370C cease budding rapidly and within 48 h convert almost totally to the multicellular form. Mutant Mc 2 differs phenotypically from Mc 3 in that the mutation carried by Mc 2 is lethal at 370C. Mutant Mc 1, unlike mutants Mc 2 and 3, continues budding for several generations during incubation at the restrictive temperature, and only about 50% of the cells become multicellular during a 48-h incubation period. Induction of the multicellular morphology by incubation of the mutants at 370C is characterized by cessation of budding, enlargement of the unbudded cells, continuation of macromolecular synthesis, and, finally, division of the cellular unit (7). The continuation of DNA synthesis and the formation of septa at sites remote from their normal position between the mother cell and daughter bud in the temperature-induced multicellular forms, as well as the presence of several nuclei in acid-induced multicellular bodies (9), suggests that nuclear division also occurs during incubation of the mutants at the restrictive temperature. This possibility was investigated
through the use of mithramycin, a fluorescent nuclear stain (2, 10). Cells of the mutants and wild type were grown at 25 and 370C in CDY as previously described (7). Samples were collected at various intervals duing incubation. Nuclei were stained according to the procedure of Slater (8). Samples were fixed by treatment for 10 min in absolute alcohol and acetone (1:1, vol/vol); fixed cells that were to be examined at a later time were stored at 40C in 25% ethanol. After removal of the ethanol, the cells were stained with mithramycin and incubated overnight at 40C. Mithramycin was a generous gift of Pfizer, Inc., Groton, Conn. Preparations were examined with a Zeiss RA/ 38 fluorescent microscope equipped with a quartz-halogen bulb, a KP 500 excitation filter, and a 530 A barrier filter. Kodak Tri-X Pan film was used for all exposures. The average number of nuclei was derived by division of the total number of nuclei observed by the number of units counted. Any cellular group that appeared by dark-field microscopy as a discrete and distinguishable entity was scored as a unit. Thus, budded and unbudded cells as well as multicellular forms were scored as single units. Values represent the average of three independent counts; for each count 120 units were scored. Microscopic examination of wild-type celLs stained with mithramycin revealed that each unit usually contained at least one nucleus (Fig. 1A). Occasionally two nuclei were observed in budded forms, one nucleus in the mother cell. and the other in the daughter bud. Most likely these forms represent cells in which nuclear division had occurred but in which cytokinesis and/or cell separation had not. In Mc 1 (Fig. 1B), approximately two to three nuclei were visible after 48 h at the restrictive temperature, and usually as many as four or five nuclei could be observed in the multicellular forms of Mc 2
1456
VOL. 137, 1979
NOTES
1457
I
FIG. 1. Mithramycin-stained cells of wild type (A) and the three mutants, Mc I (B), Mc 2 (C), and Mc 3 (D). Cultures were inoculated with log-phase yeastlike cells and incubated at 37°C for 48 h. Magnification, x3,240.
and 3 after the same period of incubation (Fig. 1C and D). Examination of individual multicellular units with both fluorescent and incandescent light revealed that each cell in the unit possessed at least one nucleus and that many of the cells contained two or more nuclei. Changes in the average number of nuclei per unit during incubation at 25 and 370C are presented in Fig. 2. In celLs of the wild type and the mutants cultured at 250C, the average number of nuclei was approximately one throughout the entire growth period (Fig. 2A). The maximum number of nuclei observed in a unit was two. Incubation of wild-type cells at 370C caused a transient increase in the number of nuclei per unit from a value of one to a value of almost two (Fig. 2B). Probably this increase was due only
to temperature shock. Increase in nuclear number was quite evident for cells of the Mc mutants cultured at 370C (Fig. 2B). The most spectacular increase was that observed for units of Mc 3. By 36 h the nuclear number had risen from a value slightly greater than one to a value of about five. Incubation of celLs of Mc 2 at 370C resulted in an increase in the number of nuclei per unit to a value slightly greater than three, but the number declined to about 2.5 during the period between 36 and 48 h. By 48 h, units of Mc 1 contained an average of about 2.5 nuclei. The maximum number of nuclei observed in units of Mc 1 was 13, whereas it was 22 for Mc 2 and 31 for Mc 3. From the results, the average time between nuclear divisions in units of Mc 3 at 370C was calculated as about 8.5 h, indicating
1458 NOTES
F
5.U
J. BACTERIOL.
AMC I OMC2
Z
D3
o MC 3
*WT
cr w
4.0
-
3.0
-
w
-J
z LL.
0 cx: w
2.0
z w
1.0
250C
~~A W
1.5-
12
24
36
48
TIME (HOURS) FIG. 2. Changes in the average number of nuclei per unit during incubation of the Mc mutants and wild type at 25°C (A) and 37°C (B). All cultures were inoculated with log-phase yeastlike cells.
that the nuclear "generation time" of this mutant is somewhat slower than the generation time of 4.5 h calculated for yeastlike cells. Increases in the average number of nuclei per unit are related to the kinetics of formnation of multicellular bodies. Cells of Mc 2 and 3, in which the average nuclear number was approximately three to five, convert rapidly and almost totally to the multicellular form, whereas units of Mc 1, in which the average nuclear number was about two or three, convert more slowly and to a lesser extent. The lethality of the mutation carried by Mc 2 is also reflected in these results; the decline in the number of nuclei between 36 and 48 h probably represents lysis of the larger multicellular forms which contain large numbers of nuclei. The increase in nuclear number also parallels the increase in the amount of radiolabeled DNA per colony-forming unit of Mc 1 and 3. After 48 h, Mc 1 is able to accumulate about
twice the amount of radiolabeled DNA that wild type is, whereas Mc 3 accumulates about 10 times more than wild type (7). Increase in radiolabeled DNA does not parallel increase in nuclear number for Mc 2; the amount of radiolabeled DNA in cultures of Mc 2 is quite high, due probably to the detection of radiolabeled DNA in nonviable units. These results and observations confirm that in mutant cells of W. dermatitidis incubated at 37°C nuclear division does indeed continue in the absence of bud formation. Thus, in several respects the Ts Mc mutants are similar to cdc 24, the Ts cell division cycle mutant of Saccharomyces cerevisiae which is unable to form a bud at the restrictive temperature (4). Both types of mutants continue macromolecular synthesis and nuclear division in the absence of bud emergence. However, the two types of mutants differ in that septation occurs in many units of the multicellular mutants incubated at 37°C, but is not observed in cells of cdc 24 incubated at the restrictive temperature. Finally, it should be noted that cells in multicellular forms may contain two or more nuclei, indicating that in W. dermatitidis nuclear division is not always followed immediately by cell division. However, our observations suggest that, as in many other cell types, nuclear division is a prerequisite for
cytokinesis. LITERATURE CITED 1. Al-Doory, Y. 1972. Chromomycosis, p. 118-146. Moun-
tain Press Publishing Co., Missoula, Mont. 2. Criseman, H. A., and R. A. Tobey. 1974. Cell cycle analysis in 20 minutes. Science 184:1297-1298. 3. Grove, S. N., K. B. Oujezdsky, and P. J. Szaniszlo. 1973. Budding in the dimorphic fungus Phialophora dermatitidis. J. Bacteriol. 115:323-329. 4. Hartwell, L. H., R. K. Mortimer, J. Culotti, and M. Culotti. 1973. Genetic control of the cell division cycle in yeast. V. Genetic analysis of cdc mutants. Genetics 74:267-286. 5. Jotisankasa, V., H. S. Nielson, and N. F. Conant. 1970. Phialophora dermatitidis; its morphology and biology. Sabouraudia 8:98-107. 6. Oujezdesky, K. B., S. N. Grove, and P. J. Szaniszlo. 1973. Morphological and structural changes during the yeast-to-mold conversion of Phialophora dermatitidis. J. Bacteriol. 113:468-477. 7. Roberts, R. L., and P. J. Szaniszlo. 1978. Temperaturesensitive multicellular mutants of Wangiella dermatitidis. J. Bacteriol. 135:622-632. 8. Slater, M. LX 1976. Rapid nuclear staining method for Saccharomyces cerevisiae. J. Bacteriol. 126:1339-1341. 9. Szaniszlo, P. J., P. H. Hsieh, and J. D. Marlowe. 1976. Induction and ultrastructure of the multicellular (sclerotic) morphology in Phialophora dermatitidis. Mycologia 68:117-130. 10. Ward, D. C., E. Reich, and I. H. Goldberg. 1965. Base specificity in the interaction of polynucleotides with antibiotic drugs. Science 149:1259-1263.
JOURNAL OF BACTERIOLOGY, Mar. 1979, p. 1456-1458 0021-9193/79/03-1456/03$02.00/0
Nuclear Division in Temperature-Sensitive Multicellular Mutants of Wangiella dermatitidis ROWENA L. ROBERTS, RUJU J. LO, AND PAUL J. SZANISZLO* Department of Microbiology, The University of Texas at Austin, Austin, Texas 78712 Received for publication 9 January 1979
Temperature-sensitive morphological mutants of Wangiella dermatitidis were stained with mithramycin, a fluorescent nuclear stain. Multicellular forms of the mutants each contained two or more nuclei, documenting that nuclear division accompanied the yeast-to-multicellular form conversion. Wangiella dermatitidis is a polymorphic zoopathogenic fungus. The vegetative.forms of this organism include yeastlike cells, multicellular forms, and moniliform and true hyphae (3, 6, 9). Multicellular forms are large, thick-walled, muriform units which resemble the tissue phase of a chromomycotic agent (1, 5). Multicellularity can be induced by culture of yeastlike cells under acidic conditions (9). Recently we reported the isolation of three temperature-sensitive (Ts) .multicellular (Mc) mutants of W. dermatitidis 8656 (7). These mutants, designated Mc 1, 2, and 3, grow in the yeastlike morphology at the permissive temperature (250C), but when cultured at near neutral pH and incubated at the restrictive temperature (370C) they express the multicellular phenotype. The parental strain grows in the yeastlike morphology at both 25 and 370C. Yeastlike cells of Mc 2 and 3 incubated at 370C cease budding rapidly and within 48 h convert almost totally to the multicellular form. Mutant Mc 2 differs phenotypically from Mc 3 in that the mutation carried by Mc 2 is lethal at 370C. Mutant Mc 1, unlike mutants Mc 2 and 3, continues budding for several generations during incubation at the restrictive temperature, and only about 50% of the cells become multicellular during a 48-h incubation period. Induction of the multicellular morphology by incubation of the mutants at 370C is characterized by cessation of budding, enlargement of the unbudded cells, continuation of macromolecular synthesis, and, finally, division of the cellular unit (7). The continuation of DNA synthesis and the formation of septa at sites remote from their normal position between the mother cell and daughter bud in the temperature-induced multicellular forms, as well as the presence of several nuclei in acid-induced multicellular bodies (9), suggests that nuclear division also occurs during incubation of the mutants at the restrictive temperature. This possibility was investigated
through the use of mithramycin, a fluorescent nuclear stain (2, 10). Cells of the mutants and wild type were grown at 25 and 370C in CDY as previously described (7). Samples were collected at various intervals duing incubation. Nuclei were stained according to the procedure of Slater (8). Samples were fixed by treatment for 10 min in absolute alcohol and acetone (1:1, vol/vol); fixed cells that were to be examined at a later time were stored at 40C in 25% ethanol. After removal of the ethanol, the cells were stained with mithramycin and incubated overnight at 40C. Mithramycin was a generous gift of Pfizer, Inc., Groton, Conn. Preparations were examined with a Zeiss RA/ 38 fluorescent microscope equipped with a quartz-halogen bulb, a KP 500 excitation filter, and a 530 A barrier filter. Kodak Tri-X Pan film was used for all exposures. The average number of nuclei was derived by division of the total number of nuclei observed by the number of units counted. Any cellular group that appeared by dark-field microscopy as a discrete and distinguishable entity was scored as a unit. Thus, budded and unbudded cells as well as multicellular forms were scored as single units. Values represent the average of three independent counts; for each count 120 units were scored. Microscopic examination of wild-type celLs stained with mithramycin revealed that each unit usually contained at least one nucleus (Fig. 1A). Occasionally two nuclei were observed in budded forms, one nucleus in the mother cell. and the other in the daughter bud. Most likely these forms represent cells in which nuclear division had occurred but in which cytokinesis and/or cell separation had not. In Mc 1 (Fig. 1B), approximately two to three nuclei were visible after 48 h at the restrictive temperature, and usually as many as four or five nuclei could be observed in the multicellular forms of Mc 2
1456
VOL. 137, 1979
NOTES
1457
I
FIG. 1. Mithramycin-stained cells of wild type (A) and the three mutants, Mc I (B), Mc 2 (C), and Mc 3 (D). Cultures were inoculated with log-phase yeastlike cells and incubated at 37°C for 48 h. Magnification, x3,240.
and 3 after the same period of incubation (Fig. 1C and D). Examination of individual multicellular units with both fluorescent and incandescent light revealed that each cell in the unit possessed at least one nucleus and that many of the cells contained two or more nuclei. Changes in the average number of nuclei per unit during incubation at 25 and 370C are presented in Fig. 2. In celLs of the wild type and the mutants cultured at 250C, the average number of nuclei was approximately one throughout the entire growth period (Fig. 2A). The maximum number of nuclei observed in a unit was two. Incubation of wild-type cells at 370C caused a transient increase in the number of nuclei per unit from a value of one to a value of almost two (Fig. 2B). Probably this increase was due only
to temperature shock. Increase in nuclear number was quite evident for cells of the Mc mutants cultured at 370C (Fig. 2B). The most spectacular increase was that observed for units of Mc 3. By 36 h the nuclear number had risen from a value slightly greater than one to a value of about five. Incubation of celLs of Mc 2 at 370C resulted in an increase in the number of nuclei per unit to a value slightly greater than three, but the number declined to about 2.5 during the period between 36 and 48 h. By 48 h, units of Mc 1 contained an average of about 2.5 nuclei. The maximum number of nuclei observed in units of Mc 1 was 13, whereas it was 22 for Mc 2 and 31 for Mc 3. From the results, the average time between nuclear divisions in units of Mc 3 at 370C was calculated as about 8.5 h, indicating
1458 NOTES
F
5.U
J. BACTERIOL.
AMC I OMC2
Z
D3
o MC 3
*WT
cr w
4.0
-
3.0
-
w
-J
z LL.
0 cx: w
2.0
z w
1.0
250C
~~A W
1.5-
12
24
36
48
TIME (HOURS) FIG. 2. Changes in the average number of nuclei per unit during incubation of the Mc mutants and wild type at 25°C (A) and 37°C (B). All cultures were inoculated with log-phase yeastlike cells.
that the nuclear "generation time" of this mutant is somewhat slower than the generation time of 4.5 h calculated for yeastlike cells. Increases in the average number of nuclei per unit are related to the kinetics of formnation of multicellular bodies. Cells of Mc 2 and 3, in which the average nuclear number was approximately three to five, convert rapidly and almost totally to the multicellular form, whereas units of Mc 1, in which the average nuclear number was about two or three, convert more slowly and to a lesser extent. The lethality of the mutation carried by Mc 2 is also reflected in these results; the decline in the number of nuclei between 36 and 48 h probably represents lysis of the larger multicellular forms which contain large numbers of nuclei. The increase in nuclear number also parallels the increase in the amount of radiolabeled DNA per colony-forming unit of Mc 1 and 3. After 48 h, Mc 1 is able to accumulate about
twice the amount of radiolabeled DNA that wild type is, whereas Mc 3 accumulates about 10 times more than wild type (7). Increase in radiolabeled DNA does not parallel increase in nuclear number for Mc 2; the amount of radiolabeled DNA in cultures of Mc 2 is quite high, due probably to the detection of radiolabeled DNA in nonviable units. These results and observations confirm that in mutant cells of W. dermatitidis incubated at 37°C nuclear division does indeed continue in the absence of bud formation. Thus, in several respects the Ts Mc mutants are similar to cdc 24, the Ts cell division cycle mutant of Saccharomyces cerevisiae which is unable to form a bud at the restrictive temperature (4). Both types of mutants continue macromolecular synthesis and nuclear division in the absence of bud emergence. However, the two types of mutants differ in that septation occurs in many units of the multicellular mutants incubated at 37°C, but is not observed in cells of cdc 24 incubated at the restrictive temperature. Finally, it should be noted that cells in multicellular forms may contain two or more nuclei, indicating that in W. dermatitidis nuclear division is not always followed immediately by cell division. However, our observations suggest that, as in many other cell types, nuclear division is a prerequisite for
cytokinesis. LITERATURE CITED 1. Al-Doory, Y. 1972. Chromomycosis, p. 118-146. Moun-
tain Press Publishing Co., Missoula, Mont. 2. Criseman, H. A., and R. A. Tobey. 1974. Cell cycle analysis in 20 minutes. Science 184:1297-1298. 3. Grove, S. N., K. B. Oujezdsky, and P. J. Szaniszlo. 1973. Budding in the dimorphic fungus Phialophora dermatitidis. J. Bacteriol. 115:323-329. 4. Hartwell, L. H., R. K. Mortimer, J. Culotti, and M. Culotti. 1973. Genetic control of the cell division cycle in yeast. V. Genetic analysis of cdc mutants. Genetics 74:267-286. 5. Jotisankasa, V., H. S. Nielson, and N. F. Conant. 1970. Phialophora dermatitidis; its morphology and biology. Sabouraudia 8:98-107. 6. Oujezdesky, K. B., S. N. Grove, and P. J. Szaniszlo. 1973. Morphological and structural changes during the yeast-to-mold conversion of Phialophora dermatitidis. J. Bacteriol. 113:468-477. 7. Roberts, R. L., and P. J. Szaniszlo. 1978. Temperaturesensitive multicellular mutants of Wangiella dermatitidis. J. Bacteriol. 135:622-632. 8. Slater, M. LX 1976. Rapid nuclear staining method for Saccharomyces cerevisiae. J. Bacteriol. 126:1339-1341. 9. Szaniszlo, P. J., P. H. Hsieh, and J. D. Marlowe. 1976. Induction and ultrastructure of the multicellular (sclerotic) morphology in Phialophora dermatitidis. Mycologia 68:117-130. 10. Ward, D. C., E. Reich, and I. H. Goldberg. 1965. Base specificity in the interaction of polynucleotides with antibiotic drugs. Science 149:1259-1263.
