Regulation of Septum Formation in Aspergillus nidulans by a DNA Damage Checkpoint Pathway

Corresponding author: Steven D. Harris, Department of Microbiology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3205, sharris{at}nso2.uchc.edu (E-mail).

Communicating editor: R. H. DAVIS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In Aspergillus nidulans, germinating conidia undergo multiple rounds of nuclear division before the formation of the first septum. Previous characterization of temperature-sensitive sepB and sepJ mutations showed that although they block septation, they also cause moderate defects in chromosomal DNA metabolism. Results presented here demonstrate that a variety of other perturbations of chromosomal DNA metabolism also delay septum formation, suggesting that this is a general cellular response to the presence of sublethal DNA damage. Genetic evidence is provided that suggests that high levels of cyclin-dependent kinase (cdk) activity are required for septation in A. nidulans. Consistent with this notion, the inhibition of septum formation triggered by defects in chromosomal DNA metabolism depends upon Tyr-15 phosphorylation of the mitotic cdk p34^nimX. Moreover, this response also requires elements of the DNA damage checkpoint pathway. A model is proposed that suggests that the DNA damage checkpoint response represents one of multiple sensory inputs that modulates p34^nimX activity to control the timing of septum formation.

THE fidelity of cellular reproduction is dependent upon the temporal coordination of cytokinesis with nuclear division. Recent evidence suggests that continuous signaling between midzone microtubules and the cellular cortex is required for the completion of cytokinesis in animal cells (WHEATLEY and WANG 1996 ; OEGEMA and MITCHISON 1997 ). A number of proteins that localize to the spindle midzone during telophase could conceivably mediate these signaling events. This includes chromosomal passenger proteins such as the inner centromere proteins (INCENPs; COOKE et al. 1987 ; EARNSHAW and MACKAY 1994 ), telophase disc proteins such as TD-60 (ANDREASSEN et al. 1991 ; MARTINEAU et al. 1995 ), kinesin-related motor proteins (WILLIAMS et al. 1995 ), and anillin (FIELD and ALBERTS 1995 ). Interactions between mitotic nuclei and the cellular cortex also appear to be required for the completion of cytokinesis in the unicellular fungi Schizosaccharomyces pombe and Saccharomyces cerevisiae (FANKHAUSER et al. 1993 ; MURONE and SIMANIS 1996 ; YANG et al. 1997 ). Notably, inappropriate activation of the spindle-assembly checkpoint appears to directly inhibit septum formation in S. pombe (MURONE and SIMANIS 1996 ).

In both animal and fungal cells, cyclin-dependent kinase (cdk) activity has been implicated in controlling the timing of cytokinesis relative to nuclear division. In Xenopus lysates, phosphorylation of three different negative regulatory sites on the 20-kD myosin light chain protein by p34^cdc2 may delay cytokinesis until the initiation of anaphase (SATTERWHITE et al. 1992 ). However, the importance of this mechanism for the temporal regulation of cytokinesis in animal cells is still open to question (FISHKIND and WANG 1995 ). In S. cerevisiae, the reorganization of the actin cytoskeleton at the incipient division site is triggered by the destruction of the Clb-Cdc28 cdk complex (LEW and REED 1993 ). The destruction of p34^cdc2 also appears to promote cytokinesis in fission yeast, since a mutation that causes premature loss of cdk activity leads to multiple rounds of septation (FANKHAUSER et al. 1993 ). Collectively, these studies suggest that cdk activity suppresses cytokinesis until the initiation of anaphase.

The multicellular filamentous fungus Aspergillus nidulans undergoes cytokinesis by forming crosswalls termed septa. The process of septum formation is tightly coordinated with nuclear division in A. nidulans hyphal cells. For example, as in animal cells, mitotic nuclei are likely to specify the division site (WOLKOW et al. 1996 ). Furthermore, recent observations indicate that continuous signaling between adjacent mitotic spindles and the cortex is required for formation of the contractile actin ring and the completion of septation (MOMANY and HAMER 1997 ). Previous results have shown that uninucleate conidia give rise to multinucleate hyphal cells (predivisional hyphal cells; HARRIS 1997 ) by delaying septum formation until they satisfy a size control and complete a subsequent mitotic division (WOLKOW et al. 1996 ). The uncoupling of cytokinesis from nuclear division in these cells has permitted the identification of a class of temperature-sensitive (TS) mutants with a distinctive phenotype suggestive of a defect in the coordination of septation with nuclear division (MORRIS 1976 ; HARRIS et al. 1994 ). Although these mutants (sepB3, sepB4, and sepJ1) grow to a sufficient extent and are capable of undergoing multiple rounds of nuclear division, they fail to form septa. In addition, sepB mutants also exhibit defects in chromosomal DNA metabolism, which lead to enhanced levels of chromosome nondisjunction and mitotic recombination (HARRIS and HAMER 1995 ). These phenotypes suggested that sepB mutants fail to form septa because of incomplete or aberrant chromosome segregation. The recent finding that the predicted sepB gene product displays similarity to an S. cerevisiae protein involved in chromosomal DNA metabolism is consistent with this hypothesis (HARRIS and HAMER 1995 ).

The sepB gene product may directly promote septum formation in addition to its functions in chromosomal DNA metabolism. Alternatively, the general accumulation of DNA damage caused by sepB mutations may trigger a regulatory response that inhibits septation. The second possibility has been investigated by testing the ability of other perturbations of chromosomal DNA metabolism to prevent septum formation. Results presented here demonstrate that septation is inhibited by a variety of treatments capable of producing sublethal amounts of DNA damage. In addition, genetic evidence is provided that suggests that septation is triggered by high levels of cdk activity in A. nidulans, and that the ability of defects in chromosomal DNA metabolism to prevent septum formation is dependent upon inhibitory Tyr-15 phosphorylation of the cdk p34^nimX. Moreover, additional genetic interactions are presented that show that the DNA damage-induced inhibition of septation is enforced by a DNA damage checkpoint response. Finally, we propose that the timing of septum formation in predivisional hyphal cells is controlled by a branched regulatory pathway.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains, media, and genetic manipulations:
All strains used in this study are described in Table 1. Media used were YGV (2% glucose, 0.5% yeast extract, 0.1% vitamin mix) and CM ± supplements (1% glucose, 0.2% peptone, 0.1% yeast extract, 0.1% casamino acids, nitrate salts, trace elements, and 0.01% vitamins, pH 6.5). Supplements, vitamin mix, trace elements, and nitrate salts are described in the appendix to KAFER 1977 . For solid media, 1.5% agar was added. Hydroxyurea (HU; Sigma Chemical Co., St. Louis) was prepared as a 2 M stock solution and added to media after autoclaving.

For the viability/aneuploidy experiment, conidia were diluted and plated for single colonies on CM plates. Plates were incubated at 42° for the indicated time period and then shifted to 28°. Control plates were incubated at 28° for the duration of the experiment.

All genetic manipulations were performed as described previously (HARRIS et al. 1994 ). sepB nimX ^cdc2AF, sepJ nimX ^cdc2AF, sepA nimX ^cdc2AF, and sepB uvs double mutants were identified as TS segregants, which were also sensitive to 5 mM HU.

Growth conditions:
For all experiments, YGV or CM+ supplements containing conidia at a density of 1–5 x 10⁴/ml was gently poured into petri dishes containing glass coverslips. The conidia settled to the bottom of the petri dish and adhered tightly to the coverslips. At the appropriate times, coverslips with adherent cells were removed and processed for microscopy.

Wild-type strains were incubated at either 28° or 37°. HU (used at concentrations ranging from 1–10 mM) and diepoxyoctane (DEO; Aldrich Chemical Co., Milwaukee, used at concentrations ranging from 0.01–0.05%) were added to media at the time of inoculation. Because hyphae treated with low concentrations of HU (5 mM) tend to recover before the formation of the first septum, a second dose of HU was added at the same concentration 6 hr after inoculation.

Strains containing TS mutations were incubated at either 43.5° (restrictive temperature) or 39° (semipermissive temperature). For strains unable to undergo nuclear division at 39°, semipermissive conditions were empirically determined as the maximum temperature allowing completion of at least two rounds of nuclear division (generally 35.5–37.5°).

Staining, microscopy, and measurements:
Fixation of samples, staining with Calcofluor and Hoechst 33258, and mounting of stained coverslips on glass slides were performed as previously described (HARRIS et al. 1994 ). Slides were observed using an Olympus (Olympus Corp., Lake Success, NY) BMAX fluorescent microscope and DPLANAPO x40 and x100 (oil immersion) objectives. Photographs were taken with Kodak Technical-Pan film (Eastman Kodak, Rochester, NY) and developed in Kodak HC-110 developer.

The septation index (SI) describes the percentage of cells possessing at least one septum within a randomly selected population of 200 cells. Cell size was determined by measuring hyphal length with a calibrated eyepiece micrometer. Since fungal hyphae grow solely at the tip, the length of a hypha generally indicates the extent to which it has grown. Under the conditions used in these experiments, all strains exhibited similar hyphal diameters (i.e., 2–3 µm). Thus, hyphal length represent a reasonable approximation of cell size.

The nonparametric Mann-Whitney test was used to determine if two strains differed significantly in cell length at the time the first septum formed (ZAR 1984 ). To use this test, measurements were ranked and the U and U' values calculated. These values take the number of measurements and the sum of the ranks into consideration. The larger of the two values was compared to the respective critical value. If this value was greater than or equal to the critical value, the null hypothesis (that no significant difference in cell size exists) was rejected.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

General perturbations of DNA metabolism inhibit septum formation:
The inability of sepB mutants to undergo septation could be because of the accumulation of DNA damage resulting from a defect in chromosomal DNA metabolism (HARRIS and HAMER 1995 ). If this notion is correct, then additional perturbations of chromosomal DNA metabolism might also be expected to inhibit or delay septum formation. Among the sep mutants, the defects caused by the sepJ1 mutation most closely resemble those observed in sepB mutants. In addition to the defect in septum formation (Figure 1B; Table 2), plating experiments showed that sepJ1 mutants behave like sepB mutants in rapidly losing viability following prolonged incubation at restrictive temperature (Figure 1A). Furthermore, the increase in proportion of aneuploid survivors (Figure 1A) suggests that the sepJ1 mutation causes defects that lead to enhanced chromosome loss and/or mitotic recombination (KAFER and UPSHALL 1973 ). When examined microscopically, sepJ1 mutants were found to possess morphologically aberrant nuclei whose appearance deteriorated with each successive round of nuclear division (Figure 1, C–F). Collectively, these phenotypes suggest that the sepJ1 mutation causes defects in chromosomal DNA metabolism, which prevent septum formation.

View larger version (40K): In this window In a new window Download PPT slide   Figure 1. —The sepJ1 mutation prevents septum formation and also causes defects in chromosomal DNA metabolism. (A) Conidia from strains A28 (wild type, triangles), ASH329 (sepJ1, circles), and AJM15 (sepB4, squares) were diluted and plated for single colonies on CM plates. Plates were incubated at 42° for the indicated time period and then shifted to 28°. The zero time point was held at 28° for the duration of the experiment. Open symbols represent the percentage of viable colonies relative to the zero time point. Closed symbols represent the percentage of viable colonies with abnormal morphologies characteristic of aneuploids (KAFER and UPSHALL 1973 ). (B) Wild-type (open circles) and sepJ1 (closed circles) hyphal cells were incubated on coverslips for 9 hr (wild type) or 14 hr (sepJ1) at 42°. Coverslips were stained with Calcofluor and Hoechst 33258 to visualize septa and nuclei, respectively. For each strain, the length and nuclear number of 25 randomly selected cells was measured and plotted as a scatter graph. In addition, the septation index (SI, the percentage of cells that possess at least one septum) was determined for a randomly selected population of 200 cells. For wild-type cells, the SI was 64%, whereas it was zero for sepJ cells. The strains analyzed were A28 (wild type) and ASH329 (sepJ1). (C–E) sepJ1 hyphal cells from this experiment. (F) Wild-type control. Arrow denotes a septum. Bar, 4 µm.

To further test the notion that defects in chromosomal DNA metabolism lead to inhibition of septum formation, the effects on septation of the replication inhibitor HU and the DNA-damaging agent DEO were examined in wild-type cells. Specifically, conidia were allowed to germinate and form hyphae in the presence of sublethal doses of these agents. Under such conditions, low levels of DNA damage would presumably accumulate while growth and nuclear division would be only modestly affected. Indeed, wild-type cells incubated in the presence of 5 mM HU or 0.025% DEO for 11 hr exceeded the size at which septation normally occurs (i.e., 50 µm, HARRIS et al. 1994 ; average cell sizes for HU-treated and DEO-treated cells were 122.1 ± 57.9 µm and 157 ± 57.9 µm, respectively) and underwent multiple rounds of nuclear division (93% of HU-treated cells and 98% of DEO-treated cells possessed 8 nuclei). However, as predicted, septum formation was significantly delayed in these cells (Figure 2). For example, in the population of cells possessing 16 nuclei, the number of untreated cells that had undergone septation was approximately eightfold greater than the number of treated cells (Figure 2A). Moreover, at this stage, untreated cells were much shorter (56.8 ± 18.1 µm) than the treated cells (Figure 2, B–D).

View larger version (45K): In this window In a new window Download PPT slide   Figure 2. —Septum formation is delayed by treatment with sublethal doses of DNA-damaging agents. Conidia of wild-type strain A28 were germinated on coverslips for 11 hr at 37°. At the time of inoculation, cultures were treated with either 5 mm HU or 0.025% DEO. A third culture was left as an untreated control. Since A. nidulans hyphal cells tend to rapidly recover from HU blocks (HARRIS et al. 1994 ), a second dose of HU (5 mm) was added to the HU-treated cells 6 hr after inoculation. Coverslips were stained with Calcofluor and Hoechst 33258 to visualize septa and nuclei, respectively. (A) For each sample, 100 cells containing the indicated number of nuclei were examined and the percent possessing at least one septum was determined. (B) Untreated cells. (C) Cells treated with 5 mm HU. (D) Cells treated with 0.025% DEO. The arrow denotes a septum. Bar, 4 µm.

Several TS mutants defective in diverse aspects of cell cycle progression have been identified and characterized in A. nidulans (MORRIS 1976 ; MORRIS and ENOS 1992 ; DOONAN 1992 ). These mutants fail to form septa at restrictive temperature because of their inability to complete a single mitotic division (MORRIS 1976 ). In contrast, because they undergo mitosis and are generally not impaired for growth, most of these mutants are expected to form septa at semipermissive temperatures. However, if a specific TS mutation causes defects in chromosomal DNA metabolism, septation might be inhibited or severely delayed under these conditions. Among this collection of mutants, cells possessing the nimL15 mutation display a variety of phenotypes consistent with a defect in chromosomal DNA metabolism. At restrictive temperature, nimL15 mutants fail to undergo nuclear division and appear to arrest in S phase (MORRIS 1976 ; DOONAN 1992 ). Furthermore, at both permissive and semipermissive temperatures, strains possessing this mutation are sensitive to HU and methyl methanesulfonate (MMS) and also exhibit enhanced levels of spontaneous mutagenesis (DOONAN 1992 ; S. HARRIS, unpublished results). When incubated at semipermissive temperature, nimL15 mutants grow to an adequate size (i.e., >50 µm long) and undergo sufficient rounds of nuclear division for septation to occur (Figure 3A and Figure B). Nonetheless, the SI for nimL15 mutants was only 4%, and those cells possessing septa were 100 µm long and possessed 14 nuclei. Similar results were obtained when cells possessing the nimP22 mutation were examined for septum formation following incubation at semipermissive temperature (Figure 3C). The nimP gene encodes the A. nidulans homologue of DNA polymerase (S. JAMES, personal communication), which is thought to be required for both chromosomal DNA replication and for replication-mediated DNA repair (STILLMAN 1994 ). In contrast, most of the remaining cell cycle mutants underwent septation with kinetics, which did not differ significantly from wild type. Thus, a diverse array of mutational (i.e., sepB3, sepB4, sepJ1, nimL15, nimP22) and pharmacological (sublethal doses of HU or DEO) perturbations of chromosomal DNA metabolism share the common effect of severely delaying or inhibiting septum formation. These observations are consistent with the notion that low levels of DNA damage inhibit septation without blocking nuclear division.

View larger version (22K): In this window In a new window Download PPT slide   Figure 3. —Mutations that cause defects in chromosomal DNA metabolism also prevent septum formation. Hyphal cells of the indicated genotypes were incubated on coverslips for 9 hr (wild type) or 14 hr (nim mutants) at 37°. Coverslips were stained with Calcofluor and Hoechst 33258 to visualize septa and nuclei, respectively. For each strain, the length and nuclear number of 25 randomly selected cells was measured and plotted as a scatter graph. In addition, the SI was determined for a randomly selected population of 200 cells. The strains analyzed were: (A) A28, (B) JD139, (C) SWJ280, and (D) SO65.

Septum formation requires high levels of p34^nimX activity:
In A. nidulans, the cdk encoded by the nimX gene (p34^nimX) is required for progression through the nuclear division cycle (OSMANI et al. 1994 ). The nimX3 mutation confers cell cycle arrest in both G₁ and G₂ with severely reduced kinase activity (OSMANI et al. 1994 ; YE et al. 1995 ). When the ability of the TS cell cycle mutants to undergo septation at semipermissive temperature was examined, it was observed that the nimX3 mutation prevented septum formation (Figure 3D). Although the majority of nimX3 cells had exceeded the size at which wild-type cells undergo septation (50 µm) and possessed four or more nuclei (Figure 3A and Figure D), they yielded an SI of only 1.5%. Furthermore, nimX3 hyphae, which did form at least one septum, were >150 µm in length and possessed >8 nuclei (n = 10). These observations suggest that p34^nimX activity is required for septation in A. nidulans.

The A. nidulans nimT gene encodes the tyrosine phosphatase which triggers cdk activation by dephosphorylating the Tyr-15 residue of p34^nimX (O'CONNELL et al. 1992 ). At restrictive temperature, nimT23 mutants arrest in G₂ with low levels of p34^nimX histone H1 kinase activity (YE et al. 1995 ). If p34^nimX activity is required for septation in A. nidulans, then the nimT23 mutation should affect septum formation in a manner similar to that observed for the nimX3 mutation. Indeed, the nimT23 mutation caused a severe delay in septum formation at semipermissive temperature (Figure 4A and B; note that Figure 4 presents data from cells possessing one septum, whereas Figure 3 presents data from a random population of cells). The majority of nimT23 hyphae did not undergo septation until they were 150 µm long and had accumulated more than eight nuclei. These results are consistent with the notion that cdk activity is required for septation in A. nidulans. Furthermore, the observation that nim X3 and nimT23 mutants are capable of completing multiple rounds of nuclear division at semipermissive temperature suggests that the level of p34^nimX activity needed to trigger septation may be higher than that required for mitosis.

View larger version (21K): In this window In a new window Download PPT slide   Figure 4. —Septum formation requires high levels of mitotic cdk activity. Hyphal cells of the indicated genotypes were incubated on coverslips for 9 hr (wild type) or 12 hr (nim mutants) at either 37° (wild type, nimX cdc2AF, and nimE6) or 39° (nimT23). Coverslips were stained with Calcofluor and Hoechst 33258 to visualize septa and nuclei, respectively. For each strain, the length and nuclear number of 25 cells possessing one septum was measured and plotted as a scatter graph. In addition, the SI was determined for a randomly selected population of 200 cells. The following strains were analyzed: (A) A28, (B) MO73, (C) FRY20, and (D) SO75.

The inability to phosphorylate the Tyr-15 residue of p34^nimX leads to elevated levels of histone H1 kinase activity and causes defects in checkpoint regulation (YE et al. 1996 , YE et al. 1997 ; OSMANI and YE 1997 ). If cdk activity plays a critical role in the temporal control of septum formation, then the increase in p34^nimX activity observed when the Tyr-15 residue cannot be phosphorylated may also promote premature septation. The nimX ^cdc2AF allele encodes a p34^nimX, which is no longer subject to inhibitory phosphorylation at Tyr-15 (YE et al. 1996 ). As predicted, hyphae possessing this mutation generally underwent septation at a smaller size and with fewer nuclei compared to wild-type cells (Figure 4A and Figure C). For example, whereas only 12% of wild-type hyphae were 50 µm long when septum formation occurred, 52% of nimX ^cdc2AF cells underwent septation within this size range. In addition, 24% of nimX ^cdc2AF cells contained four or fewer nuclei when the first septum formed, a condition never observed in wild-type cells (HARRIS et al. 1994 ). Septum formation appears to be generally deregulated in nimX ^cdc2AF mutants (Figure 5 and Figure 7C). Small cells (25 µm) containing one or more septa are observed at low frequency (7%), and a significant fraction of hyphae were found to contain nuclei bisected by a septum (16%, n = 100). Similar effects upon septation were also observed when nimA5 mutants were incubated at semipermissive temperature (WOLKOW et al. 1996 ). At restrictive temperature, the nimA5 mutation confers a G₂ arrest in which p34^nimX accumulates in a Tyr-15 dephosphorylated and fully active state (OSMANI et al. 1991 ). Taken together, these observations suggest that elevated levels of p34^nimX activity trigger premature formation of the first septum in A. nidulans hyphae.

View larger version (46K): In this window In a new window Download PPT slide   Figure 5. —Deregulation of septum formation in nimX cdc2AF mutants. Conidia of strain FRY20 were germinated on coverslips for 9 hr at 37°. Coverslips were stained with Calcofluor and Hoechst 33258 to visualize septa and nuclei, respectively. Cell 1 is binucleate and possesses a septum. Cell 2 has formed a double septum. Cell 3 possesses an anucleate compartment. Bar, 4 µm.

View larger version (32K): In this window In a new window Download PPT slide   Figure 6. —Growth phenotypes of double mutants. Conidia were point inoculated onto duplicate CM plates and a CM + 5 mm HU plate. One of the CM plates and the CM + 5 mm HU plate were incubated at 28° for 3 days, and the remaining CM plate was incubated at 42° for 2 days. Row 1 (from left to right): A28 (wild type), ASH284 (sepB3), and ASH286 (nimX cdc2AF). Row 2 (from left to right): ASH280 (sepB3 nimX cdc2AF), and ASH281 (sepB3 nimX cdc2AF). Row 3 (from left to right): ASH201 (uvsB110), and ASH206 (uvsD153). Row 4 (from left to right): ASH202 (sepB3 uvsB110), ASH203 (sepB3 uvsB110), and ASH207 (sepB3 uvsD153). The differential shading of colonies is because the presence of different conidial color markers in the strain backgrounds.

View larger version (47K): In this window In a new window Download PPT slide   Figure 7. —Cellular morphology of the double mutants. Conidia were germinated on coverslips for 9–15 hr at 42°. Coverslips were stained with Calcofluor and Hoechst 33258 to visualize septa and nuclei, respectively. (A) A28 (wild type), (B) ASH284 (sepB3), (C) ASH286 (nimX cdc2AF), (D) ASH280 (sepB3 nimX cdc2AF), (E) ASH329 (sepJ1), (F) ASH326 (sepJ1 nimX cdc2AF), (G) ASH203 (sepB3 uvsB110), and (H) ASH207 (sepB3 uvsD153). Arrows denote septa. Bar, 4 µm.

The opposing effects of the nimT23 and nimX ^cdc2AF mutations on the timing of septum formation provide genetic evidence that the formation of the first septum in A. nidulans hyphae is temporally controlled by cdk activity. To date, the only cyclin identified in A. nidulans is the B-type cyclin encoded by the nimE gene (O'CONNELL et al. 1992 ). Association of this cyclin with p34^nimX is required for progression through both G₁ and G₂ (O'CONNELL et al. 1992 ; DAYTON et al. 1997 ). If the NimE/p34^nimX complex is also required for septum formation, loss of function mutations in the nimE gene might be expected to delay or inhibit septation. In contrast, if a distinct cyclin is required for septum formation, nimE mutations would presumably have no effect upon septation. Incubation of cells possessing the nimE6 mutation at semipermissive temperature resulted in delayed septation (Figure 4D). Despite completing two to three rounds of nuclear division, hyphae formed by nimE6 mutants were typically twice as long as wild-type cells when the first septum formed. Although these results do not eliminate the possibility that additional cdk complexes promote septation, they do demonstrate that the same cdk complex that regulates mitosis also controls septum formation.

Inhibitory Tyr-15 phosphorylation of p34^nimX prevents septation when chromosomal DNA metabolism is perturbed:
Since septum formation is temporally regulated by p34^nimX activity, defects in chromosomal DNA metabolism could conceivably inhibit septation by reducing the level of active kinase. One mechanism by which this could be accomplished is through increased Tyr-15 phosphorylation of p34^nimX. To test this notion, a sepB3 nimX ^cdc2AF double mutant was constructed and tested for the ability to undergo septation at restrictive temperature. The presence of septa in the double mutant would indicate that the effects of p34^nimX on septation were epistatic to those of the sepB3 mutation. In contrast, the absence of septa would suggest that defects in chromosomal DNA metabolism influence septation at a point concomitant with or downstream of p34^nimX.

sepB3 nimX ^cdc2AF double mutants were found to be TS for growth and to display aberrant nuclear morphologies at restrictive temperature (Figure 6 and Figure 7D). Furthermore, at permissive temperature, they exhibited slower growth than either of the parental single mutants and were also sensitive to HU (Figure 6). However, despite the presence of the sepB3 mutation, the double mutants were able to undergo septation at restrictive temperature (Figure 7, A–D; Table 2). Eight sepB3 nimX ^cdc2AF double mutant isolates derived from two independent crosses were examined, and all were able to form septa at restrictive temperature. Two additional observations indicate that the ability of the nimX ^cdc2AF mutation to permit septation in the presence of sublethal DNA damage is not specific to the defects caused by the sepB3 mutation. First, sepJ1 nimX ^cdc2AF double mutants undergo septation at restrictive temperature (Figure 7E and F; Table 2), and second, cells possessing the nimX ^cdc2AF mutation were able to form septa when grown in the presence of 0.025% DEO (i.e., when incubated in DEO, 54% of cells possessing eight nuclei had undergone septation, compared to 61% for untreated cells). The possibility that these epistatic interactions reflect the ability of the nimX ^cdc2AF mutation to circumvent all of the controls which regulate septum formation was eliminated by showing that sepA1 nimX ^cdc2AF double mutants do not undergo septation at restrictive temperature (Table 2). The sepA1 mutation confers a late-acting defect in septum formation (HARRIS et al. 1994 ), and the sepA gene product is thought to be required for the organization of the actin ring at the division site (HARRIS et al. 1997 ). Collectively, these results show that inhibitory Tyr-15 phosphorylation of p34^nimX is required to prevent septation in sepB3 and sepJ1 mutants, and suggest that defects in chromosomal DNA metabolism may prevent septum formation by modulating cdk activity.

The inability of sepB3 mutants to form septa requires a functional DNA damage checkpoint pathway:
Normal function of the DNA damage checkpoint in A. nidulans requires inhibitory Tyr-15 phosphorylation of p34^nimX (YE et al. 1997 ). Since this mechanism also acts to prevent septum formation when chromosomal DNA metabolism has been perturbed, it seemed possible that the inhibition of septation under these conditions may also require other components of the DNA damage checkpoint. To examine this possibility, sepB3 uvsB110 and sepB3 uvsD153 double mutants were constructed and tested for their ability to undergo septation at restrictive temperature. Phenotypic analyses of uvsB and uvsD mutants suggests that mutations in these genes abrogate the DNA damage checkpoint (YE et al. 1997 ). Specifically, uvsB and uvsD mutants are sensitive to UV irradiation, MMS, and low levels of HU (KAFER and MAYOR 1986 ; Figure 6). In addition, they fail to delay entry into mitosis when incubated in the presence of 0.01% MMS (P. KRAUS and S. HARRIS, unpublished results).

sepB3 uvsB110 and sepB3 uvsD153 double mutants were TS for growth and displayed aberrant nuclear morphologies at restrictive temperature (Figure 6; Figure 7G and Figure H). Furthermore, they exhibited a synthetic slow growth phenotype as well as sensitivity to HU at permissive temperature (Figure 6). However, despite the presence of the sepB3 mutation, the double mutants were able to form septa at restrictive temperature (Figure 7G and H; Table 2). These observations suggest that a functional DNA damage checkpoint is required to prevent septum formation in sepB3 mutants. Moreover, this effect is not specific to the defects in chromosomal DNA metabolism caused by the sepB3 mutation, because uvsB110 and uvsD153 mutants are also able to form septa in the presence of 0.025% DEO (Table 3).

A branched pathway regulates the timing of septum formation:
Because cells possessing the nimX ^cdc2AF mutation alone undergo "premature septation" (i.e., formation of the first septum at a small size and with fewer nuclei compared to wild-type cells, Figure 4C and Figure 5), the ability of this mutation to permit septum formation in sepB3 mutants presumably reflects the bypass of most of the controls that temporally regulate this process. In contrast, the uvsB110 mutation by itself does not affect the timing of septation. The majority of cells possessing this mutation were between 50 and 75 µm in length and contained >8 nuclei when they underwent septation. Thus, whereas the regulatory pathway that prevents septum formation in the presence of DNA damage has been disabled in uvsB110 mutants, the cell size control remains functional. If this notion is correct, sepB3 uvsB110 double mutants should undergo septation within the normal size range of ~50 to 100 µm. In contrast, septum formation should occur at a smaller than normal size in cells possessing both the sepB3 and nimX ^cdc2AF mutations. We found that sepB3 uvsB110 double mutants were significantly longer than sepB3 nimX ^cdc2AF mutants when they underwent septation (i.e., 100.2 ± 40.9 µm vs. 34.8 ± 10.3 µm, = 0.001, Z = 6.05). Similar results were obtained for the analogous comparison involving sepB3 uvsD153 double mutants (i.e., 109.0 ± 42.0 µm vs. 34.8 ± 10.3 µm, = 0.001, Z = 6.01). These results suggest that the inability to phosphorylate the Tyr-15 residue of p34^nimX abolishes the regulatory mechanisms that: (i) prevent septum formation until cells have grown to the appropriate size, and (ii) inhibit septation in the presence of DNA damage. In contrast, the uvsB and uvsD gene products regulate septation only when DNA damage has been incurred. Thus, the regulatory pathway that controls the timing of septation must be branched, with different gene products controlling the responses to cell size and DNA damage.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Germinating A. nidulans conidia complete multiple rounds of nuclear division and form an elongated hyphal cell before the formation of the first septum (HARRIS et al. 1994 ; WOLKOW et al. 1996 ). Previous analyses of a specific class of septation mutants suggested that a specific regulatory mechanism prevents septation when the fidelity of the early rounds of nuclear division is compromised (HARRIS et al. 1994 ; HARRIS and HAMER 1995 ). Here, the regulatory circuit that controls the timing of septum formation in predivisional hyphal cells is characterized in greater detail. In particular, it is shown that: (i) general defects in chromosomal DNA metabolism inhibit the formation of the first septum, (ii) inhibition of septum formation requires an intact DNA damage checkpoint pathway, and (iii) distinct sensory inputs control the timing of septation by modulating the activity of an interphase cdk complex.

General defects in chromosomal DNA metabolism prevent septum formation in predivisional hyphal cells:
Phenotypic characterization of the TS sepB3 and sepB4 mutations raised the possibility that the accumulation of DNA damage may inhibit formation of the first septum in germinating A. nidulans conidia (HARRIS and HAMER 1995 ). Conceivably, this response could be triggered solely by DNA damage generated as a result of sepB mutations. However, the observation that a variety of mutational (i.e., incubation of sepJ1 mutants at restrictive temperature and nimL15 or nimP22 mutants at semipermissive temperatures) and pharmacological (i.e., treatment of wild-type cells with sublethal doses of HU or DEO) perturbations of chromosomal DNA metabolism inhibit or delay septum formation suggests that this is a general response activated by multiple types of DNA damage. In support of this notion, the genotoxic effects of low doses of HU or DEO are likely to be very different. Treatment with HU causes depletion of nucleoside triphosphate pools and presumably leads to the accumulation of base substitutions (KUNZ and KOHALMI 1991 ). In contrast, the high frequency of deletions induced by DEO in fungal cells indicates that it causes DNA strand breaks (ONG and DE SERRES 1975 ; HYNES 1979 ). Furthermore, preliminary results suggest that the effects of sepB and nimL mutations on chromosomal DNA metabolism also differ significantly. Unlike mutations in sepB, at semipermissive temperatures, the nimL15 mutation causes sensitivity to both HU and MMS and promotes increased levels of spontaneous mutagenesis (S. HARRIS, unpublished observations).

It is important to note that the DNA damage that prevents septum formation in predivisional hyphal cells does not block nuclear division. Each of the sep and nim mutants, as well as wild-type cells treated with sublethal doses of HU or DEO, typically complete at least three rounds of nuclear division following the imposition of damage-inducing conditions. Although the kinetics of nuclear division have not been systematically examined under these conditions, the sepB3 mutation appears to cause only a slight delay in mitosis (HARRIS and HAMER 1995 ). Two possible explanations could account for the differential effects of DNA damage on septum formation and nuclear division in germinating conidia. One possibility is that septation is specifically inhibited by a certain type of damage that does not affect mitosis. However, the ability of a broad range of damage-inducing conditions to prevent septum formation casts doubt upon this notion. Alternatively, the response to DNA damage in predivisional cells may be quantitative. In particular, the threshold of damage required to prevent septation may be lower than that needed to inhibit nuclear division. This idea is supported by the observation that nuclear division is inhibited by doses of HU (20 mM) or DEO (0.05%) higher than those which prevent septation. Moreover, the eventual failure of nuclear division in sepB and sepJ mutants may be caused by the gradual accumulation of irreparable DNA damage until levels exceed the threshold that prevents cell cycle progression.

Inhibition of septum formation in predivisional hyphal cells requires an intact DNA damage checkpoint:
The initial characterization of both the S-phase and DNA damage checkpoint responses in A. nidulans has recently been reported (YE et al. 1996 ; YE et al. 1997 ). The phenotypes displayed by uvsB and uvsD mutants suggest that these gene products function in each of these checkpoint responses (YE et al. 1997 ). Mutations in either of these genes confers sensitivity to UV irradiation, MMS, and HU (KAFER and MAYOR 1986 ; Figure 4). Furthermore, uvsB and uvsD mutants fail to delay mitotic entry when treated with MMS (P. KRAUS and S. HARRIS, unpublished results). By analogy to the corresponding checkpoint pathways in budding and fission yeast (ENOCH and NORBURY 1995 ), these phenotypes suggest that the uvsB and uvsD gene products are required for a signal transduction component, which underlies both checkpoint responses. The ultimate effector of the DNA damage checkpoint in A. nidulans is the mitotic cdk p34^nimX (YE et al. 1997 ). Whereas the nimX ^cdc2AF mutation abrogates the DNA damage checkpoint response and permits mitotic entry in UV-irradiated or MMS-treated cells (YE et al. 1997 ), it does not affect the checkpoint that responds to the arrest of DNA replication (YE et al. 1996 ). The later response also requires inactivation of the bimE gene product (YE et al. 1996 ), a negative regulator of mitosis, which is part of the anaphase-promoting complex ( JAMES et al. 1995 ; OSMANI and YE 1997 ).

The nimX ^cdc2AF mutation, as well as mutations in uvsB or uvsD, permits inappropriate septum formation in predivisional hyphal cells that have incurred DNA damage. Notably, each of these mutations exacerbates many of the phenotypes caused by the sepB3 mutation. For example, nimX ^cdc2AF sepB3 and uvs sepB3 double mutants display severe defects in nuclear morphology and grow slowly even at permissive temperature. Thus the ability of these double mutants to undergo septation does not reflect a general suppression of the defects in chromosomal DNA metabolism induced by the sepB3 mutation. Instead, these observations demonstrate that the DNA damage-induced inhibition of septum formation in predivisional hyphal cells is enforced by a checkpoint response.

Distinct sensory inputs control the timing of septum formation by modulating the activity of an interphase cdk complex:
The phenotypes displayed by predivisional hyphal cells possessing the nimX ^cdc2AF mutation clearly indicate that most of the controls that regulate the timing of septum formation have been abrogated. These include the controls that: (i) prevent septum formation in the presence of DNA damage, and (ii) establish the appropriate cell size at which septation occurs. In contrast, the uvsB and uvsD mutations only appear to abolish the DNA damage response (i.e., uvs sepB double mutants do not septate at small cell size). These observations suggest that the formation of the first septum in germinating conidia is temporally controlled by distinct sensory inputs that control the responses to DNA damage and cellular growth (Figure 8). Furthermore, these signals converge on the regulatory module which controls Tyr-15 phosphorylation of p34^nimX (LEW and KORNBLUTH 1996 ). This module includes the Wee1 tyrosine kinase and the Cdc25 tyrosine phosphatase, which are encoded by the ankA and nimT genes, respectively, in A. nidulans (O'CONNELL et al. 1992 ; YE et al. 1997 ). Regulatory signals which act to delay septum formation presumably lower the levels of active p34^nimX by increasing ankA^wee1 activity and/or decreasing nimT ^cdc25 activity. This response could conceivably be mediated by an activity similar to the Chk1 kinase (O'CONNELL et al. 1997 ; FURNARI et al. 1997 ; SANCHEZ et al. 1997 ). Because the ultimate effect of the regulatory pathway is the inhibition of septum formation, p34^nimX must interact with a substrate that is required for septation. Given that sepA1 nimX ^cdc2AF double mutants fail to septate, and that SepA is required for actin ring formation at the division site (HARRIS et al. 1997 ), potential substrates include SepA or the gene products that regulate its activity.

View larger version (7K): In this window In a new window Download PPT slide   Figure 8. —A branched regulatory pathway controls the timing of septum formation in predivisional hyphal cells. Distinct sensory inputs control the responses to DNA damage and cell growth. The damage response requires elements of the DNA damage checkpoint pathway (uvsB and uvsD), and is activated by a variety of defects in chromosomal DNA metabolism (including those caused by mutation in sepB and sepJ ). The response to growth conditions (i.e., cell size) remains uncharacterized. Both sensory inputs presumably converge on the regulatory module which controls the activity of the mitotic cdk p34nimX. Activated p34nimX is predicted to trigger septation by modifying a gene product which is required for the activation of the late-acting sep genes. Double mutant analyses indicate that the sepA gene product functions upstream of sepD, sepG, and sepH (MORRELL 1997 ).

Results presented here show that p34^nimX is required for both mitosis and septation in predivisional hyphal cells. Moreover, the ability of these cells to undergo mitosis in the absence of septum formation implies that these effects are separable. The existence of such a relationship would presumably be undetectable in uninucleate cells, where it would be masked by the obligatory coupling of cytokinesis to mitosis (SATTERWHITE and POLLARD 1992 ). One potential explanation for the different effects of p34^nimX activity on mitosis and septation is that there exists a "septation-specific" cyclin subunit. However, the observation that mutations in the NimE cyclin delay septum formation does not support this hypothesis. Instead, the data are consistent with a model whereby septation requires attainment of threshold levels of interphase p34^nimX activity. In particular, levels of kinase activity may be assessed at a specific point during interphase in predivisional hyphal cells. Only if activity exceeds the threshold does septation occur following the next round of mitosis. An important test of this model will be to compare cdk activity during mitoses that are followed by septation to those that are not. Moreover, the model predicts that accumulation of sublethal DNA damage inhibits septum formation by activating the DNA damage checkpoint and reducing interphase cdk activity to levels below the threshold needed for septation. Additional experiments designed to assess the effects of sepB and sepJ mutations on the Tyr-15 phosphorylation state and activity of p34^nimX should test the validity of this notion.

The timing of septum formation and its role in hyphal differentiation:
A. nidulans postdivisional hyphae are differentiated in the sense that they possess two distinct cell types (FIDDY and TRINCI 1976 ; HARRIS 1997 ; KAMINSKYJ and HAMER 1998 ): (i) a tip cell in which cell cycle progression and polarized hyphal extension continue unabated, and (ii) subapical cells, which enter a period of mitotic quiescence and growth arrest. Subapical cells are likely to serve two important functions in hyphae. First, they function as "stem cells" for the formation of asexual and sexual reproductive structures (TIMBERLAKE 1990 ). Second, the presence of nondividing nuclei in these cells appears to provide postdivisional hyphae with some degree of resistance to the lethal effects of DNA damage. Indeed, postdivisional hyphae are significantly less sensitive to the effects of both UV irradiation and loss of sepB function than are predivisional hyphal cells (S. HARRIS, unpublished observations). For these reasons, it is undoubtedly crucial that nuclei within subapical cells possess chromosomes that are intact and undamaged. Thus, the importance of the DNA damage-induced inhibition of septation in predivisional hyphal cells may lie in its ability to prevent cellularization if unrepaired chromosomes enter mitosis (because of failed repair or adaptation to the checkpoint signal, PAULOVICH et al. 1997 ). In such a context, this response may represent an important developmental checkpoint in A. nidulans.

	ACKNOWLEDGMENTS

We thank JOHN DOONAN, STEVEN JAMES, GREG MAY, PETER MIRABITO, STEPHEN OSMANI, and XIANG YE for generously providing strains; JENNIFER GITTZUS for providing the data depicted in Table 3; XIANG YE, STEPHEN OSMANI, and TOM WOLKOW for insightful discussion and the communication of unpublished results; and JOHN HAMER for a critical evaluation of the manuscript. This work was supported by a grant from the National Science Foundation (MCB-9513489).

Manuscript received September 6, 1997; Accepted for publication November 17, 1997.

	LITERATURE CITED

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