Direct Evidence for Ca2+Regulation of Hyphal Branch Induction

Fungal Genetics and Biology 22, 127–139 (1997) Article No. FG971011

Direct Evidence for Ca21 Regulation of Hyphal Branch Induction

Avie Grinberg and I. Brent Heath1 Department of Biology, York University, 4700 Keele Street, North York, Ontario, M3J 1P3, Canada

Accepted for publication August 20, 1997

Grinberg, A., and Heath, I. B. 1997. Direct evidence for Ca21 regulation of hyphal branch induction. 22, 127– 139. Irradiation of growing hyphae of Saprolegnia ferax with microbeams of UV (300–380 and 385–450 nm) light induced an increase in cytoplasmic [Ca21] followed by precocious formation of one or more branches within about 4 min. The distribution of branches was strongly skewed toward the subapical side of the irradiation site, but otherwise was apparantly random. Apical (10-mm) irradiations were more effective than subapical (50-mm) ones in that they induced branches at comparable frequencies but with lower doses, consistent with higher concentrations of putative target intracellular Ca21 storage structures in this region. Once formed, induced adjacent branches seem to compete for ‘‘resources,’’ with those closer than D50 mm inhibiting each other. The results are most consistent with Ca21-induced accumulation of branch initiating factors being the cause, not the consequence, of branch formation, thus supporting a primary role for Ca21 in regulation of hyphal tip growth. r 1997 Academic Press

Index Descriptors: branching; tip growth; Ca21; UV light; Saprolegnia. Because tip growth is fundamental to fungal growth, morphogenesis, and development, there has been considerable interest in its mechanisms. Extensive data have resulted in the formulation of a number of diverse hypotheses to account for various aspects of the process (BartnickiGarcia and Lippman, 1972; Bartnicki-Garcia et al., 1989; Wessels, 1990; Heath, 1995; Heath and Janse van Rens1

To whom correspondence should be addressed.

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berg, 1996), but, due to the complexity involved, none of these have enjoyed universal acceptance or conclusive proof, nor are any able to provide a complete account of the process. An aspect of tip growth which is relatively underinvestigated is the formation of new tips during branch initiation, yet this process is also essential to understanding colony morphogensis and may add clues to the basic puzzle of tip growth. It is clear that hyphae are capable of maintaining a well-regulated and constant relationship between tip growth and branch formation, such that there is a constant speciesand medium-specific relationship which can be seen as the hyphal growth unit (reviewed by Trinci et al., 1994). However, while the hyphal growth unit concept shows that the location of branch formation is, on average, predictable, there is, in most species, very low precision in this predictability. Thus, it is seldom possible to predict the exact site of branch formation; it appears to be random, within certain limits, along the length of a main hypha. There are a diversity of compounds, paramorphogens, which are able to induce colony hyperbranching (Trinci et al., 1994). Among these are assumed variations in cytoplasmic [Ca21] (Reissig and Kinney, 1983; Harold and Harold, 1986; Robson et al., 1991). However, none of these studies were designed to analyze the behavior of cytoplasmic [Ca21] or the localization of branch induction sites. More recently, it has been shown that irradiation of Saprolegnia hyphae with UV light (330–380 nm) induces elevation of cytoplasmic [Ca21], apparently by the release of Ca21 from intracellular stores (Hyde and Heath, 1997). Localization of this stimulus by the use of a small-diameter UV microbeam offers the opportunity to further explore the role of Ca21 in the initiation and localization of branch induction.

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In this report we show that an increase in cytoplasmic [Ca21]induced by a UV microbeam precedes the induction of branch formation, that the site of branch initiation is not random, and that when initiated, adjacent branches compete with each other for subsequent growth.

MATERIALS AND METHODS Cultures, Media, and Microscopy Hyphae of Saprolegnia ferax (ATTC No. 36051) were grown overnight on dialysis membrane overlying a growth medium designated OM (Heath and Greenwood, 1970). These membranes were cut to produce hyphal tips on 0.5 3 1.5 cm portions and mounted in liquid OM in flow-through growth chambers (Heath, 1988). Media were adjusted to pH 6.8 with KOH to match the pH of the electroporation medium and the pH of solid and liquid OM were matched to reduce the recovery time following transfer to the growth chambers. Hyphae resumed growth after 5–20 min in the chambers and were irradiated between 15 and 90 min. Controls for determination of normal branch initiation sites were grown in identical chambers, fixed at desired times by flowing through 5% formaldehyde in 60 mM, pH 7.0, Pipes buffer, and then measured with an eyepiece scale. In these colonies, the first branch sites were commonly lost within the hyphal mat; thus, we measured the branch-free lengths of peripheral hyphae. There were three types of such apices. Type 1 had swollen upon transfer to the slide chamber and then resumed growth without branching so that the tip to swelling distance indicated the minimal distance to the first branch. Type 2 could be tracked back to the first branch, in which case its distance to the tip was measured. In type 3 the first branch was obscured in the hyphal mat, so we measured the length of the hypha to the mat, which is again an underestimate of the distance to the first branch. Hyphae were observed with Nomarski differential interference contrast (DIC) optics on a Reichert Polyvar microscope using green light and a 100X 1.32 NA objective lens. Only growing, ‘‘healthy’’ (Yuan and Heath, 1991) hyphae were irradiated. Growth rates were determined from images recorded 1 min apart on film or videotape.

Microbeam Irradiations A ,19-µm-diameter microbeam was produced on the specimen by a pinhole at a conjugate focal plane in the

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Grinberg and Heath

light path from a HBO 200-W mercury vapor lamp. Irradiation employed Reichert U1 (excitation h.b.w. 330– 380 nm, dichroic mirror 420 nm, barrier .418 nm) and V2 (excitation h.b.w. 385–450 nm, dichroic mirror 460 nm, barrier .470 nm) filter sets which encompass lamp output peaks at 303, 314, 334 and 364 and 408 and 436 nm, respectively. The intensity of the microbeams was reduced with diaphragms adjacent to the pinhole and in the objective in order to reduce light scattered around the beam site and to keep the intensity of the B1 illumination (for observation of Calcium Green) below levels which induced dye fading. (There was insufficient time to adjust aperatures between UV irradiation and dye observation.) Energy in the microbeams was measured by optically connecting the objective to an UV 100 photocell (United Detector Technology, Hawthorne, CA) with immersion oil. The values, given in Table 5, were calculated using peak values of 355 and 420 nm and the manufacturer’s spectral response calibration curve. These values are approximations due to scattered light impinging on the photocell surface, but are comparable to microbeams previously used on hyphae (Table 5). The microbeams were aligned with the center of the field of view with the aid of the faint autofluorescent microbeam image of nontarget hyphae using an image intensifier (Video Scope KS 1381, Video Scope International, Washington D.C.) and enhanced using a DVS-3000 image processor (Hamamatsu Photonics, Hamamatsu City, Japan). The microbeam was then removed, and a selected hypha aligned over the center spot while viewed with green light and DIC optics and then allowed to grow until its tip reached a point 10 or 50 µm beyond the center spot. The beam was then turned on again for the desired irradiation time. The microscope was focused on the median plane of the hyphae before exposure to the microbeam to ensure that the location of the most intense part of the beam was consistent between hyphae. Irradiations were started at sites centered at 10 µm (apical) and 50 µm (subapical) from the growing tips and continued for 10 or 60 s. At 50 µm, there is very little detectable vacuolation (Yuan and Heath, 1991). Hyphae were observed after irradiation and the earliest stage of branch initiation was identified by focusing through the hyphae. Initiation time was recorded from the end of irradiation and distances were measured between centers of the branch and microbeam sites. For apical irradiations, the distance at the time of emergence was also directly measured from the tip. However, determining this distance for the longer subapical irradiations was complex

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because the hyphae grew a considerable distance (mean ,80 µm) during and after the irradiation, so that at the time of initiation the distance to the tip was large and not easily directly measured. The distance was calculated by (1) multiplying the growth rate recorded before irradiation by the time between the start of irradiation and the emergence of the branch, (2) adding 50 µm for the distance of the irradiated site from the tip at the start of the irradiation, and (3) adding (or subtracting in the case of initiation sites distal to the irradiation site) the measured distance of the initiation site from the irradiation site. This procedure overestimates the true value because it does not take into account the reduction in growth rate during the irradiation time. However, since the primary use of these values was to show that the induced branch sites were ahead of normal branch sites, this error will underestimate, and thus strengthen, the concluded difference.

cytoplasm where curvature could have introduced differences in cytoplasmic volume sampled. Control procedures included subjecting ,50-µm subapical regions of growing hyphae to (1) identical sequences of irradiations, but with no dye, to measure levels of autofluorescence before and after the microbeam and (2) two B1 irradiations, with dye, with a 1-min gap between to detect any possible fluctuations in dye distribution, internal [Ca21], or possible fading caused by the B1 irradiations alone. All figures followed by 6 are means followed by standard deviations and sample sizes.

RESULTS Microbeam Induction of Branching

[Ca21]

Measurements of Hyphae

Dialysis membrane cultures were cut as described above and floated, four per 1 ml of 10 µM Calcium Green-1 (Molecular Probes Inc., Eugene, OR) in 0.067 M Sorenson’s phosphate buffer (pH 6.8), between the electrodes in a 4-mm gap chamber of a BRL Cell-Porator (Bethesda Research Laboratories, Life Technologies Inc., Gaithersburg, MD). Electroporation was at room temperature with the 60-µF capacitor and an initial voltage of 210 V, giving calculated pulses of 476 µs and field strengths of 0.525 kv/cm (cf. Jackson and Heath, 1990b). The hyphae were left in the chamber for 15–20 min, transferred to liquid OM (pH 6.8) in embryo cups for at least 1 h to recover (Jackson and Heath, 1990b), and then slid off the dialysis membrane and mounted on slides in liquid OM (pH 6.8). Hyphae were observed and recorded with the above video system. The growth rate of each dye-loaded hypha was measured from the video screen over a 1-min interval and dye fluorescence was recorded with a B1 filter set (excitation h.b.w. 450–490 nm, dichroic mirror 510 nm, barrier 515–545 nm) for about 3 s. The hypha was then irradiated with the appropriate microbeam and immediately after dye fluorescence in the same area was again recorded for 3 s. Each filter switch took about 8 s. Fluorescence intensities were measured with an Image 1 (Universal Imaging Corp., West Chester, PA) image processor which calculated pixel intensities, averaged over approximately 3 s, in a 30 3 8 µm box centered on the microbeam site and oriented parallel to the long axis of the hyphae. The box size was selected to encompass the irradiated and closely adjacent cytoplasm, but to exclude the peripheral

In order to demonstrate specific induction of branching, it is essential to show that branch formation does not normally occur in the region of presumed induction. Saprolegnia hyphae growing on dialysis membranes on agar initiate their first branches at a mean of 162 6 51 µm behind the leading hyphal tips (Levina et al., 1995), but in the liquid medium of the slide chambers used in this work this distance was increased to more than 430 µm (Fig. 1). This figure is a minimum underestimate based on the branch-free lengths of hyphal tips because the true distance was obscured for most hyphae. The minimum and maximum branch-free distances on unirradiated hyphae increased from 150 and 800 µm after 15 min in the chambers to 410 and 1600 µm after 90 min (Fig. 1). These distances do not indicate the length to the first branch because the hyphae had not reached the length required for branch formation. However, any branches which form closer to the tips than these distances may be assumed to have been induced by the relevant treatments. A combined total of 84% of hyphae responded to apical and subapical irradiations by forming branches up to 170 µm from the irradiation site within 10 min (Fig. 2; Table 1). All of the branching sites were located well ahead of any of the indicated sites of normal branch initiation (Figs. 1 and 4 and Table 1). Most responding hyphae (70%) produced a single branch (Table 1), but others simultaneously produced two or rarely three (Tables 1 and 2). The distribution of all branching sites was strongly biased; 92% occurred at, or subapical to, their irradiation sites (Fig. 3). Interestingly, while only 6% of the subapical irradiations produced

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FIG. 1. Branch-free lengths of hyphae growing for the indicated times in the same chambers used for branch induction. (A) Data for mean lengths of all types of hyphae (open squares), minimum lengths of all types (circles), maximum lengths of all types (triangles), and calculated lengths based on a growth rate of 13 µm/min (diamonds). For comparison, the mean distances to the first branches from Table 1 (crossed squares) are shown for U1 apical irradiations (shown at 30 min), U1 subapical irradiations (shown at 60 min), and V2 subapical irradiations (shown at 90 min). (B) Data for the mean branch-free lengths of each type of hypha, as described under Materials and Methods. All types (open squares, same data shown in A), type 1 (diamonds), type 2 (circles), type 3 (triangles), and calculated lengths based on a growth rate of 13 µm/min (crossed squares). Sample sizes for each data point are indicated by the numbers on the graphs. Error bars are omitted for clarity, but for all 16 sample points, the SEM was 9.3% of the mean and for all samples where n . 10, the SEM was ,8.9%.

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Grinberg and Heath

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TABLE 1 Branch Induction by Microbeams

Subapical (50 µm, 60 s) V2 (n 5 15) U1 (n 5 24) Apical (10 µm, 10 s) U1 (n 5 16)

Growth ratea (µm/min)

Single branch formation

Multiple branch formationb

Total number of multiple branches

13.8 6 1.3 12.5 6 1.5

67 63

27 25

9 15

15.9 6 1.4

44

25

8

Branches which grewe

Initiation timec (min)

Distance between first branch and tip (µm)c,d

Total

Singles

Multiples

5.2 6 1.7 (3.3–10.0) 4.4 6 2.1 (0.08–10.4)

158 6 32 (101–237) 141 6 34 (92–217)

63 67

80 80

44 53

80 6 16 (50–100)

53

86

25

3.7 6 1.6 (2.0–6.2)

Note. All values are means or percentages. n, number of hyphae irradiated. All hyphae were not loaded with Calcium Green. Calculated only for those which branched. b The difference between the single plus multiple branch values and the total irradiated is the number which did not branch. c Figures in parentheses indicate ranges. d All values at the time of branch emergence. The subapical values were calculated as indicated under Materials and Methods and the apical ones were measured directly. e Not all branches which were initiated continued to grow; these values indicate the ones which did. a

branches ahead of the irradiation site, that region was fully competent to branch. For example, apical irradiations induced 53% of their branches in the region between the apical and subapical irradiation sites and all of their first branches emerged between 50 and 100 µm from the tip, whereas only one of the subapically induced branches appeared in this zone (Table 1). However, there was no indication of any other preferred location relative to the irradiation site. Subapical irradiations with the U1 and V2 filters were equally effective at inducing branches (Table 1), but U1 produced more in the irradiated site, whereas none were found there with V2 (Fig. 3). Absence of branching at the V2 site may be attributable to damage resulting from the higher dosage (Table 5) relative to the U1 beam. However,

FIG. 2. Illustration of the results of a single apical U1 irradiation. Numbers at lower left indicate seconds after the first image. Images were printed from a videotape sequence with all camera settings held constant for the entire sequence. A growing Calcium Green-loaded hypha was observed briefly with the B1 filter (16), then irradiated for 10 s (27), observed again with the B1 filter, and found to have a 29.9% increase (based on a preirradiation corrected pixel intensity of 28.4, cf. Table 4) in fluorescence (38) and then observed to swell slightly (108) before growing out as a normal-looking tip (189). At 321 s a branch started to form at the site of the arrow. The swelling to the right of this image aligns with the rear of the swelling seen in the 189 image, thus indicating the distance of the branch site from the apex of the hypha. The branch continued to grow and turned toward the original apex (373–499). All are 3900.

previous electron microscope studies (McKerracher and Heath, 1986) have shown that higher doses of 279-nm light did not cause detectable damage to cytoplasm, and the present observations of the U1 and V2 sites did not detect any changes. The ability to make high-resolution observations and to detect possible subtle changes during irradiation was compromised by the optical system needed to produce the microbeams. Apical cytoplasm was apparently more responsive than subapical to U1 irradiation. Subapically, a 10-s irradiation (17% of the 60-s dose) was completely noninductive (n 5 11), whereas this dose in the apex was almost as inductive as the 60-s subapical dose [69% versus 88% (Table 1)]. The noninductive apical U1 irradiations (31%) gave highly vacuolate cytoplasm within minutes after irradiation. All irradiations gave transient cessation or slowing of tip growth during the irradiation, followed by recovery to normal morphology and growth rates by approximately 1 min after the end of the irradiation. The difficulties of simultaneously observing the vicinity of the irradiation site and the tip precluded detailed recording of this behavior. Irradiations of both normal (Table 1) and Calcium Green-loaded (Fig. 2) hyphae were able to induce branching, but the frequency of branching in the dye-loaded ones seemed to be reduced. However, the technical difficulties of obtaining fast-growing loaded hyphae and successful irradiations followed by sufficient observation times to ascertain subsequent branching behavior precluded obtaining sufficient hyphae for statistical analysis of this apparent phenomenon.

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Interference between Branches Sixty-three percent of branches induced by all irradiations grew after initiation, but single initiations were more likely to grow (81%) than multiple ones (44%; Table 2). Among the latter, closely spaced branches interfered with each other, such that only when they were separated by more than about 50 µm did both grow (Table 3). Similar interference was also indicated when comparing apical versus subapical U1-induced branches. Sixty-seven percent of subapical branches grew, compared with only 53% of apical ones and of the latter, 8 of 11 that formed at .80 µm from the tip continued to grow, compared with all 4 induced between 50 and 75 µm that failed to grow. While not analyzed extensively, induced branches show a lag phase of about 3 min before reaching a linear equilibrium rate, whereas those which initiate but fail to grow show similar initial kinetics but terminate during the lag phase (Figs. 5 and 6).

Microbeam-Induced Elevation of Cytoplasmic [Ca21] Calcium Green-loaded hyphae selected for analysis (there appeared to be more hyphae which either were not growing or grew at slower rates in Calcium Green-loaded colonies relative to unloaded colonies, although this was not measured) grew at rates similar to those of unloaded hyphae (compare Tables 1 and 4) and showed specific staining, the intensity of which was substantially above autofluorescence at the wavelengths and irradiation locations used (Table 4). The staining patterns were not analyzed in detail, but appeared similar to those previously reported (Hyde and Heath, 1997), with no evidence for sequestration into vesicles or vacuoles and subapical zones of exclusion comparable to nuclei (Fig. 7). We conclude that the Calcium Green was located in the cytoplasm, where it could report cytoplasmic [Ca21]. Both the U1 and V2 microbeams induced an increase in Calcium Green fluorescence, indicating an increase in cytoplasmic [Ca21] (Figs. 2 and 7; Table 4). Increases were observed in all hyphae examined, the mean percentage increases varying from 28.8 to 41.3 (Table 4) and individual

FIG. 3. Distribution of branch initiation sites with respect to the irradiation site (0) for V2 (A) and U1 (B) subapical and U1 apical (C) irradiations. Negative values on the abcissa indicate sites ahead of the irradiation site.

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FIG. 4. Scale cartoon summarizing the relationships between the minimum, mean, and maximum branch-free lengths of all unirradiated hyphae after 15 and 90 min in the chambers (data from Fig. 1) and the irradiation sites (B) and minimum, mean, and maximum branch initiation sites produced by the U1 subapical and apical irradiations (data from Table 1).

TABLE 2 Characteristics of Those Hyphae Which Responded by Producing Two or More Branches Distances from tip (µm) to

Subapical V2 beam

U1 beam

Apical U1 beam

Irradiation sitea

First branch

Second branch

Third branch

165 115 125 193 110 103 77 72 104 116

196 130 166 237 110 103 134 187 121 92

234 147 192 276 136 135 177 241 176 116

— — — 302 — — 202 — 226 134

82 63 73 63

72 78 103 53

102 98 133 88

— — — —

Note. Numbers in bold indicate those branches which continued to grow after initiation; the others did not. a Measured in individual hyphae at the time of branch emergence from the tip to the center of the irradiation site, either from direct measurements for the apical irradiations or calculated as described under Materials and Methods for the subapical irradiations.

hyphae ranging from 13.4 to 81.8%. A 35% increase in pixel intensities in this range corresponds to an approximately 25% increase in [Ca21], based on calibrations by Dearnaley et al. (1997). This relationship is only an approximation since the calibrations were performed on different cells and with another microscope and image analysis system. However, the only interpretations based on it are that the dye was responding to cytoplasmic [Ca21] and that the increase in [Ca21] was not large. The increase in indicated cytoplasmic [Ca21] was similar following the U1 beam in both the apex and subapex, even though the total dose of energy in the apex was only 17% of that given subapically. This similarity is comparable with the nearly equal effectiveness of the apical irradiations in branch induction (Table 1). It is important to note that the

TABLE 3 Interbranch Initiation Distances (µm) and Their Subsequent Growth Growth behavior

Mean Range n

Total

Both grew

Only one grew

Neither grew

39.5 6 20.7 17.9–105 22

73.2 6 22.1 54.2–105 4

34.3 6 10.9 20.0–55.1 12

27.5 6 9.6 17.9–35.0 6

Note. Data from the 14 hyphae and 32 branches shown in Table 2. In addition to the simple interactions between adjacent pairs of branches, for those hyphae with 3 branches each, the interactions between 1 and 3 were also included.

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FIG. 6. Growth of two induced branches, one of which was a single branch which grew (squares) and the other was one of a pair of which failed to grow (diamonds). Both showed similar initial kinetics with a lag phase, but the growing one attained a linear growth rate of approximately 6 µm/min. Time 0 was when they first became detectable.

increased fluorescence was measured immediately after irradiation, whereas the first signs of branch induction were not detected until an average of about 4 min later. The increase in fluorescence was not due to the B1 irradiations used to record the Calcium Green images because the dual B1 irradiations without an intervening microbeam elicited no signal increase. These irradiations also failed to elicit either changes in growth at the tips or branch initiation. However, they did cause a small (,10%) reduction in autofluorescence when used on unloaded hyphae, which was not taken into account when correcting the before and after irradiation Calcium Green values, so that the recorded increases in Calcium Green intensities are an underestimate of the true increases.

DISCUSSION We have demonstrated that localized irradiation of growing hyphae with UV light induces initiation of one or

FIG. 5. Branch formation, from the time that it was first detectable (0) until it had developed into a tip growing at a normal steady rate. This is the same branch as that shown in Fig. 6. Times in seconds are indicated on each print. All are 3666.

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TABLE 4 Irradiation-Induced Increases in Calcium Green Fluorescence Mean pixel intensities (less autofluorescence)

Subapical V2 (n 5 2)a U1 (n 5 8) Apical U1 (n 5 9) Autofluorescence U1 (n 5 10)

Growth rate (µm/min)

Before irradiation

After irradiation

Difference

% change

12.0 6 0.7 12.7 6 0.9

41.3 6 10.7 36.2 6 9.6

53.2 6 6.2 51.2 6 10

11.9 6 4.5 15.8 6 5.5

128.8 141.3

11.6 6 0.8

38 6 14

50.1 6 19.2

12.1 6 6.7

131.8

—

54.1 6 9.0

—

—

—

Note. Before and after U1 irradiation values were significantly different (P , 0.001, paired-sample t test). There was no difference between the U1-induced increases in fluorescence intensity in the tip relative to the subapical region (t test). a V irradiation of two unloaded hyphae reduced the autofluorescence intensity by 45%; thus, the before-irradiation 2 values were reduced by 54.1 and the after values by 45% of 54.1 5 24.3.

more branches in a nonrandom location relative to the site of irradiation. With current technology, we cannot rule out the possibility that irradiation induces a generalized ‘‘stress response’’ which leads immediately to a Ca21 flux and subsequently to precocious branching, which may not be a consequence of that flux. Nevertheless, facets of our observations are consistent with previous studies on the involvement of Ca21 in tip growth. They can be most simply interpreted in ways which contribute to our understanding of the factors involved in inducing and regulating tip growth. Other facets further our understanding of cytoplasmic polarity and the extent of cytoplasmic support needed to sustain a single tip.

Mechanisms of Tip Induction Elevated cytoplasmic [Ca21] is most likely directly involved in the branch induction phenomenon and as a causative agent rather than an epiphenomenon.

The most direct evidence for elevated cytoplasmic [Ca21] is the rise in Calcium Green fluorescence which follows irradiation. Even though we were only using a single wavelength indicator dye, we believe that the rise is attributable to an increase in cytoplasmic [Ca21] because we were observing the same region of cytoplasm before and after irradiation and there was no evidence for substantial cytoplasmic rearrangements during the irradiations. This rise following UV irradiation is consistent with previous reports in which UV irradiation of both Saprolegnia and Basidiobolus hyphae induced either increased [Ca21] as measured by another Ca21-sensitive dye system (Hyde and Heath, 1997) or cytoplasmic contractions and inhibition of saltatory movements which could be related to Ca21 fluxes in the cytoplasm (Jackson and Heath, 1992; McKerracher and Heath, 1986). It is also consistent with the present observed effects on tip growth, a process previously shown to be sensitive to fluctuations in [Ca21]

TABLE 5 Relationships between Characteristics of UV Irradiations and Cytoplasmic Effects Species (reference)

Wavelength (nm)

Beam intensity (erg/s/µm2)

Total dose (erg/µm2)

Effect

S. ferax (1) S. ferax (1) S. ferax (2) S. ferax (3) S. ferax (3) S. ferax (3) B. magnus (4) B. magnus (4)

385–450 330–380 330–380 270 270 290 279 279

2.43 0.068 ? 0.22 0.14 0.33 ,0.06 ,0.1

145.8 0.68 & 4.08 ? 18.3 13.5 33.3 5 15

[Ca21] elevation, branch induction [Ca 21] elevation, branch induction [Ca21 ] elevation, cessation of growth Ca21-related contractions No contractions No contractions Ca21-related inhibition of saltations; no contractions Ca21-related inhibition of saltations and cytoplasmic contractions

Note. References: (1) present work; (2) Hyde and Heath, 1997; (3) Jackson and Heath, 1992; (4) McKerracher and Heath, 1986.

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(reviewed in Jackson and Heath, 1990a; Garrill et al., 1993; Hyde and Heath, 1997). Because the cytoplasmic [Ca21] increases are localized and precede and predict the branch induction process, it is most likely that they are a cause, rather than a consequence, of branch initiation. Certainly the timing of their formation makes it very unlikely that they have a secondary role derived from the consequence of branch induction. The mechanisms by which the increased [Ca21] could function in branch induction are unclear. Diffusion to a threshold concentration from a localized release site seems unlikely because such a process should not give the highly assymetric clustering of branches proximal to the irradiation sites. This asymmetry cannot be due to induction incompetence distal to the irradiation site, at least for the subapical irradiations, since the same distal regions were branching-competent in response to the apical irradiations. Within the hyphae, there is a gradient of organelle distributions for all organelles, but none of these patterns detectably relate to the branching asymmetries (Heath and Kaminskyj, 1989). However, hyphal cytoplasm does possess intrinsic polarity, as evidenced by tipward contractions induced by diverse agents (McKerracher and Heath, 1986; Jackson and Heath, 1992; Kaminskyj et al., 1992). The basis for this polarity is unknown, but the contractions induced by microbeam irradiation are remarkable in only occurring toward the irradiation site from the proximal cytoplasm; distal cytoplasm does not move back to the site

FIG. 7. Four examples of increases in Calcium Green fluorescence following apical U1 irradiations. In each sequence the hypha was observed to be growing with normal morphology prior to irradiation (0 s), its fluorescence was observed with the B1 filter prior to irradiation (7–13), it was irradiated in the indicated positions for 10 s (12–29), reimaged with the B1 filter (27–38), and then growth was followed (33–137). All times are indicated, in seconds, at the tops of the prints. The increase in fluorescence intensity after the irradiation was variable, being 44.8, 22.6, and 41.7% based on preirradiation-corrected pixel intensities of 13.4, 43.9, and 59 for series 1, 3, and 4 respectively (cf. Table 4). Data for series 2 were not recorded. Furthermore, the dye distributions were also typically nonhomogeneous, with zones of reduced intensity as might be expected for a dye distributed in cytoplasm with a heterogeneous population of organelles. The effects of the irradiations were also variable, with little shape change and rapid growth recovery (top series), slower growth recovery (series 2 and 4), and shape change with rapid growth recovery (series 3). All images in each series are accurately aligned at their bases, and the small crosses over the center of the subapical cytoplasm also indicate a constant position on each series. Note the absence of any indications of postirradiation cytoplasmic change in the irradiated regions. All are 3740

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and proximal cytoplasm does not ‘‘overshoot’’ the site (McKerracher and Heath, 1986; Jackson and Heath, 1992). Although the low doses used to induce branches did not induce any obvious contractions (presumably because they did not reach a threshold needed for contractions), activation of a part of a contractile system could have provided the means for transport of branch-initiation ‘‘factors’’ to accumulate from low and diffuse proximal distributions to critical concentrations predominantly only proximal to the irradiations. The nature of these ‘‘factors’’ is unknown, but plasma membrane Ca21-transmitting channels (Garrill et al., 1993), organelles involved in Ca21 homeostasis (Jackson and Heath, 1993; Allaway et al., 1997), or wall vesicles involved in tip growth (Heath et al., 1985) are attractive candidates. Induced translocation of only some subset of cytoplasmic constituents may well be undetectable relative to the more massive translocations induced by the higher intensity/dose UV microbeams (Table 5) employed previously. A second feature of the hypothesized relationship between translocation-induced accumulation of ‘‘factors’’ and branch formation is that it provides an explanation for the still stochastic pattern of branching. Evidently there are no predetermined locations for branch formation, but the random concentration of ‘‘factors’’ within the region influenced by the elevated [Ca21] would explain the observed patterns of branch induction. The target for the UV irradiations is unlikely to be a single molecule because responses which are all relatable to a similar primary mechanism can be induced by a rather wide range of wavelengths (270–450 nm, Table 5). However, it does appear that the shorter wavelengths are more effective in that smaller doses elicit similar effects and massively larger doses of the longer wavelengths are no more effective, suggesting that the targets absorb preferentially at shorter wavelengths. However, given the relatively wide transmission characteristics of the filters used in the present work and the lack of discrimination of absorption spectra of myriad biological molecules, the present techniques are unlikely to yield a precise description of the targets. However, elements of the intracellular Ca21sequestering systems (Jackson and Heath, 1993; Allaway et al., 1997) seem likely candidates. The much greater effect, in terms of response per dose, of the apical U1 irradiations may relate to the higher concentration of mitochondria in the ,10-µm subapical region (Heath and Kaminskyj, 1989). Mitochondria have been identified as Ca21 stores in these hyphae (Yuan and Heath, 1991). However, the concentration of endoplasmic

reticulum has not been described with respect to distribution along hyphae and the recently described tubular reticulum (Allaway et al., 1997) may also be enriched in the ,10-µm subapical regions. A similarly localized peak of [Ca21] occurs in hyphae growing without exogenous Ca21 and UV irradiation of entire hyphae also elevated [Ca21] more in this region than elsewhere (Hyde and Heath, 1997). Previously observed effects of UV microbeams (McKerracher and Heath, 1986; Jackson and Heath, 1992) and ionophores (Harold and Harold, 1986) have also been said to most likely involve release of Ca21 from internal stores. Irrespective of the source of released Ca21, it is interesting to compare the present release of relatively small quantities of Ca21 from a localized source, which was accompanied by branching, with the apparently larger release resulting from irradiation of much larger regions of hyphae which failed to induce branching (Hyde and Heath, 1997). Consistent with the hypothesized larger release resulting from the larger target was the cessation of tip growth for much longer (,20 min), indicating a much reduced capability for restoring Ca21 homeostasis. With respect to the mode of action of the Ca21, it is interesting to note that, based on previous observations of the rate of spreading of inhibition of saltatory movements induced by a UV microbeam (,50 µm/min; McKerracher and Heath, 1986), one might predict that Ca21 released from the irradiation site would have reached the furthest site of branch initiation (170 µm) within approximately 3 min, before branches typically emerge. This suggests that the Ca21 does indeed trigger the establishment of the tip growth apparatus which only later leads to the formation of visible signs of tip emergence.

Cytoplasmic ‘‘Support’’ of Hyphal Tips As noted previously, Trinci and colleagues (Trinci et al., 1994) demonstrated a relationship between hyphal length and branch formation, the hyphal growth unit, which indicates that hyphal organisms have some specific length of hypha which is needed to sustain a tip. The present observations both support and refine this concept. The ability of the irradiations to induce branches much closer to tips which continue normal growth rates shows that there is no absolute requirement for the normal length of hypha which relates to each tip. This is consistent with previous observations concerning paramorphogens (Trinci et al., 1994) and the general phenomenon of hyperbranching in response to many treatments. However, at a finer level, there is competition between branches. This is seen

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in the facts that (1) when multiple branches were induced, only if they were further than about 50 µm apart would both grow; closer than this, either only one or neither grew (Table 3) and (2) branches induced close to an existing tip (by apical irradiations) were overall less likely to grow, especially if more than one was induced (Table 1). Competition between branches, presumably for cytoplasmic ‘‘support’’ is intrinsically likely. Limited supplies of energy from mitochondria and materials, such as wall vesicles, from the endomembrane system are obvious components needed to ‘‘support’’ a growing tip. However, the competition my be more mechanical than this. A growing tip in oomycete hyphae utilizes an apical ‘‘cap’’ of F-actin which extends subapically along the plasma membrane and one of its roles appears to be moving wall vesicles to the tip (Heath and Kaminskyj, 1989). The length of this ‘‘cap’’ is proportional to the rate of growth (Jackson and Heath, 1990a), suggesting that faster growing hyphae ‘‘harvest’’ vesicles from a larger volume of subapical cytoplasm. At growth rates of ,13 µm/min (Table 1), ‘‘caps’’ are ,30 µm long (this is a minimum value, determined for purposes of measurement; the effective length is undoubtedly longer; Jackson and Heath, 1990a; Heath and Kaminskyj, 1989). If this length of hypha is needed to ‘‘support’’ a growing tip, a comparable length might be needed to ‘‘support’’ a branch, and in this case it could be predicted to be composed of ,15 µm from either side of the base of the ,10-µm-diameter (20 µm from the center) branch. Such a concept indicates that at a growing tip, a branch would not be able to grow if its center were within ,30 µm (for the main tip) 1 ,20 µm (for the distal region ‘‘feeding’’ the branch) 5 50 µm from the tip. Apical irradiations induced branches at 50–100 µm from the tips (Table 1), but only those .80 µm grew, in reasonably close agreement with the above hypothesis. Similarly, for induced branches closer than 40 µm center to center, both would not be predicted to grow, in remarkably close agreement with the interference shown in Table 3. The formation of radial arrays of F-actin around the bases of branches has been shown previously for developing branches (Heath, 1987) and more recently for initiating branches (Bachewich and Heath, 1997). Overall, our results show that UV microirradiations are able to produce increases in cytoplasmic [Ca21] which are followed by precocious branch induction. Exploration of these, and previous UV microirradiation, results suggests that small Ca21 fluxes are capable of inducing the assembly of the tip growth apparatus and are thus also likely to play a

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Grinberg and Heath

role in the regulation of this apparatus during equilibrium hyphal growth.

ACKNOWLEDGMENTS This work was supported by the Natural Sciences and Engineering Research Council of Canada. The assistance, advice, and constructive comments of Cathy Bachewich, Gagan Gupta, and Geoff Hyde during phases of the work are gratefully acknowledged.

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