Cytoplasmic migrations and vacuolation are associated with growth recovery in hyphae of Saprolegnia, and are dependent on the cytoskeleton

Mycol. Res. 103 (7) : 849–858 (1999)

849

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Cytoplasmic migrations and vacuolation are associated with growth recovery in hyphae of Saprolegnia, and are dependent on the cytoskeleton

C A T H E R I N E B A C H E W I C H A N D I. B R E N T H E A T H Department of Biology, York University, 4700 Keele Street, Toronto, Ontario, M3J 1P3, Canada

Hyphae of Saprolegnia ferax recovered from intracellular acidification with variable degrees of apical vacuolation and migration of cytoplasm out of the apex, prior to apical refilling and resumption of growth. The response was not due to alkalinization, but was generalized to recovery from diverse growth inhibitors including procaine, latrunculin B and the kinase inhibitor 6dimethylaminopurine. The vacuoles apparently arise from expansion and fusion of apical tubular vacuoles. This process is mediated by microtubules, since their disruption suppresses apical vacuolation. The shape of the vacuoles appears to be F-actin imposed, because their expansion is enhanced by F-actin disruption. The accompanying, often bidirectional, cytoplasmic migrations are independent of tip growth, require F-actin, and probably represent part of a hypothetical generalized cytoplasmic avoidance response.

Cytoplasmic migration and vacuolation are vital aspects of hyphal tip growth, the polarized mode of expansion utilized by filamentous fungi, as well as other cell types (Heath, 1994). As such, the cytoplasm and vacuoles demonstrate polarity in mobility and organization. As the hypha extends, bulk cytoplasm migrates forward, maintaining its position relative to the expanding tip. In many hyphae, this process produces an increasingly vacuolate subapical region, containing a thin layer of cortical cytoplasm. The vacuole population consists of a dynamic system of large spherical vesicles and tubules (Shepherd, Orlovich & Ashford, 1993 a). In oomycetes, the vacuoles change from the large, subapical bodies in the subapex to a tubular reticulum that pervades the apical cytoplasm (Allaway et al., 1997). Thus, to some extent the distribution and migration of vacuoles and cytoplasm are reciprocal, and their behaviour must be coordinately generated and regulated. Despite their importance to tip growth, the organization and migration of cytoplasm and vacuoles are not fully understood. Cytoplasmic migration appears to be an active process, not due to a ‘ push ’ from subapical expanding vacuoles, since there is no clear division between the vacuoles and bulk cytoplasm (Heath & Heath, 1979 ; McKerracher & Heath, 1987 ; Rees, Shepherd & Ashford, 1994 ; Kaminskyj & Heath, 1996 ; Allaway et al., 1997), no pressure gradient (Money, 1990), and the rate of vacuolar motility in filamentous fungi is independent of the rate and direction of cytoplasmic streaming (Shepherd et al., 1993 a). It has been suggested that cytoplasmic migration in hyphae is somewhat analogous to amoeboid motion (Reinhardt, 1892 ; Issac, 1964 ; McKerracher & Heath, 1987 ; Jackson & Heath, 1993 a ; Heath, 1994 ; Kaminskyj & Heath, 1996), since Ca#+-dependent cytoplasmic

contractions towards the hyphal tip can be induced in Saprolegnia (Kaminskyj, Jackson & Heath, 1992 ; Jackson & Heath, 1993 a) and Basidiobolus (McKerracher & Heath, 1986), hyphal cytoplasm contains F-actin (Jackson & Heath, 1993 b ; Heath, 1987) and myosin homologues (Kaminskyj & Heath, 1995 ; McGoldrick, Gruver & May, 1995), and the cytoplasm appears to be organized into a static, cortical ectoplasmic layer, over which a more fluid central endoplasmic layer migrates (Kaminskyj et al., 1992 ; Kaminskyj & Heath, 1996 ; Bachewich & Heath, 1997 a). Abundant adhesions between the cortical F-actin population and the cell wall along the whole length of hyphae were recently demonstrated, providing a basis against which central cytoplasm could exert force for movement (Bachewich & Heath, 1997 a). More abundant adhesions in the apex (Kaminskyj & Heath, 1995 ; Bachewich & Heath, 1997 a) could also ensure tip-ward cytoplasmic migration, especially if they interact with the central actin population (Bachewich & Heath, 1997 a). Thus, cytoplasmic migration in hyphae appears to be an active process, likely involving contractions similar to amoeboid cells. Consistent with this are the recent demonstrations of noninduced cytoplasmic contractions that precede branching in Aspergillus (Reynaga-Pena & Bartnicki-Garcia, 1996), and pulsed growth in fungal hyphae (Lopez-Franco, BartnickiGarcia & Bracker, 1994) and other tip-growing cells (Pierson et al., 1996). Cytoplasmic migration appears to be a separate process, however, that must be coordinated with hyphal extension. Indeed, the Ca#+-dependent induced contractions occur after growth has stopped (Kaminskyj et al., 1992 ; Jackson & Heath, 1993 a), but these are abnormal examples of ‘ migrating ’ cytoplasm. Since actin is an underlying regulator of many aspects of tip growth it is, therefore, difficult to

Cytoplasmic migrations and vacuolation in hyphae analyse actin’s role in cytoplasmic migration in isolation. For example, anti-actin drugs and treatments which stop cytoplasmic migration inhibit hyphal extension as a whole (Jackson & Heath, 1990, 1993 a ; Gupta & Heath, 1997). To date, an analysis of the migratory properties and regulation of hyphal cytoplasm alone has not been performed. The fungal vacuole population is important for macromolecular degradation, metabolic storage, and cytosolic ion homeostasis (Klionsky, Herman & Emr, 1990). In filamentous fungi, the vacuole system is composed of tubules and large vesicles, which frequently change shape (Shepherd et al., 1993 a ; Rees et al., 1994). In oomycetes, the network demonstrates a true polarity in organization, with tubule-like vacuoles pervading the apical cytoplasm, and large, more spherical vacuoles occupying subapical regions (Rees et al., 1994 ; Allaway et al., 1997). Due to their motility and potential peristaltic transfer of components along the network, the vacuolar system has been proposed to be important for long distance transport of molecules in hyphae (Shepherd et al., 1993 b ; Hyde & Ashford, 1997). Microtubules and microfilaments are associated with the vacuoles in filamentous fungi, and microtubules were suggested to be important for the motility and integrity of the network (Shepherd et al., 1993 a ; Allaway et al., 1997). Since vacuolar movements are independent of the rate and direction of cytoplasmic streaming (Shepherd et al., 1993 a), different regulatory systems must be responsible for these two motile processes. In a recent report, we described a phenomenon of abnormal apical vacuolation and bulk cytoplasmic migration in nonextending hyphae, during recovery from lowered intracellular pH (Bachewich & Heath, 1997 b). We have investigated this recovery response further in order to determine its relevance to the growth recovery process in general, and to elucidate the mechanisms underlying vacuole structure and cytoplasmic migration, two important aspects of hyphal growth. MATERIALS AD METHODS Hyphae of Saprolegnia ferax (Gruith.) Thur. (ATCC No. 36051) were grown overnight on dialysis tubing overlying an organic medium, designated as OM (Heath & Greenwood, 1970), supplemented with agar. Colonies were cut into 1i1 cm sections, and allowed to recover for 1 h, prior to mounting in flow-through slide chambers (Heath, 1988), as previously describe (Bachewich & Heath, 1997 b). Hyphae were exposed to growth-inhibiting concentrations of weak acids which lower intracellular pH, including 1n1 m acetic, propionic, and butyric acids in OM at pH 4n6 (Bachewich & Heath, 1997 b), for 10 min. Up to 500 µl of solution was washed through the slide chamber during this time period. Hyphae were then induced to recover by continuously flowing fresh OM, pH 5n3, through the chamber, until hyphal growth resumed. Hyphal responses during recovery, including the degree of apical vacuolation and cytoplasmic migration, were recorded. To determine whether the recovery responses following a decrease in intracellular pH were due to intracellular alkalinization, cytoplasmic pH was increased using two structurally different weak bases, procaine (pKa 9) (ICN

850 Biomedicals Inc., Aurora, OH) (Kropf, Henry & Gibbon, 1995) and NH Cl (pKa 9n3). The bases were dissolved in OM % buffered with 50 m HEPES (Sigma, St Louis, MO), pH 7n8–8). A high external medium pH was required to produce a large percentage of neutral membrane-permeable ammonium group, due to the high pKa values of the bases. Upon entering a lower pH environment in the cell, the bases would bind free H+, thereby alkalinizing the cytoplasm (Guern et al., 1991). Hyphae initially stopped growing in response to high pH medium and, therefore, needed to adapt for at least 10–20 min in the medium prior to mounting in the slide chambers. Various concentrations were utilized to determine the optimum growth-inhibiting concentration. 4 m procaine and 40 m NH Cl inhibited growth completely, while growth % was able to resume at lower rates with half these concentrations. A higher concentration of NH Cl was required % in part because of its higher pKa value. To determine if the growth recovery response following weak acid treatment was a general phenomenon, recovery from other growth-inhibiting agents was observed. Hyphae were exposed to (i) 4 m procaine, pH 7n8 for 10 min, then washed with HEPES buffered OM, pH 7n8, until growth resumed, (ii) 0n5 µg ml−" of the actin inhibitor latrunculin B (Calbiochem, Markham, ONT.) (Gupta & Heath, 1997 ; Bachewich & Heath, 1998) for 10 min, followed by continuous washing with OM, pH 5n3, and (iii) the general kinase inhibitor 6-dimethylaminopurine (DMAP) (Sigma). 1 m in OM, pH 5n3, was determined to inhibit growth completely, while growth resumed in 0n5 m. After 10 min in 1 m, hyphae were washed with OM pH 5n3, until growth resumed. Hyphal responses during growth inhibition and recovery were recorded for all treatments. To determine the importance of actin in the recovery response, hyphae pretreated with 1 m acetic acid for 10 min were washed with OM containing 0n5 µg ml−" latrunculin B for 30–50 min. The importance of microtubules was determined by incubating hyphae for 2 h in 2 µg ml−" nocodazole, followed by exposure to 1n1 m acetic acid for 10 min, and subsequent washing with OM, in the continuous presence of nocodazole. The long preincubation in nocodazole was required because the effects of nocodazole take at least 1 h to be manifested (Heath, 1982). The importance of microtubules in apical vacuole formation was investigated by preincubating hyphae in 2 µg ml−" nocodazole for 2 h, followed by coincubation in nocodazole and 0n5 µg ml−" latrunculin B, which induces apical vacuolation on its own (Gupta & Heath, 1997), for 20–60 min. To visualize the normal apical vacuole network, vacuoles were stained with 6-carboxy fluorescein diacetate (6-CFDA ; Sigma) using the method of Shepherd et al. (1993 a). Hyphal colonies were incubated in 1 ml of 25 µg ml−" 6-CFDA in OM, pH 5n3, in the dark, for 10 min, after which hyphae were allowed to recover in fresh OM alone for at least 30 min, prior to mounting in the slide chambers. The effects of various growth inhibitors on vacuole structure were analysed by exposing 6-CFDA-loaded hyphae to 4 m procaine, 1 m acetic acid, or 0n5 µg ml−" latrunculin B for 10 min. To determine the effects of microtubule disruption on the vacuole network, hyphae were pretreated for 2 h in

Catherine Bachewich and I. Brent Heath

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2 µg ml−" nocodazole, followed by loading with 25 µg ml−" 6-CFDA and recovery in OM plus nocodazole, as described. Hyphae preincubated in 1 m DMAP for 10 min were fixed and stained with RP to visualize actin, as previously described (Bachewich & Heath, 1997 b). Hyphae were examined with Nomarski differential interference contrast optics, using a i100, 1n32 NA objective. Images were either photographed, or recorded on videotape, using a Hamamatsu C2400 SIT camera and a Hamamatsu DVS3000 image enhancement system. Epifluorescence was used for analysing 6-CFDA-stained vacuoles and RP-stained actin, employing B1 and G2 filter sets, respectively.

RESULTS As previously described, recovery of growth in hyphae pretreated with weak acids is characterized by increased apical vacuolation, followed by cytoplasmic re-occupation of the apex, and subsequent growth recovery (Bachewich & Heath, 1997 b). We have further analysed the events related to these ‘ recovery ’ conditions. These events are described quantitatively in Table 1, and descriptively below.

Apical vacuolation and cytoplasmic migration occur during recovery from intracellular acidification Lowering intracellular pH with weak acids altered cytoplasmic organization, but did not induce any detectable apical vacuolation (see Bachewich & Heath, 1997 b for details). Within a few minutes of washing hyphae pre-treated for 10 min with 1 m acetic, propionic or butyric acids, pH 4n6, the cytoplasm appeared more homogeneous as previously-contrasted

A

C c c v

B

D v c

v

Fig. 1. Diagrammatic summary of the maximum degrees of visible vacuolation associated with growth recovery from weak acids. (A) The apical cytoplasm (c) of hyphae prior to washing did not contain any vacuoles detected by DIC optics (Table 1, col. 1, lines 1, 5, 6). (B) Small, elongate vacuoles (v) appeared throughout the cytoplasm upon initial washing (Table 1, col. 4, lines 1, 2, 6). (C) Continued washing created larger tubular and spherical vacuoles in the cytoplasm and at the apex in some hyphae (Table 1, col. 5, lines 1, 2, 3, 4, 6), while vacuoles enlarged from this stage to occupy the majority of the apex, with some cortical cytoplasm remaining (D), in other hyphae (Table 1, col. 6, lines 1, 2, 3, 4, 6). In extreme cases, the cytoplasmic layer was no longer detectable. Although the vacuole patterns were grouped into these categories, variations were present.

organelles lost visibility. Increased vacuolation was then initiated either posterior to, or throughout the apical mass of cytoplasm, with vacuole shapes ranging from elongate and tubular to spherical (Table 1, line 1 ; Figs 1, 2). Continued, increasing vacuolation cleaved through the cytoplasm randomly, often reaching into or simultaneously forming at the extreme apex. The vacuoles were dynamic, changing in shape and positioning, and they increased in size to variable degrees. The initial vacuoles often transformed from tubular

Table 1. Vacuolation and cytoplasmic migration associated with growth recovery (%) Maximum degree of vacuolation Presence of vacuolation Linear After# tubules (Fig. Before" Throughout Posterior 1 B) (1) AcOH, OM recovery 7 (n l 27) (2) Procaine, OM recovery 25 (n l 8) (3) LAT, OM recovery 100 (n l 7) (4) DMAP, OM recovery 67 (n l 6) (5) AcOH, Nocodazole 0 recovery (n l 9) (6) AcOH, LAT recovery 0 (n l 9)

Tubules and apical (Fig. 1 C)

Cytoplasmic migration Large central During Regrowth (Fig. initial Complete (minp 1 D) Absent Bulk$ vacuolation Bidirectional refill ...)

59

41

7

22

70

11

100

74

48

71

21p2

88

12

25

50

25

12

86

75

38

100

10p2

86

14

0

43

57

14

67

71

0

14

15p2

83

17

0

67

33

17

80

33

0

0

12p4

0

0

0

0

0

100

0

0

0

n.d.%

17p2

55

44

11

67

33

33

0

56

11

0

0

" Represents the presence of vacuoles during the initial treatment, before or after the wash was initiated. # Vacuoles initially appeared either throughout or in the posterior of the apical cytoplasm. $ Total hyphae (%) that showed bulk and local cytoplasmic migrations during vacuolation and during the ‘ refilling ’ stage. Excludes hyphae that showed

only transient or very localized movements. % Hyphae did not vacuolate or empty of cytoplasm, therefore ‘ refilling ’ was not observed.

Cytoplasmic migrations and vacuolation in hyphae

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Fig. 3. Transition in vacuole shape during growth recovery from 1n1 m acetic acid. (A) After approximately 7 min of washing away acetic acid, little vacuolation was observed in the cytoplasm, but after 10 min (B), linear vacuoles appeared throughout the cytoplasm (arrow in B), as well as more spherical vacuoles in the extreme apex. (C–F) As the vacuoles increased in size, they became less linear, and occupied the central region of the hypha, while a small cortical layer of cytoplasm remained. Bar l 10 µm.

Fig. 2. Apical vacuolations and cytoplasmic migrations associated with recovery from lowered intracellular pH. (A) A hypha previously treated with 1n1 m butyric acid (pH 4n6) for 10 min, at the beginning of a wash with growth medium alone. (B–F) Spherical and more linear vacuoles (arrow in F) formed throughout the cytoplasm during the wash, changing their shape over time. (G–M) By approx. 10 min, the apical cytoplasm (arrows) started to migrate backwards, as larger vacuoles appeared at the extreme apex. (M) This hypha vacuolated to an extreme extent, such that little visible cytoplasm remained in the tip. (N–R) After approx. 20 min, cytoplasm migrated back into the hypha, dissipating the vacuoles (The rear of the hypha moved into a different focal plane during this process, creating the out of focus blur.). (S, T) The hypha then resumed growth by producing a branch directly behind the main tip, straight up towards the objective (arrow), making it difficult to detect the actual time of growth resumption. Bar l 10 µm.

to spherical units (Fig. 3). The maximum degree of vacuolation in a given hypha ranged from small linear elements, or tubules, to larger tubules and more spherical apical vacuoles, to a large central vacuole, occupying most or all of the apical tube (Figs 1, 2 M, 3 F, 6 H). Only a thin peripheral layer of cytoplasm, which in extreme cases was no longer visible, remained in the hyphal apex with the highest degree of vacuolation. The vacuoles in the apical cytoplasm apparently originated from the pre-existing reticulate tubular vacuoles, which were previously visualized by loading with the dye 6-CFDA (Figs 4 A–D) (Shepherd et al., 1993 a ; Rees et al., 1994). Tubular vacuoles loaded with the dye could not be continuously

observed throughout the recovery process because of sensitivity to the excitation light (Rees et al., 1994 ; Allaway et al., 1997). Different hyphae with dye-loaded vacuoles were, therefore, observed at different stages of recovery. The dye was initially contained in tubular, albeit thicker vacuoles during acetic acid treatment, although many hyphae contained dye in the cytoplasm, making it difficult to visualize any pattern of vacuoles (Figs 4 E–H). The dye was then found in the enlarging apical vacuoles during the wash (Figs 4 I, J). This trend was supported by one hypha which unaccountably withstood two brief exposures to excitation light and showed tubules during acetic acid treatment replaced by dye-loaded large apical vacuoles during recovery. It is likely, therefore, that the apical vacuoles formed during recovery originate from the apical tubule network. This conclusion is supported by the observation that when dye-loaded, growing hyphae were exposed to excitation light for 1–2 min, the tubules retracted back from the tip, thickened and fused, forming 1–2 large vacuoles in the apex (Fig. 5). This demonstrates the inherent ability of the tubular vacuoles to fuse and enlarge. Cytoplasmic migrations, observed as coordinated movements of cytoplasm and organelles, typically, but not always, accompanied vacuole formation. Cytoplasmic migration ranged from large bulk movements wherein large masses moved rapidly over long distances (Figs 2 F–M, 3 E, F, 6) to more local movements where smaller portions of cytoplasm moved much more slowly over shorter distances. The migrations sometimes (2\24 hyphae) included rapid contraction forward into a condensed mass in the tip, after which the cytoplasm moved back away from the tip. Migrations were either unidirectional or bidirectional, the latter for a particular mass of cytoplasm or in adjacent masses (Fig. 6). Net movement of bulk migrations occurred in a subapical direction. In hyphae without cytoplasmic migrations during initial vacuole formation, migrations were often observed at later stages in the recovery process. After reaching maximum vacuolation, cytoplasm refilled the hyphae to various degrees prior to resumption of growth (Figs 2 N–R). Migration of cytoplasm was not always visible,

Catherine Bachewich and I. Brent Heath

Figs 4–5. Fig. 4. 6-CFDA-loaded vacuoles in apical regions of growing hyphae (A–D), acetic acid-treated hyphae (E–H) and hyphae washed of acetic acid (I, J). Growing hyphae contain a fine reticulate network of vacuoles in the apical region (B), while the subapical region contains larger vacuoles (C D). Note the reticulum in the branch emerging from a subapical region (D). Hyphae treated with acetic acid for 10 min often contained dye in the cytoplasm, resulting in a homogeneous appearance (F). A tubule-like pattern of vacuoles, albeit thicker than normal, was observed in other hyphae (H). Upon washing, the dye was found in the large vacuoles that formed (J). Bar l 10 µm. Fig. 5. Transition in apical network, stained with 6CFDA, in response to excitation light. (A, B) Apical vacuoles in growing hyphae are reticulate, but with exposure to excitation light over a period of a few minutes, the tubules retract from the tip (C) and appear to fuse (D, E, F), forming large central vacuoles. Bar l 10 µm.

853 as the vacuoles simply seemed to disappear, especially when initial vacuolation was less extensive, but cytoplasm moved forward en masse from the subapex in many hyphae that showed extensive vacuolation, as previously described (Bachewich & Heath, 1997 b). Cortical cytoplasm moved predominantly forward first. As more cytoplasm filled the tip, the central cytoplasm at the apex moved back, as if the incoming peripheral layers were pushing it back, reminiscent of fountain streaming. Vacuoles could transiently reappear at this stage, but most hyphae completely refilled with cytoplasm prior to resuming growth after about 20 min. Since many cells become alkaline upon recovery from acidification (Guern et al., 1991), we tested the possibility that intracellular alkalinization triggered the vacuolation response. The weak bases procaine and NH Cl were used at 4 m (n l % 10) and 40 m (n l 6) and pH 7n8 respectively, since these concentrations inhibited growth completely after a few minutes of application, while 2 m (n l 16) and 20 m (n l 6) inhibited growth only transiently. Neither base induced the same vacuolation response as seen during acid recovery. A few vacuoles were observed in a few hyphae, but they were small and spherical and did not resemble either the pattern or degree of vacuolation observed during acid recovery. This result was also supported by the observation that 6-CFDAloaded tubular vacuoles retained their shapes, although they appeared somewhat thicker, following 10 min exposures to 4 m procaine (n l 8 slides) (Figs 7 A–D). The shape and size of subapical vacuoles was, however, affected by alkalinization, since many became smaller, spherical, and more separated (Figs 7 E–G). This was only observed with the higher concentrations of bases that inhibited growth completely. Alkalinization induced no obvious changes in cytoplasmic migration. Therefore, although cytoplasmic alkalinization can affect sub-apical vacuole shape, it is not likely to be the main factor that triggers either the vacuole or cytoplasmic migration responses associated with growth recovery from acidification. Apical vacuolation is a general response associated with growth recovery

Fig. 6. Bidirectional cytoplasmic movements in a hyphae recovering from 1n1 m acetic acid. (A–H) After large central vacuoles formed by 9 min of washing, cortical clumps of cytoplasm moved in opposite directions. One clump (arrowhead) moved forward (A–C), then reversed direction and moved backward (D–H). A different cytoplasmic clump (arrow) simultaneously moved backward (A–D) while the first clump moved forward. Bar l 10 µm.

To determine whether apical vacuolation was a general feature associated with growth recovery, the process was observed after inhibition by diverse agents. Hyphae recovering from 10 min in 4 m procaine vacuolated similarly to those recovering from acid treatments (Table 1, line 2). The range of vacuoles was similar, although there was a decrease in the proportion of large, central vacuoles. Cytoplasmic migrations and hyphal tip refilling were also within a similar range, but growth resumed in a shorter period of time. Analysis of recovery from latrunculin B, an actin inhibitor (Gupta & Heath, 1997 ; Bachewich & Heath, 1998) was complicated by the formation of vacuoles during initial treatment (Table 1, line 3). During exposure to 0n5 µg ml−" latrunculin B for 10 min, large vacuoles formed in most swollen apices (n l 7) (as previously described [Gupta & Heath, 1997]) and some formed further back in some hyphae (Fig. 8 A). This was also demonstrated by loading the apical tubule network with 6-CFDA, which increasingly thickened

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10

Fig. 7. Apical (A–D) and subapical (E–G) vacuoles in response to 4 m procaine. After 10 min, apical vacuoles loaded with 6-CFDA remained tubular, albeit thicker than normal (cf Fig. 4 B). Conversely, the subapical vacuoles become smaller, more round and separated (cf Fig. 4 D). (E, F) The same region of a hypha 2 min (E) and 4 min (F) after addition of procaine. (G) Two different hyphae 6 min after addition off procaine. Bar l 10 µm.

with time in 0n5 µg ml−" latrunculin B (n l 3 slides, different hyphae observed with brief light exposures at various times over 10 min) and the large spherical apical vacuoles seen at the termination of growth with DIC optics were dye-loaded (Fig. 9). In non-vacuolated parts of the apical cytoplasm, however, additional vacuolation, comparable to that seen following acid treatment, occurred during recovery (Figs 8 B, C). Tubular vacuoles pervaded the cytoplasm, and the large apical vacuoles enlarged. The maximum degree of vacuolation was similar to recovery from weak acids, but there were no hyphae with only tubular vacuoles. Cytoplasmic movements were similar to those observed during recovery from acids, but bidirectional movements were absent and the hyphae did not refill with cytoplasm to the same extent prior to regrowth. Regrowth was not delayed but new tips formed from the sides of the still-vacuolate swollen apices (Figs

Figs 8–11. Fig. 8. Vacuolation associated with recovery from latrunculin B. (A) A hypha treated with 0n5 µg ml−" for 10 min contains an apical vacuole (arrow) and some vacuolation (arrowhead) throughout the cytoplasm. (B–D) Upon washing out latrunculin, vacuolation increased, and growth resumed from the swollen tip. Note that growth initially resumed at the tip containing very little cytoplasm (C). More cytoplasm flowed into the tip as it grew (D). Bar l 10 µm. Fig. 9. Latrunculin-induced actin disruption alters the apical vacuole pattern. (A–D) paired DIC and epifluorescence images of hyphae preloaded with 6-CFDA and exposed to 0n5 µg ml−" latrunculin B for 7 (A, B) and 12 (C, D) min, respectively. Note the loss of reticulate vacuoles, and formation of large spherical ones in the extreme apex (cf with Fig. 8 A). Bar l 10 µm. Fig. 10. 6-DMAP disrupts normal subapical actin and cytoplasmic organization in hyphae. (A, B) Normal hyphae fixed and stained with RP contain a high density of actin cables and plaques in the cortex (A), and little actin in central regions (B, a separate hypha). Incubating hyphae in 1 m 6 DMAP for 10 min (C–E) led to a decrease in, and disorganization of, actin in the cortex (C), and increase in the amount of central actin (D, same hypha). Note the disorganization of cytoplasm in the corresponding DIC image (E) of the central region of the hypha (cf. Fig. 4 C). Bar l 10 µm. Fig. 11. Actin disruption with latrunculin in the absence of normal microtubules does not produce apical vacuolation. (A) A growing hypha preincubated for 3 h in 2 µg ml−" nocodazole prior to the addition of latrunculin B. (B) The same hypha incubated in nocodazole and 0n5 µg ml−" latrunculin B for 20 min. Note the absence of vacuolation, compared to when hyphae are incubated in latrunculin alone (cf Figs 8 A, 9). (C, D) Different hyphae after 45 min in latrunculin B, plus nocodazole. Major apical vacuolation is still not visible. Bar l 10 µm.

8 C, D). Even though vacuoles were already present within latrunculin B-treated hyphae, therefore, subsequent washing induced additional vacuolation similar to that seen during acid recovery.

Catherine Bachewich and I. Brent Heath

855 but hyphae resumed growth in 0n5 m (n l 8). The apical cytoplasm looked normal, but the subapical cytoplasm became very disorganized over time. RP staining indicated that the normal cortical F-actin population partially collapsed into the central cytoplasm (Fig. 10). There was also an initial transient (lasting a few minutes) increase in subapical cytoplasmic migration which was often bidirectional, with cytoplasm on opposite sides of a hyphae moving in opposite directions. However, these movements were not observed in every hypha within each of the 13 colonies analysed. Upon washing DMAP out with OM, many hyphae burst or their cytoplasm contracted violently and they were not observed further. Those that did not burst demonstrated increased vacuolation throughout the cytoplasm, comparable to the other treatments. Similar to recovery from latrunculin, all hyphae vacuolated beyond the initial ‘ tubules only ’ stage, bidirectional cytoplasmic migrations were absent and all hyphae failed to completely refill with cytoplasm prior to regrowth at normal times. Apical vacuolation requires microtubules, while cytoplasmic migrations require F-actin

Figs 12, 13. Fig. 12. A hypha recovering from acetic acid, in the presence of the microtubule inhibitor nocodazole. (A) A growing hypha, preincubated in 2 µg ml−" nocodazole for 2 h, prior to addition of 1n1 m acetic acid, pH 4n6. Note the wavy fashion in which the hypha grew. (B) After 10 min in acetic acid (plus nocodazole), growth was disrupted, and no apical vacuoles are present. (C–E) Upon washing with growth medium plus nocodazole, no vacuolation or cytoplasmic migration was observed, prior to resumption of growth (F, G) (The hypha was shifted back in (G) to show the swollen tip). Bar l 10 µm. Fig. 13. A hypha recovering from acetic acid, in the presence of the actin inhibitor latrunculin B. (A) A hypha previously incubated in 1n1 m acetic acid (pH 4n6) for 10 min, at the beginning of a wash in growth medium plus 0n5 µg ml−" latrunculin B. (B, C) Small vacuoles (arrows) formed throughout the cytoplasm during early stages of the wash. (D–G) The degree of vacuolation increased to occupy the apex, comparable to the wash without latrunculin, but bulk migrations of cytoplasm were not present, only local movements. The cytoplasm was also more clumped compared to the control situation (cf Figs 2–H–M). Note the similarity in vacuole and cytoplasmic pattern over 15 min (C–G). Cytoplasm did not refill the tube during exposure to latrunculin, nor did the hypha resume growth. (H) Upon removing latrunculin from the wash, the cytoplasm became less clumped and spread into the apex. (I) Growth resumed far from the tip in the form of subapical branches, in contrast to growth resumption at the tip when latrunculin is not included in the wash (cf. Figs 2 S, T). Similar results were obtained when the wash with latrunculin was extended to 40 or 50 min. Bar l 10 µm.

Recovery from growth inhibition by the general kinase inhibitor DMAP was also similar to acid recovery (Table 1, line 4). Because the effects of the drug itself have not been previously reported for hyphae, they will be briefly described first. DMAP, 1n0 m (n l 15), induced permanent growth inhibition, tip swelling and vacuolation (in 67 % of hyphae), similar to the effects of latrunculin B (Gupta & Heath, 1997),

The possible role of microtubules in apical vacuolation and cytoplasmic migrations was investigated by pretreating hyphae with 2 µg ml−" nocodazole for 2 h, then subjecting them to growth inhibition by, and recovery from, acetic acid, all in the continued presence of nocodazole (Table 1, line 5). Nocodazole-treated hyphae grew at reduced rates (Heath, 1982), became very wavy and contained clustered, unidentified organelles which normally display saltatory movements (Heath, 1988), but showed no apical vacuolation. The hyphae also contained apical tubular vacuoles as shown by 6-CDFA staining, but the dye signal was extremely low, and many hyphae from all slides analysed (n l 10) did not demonstrate any vacuole staining. Growth recovery occurred within a similar amount of time as recovery from weak acids, but was not accompanied by either apical vacuolation or cytoplasmic migration (Fig. 12). The apical vacuolation induced by latrunculin B treatment was also suppressed when microtubules were disrupted with nocodazole. Hyphae preincubated in nocodazole and exposed to both nocodazole and latrunculin B (n l 4 slides) stopped growing and swelled, but the apical vacuoles normally seen in the apex with latrunculin B alone did not form, even after 1 h (Fig. 11). Thus apical vacuole formation induced by two different treatments requires microtubules. F-actin is not required for vacuolation during growth recovery. F-actin disruption by inclusion of 0n5 µg ml−" latrunculin B in the recovery solution (Gupta & Heath, 1997 ; Bachewich & Heath, 1998) allowed the normal production of vacuoles in the apical cytoplasm, although fewer hyphae vacuolated to the extent of containing a large central vacuole (Table 1, line 6). Cytoplasmic migration during acid recovery was, however, reduced (Fig. 13). Bulk migrations were absent and the lesser movements were more localized, transient and slow. The cytoplasm also appeared to be more clumped, and less fluid. Latrunculin B suppressed refilling of the hyphal apices and delayed resumption of growth during the average

Cytoplasmic migrations and vacuolation in hyphae 40 min observation period, but even up to 50 min. The hyphae were still viable, however, because they resumed growth upon washing out the latrunculin. During this resumption, the cytoplasm became less clumped and apical vacuolation was reduced to some extent, but the majority of hyphae did not completely refill with cytoplasm, and growth resumed in subapical regions, far from the main tip (Bachewich & Heath, 1998). DISCUSSION We demonstrate a common response, abnormal apical vacuolation and cytoplasmic migration, in hyphae recovering from diverse growth-inhibitions. This response appears to be related to cytoplasmic homeostasis and a possibly generalized eukaryotic avoidance response. They also indicate roles for actin in vacuole shape maintenance and cytoplasmic migration, and microtubules in vacuole fusion and migration. Apical vacuoles develop from expansion/fusion of preexisting tubular vacuoles In the initial report of the acid-recovery vacuolation response (Bachewich & Heath, 1997 b), we suggested that the vacuoles invaded the apices from the subapex. Our further analysis indicates that it is more likely that the apical vacuoles primarily originate from the fusion and\or expansion of the preexisting tubular vacuoles, based on the following : (i) Apical vacuolation involved sequential formation of generally tubular then increasingly larger, somewhat spherical vacuoles, (ii) 6-CFDA-loaded tubules appeared to transform into large dye-loaded apical vacuoles during recovery and (iii) the tubular vacuoles were capable of enlarging and fusing into large spherical vacuoles, as shown by the reaction of the 6CFDA-loaded tubules to excitation light. Allaway et al. (1997) have also shown the enlargement of tubular apical vacuoles following hyphal damage. In addition, dynamic vacuole fusion occurs in growing hyphae, the activity of which varied with environmental conditions (Hyde & Ashford, 1997). Vacuole fusion and fission also occur in fission yeast in response to osmotic stress, a process which involves MAP kinase signalling (Bone et al., 1998). Vacuole expansion in a constant cellular volume (all vacuolation occurred after growth cessation) necessitates either transfer of water from the cytoplasm, or reciprocal cytoplasm\vacuole displacements. In the case of the former, an increase in cytoplasmic dry matter is expected, unless concomitant dry matter transfer from the cytoplasm occurs. Although DIC imaging did not reveal any obvious increase in cytoplasmic dry matter during vacuolation, non-detectable changes could have occurred. In order for vacuoles to take up water from the cytoplasm, changes in vacuolar osmotic content must be taking place. Since Cole et al. (1997) indicated the presence of a nonspecific anion transporter in hyphal tonoplasts, uptake of acetate anions could be a contributing factor, as well as uptake of other ions and molecules. Reciprocal cytoplasm\vacuole displacements could also have taken place, however, since cytoplasmic migration occurred simultaneously with apical vacuole formation in many hyphae. If the vacuolar tubular network in the apex is connected with

856 larger subapical vacuoles, cytoplasm moving into the subapical region could deform the larger vacuoles, inducing water transfer to, and expansion of, the apical tubules. The absence of cytoplasmic migration during vacuolation in some hyphae could reflect non-detection of cytoplasmic movements occurring in regions outside of the plane of focus. Peristaltic movement of fluorescent dye along tubular vacuoles, and transfer of dye from one vacuole to another via the tubules, were previously shown in hyphae of Pisolithus (Shepherd et al., 1993 b), supporting the idea that the water content of subapical vacuoles could be transferred to the apical vacuoles. Cytoskeletal determination of tubular vacuole shape and organization The shape and distribution of the apical tubular vacuoles necessitates a system to impose such asymmetry and order. The cytoskeleton, specifically F-actin, is likely to be part of this system. Latrunculin-induced actin disruption led to loss of tubule shape, producing thickened tubules and large spherical vacuoles in the apex (Gupta & Heath, 1997). Long term Factin disruption produces spherical vacuolation throughout the cytoplasm (Bachewich & Heath, 1998), abnormal vacuolation occurs under conditions that perturb actin in Saccharomyces (Raymond et al., 1992), and F-actin-selective uv microbeams induced apical hyphal vacuolation (Jackson & Heath, 1993 a). The contrary observation of general maintenance of tubular vacuole shape following F-actin disruption by procaine and acetic acid (Bachewich & Heath, 1997 b ; Bachewich & Heath, unpublished results) could be due to the lower degree of disruption relative to latrunculin (Gupta & Heath, 1997) and\or pH-induced alteration of the H+ gradient across the vacuolar membrane, a feature that is important for vacuolar fusion (Haas, Conradt & Wichner, 1994). Disruptions of vacuole-associated actin during the recovery response could, therefore, contribute to the abnormal apical vacuolations. On the other hand, microtubules do not appear to be essential for tubular vacuole morphology in Saprolegnia because the vacuoles survive concentrations of nocodazole which disrupt microtubules (Heath, 1982 ; unpublished results). Microtubules are, however, apparently important to the processes of vacuole enlargement and fusion since the removal of microtubules with nocodazole suppressed both latrunculininduced and acid-recovery-induced vacuolation, consistent with previous suggestions of microtubule involvement in fungal vacuolation (Guthrie & Wichner, 1988 ; Allaway et al., 1997). The ways in which F-actin and microtubules interact in regulating the organization of vacuoles remain unclear, but since the apical tubular vacuole system is in fact made up of numerous separate tubular components which parallel and associate with the microtubules (Allaway et al., 1997), the microtubules may be essential for translocation of the tubular vesicles to facilitate their fusions. However, the specificity of the inhibitory actions of either nocodazole or latrunculin should not be taken for granted. While they do indeed alter their best known targets, the fact that nocodazole-treated hyphae showed very low intensity 6-CFDA staining of tubular vacuoles suggests effects other than on microtubules.

Catherine Bachewich and I. Brent Heath These unknown effects could also explain the suppression of predicted vacuolations. The postulated loss of F-actin-based restraint on vacuole shape during acid-recovery is likely to be mediated by alterations in intracellular ion concentrations. Since hyphae were recovering from altered intracellular ion content, ionic ‘ adjustments ’ are predicted to occur. These ‘ adjustments ’ are not likely to be restricted to the perturbed ions (H+) since interactions between diverse ions (e.g. H+ and Ca#+) are known (Felle, 1988 ; Plieth, Sattelmacher & Hansen, 1997). In support of this, cytoplasmic contractions towards the tip, which were previously demonstrated to be Ca#+-dependent (McKerracher & Heath, 1986 ; Kaminskyj et al., 1992 ; Jackson & Heath, 1993 a), and initiated by treatments which potentially altered bulk intracellular ion concentrations (Bachewich & Heath, 1997 a), took place in some hyphae during recovery. Therefore, upon washing away the various growth inhibitors, intracellular ion adjustments are suggested to transiently disrupt cytoskeletal restraints of the tubular vacuoles, permitting their subsequent expansion and fusion. Cytoplasmic migration in non-extending hyphae is Factin-based, and can be bidirectional The recovery response showed cytoplasmic migration in the absence of hyphal growth, indicating that it is an independent process that must normally be coordinated with extension. The refilling of vacuolated hyphal apices with cytoplasm also demonstrated that cytoplasm is capable of migrating towards the hyphal tip in the absence of apical cytoplasm\wall adhesions. The cytoplasm could therefore exert force along the cortical regions of the hypha to propel itself towards the tip. Since latrunculin B disorganized F-actin, and severely reduced the movements of cytoplasm, migration was presumably actin-dependent. In contrast to the normal migrations, however, those seen during recovery were often bidirectional, indicating changes in the normally apex-directed polarized behaviour of the F-actin system. Such changes could derive from alterations in presumed F-actin polarity, but could also relate to changes in filament interactions with associated ‘ motors ’ such as myosin. Induction of bidirectional migrations by DMAP, a kinase inhibitor, could be due to alterations in the activity of myosin. However, the disrupting effects of DMAP on cortical actin and cytoplasmic organization could allow for bidirectional migration of isolated regions of cytoplasm. The more general bidirectional movements observed during recovery most likely relate to changes in cytoplasmic ion concentrations as discussed above. Cytoplasmic movements may be part of an ‘ avoidance response ’, and support actin’s function as a targeting component for polar growth In hyphal organisms, entry into new environments is mediated by tip growth. Such environments may be either positive or negative, the sensing of which is most likely to be located in the entering hyphal tips. An avoidance response, such as that shown by retraction of amoeboid pseudopods (Bray, 1992), wherein the cytoplasm and growth-sustaining cellular ma-

857 chinery is displaced away from the negative stimulus for possible new growth in a more favourable environment, would have survival value. The various treatments used here could be perceived by the hyphae as ‘ negative environments ’, prompting the displacement of cytoplasm and resources away from the sensing hyphal tips, as vacuoles occupy the apical volume. Re-entry of the cytoplasm into the previously colonized area (i.e. the ‘ empty ’ hyphal tips) after the removal of the negative stimulus would be an efficient recovery response. In support of this, hyphae recovering from acetic acid normally regrew from the old tips after cytoplasmic reentry. The absence of this ‘ avoidance ’ response during the treatments themselves could reflect their harsh effects, impairing the hypha’s ability to respond until removal of the treatment is initiated. Actin has been proposed to mark cortical sites and function as a targeting component for polar growth in Saccharomyces (Drubin & Nelson, 1996) and other tip-growing organisms (Shaw & Quatrano, 1996). Cytoplasmic reoccupation of the apex and subsequent growth from the original tip during the recovery response in Saprolegnia could reflect the maintenance of some original growth-related targeting machinery in the tip, such as actin, supporting this idea. When actin was disrupted with latrunculin for long periods of time following acetic acid treatment, cytoplasm did not reoccupy the tip, and the tip did not regrow, during exposure to, and upon removal of, latrunculin. The original polarity-related actin is likely to have been disrupted, preventing cytoplasmic reoccupation and growth of the original apex, compared to when latrunculin was not included in the wash. Consistent with this, buds of the yeast Saccharomyces were abandoned, and new ones formed, upon removing cells from latrunculin A (Ayscough et al., 1997). Resumption of growth of the main tip following short term exposures to latrunculin could indicate that some targeting-related actin remained in the original apex. In summary, we propose the following model to explain the recovery response. Upon washing out acetic acid or other ‘ harmful ’ agents, cytoplasm moves away from the main tip, and is replaced by enlarged vacuoles. The vacuoles help to restore cytoplasmic ‘ normality ’ by sequestering the agents. Changes in intracellular ion concentrations trigger disruption of actin-based cytoskeletal restraints on the vacuoles, allowing them to expand and fuse. The ionic changes also alter cytoplasmic F-actin, allowing the abnormal contractions, bidirectional migrations, and bulk migrations away from the tip. The degree of F-actin disruption determines the extent of cytoplasmic migration. Reestablishing normal ionic conditions, and maintenance of actin-based polarity signals in the original tip, facilitate cytoplasmic reoccupation of the hyphae, and resumption of apical growth. This work was supported by the Natural Sciences and Engineering Research Council of Canada. REFERENCES Allaway, W. G., Ashford, A. E., Heath, I. B. & Hardham, A. R. (1997). Vacuolar reticulum in oomycete hyphal tips : An additional component of the Ca#+ Regulatory system ? Fungal Genetics and Biology 22, 209–220.

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