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The interrelationships of actin and hyphal tip growth in the ascomycete Geotrichum candidum
Fungal Genetics and Biology 38 (2003) 85–97 www.elsevier.com/locate/yfgbi
The interrelationships of actin and hyphal tip growth in the ascomycete Geotrichum candidum I. Brent Heath,* Michael Bonham, Ali Akram, and Gagan D. Gupta Biology Department, York University, 4700 Keele Street, Toronto, Ont. M3J1P3, Canada Received 30 August 2001; accepted 30 July 2002
Abstract Geotrichum candidum is unusual among reported hyphal ascomycetes in that its hyphae readily stain with phalloidin to reveal actin concentrated in the Spitzenk€ orper (SPK) and plaques associated with the plasma membrane (PM). Loss of SPK actin, but not the PM plaques, following latrunculin B treatment produces tip swelling, consistent with actin restraining tip morphology or localizing vesicle exocytosis. Tip morphogenesis may also involve a spectrin-like protein which concentrates at the apical PM in plaques unassociated with the actin plaques. Branch formation occurs with growth rates initially about 20% those of leading tips, and does not involve a morphologically detectable SPK, nor SPK-like actin ensembles, indicating the dispensibility of this structure in tip growth. Surprisingly, new tubular tips can form in the continued presence of latrunculin, consistent with alternative cellular systems, such as the spectrin-like protein, substituting for actin’s critical functions. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Geotrichum candidum; Actin; Tip growth; Latrunculin; Spitzenk€ orper; Cytoskeleton; Exocytosis; Spectrin
1. Introduction Hyphal tip growth involves many intimately related processes which must be very precisely regulated to produce the typically uniform diameter tube characteristic of hyphal organisms. Among these processes are regulation of tip extensibility and localized vesicle exocytosis. The vesicles deliver cell wall components and plasma membrane (PM) (Fevre and Rougier, 1982; Grove and Bracker, 1970; Grove et al., 1970; Heath et al., 1971; Heath et al., 1985) and possibly also cell wall softening enzymes (Bartnicki-Garcia and Lippman, 1972). A common, but not universal, component of growing tips is the Spitzenk€ orper (SPK) (Brunswick, 1924; Girbardt, 1957), the behaviour of which correlates well with tip growth (Bracker et al., 1997; L opez-Franco and Bracker, 1996; Reynaga-Pe~ na and Bartnicki-Garcia, 1997; Riquelme et al., 1998, 2000) and which may play some role in regulating exocytosis (Bracker et al., 1997). Vesicles aggregate as part of the SPK (Girbardt, * Corresponding author. Fax: +1-416-736-5698. E-mail address: [email protected] (I. Brent Heath).
1969) and it is possible to model tip morphogenesis on the assumption that it regulates the sites of exocytosis by directing the sites of impact of the vesicles at the PM (Bartnicki-Garcia et al., 1989; Gierz and BartnickiGarcia, 2001). The composition of the SPK is largely unknown. Its observed movements (L opez-Franco and Bracker, 1996; Reynaga-Pe~ na and Bartnicki-Garcia, 1997) and postulated roles in vesicle behaviour indicate a force generating component. Actin (as well as microtubules [McDaniel and Roberson, 1998, 2000; Riquelme et al., 1998]) may be part of such a component, but there are conflicting reports concerning its presence. It has been reported in a few species (Bourett and Howard, 1991; Czymmek et al., 1995; Degousee et al., 2000; Heath et al., 2000; McDaniel and Roberson, 2000; Srinivasan et al., 1996), but there are more reports of actin localizations in species likely to contain a SPK, yet presenting no images interpretable as SPK actin (Akashi et al., 1994; Anderson and Soll, 1986; Ayad-Durieux et al., 2000; Fischer and Timberlake, 1995; Heath, 1990; Roberson, 1992; Runeberg et al., 1986; Salo et al., 1989; Tinsley et al., 1996; Torralba et al., 1998; Tsukamoto
1087-1845/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 8 7 - 1 8 4 5 ( 0 2 ) 0 0 5 1 1 - X
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et al., 1996). In the latter reports, the tip-rich actin predominantly forms plaques in the peripheral cytoplasm. The relationship, if any, between plaques and SPK is obscure, as are the functions and behaviour of the plaques. In species lacking a SPK, such as the oomycetes, the apical organization of actin differs from the eufungi. It has been suggested that this actin contributes to a membrane skeleton that regulates the extensibility, and thus shape, of the hyphal tips (Gupta and Heath, 1997; Heath, 1990, 1995; Jackson and Heath, 1990; Kaminskyj and Heath, 1996). A similar actin-containing membrane skeleton, with a similar function, may exist in eufungi (Heath, 2000, 2001; Kaminskyj and Heath, 1996). Evidence for membrane skeleton-based morphogensis is hyphal tip swelling following actin disruption by cytochalasins (reviewed in Grove and Sweigard, 1996; Heath, 1990), but cytochalasins are known to (Lin and Spudich, 1974), or may (Treves et al., 1987), lack specificity for actin. Latrunculins, such as latrunculin B (LatB), are well characterized as targeting actin in fungi (Ayscough et al., 1997). Although other targets have not been ruled out, their effect at lower concentrations than cytochalasins supports a direct morphogenic role for actin in an oomycete (Gupta and Heath, 1997). They have not been employed in any extensive analysis of eufungi, although preliminary observations of Neurospora crassa indicated responses differing from those of oomycetes (Heath et al., 2000). Similar effects from different inhibitors sharing a known target are strong support for a role for that target in the tip growth process. Equally, different responses to the same inhibitor in different species would argue for differences in the contributions of the target. The purpose of this study was to further investigate the possible roles of actin in tip growth of a eufungus. Geotrichum candidum was initially selected because it is an ascomycete which has an unusual feature of normally producing its leading branches in a predictable location immediately subjacent to the most apical septum, thus facilitating analysis of branch initiation. Early observations demonstrated a fortuitous bonus in that its hyphal actin readily stained with rhodamine-labelled phalloidin (RP). The results obtained further refine our understanding of the organization and functions of hyphal actin and the SPK.
2. Materials and methods 2.1. Organism and cultures Geotrichum candidum (Galactomyces geotrichum (Butler & Petersen) Redhead & Malloch) was generously supplied by Dr. A.P.J. Trinci, University of Manchester. All cultures were grown on a complex medium designated OM (Heath and Greenwood, 1970). Unless noted
to the contrary, hyphae were grown on dialysis membrane overlying OM agar at 22 °C. Typically 1 cm2 rectangles of membrane plus hyphae were cut from edges of colonies, allowed to recover on the agar for 30– 60 min then either mounted in flow-through chambers (Heath, 1988) for observation of living hyphae, or irrigated with various treatment solutions while lying on top of the agar (with frequent addition of fresh medium to enhance constant drug concentration and prevent drying) or in the bottom of Petri dishes. LatB (Sigma, Oakville, ON) was made as a 1 mg ml1 stock solution in dimethyl sulphoxide (DMSO) and freshly diluted in OM to give working concentrations of 0.1, 0.5, 1.0, 5.0, and 10.0 lg ml1 (1.0 lg ml1 ¼ 2:52 lM) LatB, and 0.01– 1.0% DMSO. Control experiments using 1.0% DMSO alone showed no observed effects on hyphae. 2.2. Growth rates and hyphal swelling Hyphae were mounted in the growth chambers with 15 ll of OM and allowed to recover for about 10 min. To observe the effects of LatB, growing tips were videotaped for 6–8 min, then 50 ll of LatB was drawn across the chamber, with continuous recording, and the hyphae were recorded for a further 5–6 min. Growth was measured at 1 or 2 min intervals with a ruler on the screen at a magnification of 1 mm ¼ 0.8 lm. Growth rates were determined from the best fit lines over 5 or 6 min before and after addition of LatB. In other experiments, growth rates were determined from prints taken at various time intervals at magnifications where 1 mm ¼ 0.56–1.1 lm. Growth rates were also measured from colony diameters in liquid OM in Petri dishes. Hyphal swelling was measured from thermal prints from video recordings for up to 20 min after LatB addition, at a magnification where 1 mm ¼ 2 lm. Hyphae of both G. candidum and N. crassa (Heath et al., 2000) observed and recorded for the presence of SPK (Figs. 1–8) were gently detached from the dialysis membrane into liquid OM, mounted under a coverslip and allowed to recover growth prior to observation under exactly the same conditions as each other. 2.3. Cytochemistry Hyphae to be stained only with rhodamine-labelled phalloidin (RP, Molecular Probes, Eugene, OR) were fixed by the addition of 1 ml of 2% formaldehyde and 0.025% glutaraldehyde in 60 mM PIPES buffer [pH 7.0] containing 5 mM MgCl2 , 5 mM EGTA, and 0.05% Triton X100 to the hyphae in their growth environment for 4 min. The hyphae were then rinsed in 4 ml of the above buffer solution (without Triton or fixatives), transferred to a humid Petri dish with 5 or 10 ll of 5 lg ml1 RP in PIPES buffer per colony and left for 15– 25 min. The colonies were then rinsed 2–3 in 150 ll
I. Brent Heath et al. / Fungal Genetics and Biology 38 (2003) 85–97
of PIPES buffer, removed from their dialysis membrane (if present) and mounted in 30 ll of Prolong (Molecular Probes) mounting medium.
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Immunocytochemical staining of hyphae employed 200 ll of the same fixation solution as above, followed by a 30 s rinse in 60 mM PIPES buffer, cell wall digestion in 9 mg ml1 Dreiselase (Kyama Hakh Kogyo, Tokyo) and 1 mg ml1 Novozyme-234 (InterSpex Products, Foster City, CA) in 30 mM MES (pH 5.5) containing 1% BSA, 5 mM MgSO4 , and 5 mM CaCl2 for 5–7 min, a rinse in PIPES, incubation in polyclonal rabbit antichicken a and b spectrin (lot #054H4830, used at 1/100 in 60 mM PIPES [pH 7.5] with 0.5% BSA and 0.01% NaN3 , Sigma) for 1 h, rinsing for 30 s in 60 mM PIPES buffer, incubation in 200 ll of FITC-labelled goat antirabbit secondary antibody (1/600 in above antibody solution, Cedarlane, Hornby, ON) for 30 min, rinsing in PIPES, and mounting in Prolong, after removal from the dialysis membrane. For double staining with antibody and RP, 10 ll of RP was added for the last 15 min of the secondary antibody incubation. All fixation and staining procedures were carried out at 22 °C. Hyphae were observed with Nomarski differential interference contrast (DIC), phase contrast, or epifluorescence optics on a Reichert Polyvar microscope using B1 (FITC) and G2 (rhodamine) filter combinations. Objectives were 40 and 100 with NAs of 0.9 and 1.32, respectively. Images were recorded on film (Kodak T400 CN), VHS tape or with a digital camera (MicroMax 1300Y, Roper Scientific, Trenton, NJ). All
b Figs. 1–17. Median DIC optical sections of a hyphal tip of N. crassa growing at 6.2 lm min1 showing clear SPK (arrow) (Fig. 1), compared with similarly imaged tips of G. candidum growing at similar rates (6.4, 1.8, and 2 lm min1 , respectively) and showing no similarly clear SPK (Figs. 2–4). Phase contrast images of growing G. candidum hyphae similarly show no consistent evidence of a phase dark SPK among the diversity of tip cytoplasmic patterns (Figs. 5a–e). Branches form proximal to the most apical septa and grow forward much slower than hyphal tips. In the sequence seen in Fig. 6 (a and b, t ¼ 0, 13 min), the rate was 0.23 lm min1 , whereas in a similar sequence (Fig. 7a and b, t ¼ 0, 17.4 min) it was 0.5 lm min1 . Septal ingrowth was observed in a septum developing adjacent to an already formed septum (Fig. 8a– c, t ¼ 0, 25, 43 min). It had already started to grow (a) and closed completely (c). For all Figs. 1–8, the hyphae were mounted in the culture chambers without dialysis membrane. In control hyphae, comparable to those seen in Figs. 2–8 but stained with RP, tips showed prominent SPK-like staining subtended by peripheral plaques, which were typically rare in the most apical 3–4 lm (Figs. 9, 11, 12, 14, and 15). In straight hyphae, the SPK-like staining was central (Figs. 9, 11, and 15), whereas in curved hyphae, it was located eccentrically, towards the direction of growth (Figs.11 and 12). An oblique, near face view of three adjacent tips emphasizes the location of the SPK-like staining and the surrounding region clear of plaques (Fig. 14). In contrast to the leading hyphal tips, branch tips consistently lacked any sign of SPK-like staining, instead the young tips were covered with plaques over the entire tip (Figs. 16 and 17). Two tips forming at slightly different stages from adjacent septa are seen in Fig. 17. Mature septa are accompanied by a double layer of plaques (Fig. 10), whereas a presumed forming septa, viewed obliquely, showed a ring of filaments (Fig. 13). Scale on Fig. 2 also for Figs. 3–5 and that on Fig. 16 for Figs. 14 and 15. Scale bars in all micrographs ¼ 5 lm.
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cytochemical conclusions are based on subjective observations of many hyphae in one or more colonies, and recorded representative images from those hyphae. All figures are of G. candidum and all scale bars indicate approximately 5 lm unless noted to the contrary.
3. Results 3.1. Control hyphae The dominant hyphal growth pattern is straight tapered leading tips (mean diameter at the base of the taper ¼ 4.7 lm 1:6, n ¼ 92) (Figs. 2–5) which grew at a mean of 1.3 lm min1 (0.79, range 0.1–6.4, n ¼ 140) in the growth chambers and somewhat faster (1.96 and 2.08 lm min1 , two colony radii) in Petri dishes. Variation in growth rates did not correlate with hyphal diameters. The hyphae lack a detectable SPK when examined with DIC (Figs. 2–4), using the same optics that reveal clear SPK in Neurospora. (Fig. 1) (Heath et al., 2000), or with phase contrast (Fig. 5). This statement is also based on unrecorded observations of many growing hyphae with growth rates covering the full range observed. The first branch normally forms immediately subjacent to the first septum (Figs. 6 and 7), but the time of initiation is variable. Branch length did not correlate (r2 ¼ 0:09, n ¼ 33) with the length of the apical cell (a proxy measure of both the age of the septum and the apical cell) and the earliest detectable branches were on hyphae with a 7% standard deviation in the latter measurement (n ¼ 5). Branches between 4.4 (earliest recorded) and 12.2 lm grew much slower (mean 0.29 lm min1 0:18, n ¼ 3; Figs. 6 and 7) than hyphal tips and did not accelerate over a maximum of 17 min of observation. In one simultaneous recording, the branch also grew slower than its associated main tip (0.15 vs 0.7 lm min1 ). Recorded branches were also thinner than hyphal tips (mean diameter 3.75 lm 0:45, n ¼ 3) (Figs. 6 and 7). On one fortuitous occasion, a septum was observed during its growth (Fig. 8). When first observed, its aperture diameter was 4.6 lm and the hyphal outer diameter was 7.2 lm. Over 42 min it grew steadily centripetally at an edge advance of 0.05 lm min1 , while the tip of the same hypha extended at 0.28 lm min1 . RP staining is predicted to stain polymeric actin (discussed in references in the Introduction and Discussion), an assumption in the following results. Actin in leading hyphal tips concentrates in a structure approximately the same size and location as expected of a SPK (Figs. 9, 11, 12, 14, 15, and 18). Hyphae apparently changing growth direction at the time of fixation contained eccentrically located SPK-like actin towards the new growth direction (Figs. 11 and 12). The SPK-like actin is the only detectable concentration in the most
apical 3–4 lm (Figs. 9, 11, 12, 14, 15, and 18). Below this, there is a ring of fine (0.5 lm diameter) PM-associated actin plaques whose concentration declines steeply sub-apically to a uniform low level throughout the rest of the hyphae. The sub-apical plaques were variable and not always detectable. Sub-apically there are also occasional rings of actin which seem to coincide with the very early stages of septum formation (Fig. 13). At mature septa, the actin forms two plates of plaques (Fig. 10), apparently corresponding to the PM on each side. There were also occasionally fine actin filaments, often associated with septa, parallel to the long axis of the hyphae. Actin in young branches (length < hyphal diameter, 8 documented, many others observed), apparently form-
Figs. 18–19. Anti-spectrin staining of hyphal tips. In Fig. 18, a group of tips (DIC image in e) were double stained with RP (a, c) and antispectrin antibody (b, d) and imaged at predominantly median (a, b) and surface (c, d) focal planes. The apical SPK-like and plaque staining characteristic of growing tips clearly differs from the peripheral plaques of spectrin staining that occur over the entire apex. Careful comparison of the actin and spectrin images shows that the plaques are not coincident. In Fig. 19, a spectrin-stained tip plasmolysed slightly during processing and shows that the peripheral plaques have retracted with the PM, leaving a few remaining on strands of PM extending out to the cell wall (arrows).
I. Brent Heath et al. / Fungal Genetics and Biology 38 (2003) 85–97
ing at the time of fixation, was never concentrated into SPK-like organization. All contained only tip-high concentrations of plaques over their entire tips (Figs. 16 and 17). The anti-spectrin antibody will, for convenience, be described as localizing spectrin, which does not co-localize with actin. It is exclusively at the PM, forming fine (0.5 lm diameter) patches covering the entire apex; either in uniform density along the PM, or a
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slightly tip-high gradient (Fig. 18). Where both actin and spectrin are co-localized, the similarly sized plaques and patches are mostly not superimposable (Fig. 18). In hyphae inferred to be growing at the time of fixation, because they contain SPK-like actin, the spectrin covers the entire tip (Fig. 18). The spectrin patches do not appear to be located in or on the cell wall because they retract with the PM during plasmolysis (Fig. 19). 3.2. Latrunculin B effects 3.2.1. Actin disruption Actin disruption was primarily analyzed in 1 lm ml1 LatB, the concentration most effective in inducing swelling and comparable to other concentrations in early effects on tip growth. Changes in apical actin organization were observed as early as 1 min after treatment. Initially, the plaques retained their normal exclusion from the most apical 3– 4 lm, but there was no SPK-like staining (Fig. 20). These tips appeared to be rather more blunt than normal growing tips, but swelling only became evident by 5 min, and then it was not as extensive or consistent under the culture regime used for these hyphae relative to that used for the quantitative results discussed below. In all hyphae observed after >5 min in LatB, the tips and young branches contained plaques which were PMassociated and most concentrated at the extreme tips (Figs. 27, 22–24). No SPK-like staining was observed. Sub-apically the plaques appeared normal. At 0.5 and 5.0 lg ml1 , actin patterns were only observed after >5 min and in all cases, irrespective of the degree of hyphal tip swelling, the patterns were the same as those of the >5 min hyphae described above. Actin patterns were not investigated for 0.1 and 10 lg ml1 .
b Figs. 20–32. The effects of LatB on the actin organization (RP images in Figs. 20–24, 27) and morphology of hyphal tips. Tips treated with 1 lg ml1 for 1 (Fig. 20), 5 (Figs. 21 and 22), and 10 (Figs. 23 and 24) min show that even at this earliest time, the SPK-like staining is lost, but the plaques retain their pre-treatment sub-apical distribution, remaining absent from the most apical 3–4 lm (seen in the series of focal planes extending from the upper through median to lower surface in Fig. 20a–d). At later times, the plaques extend over the entire apices (Figs. 21–24), as seen especially in the three focal planes of the adjacent hyphae in Fig. 23a–c. Application of 5 lg ml1 (Figs. 25–32) for 22 (Fig. 27), 60 (Figs. 25 and 26), and 120 (Figs. 28–31) min produces swollen tips which rapidly attain a maximum diameter and contain peripheral actin plaques located over the entire apex (Fig. 27). However, after prolonged incubation in the drug, localized new outgrowths are produced from the swollen regions (Figs. 29–31) and branches become swollen (Fig. 28), a feature not observed at earlier times. After 120 min in the drug followed by its replacement with normal medium for 60 min, numerous tubular outgrowths (branches) are produced, primarily from the swollen apical regions (Fig. 32). Scale on Fig. 27 also for Figs. 26, 28–30, and 32.
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3.2.2. Tip expansion To explore previously reported (in another species, (Gupta and Heath, 1997)) transient growth acceleration upon addition of LatB, we compared the rate of growth in the 2 min interval prior to LatB addition with that 2 min after. This was the shortest interval capable of giving a consistent and reliable measure of hyphal extension. In both controls and LatB treatments there was a transient reduction of extension in about 70% of hyphae (Fig. 33; Table 1). In controls, but not LatB, this was followed by increased rates in the subsequent 4 min, so that after 5– 6 min treatment there was a concentration-dependent reduction in forward growth rate, as shown in Table 2. As forward growth slowed, hyphae swelled sub-apically to approximately apical spheres (Figs. 25–31). Swelling was time and concentration-dependent (Fig. 34) and involved both leading hyphal tips and short branches (Fig. 28). At all concentrations it was initially rapid then slowed as maximum swelling was approached (Fig. 34). The initial rate of swelling did not correlate with either pre-addition growth rate or hyphal diameter, but was very sensitive to LatB concentration, peaking at 1.0 lg min1 (Fig. 34). Swelling may involve surface area (cell wall and PM) synthesis as well as shape change. To separate these components, we calculated the predicted surface increase,
based on the mean control rate of tip growth and hyphal diameter, and compared this with the observed increase based on the diameter of the swollen tips (Table 3). The ratio between these values would be 100% if the normal rate of increase was maintained and only the shape changed, but in fact the ratios for 0.5, 1.0, and 5.0 lg ml1 were initially higher, dropping below 100% after 10– 15 min (Fig. 35). Thus initial swelling was due to either greater than predicted surface synthesis or swelling with the normal rate of synthesis. Conversely, both 0.1 and 10.0 lg min1 were below 100% at all times, indicating less than predicted synthesis or expansion (Fig. 35). The maximum swollen diameter was about 2–2.5 times normal hyphal diameter, both in slide chambers and Petri dishes, irrespective of incubation time or LatB concentration. The only indication of greater ‘‘expansion’’ was occasional tip bursting. In the slide chambers, only hyphae incubated in 5 lg ml1 burst; by 20 min, 40% had done so. Among the Petri dish cultures, bursting was observed rarely in 0.5 lg ml1 , more frequently in 1.0 lg ml1 and maximally in 5 lg ml1 . Bursting frequency in the dish cultures was estimated at about 30% in 5 lg ml1 by 60 min, comparable to the frequency measured in the slide chambers. In both slide chambers and Petri dishes, hyphae apparently adapted to the continual presence of the drug.
Table 1 Growth rate changes in first interval after adding LatB Treatment
LatB
Controls
Increased 1
Unchanged
Decreased
Increased
Unchanged
Decreased
0.1 lg ml 0.5 lg ml1 1.0 lg ml1 5.0 lg ml1 l 10.0 lg ml1
0 1 0 0 0
9 3 1 1 4
3 8 7 9 20
2 1 0 0 2
1 0 2 4 1
4 6 6 7 3
Totals Total (%)
1 2
18 27
47 71
5 13
8 21
26 67
Note. Examples of these change designations are given in Fig. 33. Sensitivity of the observations is indicated by the fact that during the 2 min interval after adding LatB, assuming the control growth rate of 1.62 lg ml1 , one would see length change of 3.24 lm ¼ 4 mm on the screen. Even for 10 lg ml1 , which had the slowest mean growth rate after addition, one would get 0:59 2 lm ¼ 1:5 mm, which was easily measurable. Table 2 Growth rates before and after addition of LatB Treatment
Rate before (lm min1 )
Rate after (lm min1 )
Rate reduction after LatB (%)
n
‘‘p’’ value
Outgrowth time (min)
Control 0.1 lg ml1 0.5 lg ml1 1.0 lg ml1 5.0 lg ml1 l 10.0 lg ml1
1:62 0:48 0:89 0:29 1:27 0:29 1:60 0:60 2:04 1:19 0:95 0:30
1:62 0:48 0:66 0:10 0:82 0:31 0:65 0:64 0:67 0:64 0:59 0:17
0 26 35 59 67 38
39 12 12 8 10 24
>0.5 <0.005 <0.01 <0.01 <0.005 <0.001
na 5:8 1:2 11:6 2:7 17:3 5:9 19:5 0:6 na
Note. Growth rates were determined by measuring changes in hyphal length at 2 min intervals over 5 or 6 min before and after the addition of LatB and calculating the mean rates from the linear regression lines. Values are means SD. Measurement accuracy was found to be approximately 1 mm on the sreen ¼ 0.8 lm. Separate controls were performed for each concentration and found by paired sample t test not to differ between before and after, hence all were combined as a single control. Before and after rates were compared by paired sample t test and gave the presented ‘‘p’’ values. ‘‘Outgrowth time’’ indicates the time taken to form a new tip from swollen tips in the continued presence of LatB. These means increased linearly with the log of LatB concentration (r2 ¼ 0:926).
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In slide chambers, after tip swelling, one or more new tips (termed outgrowths), with diameters similar to normal tips, were produced (Figs. 29–31). Times for
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outgrowth formation increased linearly with the log of LatB concentration (Table 1). No recovery was observed in 10 l g ml1 , possibly due to observation for less
Figs. 33–35. Growth responses of hyphae to addition of LatB. Representative growth curves (Fig. 33) of three individual hyphae followed before and after the addition (indicated by arrows) of 0.1 (‘‘unchanged’’ and ‘‘slowed’’) and 0.5 (‘‘increased’’) lg ml1 LatB. The designations of changes, or otherwise, in growth rate after LatB addition do not indicate statistically significant changes, but show the criteria for the designations in Table 1. In Fig. 34, the mean percent by which individual hyphae swelled in different concentrations of LatB (bold underlined numbers, in lg ml1 ) is shown, with the sample sizes (plain numbers, 8 at all times for 1.0 lg ml1 ) indicated at each time point. The ‘‘B’’ numbers on the 5 lg ml1 line indicate the number of hyphae which burst between time points, thus explaining the drop in the mean curve. Standard deviations for each data point varied between 29–42 (0.1 lg ml1 ), 25–49 (0.5 lg ml1 ), 25–52 (1.0 lg ml1 ), 16–50 (5.0 lg ml1 ), and 4–43 (10.0 lg ml1 )%. Fig. 35 plots the O/P% swelling parameter from Table 3 over time at the different concentrations. If swelling were due solely to the unrestrained (i.e., not in the normal tubular shape) formation of the predicted amount of new cell surface at the indicated time intervals, then the ‘‘predicted’’ line would be seen. Values above the ‘‘predicted’’ line represent more surface formation than predicted by pre-drug-application growth rates and those below indicate less expansion. Thus, at the intermediate concentrations of 0.5 and 1.0 lg ml1 (and probably insignificantly at 5.0), the initial rate of swelling was greater than predicted by normal growth rates, whereas at the lower and higher concentrations there was never the predicted rate of expansion, and at all concentrations, the rate of swelling fell below predictions by 10–15 min after drug addition.
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cies it was only observed in 12% of tips (Srinivasan et al., 1996). The strong and consistent SPK-like RP staining in G. candidum, and its sensitivity to LatB, supports its reality as a major SPK component. The negative reports most likely indicate actin’s lability. The specificity of LatB (Ayscough et al., 1997) and its rapid disruption of the SPK suggests that actin is important to SPK integrity and functions. The latter may include SPK motility (L opez-Franco and Bracker, 1996) for which actin is well suited. Myosin, the most common type of force generating protein interacting with actin, is also concentrated in hyphal tips, at least of Aspergillus (McGoldrick et al., 1995). Another putative function is in vesicle distribution. Vesicles cluster around the SPK (Grove and Bracker, 1970), hyphal morphogenesis can be modeled assuming that the SPK functions in vesicle distribution (Bartnicki-Garcia et al., 1989; Gierz and Bartnicki-Garcia, 2001) and SPK manipulation can relocate expansion, most likely indicative of altered vesicle distribution (Bracker et al., 1997). The limited data implicating actin in fungal vesicle transport has been recently reviewed (Heath, 2000). While actin-based SPK organization is likely, it may not be the only structural component since SPK disruption with cytochalasin leaves a residual fibrillar core (Grove and Sweigard,
than 25 min. Similar recovery occurred in Petri dishes, but took longer since, at 5 lg ml1 (the concentration monitored in detail) the outgrowths appeared between 30 and 40 min, compared with 20 min in slide chambers. In Petri dish cultures, outgrowths remained pointed, but did not grow for up to 120 min, although by 180 min some were swollen. Cessation of tip growth and subsequent swelling were fully reversible. Colonies inhibited for 180 min in Petri dishes, then transferred to drug-free medium for 60 min, produced branches with normal diameters and morphology (Fig. 32). The rate of tip growth, calculated from the extensions beyond the swollen regions in 60 min, was 1.8 lm min1 (n ¼ 3) in 0.5 lg ml1 , comparable to normal rates, but only 0.22 lm min1 (n ¼ 9) in 5.0 lg ml1 . The slower rate may reflect the untested possibility of a longer lag time prior to regrowth.
4. Discussion 4.1. SPK actin As listed in Section 1, there is uncertainty over the presence of actin in the SPK. Even in one positive spe-
Table 3 Surface area increases after adding LatB Time after adding LatB (min) 0 a
Length increase ðlmÞ 0.1 lg ml1
5 8.1 2.51 92 68 74
10
15
20
16.2 2.65 183 78 42
24.3 2.8 275 88 32
32.4 2.9 366 96 26
rb Pc Od O/P%
1.8
0.5 lg ml1
r P O O/P%
2.05
3.3 104 124 119
4.05 208 193 93
4.2 312 209 67
4.3 417 219 56
1.0 lg ml1
r P O O/P%
2.05
3.65 104 154 148
4.55 208 247 118
4.9 312 289 93
5.15 417 320 77
5.0 lg ml1
r P O O/P%
3.85
4.55 196 214 109
5.55 392 341 87
5.35 588 313 53
—
r P O O/P%
2.1
2.2 107 47 44
2.65 214 74 35
2.9 321 92 29
3.2 428 115 27
1
10.0 lg ml
a
— — —
Length increase calculated from average control growth rate from Table 1 multiplied by time. Mean radius measured at indicated time in lm. c Predicted increase in surface area based on mean diameter prior to LatB addition multiplied by predicted increase in length (as shown in footnote a). d Observed surface area based on observed diameter sphere less basal circle determined from cross-sectional area of hyphae prior to LatB addition. b
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1996). Assuming cytochalasin and LatB disruption are similar, the absence of a residual RP-positive core in G. candidum suggests the core contains other fibrils. In spite of reported (see Section 1) tight relationships between SPK and hyphal growth, present observations show that its normal actin organization is dispensable for initiation and production of tubular hyphae. Renewed pointed outgrowths, and branch formation, in the presence of sufficient LatB to disrupt hyphal growth and cause loss of SPK actin in the first place indicate this point. This result could be explained by ‘‘adaptation’’ to
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LatB, although the facts that the outgrowths cease further growth and much later swell argue against this possibility. More compelling is the absence of detectable SPK-like actin in forming and young branches and the substitution of apical plaques in its place. The growth rates of the hyphal outgrowths in LatB, and normal branches, are much lower than those of leading tips. We suggest that the normal SPK actin complement is one end of a spectrum of actin recruitment and organization facilitating fast tip growth; alternative smaller organizations suffice to initiate and sustain slow growth.
Table 4 Variations in amino acid sequences in the primary phalloidin-binding domain of actin
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4.2. Actin as morphogen The net-like apical actin in oomycete hyphae (Heath, 1987), which lack a SPK, apparently functions, at least in part, to mechanically regulate extension of the hyphal tip (Gupta and Heath, 1997; Heath, 1995; Jackson and Heath, 1990; Kaminskyj and Heath, 1996). SPK actin in G. candidum is not well organized for such a role, but may not be the only apical actin (reviewed in Heath, 2000, 2001). We suggest that SPK actin is the focus of a less concentrated radial array connected to a membrane skeleton containing both actin and a spectrin-like protein (Degousee et al., 2000). This model provides a mechanism for SPK mobility, and vesicle distribution, as well as a mechanical role in tip morphogenesis. The concentration of actin plaques at the shoulders of the growing tips is well suited to anchor both the SPK and other hypothetical morphogenic apical actin, assuming that they do attach to the PM and cell wall, as discussed below. The identity of the protein stained by the antispectrin antibody is not yet clear, but comparison with Neurospora (Degousee et al., 2000) suggests that it has some properties of spectrin, and thus the potential to contribute to a membrane skeleton. Its apical organization differs from that in Neurospora (Degousee et al., 2000); it is more similar to that in Saprolegnia (Kaminskyj and Kaminskyj and Heath, 1995), suggesting diversity in organization of the putative membrane skeleton. Nevertheless, formation of new tips in the continued presence of LatB is consistent with another cellular component taking over an essential role of actin, as previously suggested (Heath, 2000). Critical to the hypothesized role of actin in morphogenesis of eufungal hyphae is LatB-induced swelling, comparable to previous reports of swelling caused by both latrunculins (Ayscough et al., 1997; Heath et al., 2000) and cytochalasins (reviewed in Heath, 1990 and Grove and Sweigard, 1996; Herr and Heath, 1982; Heath et al., 2000). LatB-induced sub-apical swelling in G. candidum is consistent with actin disruption permitting abnormal expansion and delocalizing exocytosis. Evidence for abnormal expansion comes from the initial rate of swelling being greater than that predicted by the normal continued rate of cell surface synthesis (ratios > 100% in Fig. 35). This expansion is only seen at intermediate LatB concentrations. Presumably lower concentrations produce insufficient weakening of the putative morphogenic actin and higher concentrations disrupt exocytosis, an undeniable part of the growth and expansion process. However, relative to the initial tip acceleration of the oomycetes (Gupta and Heath, 1997), growth slowing followed by expansion indicates differences between the hyphae and their differing actin organizations.
Sub-apical swelling may also indicate delocalized, sub-apical exocytosis, previously reported as a LatB response of oomycete hyphae (Bachewich and Heath, 1998; Gupta and Heath, 1997), thus indicating similarities in hyphal functions between phylogenetically very distant species. However, the delocalized synthesis in the oomycete produced more wall material deposited throughout the sub-apical regions (Bachewich and Heath, 1998), which may relate to the oomycete wound response system (Levina et al., 2000). However, in both oomycetes and eufungi, sub-apical swelling presumably involves release of wall softening enzymes, postulated to be components of the normally apically exocytosed vesicles (Bartnicki-Garcia and Lippman, 1972). 4.3. Actin plaques Contrary to previous reports in which the hyphae of fungi contain plaques clustered over their extreme tips (reviewed in Heath, 1990, see also Akashi et al., 1994; Ayad-Durieux et al., 2000; Fischer and Timberlake, 1995; Li and Heath, 1994; Tsukamoto et al., 1996), the absence of such plaques at the extreme tips of G. candidum hyphae indicates that they are inessential for normal tip growth. A similar absence was reported by Roberson (1992), Srinivasan et al. (1996), and Torralba et al. (1998). Thus, it is unlikely that their presence in young branches indicates an obligatory role there either. Further evidence against a direct role of plaques in tip growth is that growth and SPK actin, are both rapidly disrupted at 0.5 lg ml1 LatB, whereas plaques survive prolonged incubation in at least 5.0 lg ml1 . Most likely, their presence in slow (branches), but not fast (leading hyphae), growing tips is due to the time required for their assembly. Consistent with this, growth rates of 2 lm min1 suggest their assembly by 3–4 lm behind the leading tips reqiures 2 min. Following LatB growth reduction, they are absent at 1 min, but occupy entire tips by 5 min, i.e., formation takes between 1 and 5 min. While plaques are superficially similar in diverse species, their composition must differ. In Saccharomyces, plaque elimination was only demonstrated at 80 lg ml1 LatB (Ayscough et al., 1997), G. candidum, requires >5.0 lg ml1 and in the oomycete, Saprolegnia, <0.5 lg ml1 (Bachewich and Heath, 1998; Gupta and Heath, 1997), suggesting differences in composition (most likely actin binding proteins) and functions. In ascomycetes, and basidiomycetes, plaques probably correspond to filasomes (Bourett and Howard, 1991; Corr^ea and Hoch, 1993; Howard, 1981; Kanbe et al., 1989; Roberson, 1992; Srinivasan et al., 1996). Such is likely in G. candidum too, but the functions and behaviour of filasomes remain elusive. Oomycete plaques appear to have an adhesive role (Bachewich and Heath, 1997), but differences in sensitivity to LatB mentioned above, and the absence of filasomes in oomycetes, make
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functional comparisons between the species difficult. However, filasomes are adjacent to the PM, with which they could interact. 4.4. Phallotoxin staining Fungi seem to fall into two groups, those, like plants and animals, whose actin readily stains strongly with phallotoxins and those which remain essentially unstained using similar protocols (Table 4). The phalloidin binding domain of Saccharomyces actin critically involves amino acids 158, 177, and 179 (Belmont et al., 1999; Drubin et al., 1993), but these are conserved between staining and non-staining species (Table 4). Elsewhere in this region, there are no consistent differences between these two groups, except at 178 and 180. At 178, all non-staining species have a valine, whereas all staining species have either a leucine or isoleucine and at 180 the difference is methionine vs leucine. The sole exception to this trend is Absida, a mucoralean zygomycete which also has a methionine at 180 and is predicted to be RP positive. However, this prediction may be invalid, being based only on two Enteromophthoralean species (Butt and Heath, 1988; Heath, 1990) that are not closely related to the Mucorales. We conclude that differential RP staining is due to differential phalloidin binding to actin, most likely mediated by the differences at positions 178 and 180. The amino acids at 176, 178, and 180 underlie the arginine and aspartate that are totally conserved (Table 4) and critical to RP binding (Belmont et al., 1999; Drubin et al., 1993). Changes in these three amino acids would alter the disposition of the upper two and influence the phalloidin binding site. However, the changes are likely to be fairly small scale such that further modelling attempts to refine understanding of the phalloidin/actin interaction will not be definitive with current technology. While we have focussed on the amino acids previously identified as important in phallotoxin binding, comparing the rest of the sequences of Saccharomyces, Candida, Schizosaccharomyces, Schizophyllum, Suillius, Coprinus, and Puccinia with Aspergillus, Neurospora, and Trichoderma revealed other differential single changes: V45I, I75V, SGMT194A, ML227I, E259EKQ, A272G, Y279F, and A312S. The apparent differences in phallotoxin binding are most likely to have occurred during the evolution of the hyphal ascomycetes, because all known non-staining species (Magnaporthe [Bourett and Howard, 1992], Trichoderma [Czymmek et al., 1995], Sordaria [Thompson-Coffe and Zickler, 1993], and Neurospora and Aspergillus [Heath, unpublished]) are Pyrenomycetales or Plectomycetales (Alexopoulos et al., 1996), whereas those reported to stain are among the Saccharomycetales (Saccharomyces, e.g. [Adams and Pringle, 1984],
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Candida [Anderson and Soll, 1986], Ashbya [AyadDurieux et al., 2000] and presently G. candidum), Archiascomycetes (Schizosaccharomyces e.g. [Marks and Hyams (1985)]), Chytridiomycetes (Li and Heath, 1994), Zygomycetes (Butt and Heath, 1988; Heath, 1990), and Basidiomycetes (Hoch and Staples, 1983; Raudaskoski et al., 1988; Salo et al., 1989). Loss of phallotoxin binding may have occurred as one or two neutral mutations in the hyphal ascomycete line, a relatively recent event (Heckman et al., 2001).
Acknowledgments This work was supported by a gratefully acknowledged research grant (IBH) and scholarships (MB and GDG) from NSERC. Dr. A.P.J. Trinci very kindly suggested the use of, and supplied, G. candidum. The modeling observations were generously made by Dr. L. Donaldson, York University.
References Adams, A.E.M., Pringle, J.R., 1984. Relationship of actin and tubulin distribution to bud growth in wild-type and morphogenetic mutant Saccharomyces cerevisiae. J. Cell Biol. 98, 934–945. Akashi, T., Kanbe, T., Tanaka, K., 1994. The role of the cytoskeleton in polarized growth of the germ tube in Candida albicans. Microbiology 140, 271–280. Alexopoulos, C.J., Mims, C.W., Blackwell, M., 1996. Introductory Mycology. Wiley, New York. Anderson, J.M., Soll, D.R., 1986. Differences in actin localization during bud and hypha formation in the yeast Candida albicans. J. Gen. Microbiol. 132, 2035–2047. Ayad-Durieux, Y., Knechtle, P., Goff, S., Dietrich, F., Philippsen, P., 2000. A PAK-like kinase is required for maturation of young hyphae and septation in the filamentous ascomycete Ashbya gossypii. J. Cell Sci. 113, 4563–4575. Ayscough, K.R., Stryker, J., Pokala, N., Sanders, M., Crews, P., Drubin, D.G., 1997. High rates of actin filament turnover in budding yeast and roles for actin in establishment and maintenance of cell polarity revealed using the actin inhibitor latrunculin-A. J. Cell Biol. 137, 399–416. Bachewich, C.L., Heath, I.B., 1997. Differential cytoplasm-plasma membrane-cell wall adhesion patterns and their relationships to hyphal tip growth and organelle motility. Protoplasma 200, 71–86. Bachewich, C.L., Heath, I.B., 1998. Radial F-actin arrays precede new hypha formation in Saprolegnia: implications for establishing polar growth and regulating tip morphogenesis. J. Cell Sci. 111, 2005– 2016. Bartnicki-Garcia, S., Hergert, F., Gierz, G., 1989. Computer simulation of fungal morphogenesis and the mathematical basis for hyphal (tip) growth. Protoplasma 153, 46–57. Bartnicki-Garcia, S., Lippman, E., 1972. The bursting tendency of hyphal tips of fungi: presumptive evidence for a delicate balance between wall synthesis and wall lysis in apical growth. J. Gen. Microbiol. 73, 487–500. Belmont, L.D., Patterson, G.M.L., Drubin, D.G., 1999. New actin mutants allow further characterization of the nucleotide binding cleft and drug binding sites. J. Cell Sci. 112, 1325–1336.
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Bourett, T.M., Howard, R.J., 1991. Ultrastructural immunolocalization of actin in a fungus. Protoplasma 163, 199–202. Bourett, T.M., Howard, R.J., 1992. Actin in penetration pegs of the fungal rice blast pathogen, Magnaporthe grisea. Protoplasma 168, 20–26. Bracker, C.E., Murphy, D.J., L opez-Franco, R., 1997. Laser microbeam manipulation of cell morphogenesis in growing fungal hyphae. Proc. Int. Soc. Photo-Opt. Instrum. Engrg. 2983, 67– 80. Brunswick, H., 1924. Untersuchungen uber Geschlechts- und Kernverhaltnisse bei der Hymenomzetengattung Coprinus. Gustav Fischer, Jena. Butt, T.M., Heath, I.B., 1988. The changing distribution of actin and nuclear behavior during the cell cycle of the mite-pathogenic fungus Neozygites sp.. Eur. J. Cell Biol. 46, 499–505. Corr^ea, A., Hoch, H.C., 1993. Microinjection of urediospore germlings of Uromyces appendiculatus. Exp. Mycol. 17, 253–273. Czymmek, K.J., Bourett, T.M., Howard, R.J., 1995. Immunolocalization of tubulin and actin in thick-sectioned fungal hyphae after freeze-substitution fixation and methacrylate de-embedment. J. Microsc. 181, 153–161. Degousee, N., Gupta, G.D., Lew, R.R., Heath, I.B., 2000. A putative spectrin-containing membrane skeleton in hyphal tips of Neurospora crassa. Fungal Genet. Biol. 30, 33–44. Drubin, D.G., Jones, H.D., Wertman, K.F., 1993. Actin structure and function: roles in mitochondrial organization and morphogenesis in budding yeast and identification of the phalloidin-binding site. Mol. Biol. Cell 4, 1277–1294. Fevre, M., Rougier, M., 1982. Autoradiographic study of hyphal cell wall synthesis of Saprolegnia. Arch. Microbiol. 131, 212– 215. Fischer, R., Timberlake, W.E., 1995. Aspergillus nidulans apsA (anucleate primary sterigmata) encodes a coiled-coil protein required for nuclear positioning and completion of asexual development. J. Cell Biol. 128, 485–498. Gierz, G., Bartnicki-Garcia, S., 2001. A three-dimensional model of fungal morphogenesis based on the vesicle supply center concept. J. Theoret. Biol. 208, 151–164. Girbardt, M., 1957. Der Spitzenk€ orper von Polystictus versicolor (L.). Planta 50, 47–50. Girbardt, M., 1969. Die Ultrastruktur der Apikalregion von Pilzhyphen. Protoplasma 67, 413–441. Grove, S.N., Bracker, C.E., 1970. Protoplasmic organization of hyphal tips among fungi: vesicles and Spitzenk€ orper. J. Bacteriol. 104, 989–1009. Grove, S.N., Bracker, C.E., Morre, D.J., 1970. An ultrastructural basis for hyphal tip growth in Pythium ultimum. Am. J. Bot. 57, 245– 266. Grove, S.N., Sweigard, J.A., 1996. Apical cytoplasmic organization and polar growth in Rhizoctonia solani are modified by cytochalasins. WWW J. Biol. 2: Unstated. Available: http://epress.com/ w3jbio/vol2/grove/grove.htm. Gupta, G., Heath, I.B., 1997. Actin disruption by latrunculin B causes turgor-related changes in tip growth of Saprolegnia hyphae. Fungal Genet. Biol. 21, 64–75. Heath, I.B., 1987. Preservation of a labile cortical array of actin filaments in growing hyphal tips of the fungus Saprolegnia ferax. Eur. J. Cell Biol. 44, 10–16. Heath, I.B., 1988. Evidence against a direct role for cortical actin arrays in saltatory organelle motility in hyphae of the fungus Saprolegnia ferax. J. Cell Sci. 91, 41–47. Heath, I.B., 1990. The roles of actin in tip growth of fungi. Int. Rev. Cytol. 123, 95–127. Heath, I.B., 1995. Integration and regulation of hyphal tip growth. Can. J. Bot. 73 (Suppl. 1), S131–S139. Heath, I.B., 2000. Organization and function of actin in hyphal tip growth. In: Staiger, C.J., Baluska, F., Volkmann, D., Barlow, P.W.
(Eds.), Actin: a dynamic framework for multiple plant cell functions. Kluwer Academic Publishers, Dordrecht, pp. 275–300. Heath, I.B., 2001. Bridging the divide, cytoskeleton-plasma membrane-cell wall interactions in growth and development. In: Howard, R.J., Gow, N.A.R. (Eds.), The Mycota VIII: Biology of the Fungal Cell. Springer, Heidelberg, pp. 201–223. Heath, I.B., Gay, J.L., Greenwood, A.D., 1971. Cell wall formation in the Saprolegniales: cytoplasmic vesicles underlying developing walls. J. Gen. Microbiol. 65, 225–232. Heath, I.B., Greenwood, A.D., 1970. The structure and formation of lomasomes. J. Gen. Microbiol. 62, 129–137. Heath, I.B., Gupta, G., Bai, S., 2000. Plasma membrane-adjacent actin filaments, but not microtubules, are essential for both polarization and hyphal morphogenesis in Saprolegnia ferax and Neurospora crassa. Fungal Genet. Biol. 30, 45–62. Heath, I.B., Rethoret, K., Arsenault, A.L., Ottensmeyer, F.P., 1985. Improved preservation of the form and contents of wall vesicles and the Golgi apparatus in freeze substituted hyphae of Saprolegnia. Protoplasma 128, 81–93. Heckman, D.S., Geiser, D.M., Eidell, B.R., Stauffer, R.L., Kardos, N.L., Hedges, S.B., 2001. Molecular evidence for the colonization of land by fungi and plants. Science 293, 1129–1133. Herr, F.B., Heath, M.C., 1982. The effects of antimicrotubule agents on organelle positioning in the cowpea rust fungus, Uromyces phaseoli var. vignae. Exp. Mycol. 6, 15–24. Hoch, H.C., Staples, R.C., 1983. Visualization of actin in situ by rhodamine-conjugated phalloidin in the fungus Uromyces phaseoli. Eur. J. Cell Biol. 32, 52–58. Howard, R.J., 1981. Ultrastructural analysis of hyphal tip cell growth in fungi: Spitzenk€ orper, cytoskeleton and endomembranes after freeze- substitution. J. Cell Sci. 48, 89–103. Jackson, S.L., Heath, I.B., 1990. Evidence that actin reinforces the extensible hyphal apex of the oomycete Saprolegnia ferax. Protoplasma 157, 144–153. Kaminskyj, S.G.W., Heath, I.B., 1995. Integrin and spectrin homologues, and cytoplasm-wall adhesion in tip growth. J. Cell Sci. 108, 849–856. Kaminskyj, S.G.W., Heath, I.B., 1996. Studies on Saprolegnia ferax suggest the general importance of the cytoplasm in determining hyphal morphology. Mycologia 88, 20–37. Kanbe, T., Kobayashi, I., Tanaka, K., 1989. Dynamics of cytoplasmic organelles in the cell cycle of the fission yeast Schizosaccharomyces pombe. J. Cell Sci. 94, 647–656. Levina, N.N., Heath, I.B., Lew, R.R., 2000. Rapid wound responses of Saprolegnia hyphae depend upon actin and Ca2þ -involving deposition of callose plugs. Protoplasma 214, 199–209. Li, J., Heath, I.B., 1994. The behaviour of F-actin during the zoosporic phases of the chytridiomycete gut fungi Neocallimastix and Orpinomyces. Exp. Mycol. 18, 57–69. Lin, S., Spudich, J., 1974. Biochemical studies on the mode of action of cytochalasin B. J. Biol. Chem. 249, 5778–5783. L opez-Franco, R., Bracker, C.E., 1996. Diversity and dynamics of the Spitzenk€ orper in growing hyphal tips of higher fungi. Protoplasma 195, 90–111. Marks, J., Hyams, J.S., 1985. Localization of F-actin through the cell division cycle of Schizosaccharomyces pombe. Eur. J. Cell Biol. 39, 27–32. McDaniel, D., Roberson, R., 2000. Microtubules are required for motility and positioning of vesicles and mitochondria in hyphal tip cells of Allomyces macrogynus. Fungal Genet. Biol. 31, 233– 244. McDaniel, D.P., Roberson, R.W., 1998. c-Tubulin is a component of the Spitzenk€ orper and centrosomes in hyphal-tip cells of Allomyces macrogynus. Protoplasma 203, 118–123. McGoldrick, C.A., Gruver, C., May, G.S., 1995. myoA of Aspergillus nidulans encodes an essential myosin I required for secretion and polarized growth. J. Cell Biol. 128, 577–587.
I. Brent Heath et al. / Fungal Genetics and Biology 38 (2003) 85–97 Raudaskoski, M., Salo, V., Nini, S.S., 1988. Structure and function of the cytoskeleton in filamentous fungi. Karstenia 28, 49–60. Reynaga-Pe~ na, C.G., Bartnicki-Garcia, S., 1997. Apical branching in a temperature sensitive mutant of Aspergillus niger. Fungal Genet. Biol. 22, 153–167. Riquelme, M., Gierz, G., Bartnicki-Garcıa, S., 2000. Dynein and dynactin deficiencies affect the formation and function of the Spitzenk€ orper and distort hyphal morphogenesis of Neurospora crassa. Microbiology 146, 1743–1752. Riquelme, M., Reynaga-Pe~ na, C.G., Gierz, G., Bartnicki-Garcia, S., 1998. What determines growth direction in fungal hyphae? Fungal Genet. Biol. 24, 101–109. Roberson, R.W., 1992. The actin cytoskeleton in hyphal cells of Sclerotium rolfsii. Mycologia 84, 41–51. Runeberg, P., Raudaskoski, M., Virtanen, I., 1986. Cytoskeletal elements in the hyphae of the homobasidiomycete Schizophyllum commune visualized with indirect immunofluorescence and NBDphallacidin. Eur. J. Cell Biol. 41, 25–32. Salo, V., Niini, S.S., Virtanen, I., Raudaskoski, M., 1989. Comparative immunocytochemistry of the cytoskeleton of filamentous fungi with dikaryotic and multinucleate hyphae. J. Cell Sci. 94, 11– 24.
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Srinivasan, S., Vargas, M.M., Roberson, R.W., 1996. Functional, organizational, and biochemical analysis of actin in hyphal tip cells of Allomyces macrogynus. Mycologia 88, 57–70. Thompson-Coffe, C., Zickler, D., 1993. Cytoskeletal interactions in the ascus development and sporulation of Sordaria macrospora. J. Cell Sci. 104, 883–898. Tinsley, J.H., Minke, P.F., Bruno, K.S., Plamann, M., 1996. p150glued , the largest subunit of the dynactin complex, is nonessential in Neurospora but required for nuclear distribution. Mol. Biol. Cell 7, 731–742. Torralba, S., Raudaskoski, M., Pedregosa, A.M., Laborda, F., 1998. Effect of cytochalasin A on apical growth, actin cytoskeleton organization and enzyme secretion in Aspergillus nidulans. Microbiology 144, 45–53. Treves, S., Di Virgilio, F., Vaselli, G.M., Pozzan, T., 1987. Effect of cytochalasins in cytosolic-free calcium concentration and phosphoinositide metabolism in leukocytes. Exp. Cell Res. 168, 285– 298. Tsukamoto, M., Yamamoto, M., Tanabe, S., Kamada, T., 1996. Cytochalasin-E-resistant mutants of Coprinus cinereus: isolation and genetic, biochemical, and cytological analyses. Fungal Genet. Biol. 20, 52–58.
The interrelationships of actin and hyphal tip growth in the ascomycete Geotrichum candidum I. Brent Heath,* Michael Bonham, Ali Akram, and Gagan D. Gupta Biology Department, York University, 4700 Keele Street, Toronto, Ont. M3J1P3, Canada Received 30 August 2001; accepted 30 July 2002
Abstract Geotrichum candidum is unusual among reported hyphal ascomycetes in that its hyphae readily stain with phalloidin to reveal actin concentrated in the Spitzenk€ orper (SPK) and plaques associated with the plasma membrane (PM). Loss of SPK actin, but not the PM plaques, following latrunculin B treatment produces tip swelling, consistent with actin restraining tip morphology or localizing vesicle exocytosis. Tip morphogenesis may also involve a spectrin-like protein which concentrates at the apical PM in plaques unassociated with the actin plaques. Branch formation occurs with growth rates initially about 20% those of leading tips, and does not involve a morphologically detectable SPK, nor SPK-like actin ensembles, indicating the dispensibility of this structure in tip growth. Surprisingly, new tubular tips can form in the continued presence of latrunculin, consistent with alternative cellular systems, such as the spectrin-like protein, substituting for actin’s critical functions. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Geotrichum candidum; Actin; Tip growth; Latrunculin; Spitzenk€ orper; Cytoskeleton; Exocytosis; Spectrin
1. Introduction Hyphal tip growth involves many intimately related processes which must be very precisely regulated to produce the typically uniform diameter tube characteristic of hyphal organisms. Among these processes are regulation of tip extensibility and localized vesicle exocytosis. The vesicles deliver cell wall components and plasma membrane (PM) (Fevre and Rougier, 1982; Grove and Bracker, 1970; Grove et al., 1970; Heath et al., 1971; Heath et al., 1985) and possibly also cell wall softening enzymes (Bartnicki-Garcia and Lippman, 1972). A common, but not universal, component of growing tips is the Spitzenk€ orper (SPK) (Brunswick, 1924; Girbardt, 1957), the behaviour of which correlates well with tip growth (Bracker et al., 1997; L opez-Franco and Bracker, 1996; Reynaga-Pe~ na and Bartnicki-Garcia, 1997; Riquelme et al., 1998, 2000) and which may play some role in regulating exocytosis (Bracker et al., 1997). Vesicles aggregate as part of the SPK (Girbardt, * Corresponding author. Fax: +1-416-736-5698. E-mail address: [email protected] (I. Brent Heath).
1969) and it is possible to model tip morphogenesis on the assumption that it regulates the sites of exocytosis by directing the sites of impact of the vesicles at the PM (Bartnicki-Garcia et al., 1989; Gierz and BartnickiGarcia, 2001). The composition of the SPK is largely unknown. Its observed movements (L opez-Franco and Bracker, 1996; Reynaga-Pe~ na and Bartnicki-Garcia, 1997) and postulated roles in vesicle behaviour indicate a force generating component. Actin (as well as microtubules [McDaniel and Roberson, 1998, 2000; Riquelme et al., 1998]) may be part of such a component, but there are conflicting reports concerning its presence. It has been reported in a few species (Bourett and Howard, 1991; Czymmek et al., 1995; Degousee et al., 2000; Heath et al., 2000; McDaniel and Roberson, 2000; Srinivasan et al., 1996), but there are more reports of actin localizations in species likely to contain a SPK, yet presenting no images interpretable as SPK actin (Akashi et al., 1994; Anderson and Soll, 1986; Ayad-Durieux et al., 2000; Fischer and Timberlake, 1995; Heath, 1990; Roberson, 1992; Runeberg et al., 1986; Salo et al., 1989; Tinsley et al., 1996; Torralba et al., 1998; Tsukamoto
1087-1845/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 8 7 - 1 8 4 5 ( 0 2 ) 0 0 5 1 1 - X
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et al., 1996). In the latter reports, the tip-rich actin predominantly forms plaques in the peripheral cytoplasm. The relationship, if any, between plaques and SPK is obscure, as are the functions and behaviour of the plaques. In species lacking a SPK, such as the oomycetes, the apical organization of actin differs from the eufungi. It has been suggested that this actin contributes to a membrane skeleton that regulates the extensibility, and thus shape, of the hyphal tips (Gupta and Heath, 1997; Heath, 1990, 1995; Jackson and Heath, 1990; Kaminskyj and Heath, 1996). A similar actin-containing membrane skeleton, with a similar function, may exist in eufungi (Heath, 2000, 2001; Kaminskyj and Heath, 1996). Evidence for membrane skeleton-based morphogensis is hyphal tip swelling following actin disruption by cytochalasins (reviewed in Grove and Sweigard, 1996; Heath, 1990), but cytochalasins are known to (Lin and Spudich, 1974), or may (Treves et al., 1987), lack specificity for actin. Latrunculins, such as latrunculin B (LatB), are well characterized as targeting actin in fungi (Ayscough et al., 1997). Although other targets have not been ruled out, their effect at lower concentrations than cytochalasins supports a direct morphogenic role for actin in an oomycete (Gupta and Heath, 1997). They have not been employed in any extensive analysis of eufungi, although preliminary observations of Neurospora crassa indicated responses differing from those of oomycetes (Heath et al., 2000). Similar effects from different inhibitors sharing a known target are strong support for a role for that target in the tip growth process. Equally, different responses to the same inhibitor in different species would argue for differences in the contributions of the target. The purpose of this study was to further investigate the possible roles of actin in tip growth of a eufungus. Geotrichum candidum was initially selected because it is an ascomycete which has an unusual feature of normally producing its leading branches in a predictable location immediately subjacent to the most apical septum, thus facilitating analysis of branch initiation. Early observations demonstrated a fortuitous bonus in that its hyphal actin readily stained with rhodamine-labelled phalloidin (RP). The results obtained further refine our understanding of the organization and functions of hyphal actin and the SPK.
2. Materials and methods 2.1. Organism and cultures Geotrichum candidum (Galactomyces geotrichum (Butler & Petersen) Redhead & Malloch) was generously supplied by Dr. A.P.J. Trinci, University of Manchester. All cultures were grown on a complex medium designated OM (Heath and Greenwood, 1970). Unless noted
to the contrary, hyphae were grown on dialysis membrane overlying OM agar at 22 °C. Typically 1 cm2 rectangles of membrane plus hyphae were cut from edges of colonies, allowed to recover on the agar for 30– 60 min then either mounted in flow-through chambers (Heath, 1988) for observation of living hyphae, or irrigated with various treatment solutions while lying on top of the agar (with frequent addition of fresh medium to enhance constant drug concentration and prevent drying) or in the bottom of Petri dishes. LatB (Sigma, Oakville, ON) was made as a 1 mg ml1 stock solution in dimethyl sulphoxide (DMSO) and freshly diluted in OM to give working concentrations of 0.1, 0.5, 1.0, 5.0, and 10.0 lg ml1 (1.0 lg ml1 ¼ 2:52 lM) LatB, and 0.01– 1.0% DMSO. Control experiments using 1.0% DMSO alone showed no observed effects on hyphae. 2.2. Growth rates and hyphal swelling Hyphae were mounted in the growth chambers with 15 ll of OM and allowed to recover for about 10 min. To observe the effects of LatB, growing tips were videotaped for 6–8 min, then 50 ll of LatB was drawn across the chamber, with continuous recording, and the hyphae were recorded for a further 5–6 min. Growth was measured at 1 or 2 min intervals with a ruler on the screen at a magnification of 1 mm ¼ 0.8 lm. Growth rates were determined from the best fit lines over 5 or 6 min before and after addition of LatB. In other experiments, growth rates were determined from prints taken at various time intervals at magnifications where 1 mm ¼ 0.56–1.1 lm. Growth rates were also measured from colony diameters in liquid OM in Petri dishes. Hyphal swelling was measured from thermal prints from video recordings for up to 20 min after LatB addition, at a magnification where 1 mm ¼ 2 lm. Hyphae of both G. candidum and N. crassa (Heath et al., 2000) observed and recorded for the presence of SPK (Figs. 1–8) were gently detached from the dialysis membrane into liquid OM, mounted under a coverslip and allowed to recover growth prior to observation under exactly the same conditions as each other. 2.3. Cytochemistry Hyphae to be stained only with rhodamine-labelled phalloidin (RP, Molecular Probes, Eugene, OR) were fixed by the addition of 1 ml of 2% formaldehyde and 0.025% glutaraldehyde in 60 mM PIPES buffer [pH 7.0] containing 5 mM MgCl2 , 5 mM EGTA, and 0.05% Triton X100 to the hyphae in their growth environment for 4 min. The hyphae were then rinsed in 4 ml of the above buffer solution (without Triton or fixatives), transferred to a humid Petri dish with 5 or 10 ll of 5 lg ml1 RP in PIPES buffer per colony and left for 15– 25 min. The colonies were then rinsed 2–3 in 150 ll
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of PIPES buffer, removed from their dialysis membrane (if present) and mounted in 30 ll of Prolong (Molecular Probes) mounting medium.
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Immunocytochemical staining of hyphae employed 200 ll of the same fixation solution as above, followed by a 30 s rinse in 60 mM PIPES buffer, cell wall digestion in 9 mg ml1 Dreiselase (Kyama Hakh Kogyo, Tokyo) and 1 mg ml1 Novozyme-234 (InterSpex Products, Foster City, CA) in 30 mM MES (pH 5.5) containing 1% BSA, 5 mM MgSO4 , and 5 mM CaCl2 for 5–7 min, a rinse in PIPES, incubation in polyclonal rabbit antichicken a and b spectrin (lot #054H4830, used at 1/100 in 60 mM PIPES [pH 7.5] with 0.5% BSA and 0.01% NaN3 , Sigma) for 1 h, rinsing for 30 s in 60 mM PIPES buffer, incubation in 200 ll of FITC-labelled goat antirabbit secondary antibody (1/600 in above antibody solution, Cedarlane, Hornby, ON) for 30 min, rinsing in PIPES, and mounting in Prolong, after removal from the dialysis membrane. For double staining with antibody and RP, 10 ll of RP was added for the last 15 min of the secondary antibody incubation. All fixation and staining procedures were carried out at 22 °C. Hyphae were observed with Nomarski differential interference contrast (DIC), phase contrast, or epifluorescence optics on a Reichert Polyvar microscope using B1 (FITC) and G2 (rhodamine) filter combinations. Objectives were 40 and 100 with NAs of 0.9 and 1.32, respectively. Images were recorded on film (Kodak T400 CN), VHS tape or with a digital camera (MicroMax 1300Y, Roper Scientific, Trenton, NJ). All
b Figs. 1–17. Median DIC optical sections of a hyphal tip of N. crassa growing at 6.2 lm min1 showing clear SPK (arrow) (Fig. 1), compared with similarly imaged tips of G. candidum growing at similar rates (6.4, 1.8, and 2 lm min1 , respectively) and showing no similarly clear SPK (Figs. 2–4). Phase contrast images of growing G. candidum hyphae similarly show no consistent evidence of a phase dark SPK among the diversity of tip cytoplasmic patterns (Figs. 5a–e). Branches form proximal to the most apical septa and grow forward much slower than hyphal tips. In the sequence seen in Fig. 6 (a and b, t ¼ 0, 13 min), the rate was 0.23 lm min1 , whereas in a similar sequence (Fig. 7a and b, t ¼ 0, 17.4 min) it was 0.5 lm min1 . Septal ingrowth was observed in a septum developing adjacent to an already formed septum (Fig. 8a– c, t ¼ 0, 25, 43 min). It had already started to grow (a) and closed completely (c). For all Figs. 1–8, the hyphae were mounted in the culture chambers without dialysis membrane. In control hyphae, comparable to those seen in Figs. 2–8 but stained with RP, tips showed prominent SPK-like staining subtended by peripheral plaques, which were typically rare in the most apical 3–4 lm (Figs. 9, 11, 12, 14, and 15). In straight hyphae, the SPK-like staining was central (Figs. 9, 11, and 15), whereas in curved hyphae, it was located eccentrically, towards the direction of growth (Figs.11 and 12). An oblique, near face view of three adjacent tips emphasizes the location of the SPK-like staining and the surrounding region clear of plaques (Fig. 14). In contrast to the leading hyphal tips, branch tips consistently lacked any sign of SPK-like staining, instead the young tips were covered with plaques over the entire tip (Figs. 16 and 17). Two tips forming at slightly different stages from adjacent septa are seen in Fig. 17. Mature septa are accompanied by a double layer of plaques (Fig. 10), whereas a presumed forming septa, viewed obliquely, showed a ring of filaments (Fig. 13). Scale on Fig. 2 also for Figs. 3–5 and that on Fig. 16 for Figs. 14 and 15. Scale bars in all micrographs ¼ 5 lm.
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cytochemical conclusions are based on subjective observations of many hyphae in one or more colonies, and recorded representative images from those hyphae. All figures are of G. candidum and all scale bars indicate approximately 5 lm unless noted to the contrary.
3. Results 3.1. Control hyphae The dominant hyphal growth pattern is straight tapered leading tips (mean diameter at the base of the taper ¼ 4.7 lm 1:6, n ¼ 92) (Figs. 2–5) which grew at a mean of 1.3 lm min1 (0.79, range 0.1–6.4, n ¼ 140) in the growth chambers and somewhat faster (1.96 and 2.08 lm min1 , two colony radii) in Petri dishes. Variation in growth rates did not correlate with hyphal diameters. The hyphae lack a detectable SPK when examined with DIC (Figs. 2–4), using the same optics that reveal clear SPK in Neurospora. (Fig. 1) (Heath et al., 2000), or with phase contrast (Fig. 5). This statement is also based on unrecorded observations of many growing hyphae with growth rates covering the full range observed. The first branch normally forms immediately subjacent to the first septum (Figs. 6 and 7), but the time of initiation is variable. Branch length did not correlate (r2 ¼ 0:09, n ¼ 33) with the length of the apical cell (a proxy measure of both the age of the septum and the apical cell) and the earliest detectable branches were on hyphae with a 7% standard deviation in the latter measurement (n ¼ 5). Branches between 4.4 (earliest recorded) and 12.2 lm grew much slower (mean 0.29 lm min1 0:18, n ¼ 3; Figs. 6 and 7) than hyphal tips and did not accelerate over a maximum of 17 min of observation. In one simultaneous recording, the branch also grew slower than its associated main tip (0.15 vs 0.7 lm min1 ). Recorded branches were also thinner than hyphal tips (mean diameter 3.75 lm 0:45, n ¼ 3) (Figs. 6 and 7). On one fortuitous occasion, a septum was observed during its growth (Fig. 8). When first observed, its aperture diameter was 4.6 lm and the hyphal outer diameter was 7.2 lm. Over 42 min it grew steadily centripetally at an edge advance of 0.05 lm min1 , while the tip of the same hypha extended at 0.28 lm min1 . RP staining is predicted to stain polymeric actin (discussed in references in the Introduction and Discussion), an assumption in the following results. Actin in leading hyphal tips concentrates in a structure approximately the same size and location as expected of a SPK (Figs. 9, 11, 12, 14, 15, and 18). Hyphae apparently changing growth direction at the time of fixation contained eccentrically located SPK-like actin towards the new growth direction (Figs. 11 and 12). The SPK-like actin is the only detectable concentration in the most
apical 3–4 lm (Figs. 9, 11, 12, 14, 15, and 18). Below this, there is a ring of fine (0.5 lm diameter) PM-associated actin plaques whose concentration declines steeply sub-apically to a uniform low level throughout the rest of the hyphae. The sub-apical plaques were variable and not always detectable. Sub-apically there are also occasional rings of actin which seem to coincide with the very early stages of septum formation (Fig. 13). At mature septa, the actin forms two plates of plaques (Fig. 10), apparently corresponding to the PM on each side. There were also occasionally fine actin filaments, often associated with septa, parallel to the long axis of the hyphae. Actin in young branches (length < hyphal diameter, 8 documented, many others observed), apparently form-
Figs. 18–19. Anti-spectrin staining of hyphal tips. In Fig. 18, a group of tips (DIC image in e) were double stained with RP (a, c) and antispectrin antibody (b, d) and imaged at predominantly median (a, b) and surface (c, d) focal planes. The apical SPK-like and plaque staining characteristic of growing tips clearly differs from the peripheral plaques of spectrin staining that occur over the entire apex. Careful comparison of the actin and spectrin images shows that the plaques are not coincident. In Fig. 19, a spectrin-stained tip plasmolysed slightly during processing and shows that the peripheral plaques have retracted with the PM, leaving a few remaining on strands of PM extending out to the cell wall (arrows).
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ing at the time of fixation, was never concentrated into SPK-like organization. All contained only tip-high concentrations of plaques over their entire tips (Figs. 16 and 17). The anti-spectrin antibody will, for convenience, be described as localizing spectrin, which does not co-localize with actin. It is exclusively at the PM, forming fine (0.5 lm diameter) patches covering the entire apex; either in uniform density along the PM, or a
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slightly tip-high gradient (Fig. 18). Where both actin and spectrin are co-localized, the similarly sized plaques and patches are mostly not superimposable (Fig. 18). In hyphae inferred to be growing at the time of fixation, because they contain SPK-like actin, the spectrin covers the entire tip (Fig. 18). The spectrin patches do not appear to be located in or on the cell wall because they retract with the PM during plasmolysis (Fig. 19). 3.2. Latrunculin B effects 3.2.1. Actin disruption Actin disruption was primarily analyzed in 1 lm ml1 LatB, the concentration most effective in inducing swelling and comparable to other concentrations in early effects on tip growth. Changes in apical actin organization were observed as early as 1 min after treatment. Initially, the plaques retained their normal exclusion from the most apical 3– 4 lm, but there was no SPK-like staining (Fig. 20). These tips appeared to be rather more blunt than normal growing tips, but swelling only became evident by 5 min, and then it was not as extensive or consistent under the culture regime used for these hyphae relative to that used for the quantitative results discussed below. In all hyphae observed after >5 min in LatB, the tips and young branches contained plaques which were PMassociated and most concentrated at the extreme tips (Figs. 27, 22–24). No SPK-like staining was observed. Sub-apically the plaques appeared normal. At 0.5 and 5.0 lg ml1 , actin patterns were only observed after >5 min and in all cases, irrespective of the degree of hyphal tip swelling, the patterns were the same as those of the >5 min hyphae described above. Actin patterns were not investigated for 0.1 and 10 lg ml1 .
b Figs. 20–32. The effects of LatB on the actin organization (RP images in Figs. 20–24, 27) and morphology of hyphal tips. Tips treated with 1 lg ml1 for 1 (Fig. 20), 5 (Figs. 21 and 22), and 10 (Figs. 23 and 24) min show that even at this earliest time, the SPK-like staining is lost, but the plaques retain their pre-treatment sub-apical distribution, remaining absent from the most apical 3–4 lm (seen in the series of focal planes extending from the upper through median to lower surface in Fig. 20a–d). At later times, the plaques extend over the entire apices (Figs. 21–24), as seen especially in the three focal planes of the adjacent hyphae in Fig. 23a–c. Application of 5 lg ml1 (Figs. 25–32) for 22 (Fig. 27), 60 (Figs. 25 and 26), and 120 (Figs. 28–31) min produces swollen tips which rapidly attain a maximum diameter and contain peripheral actin plaques located over the entire apex (Fig. 27). However, after prolonged incubation in the drug, localized new outgrowths are produced from the swollen regions (Figs. 29–31) and branches become swollen (Fig. 28), a feature not observed at earlier times. After 120 min in the drug followed by its replacement with normal medium for 60 min, numerous tubular outgrowths (branches) are produced, primarily from the swollen apical regions (Fig. 32). Scale on Fig. 27 also for Figs. 26, 28–30, and 32.
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3.2.2. Tip expansion To explore previously reported (in another species, (Gupta and Heath, 1997)) transient growth acceleration upon addition of LatB, we compared the rate of growth in the 2 min interval prior to LatB addition with that 2 min after. This was the shortest interval capable of giving a consistent and reliable measure of hyphal extension. In both controls and LatB treatments there was a transient reduction of extension in about 70% of hyphae (Fig. 33; Table 1). In controls, but not LatB, this was followed by increased rates in the subsequent 4 min, so that after 5– 6 min treatment there was a concentration-dependent reduction in forward growth rate, as shown in Table 2. As forward growth slowed, hyphae swelled sub-apically to approximately apical spheres (Figs. 25–31). Swelling was time and concentration-dependent (Fig. 34) and involved both leading hyphal tips and short branches (Fig. 28). At all concentrations it was initially rapid then slowed as maximum swelling was approached (Fig. 34). The initial rate of swelling did not correlate with either pre-addition growth rate or hyphal diameter, but was very sensitive to LatB concentration, peaking at 1.0 lg min1 (Fig. 34). Swelling may involve surface area (cell wall and PM) synthesis as well as shape change. To separate these components, we calculated the predicted surface increase,
based on the mean control rate of tip growth and hyphal diameter, and compared this with the observed increase based on the diameter of the swollen tips (Table 3). The ratio between these values would be 100% if the normal rate of increase was maintained and only the shape changed, but in fact the ratios for 0.5, 1.0, and 5.0 lg ml1 were initially higher, dropping below 100% after 10– 15 min (Fig. 35). Thus initial swelling was due to either greater than predicted surface synthesis or swelling with the normal rate of synthesis. Conversely, both 0.1 and 10.0 lg min1 were below 100% at all times, indicating less than predicted synthesis or expansion (Fig. 35). The maximum swollen diameter was about 2–2.5 times normal hyphal diameter, both in slide chambers and Petri dishes, irrespective of incubation time or LatB concentration. The only indication of greater ‘‘expansion’’ was occasional tip bursting. In the slide chambers, only hyphae incubated in 5 lg ml1 burst; by 20 min, 40% had done so. Among the Petri dish cultures, bursting was observed rarely in 0.5 lg ml1 , more frequently in 1.0 lg ml1 and maximally in 5 lg ml1 . Bursting frequency in the dish cultures was estimated at about 30% in 5 lg ml1 by 60 min, comparable to the frequency measured in the slide chambers. In both slide chambers and Petri dishes, hyphae apparently adapted to the continual presence of the drug.
Table 1 Growth rate changes in first interval after adding LatB Treatment
LatB
Controls
Increased 1
Unchanged
Decreased
Increased
Unchanged
Decreased
0.1 lg ml 0.5 lg ml1 1.0 lg ml1 5.0 lg ml1 l 10.0 lg ml1
0 1 0 0 0
9 3 1 1 4
3 8 7 9 20
2 1 0 0 2
1 0 2 4 1
4 6 6 7 3
Totals Total (%)
1 2
18 27
47 71
5 13
8 21
26 67
Note. Examples of these change designations are given in Fig. 33. Sensitivity of the observations is indicated by the fact that during the 2 min interval after adding LatB, assuming the control growth rate of 1.62 lg ml1 , one would see length change of 3.24 lm ¼ 4 mm on the screen. Even for 10 lg ml1 , which had the slowest mean growth rate after addition, one would get 0:59 2 lm ¼ 1:5 mm, which was easily measurable. Table 2 Growth rates before and after addition of LatB Treatment
Rate before (lm min1 )
Rate after (lm min1 )
Rate reduction after LatB (%)
n
‘‘p’’ value
Outgrowth time (min)
Control 0.1 lg ml1 0.5 lg ml1 1.0 lg ml1 5.0 lg ml1 l 10.0 lg ml1
1:62 0:48 0:89 0:29 1:27 0:29 1:60 0:60 2:04 1:19 0:95 0:30
1:62 0:48 0:66 0:10 0:82 0:31 0:65 0:64 0:67 0:64 0:59 0:17
0 26 35 59 67 38
39 12 12 8 10 24
>0.5 <0.005 <0.01 <0.01 <0.005 <0.001
na 5:8 1:2 11:6 2:7 17:3 5:9 19:5 0:6 na
Note. Growth rates were determined by measuring changes in hyphal length at 2 min intervals over 5 or 6 min before and after the addition of LatB and calculating the mean rates from the linear regression lines. Values are means SD. Measurement accuracy was found to be approximately 1 mm on the sreen ¼ 0.8 lm. Separate controls were performed for each concentration and found by paired sample t test not to differ between before and after, hence all were combined as a single control. Before and after rates were compared by paired sample t test and gave the presented ‘‘p’’ values. ‘‘Outgrowth time’’ indicates the time taken to form a new tip from swollen tips in the continued presence of LatB. These means increased linearly with the log of LatB concentration (r2 ¼ 0:926).
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In slide chambers, after tip swelling, one or more new tips (termed outgrowths), with diameters similar to normal tips, were produced (Figs. 29–31). Times for
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outgrowth formation increased linearly with the log of LatB concentration (Table 1). No recovery was observed in 10 l g ml1 , possibly due to observation for less
Figs. 33–35. Growth responses of hyphae to addition of LatB. Representative growth curves (Fig. 33) of three individual hyphae followed before and after the addition (indicated by arrows) of 0.1 (‘‘unchanged’’ and ‘‘slowed’’) and 0.5 (‘‘increased’’) lg ml1 LatB. The designations of changes, or otherwise, in growth rate after LatB addition do not indicate statistically significant changes, but show the criteria for the designations in Table 1. In Fig. 34, the mean percent by which individual hyphae swelled in different concentrations of LatB (bold underlined numbers, in lg ml1 ) is shown, with the sample sizes (plain numbers, 8 at all times for 1.0 lg ml1 ) indicated at each time point. The ‘‘B’’ numbers on the 5 lg ml1 line indicate the number of hyphae which burst between time points, thus explaining the drop in the mean curve. Standard deviations for each data point varied between 29–42 (0.1 lg ml1 ), 25–49 (0.5 lg ml1 ), 25–52 (1.0 lg ml1 ), 16–50 (5.0 lg ml1 ), and 4–43 (10.0 lg ml1 )%. Fig. 35 plots the O/P% swelling parameter from Table 3 over time at the different concentrations. If swelling were due solely to the unrestrained (i.e., not in the normal tubular shape) formation of the predicted amount of new cell surface at the indicated time intervals, then the ‘‘predicted’’ line would be seen. Values above the ‘‘predicted’’ line represent more surface formation than predicted by pre-drug-application growth rates and those below indicate less expansion. Thus, at the intermediate concentrations of 0.5 and 1.0 lg ml1 (and probably insignificantly at 5.0), the initial rate of swelling was greater than predicted by normal growth rates, whereas at the lower and higher concentrations there was never the predicted rate of expansion, and at all concentrations, the rate of swelling fell below predictions by 10–15 min after drug addition.
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cies it was only observed in 12% of tips (Srinivasan et al., 1996). The strong and consistent SPK-like RP staining in G. candidum, and its sensitivity to LatB, supports its reality as a major SPK component. The negative reports most likely indicate actin’s lability. The specificity of LatB (Ayscough et al., 1997) and its rapid disruption of the SPK suggests that actin is important to SPK integrity and functions. The latter may include SPK motility (L opez-Franco and Bracker, 1996) for which actin is well suited. Myosin, the most common type of force generating protein interacting with actin, is also concentrated in hyphal tips, at least of Aspergillus (McGoldrick et al., 1995). Another putative function is in vesicle distribution. Vesicles cluster around the SPK (Grove and Bracker, 1970), hyphal morphogenesis can be modeled assuming that the SPK functions in vesicle distribution (Bartnicki-Garcia et al., 1989; Gierz and Bartnicki-Garcia, 2001) and SPK manipulation can relocate expansion, most likely indicative of altered vesicle distribution (Bracker et al., 1997). The limited data implicating actin in fungal vesicle transport has been recently reviewed (Heath, 2000). While actin-based SPK organization is likely, it may not be the only structural component since SPK disruption with cytochalasin leaves a residual fibrillar core (Grove and Sweigard,
than 25 min. Similar recovery occurred in Petri dishes, but took longer since, at 5 lg ml1 (the concentration monitored in detail) the outgrowths appeared between 30 and 40 min, compared with 20 min in slide chambers. In Petri dish cultures, outgrowths remained pointed, but did not grow for up to 120 min, although by 180 min some were swollen. Cessation of tip growth and subsequent swelling were fully reversible. Colonies inhibited for 180 min in Petri dishes, then transferred to drug-free medium for 60 min, produced branches with normal diameters and morphology (Fig. 32). The rate of tip growth, calculated from the extensions beyond the swollen regions in 60 min, was 1.8 lm min1 (n ¼ 3) in 0.5 lg ml1 , comparable to normal rates, but only 0.22 lm min1 (n ¼ 9) in 5.0 lg ml1 . The slower rate may reflect the untested possibility of a longer lag time prior to regrowth.
4. Discussion 4.1. SPK actin As listed in Section 1, there is uncertainty over the presence of actin in the SPK. Even in one positive spe-
Table 3 Surface area increases after adding LatB Time after adding LatB (min) 0 a
Length increase ðlmÞ 0.1 lg ml1
5 8.1 2.51 92 68 74
10
15
20
16.2 2.65 183 78 42
24.3 2.8 275 88 32
32.4 2.9 366 96 26
rb Pc Od O/P%
1.8
0.5 lg ml1
r P O O/P%
2.05
3.3 104 124 119
4.05 208 193 93
4.2 312 209 67
4.3 417 219 56
1.0 lg ml1
r P O O/P%
2.05
3.65 104 154 148
4.55 208 247 118
4.9 312 289 93
5.15 417 320 77
5.0 lg ml1
r P O O/P%
3.85
4.55 196 214 109
5.55 392 341 87
5.35 588 313 53
—
r P O O/P%
2.1
2.2 107 47 44
2.65 214 74 35
2.9 321 92 29
3.2 428 115 27
1
10.0 lg ml
a
— — —
Length increase calculated from average control growth rate from Table 1 multiplied by time. Mean radius measured at indicated time in lm. c Predicted increase in surface area based on mean diameter prior to LatB addition multiplied by predicted increase in length (as shown in footnote a). d Observed surface area based on observed diameter sphere less basal circle determined from cross-sectional area of hyphae prior to LatB addition. b
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1996). Assuming cytochalasin and LatB disruption are similar, the absence of a residual RP-positive core in G. candidum suggests the core contains other fibrils. In spite of reported (see Section 1) tight relationships between SPK and hyphal growth, present observations show that its normal actin organization is dispensable for initiation and production of tubular hyphae. Renewed pointed outgrowths, and branch formation, in the presence of sufficient LatB to disrupt hyphal growth and cause loss of SPK actin in the first place indicate this point. This result could be explained by ‘‘adaptation’’ to
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LatB, although the facts that the outgrowths cease further growth and much later swell argue against this possibility. More compelling is the absence of detectable SPK-like actin in forming and young branches and the substitution of apical plaques in its place. The growth rates of the hyphal outgrowths in LatB, and normal branches, are much lower than those of leading tips. We suggest that the normal SPK actin complement is one end of a spectrum of actin recruitment and organization facilitating fast tip growth; alternative smaller organizations suffice to initiate and sustain slow growth.
Table 4 Variations in amino acid sequences in the primary phalloidin-binding domain of actin
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4.2. Actin as morphogen The net-like apical actin in oomycete hyphae (Heath, 1987), which lack a SPK, apparently functions, at least in part, to mechanically regulate extension of the hyphal tip (Gupta and Heath, 1997; Heath, 1995; Jackson and Heath, 1990; Kaminskyj and Heath, 1996). SPK actin in G. candidum is not well organized for such a role, but may not be the only apical actin (reviewed in Heath, 2000, 2001). We suggest that SPK actin is the focus of a less concentrated radial array connected to a membrane skeleton containing both actin and a spectrin-like protein (Degousee et al., 2000). This model provides a mechanism for SPK mobility, and vesicle distribution, as well as a mechanical role in tip morphogenesis. The concentration of actin plaques at the shoulders of the growing tips is well suited to anchor both the SPK and other hypothetical morphogenic apical actin, assuming that they do attach to the PM and cell wall, as discussed below. The identity of the protein stained by the antispectrin antibody is not yet clear, but comparison with Neurospora (Degousee et al., 2000) suggests that it has some properties of spectrin, and thus the potential to contribute to a membrane skeleton. Its apical organization differs from that in Neurospora (Degousee et al., 2000); it is more similar to that in Saprolegnia (Kaminskyj and Kaminskyj and Heath, 1995), suggesting diversity in organization of the putative membrane skeleton. Nevertheless, formation of new tips in the continued presence of LatB is consistent with another cellular component taking over an essential role of actin, as previously suggested (Heath, 2000). Critical to the hypothesized role of actin in morphogenesis of eufungal hyphae is LatB-induced swelling, comparable to previous reports of swelling caused by both latrunculins (Ayscough et al., 1997; Heath et al., 2000) and cytochalasins (reviewed in Heath, 1990 and Grove and Sweigard, 1996; Herr and Heath, 1982; Heath et al., 2000). LatB-induced sub-apical swelling in G. candidum is consistent with actin disruption permitting abnormal expansion and delocalizing exocytosis. Evidence for abnormal expansion comes from the initial rate of swelling being greater than that predicted by the normal continued rate of cell surface synthesis (ratios > 100% in Fig. 35). This expansion is only seen at intermediate LatB concentrations. Presumably lower concentrations produce insufficient weakening of the putative morphogenic actin and higher concentrations disrupt exocytosis, an undeniable part of the growth and expansion process. However, relative to the initial tip acceleration of the oomycetes (Gupta and Heath, 1997), growth slowing followed by expansion indicates differences between the hyphae and their differing actin organizations.
Sub-apical swelling may also indicate delocalized, sub-apical exocytosis, previously reported as a LatB response of oomycete hyphae (Bachewich and Heath, 1998; Gupta and Heath, 1997), thus indicating similarities in hyphal functions between phylogenetically very distant species. However, the delocalized synthesis in the oomycete produced more wall material deposited throughout the sub-apical regions (Bachewich and Heath, 1998), which may relate to the oomycete wound response system (Levina et al., 2000). However, in both oomycetes and eufungi, sub-apical swelling presumably involves release of wall softening enzymes, postulated to be components of the normally apically exocytosed vesicles (Bartnicki-Garcia and Lippman, 1972). 4.3. Actin plaques Contrary to previous reports in which the hyphae of fungi contain plaques clustered over their extreme tips (reviewed in Heath, 1990, see also Akashi et al., 1994; Ayad-Durieux et al., 2000; Fischer and Timberlake, 1995; Li and Heath, 1994; Tsukamoto et al., 1996), the absence of such plaques at the extreme tips of G. candidum hyphae indicates that they are inessential for normal tip growth. A similar absence was reported by Roberson (1992), Srinivasan et al. (1996), and Torralba et al. (1998). Thus, it is unlikely that their presence in young branches indicates an obligatory role there either. Further evidence against a direct role of plaques in tip growth is that growth and SPK actin, are both rapidly disrupted at 0.5 lg ml1 LatB, whereas plaques survive prolonged incubation in at least 5.0 lg ml1 . Most likely, their presence in slow (branches), but not fast (leading hyphae), growing tips is due to the time required for their assembly. Consistent with this, growth rates of 2 lm min1 suggest their assembly by 3–4 lm behind the leading tips reqiures 2 min. Following LatB growth reduction, they are absent at 1 min, but occupy entire tips by 5 min, i.e., formation takes between 1 and 5 min. While plaques are superficially similar in diverse species, their composition must differ. In Saccharomyces, plaque elimination was only demonstrated at 80 lg ml1 LatB (Ayscough et al., 1997), G. candidum, requires >5.0 lg ml1 and in the oomycete, Saprolegnia, <0.5 lg ml1 (Bachewich and Heath, 1998; Gupta and Heath, 1997), suggesting differences in composition (most likely actin binding proteins) and functions. In ascomycetes, and basidiomycetes, plaques probably correspond to filasomes (Bourett and Howard, 1991; Corr^ea and Hoch, 1993; Howard, 1981; Kanbe et al., 1989; Roberson, 1992; Srinivasan et al., 1996). Such is likely in G. candidum too, but the functions and behaviour of filasomes remain elusive. Oomycete plaques appear to have an adhesive role (Bachewich and Heath, 1997), but differences in sensitivity to LatB mentioned above, and the absence of filasomes in oomycetes, make
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functional comparisons between the species difficult. However, filasomes are adjacent to the PM, with which they could interact. 4.4. Phallotoxin staining Fungi seem to fall into two groups, those, like plants and animals, whose actin readily stains strongly with phallotoxins and those which remain essentially unstained using similar protocols (Table 4). The phalloidin binding domain of Saccharomyces actin critically involves amino acids 158, 177, and 179 (Belmont et al., 1999; Drubin et al., 1993), but these are conserved between staining and non-staining species (Table 4). Elsewhere in this region, there are no consistent differences between these two groups, except at 178 and 180. At 178, all non-staining species have a valine, whereas all staining species have either a leucine or isoleucine and at 180 the difference is methionine vs leucine. The sole exception to this trend is Absida, a mucoralean zygomycete which also has a methionine at 180 and is predicted to be RP positive. However, this prediction may be invalid, being based only on two Enteromophthoralean species (Butt and Heath, 1988; Heath, 1990) that are not closely related to the Mucorales. We conclude that differential RP staining is due to differential phalloidin binding to actin, most likely mediated by the differences at positions 178 and 180. The amino acids at 176, 178, and 180 underlie the arginine and aspartate that are totally conserved (Table 4) and critical to RP binding (Belmont et al., 1999; Drubin et al., 1993). Changes in these three amino acids would alter the disposition of the upper two and influence the phalloidin binding site. However, the changes are likely to be fairly small scale such that further modelling attempts to refine understanding of the phalloidin/actin interaction will not be definitive with current technology. While we have focussed on the amino acids previously identified as important in phallotoxin binding, comparing the rest of the sequences of Saccharomyces, Candida, Schizosaccharomyces, Schizophyllum, Suillius, Coprinus, and Puccinia with Aspergillus, Neurospora, and Trichoderma revealed other differential single changes: V45I, I75V, SGMT194A, ML227I, E259EKQ, A272G, Y279F, and A312S. The apparent differences in phallotoxin binding are most likely to have occurred during the evolution of the hyphal ascomycetes, because all known non-staining species (Magnaporthe [Bourett and Howard, 1992], Trichoderma [Czymmek et al., 1995], Sordaria [Thompson-Coffe and Zickler, 1993], and Neurospora and Aspergillus [Heath, unpublished]) are Pyrenomycetales or Plectomycetales (Alexopoulos et al., 1996), whereas those reported to stain are among the Saccharomycetales (Saccharomyces, e.g. [Adams and Pringle, 1984],
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Candida [Anderson and Soll, 1986], Ashbya [AyadDurieux et al., 2000] and presently G. candidum), Archiascomycetes (Schizosaccharomyces e.g. [Marks and Hyams (1985)]), Chytridiomycetes (Li and Heath, 1994), Zygomycetes (Butt and Heath, 1988; Heath, 1990), and Basidiomycetes (Hoch and Staples, 1983; Raudaskoski et al., 1988; Salo et al., 1989). Loss of phallotoxin binding may have occurred as one or two neutral mutations in the hyphal ascomycete line, a relatively recent event (Heckman et al., 2001).
Acknowledgments This work was supported by a gratefully acknowledged research grant (IBH) and scholarships (MB and GDG) from NSERC. Dr. A.P.J. Trinci very kindly suggested the use of, and supplied, G. candidum. The modeling observations were generously made by Dr. L. Donaldson, York University.
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