Apical cytoplasmic organization and polar growth in Rhizoctonia solani are modified by cytochalasins

Stanley N. Grove and James A. Sweigard
Department of Biology, Goshen College, Goshen, IN 46526
Central Research and Development Department, DuPont Experiment Station,
Wilmington, DE 19898


Correspondence should be addressed to: Stanley N. Grove, PhD.
Email:stanng@goshen.edu

Submitted for publication: October 1996

Keywords: Filamentous fungi, tip growth, polar growth, apical growth, hyphae, Spitzenkörper, actin, cytochalasin, exocytosis, phase-contrast microscopy, electron microscopy, endoplasmic reticulum; morphogenesis

Abbreviations and chemicals: Cytochalasin A (CA); Cytochalasin E (CE); Dimethyl sulfoxide (DMSO); Endoplasmic reticulum (ER); N-ethyl-maleimide (NEM ); p-chloro-mercuribenzoic acid (PCMB)


Title Page Abstract Introduction Materials and Methods Results
Discussion Conclusions Acknowledgements References Table of Contents

Figure 1 Figure 2 Figure 3 Figure 4 Figure 5
Figure 6 Figure 7 Figure 8 Figure 9 Figure 10


ABSTRACT

Hyphae of Rhizoctonia solani treated with cytochalasin show these changes 1) immediate reduction in polar growth rate, 2) the typical phase-contrast dark portion of the Spitzenkörper disappears in <1 min, leaving a persistent phase light core, 3) the mitochondria lose their typical orientation, 4) continued nonpolar growth forms a bulbous hyphal apex, 5) correlated light and electron microscopy of the same hyphal tip reveals the persistent core disengaged from the usual central location and the typical cluster of apical vesicles, and 6) clusters of smooth ER are found in the apical cytoplasm. The changes observed support the suggestion that apical growth and maintenance of a Spitzenkörper are both actin-dependent phenomena. It is clear that the phase-contrast bright core is a complex feature of the cytoplasm that interacts with and contains elements of smooth endoplasmic reticulum.



INTRODUCTION

Growth in the filamentous fungi typically involves a highly polarized system of exocytosis(1, 2). Tips of growing septate hyphae contain a Spitzenkörper ("apical body") composed, in part, of a cluster of cytoplasmic vesicles which seems to participate in apical growth and fungal morphogenesis (3, 4, 1). Rhizoctonia solani has a bipartite Spitzenkörper displaying a phase-contrast light inner zone (core) surrounded by a phase-contrast dark region (5, 6, 7, 8). Electron micrographs of chemically fixed (5) or freeze-substituted (9, 2, 10) hyphal tips often show an inner zone or core devoid of secretory vesicles but containing fibrillar material, microvesicles, small membranous tubules, and ribosomes. However, the nature and function of this inner region remains unclear. Mitochondria in the subapical cytoplasm are also characteristically arranged parallel to the long axis of the growing hyphae thereby imparting a typical organization not found in hyphae where apical growth has ceased (11, 5). Phase contrast microscopy readily reveals these features along with the linear growth rate of individual hyphae. What is missing, as noted by Howard (2), is the simultaneous comparison of a light-microscopic image of a living hyphal apex with an electron-microscopic image of the same region in the same cell.

Our studies of the effects of cytochalasins on hyphal tip growth have included direct comparisons of light microscopic and electron microscopic observations of the same fixed hypha. A previous report shows that the cytochalasins inhibit sporangiospore germination and hyphal growth in a non-septate fungus (12). In a preliminary communication (6) we have shown that the response to cytochalasins in hyphal tips of R. solani includes inhibition of tip growth and loss of a typical Spitzenkörper, but persistence of an inner core. Recently this persistence has been independently confirmed by video-enhanced microscopy (7, 8). Since the cytochalasins inhibit actin polymerization in vitro, (13, 14) and characteristic microfilamentous structures can be demonstrated in growing hyphal tips (9, 2, 15, 16, 17, 18, 19, 20, 21) we have examined the hypothesis that an actin-dependent process is required for apical growth in R. solani. It seems highly significant that actin has been demonstrated by ultrastructural immunolocalization in the core-region of the Spitzenkörper in Magnaporthe grisea (21) and that an essential myosin I, which probably acts by binding to actin, is required for polarized growth in Aspergillus nidulans (22).

In this report we describe the rapid changes in mode of growth, position of the Spitzenkörper, and distribution of mitochondria caused by treatment with cytochalasins. We identify apical growth and maintenance of a Spitzenkörper as actin-dependent phenomena. Further, we describe the phase-contrast bright core as a stable feature with fiborous components and closely associated tubular ER.




MATERIALS AND METHODS

Organism and Media:

An isolate of Rhizoctonia solani Kuehn was obtained from Dr. C. E. Bracker, Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN. Cultures were grown on yeast-peptone-glucose (YPG) and glucose-asparagine (GA) media as described previously (12).

Preparation and Observation of Slide Cultures

Slide cultures employed the methods of Howard and Aist (23). Slides thinly coated with GA agarose medium (1.5% agarose; Type 1, Sigma Chemical Company, St. Louis, MO) were inoculated in the center with 5 mm mycelial strips cut from just behind the growing edge of R. solani YPG cultures. The slide cultures were grown in humidity chambers for 16-28 hours at 25 ° C in the dark.

Preparation of the slides for observation followed the perfusion technique ("An alternative method . . . p. 198) of Howard and Aist (23). Briefly, channels (1-2 mm) were cut in ca. 15 x 15 mm squares containing hyphae ca. 10 mm long. A coverslip was gently placed over the remaining agarose strips and the sides of the coverslip parallel to the channels were sealed with a 1:1 mixture of paraffin and petroleum jelly. Growing hyphae displaying typical zonation (11) and located within 200 µm of a channel were selected for observation.

Liquid media containing the appropriate test chemical was added to the edge of the cover glass and allowed to perfuse around the hyphal tip. Living tip cells were observed continually under constant light exposure in a chamber held at 27.5 ° C. Linear growth rates were determined using an ocular micrometer. Controls receiving broth with 0.1% DMSO and treated hyphae receiving broth with cytochalasin (A or E at 1µg/ml) and 0.1% DMSO were observed alternately. When direct flow of the introduced medium occurred between the coverslip and the agarose, the observation was terminated because we have found that very slight environmental changes can cause growth to slow.

Direct comparison of light and electron microscope images

A direct comparison of the light and electron microscopic images of the same hypha was necessary to identify the ultrastructural equivalents of components seen in living hyphae, including clues for the composition of the Spitzenkörper. Since freeze substitution does not lend itself to the direct microscopic observation of specimens during the fixation process or to direct before and after comparisons of the same hyphae, we have used very high quality chemically fixed hyphae to obtain ultrastructural images.

To compare the light and electron microscopic images, a different slide culture technique was used (Fig. 1). Slide cultures were grown as above but trimmed and mounted so hyphal tips for examination were approaching a fresh cut edge of the agar medium beneath a coverglass attached by drops of wax. This chamber allowed the living tip to be photographed, perfused with experimental liquid medium, then perfused a second time with fixative fluid, photographed again, removed from the slide culture and processed for electron microscopy. A suitable growing tip within 350 µm of the edge was selected, photographed live, and irrigated with a drop of liquid medium containing 0.1% DMSO or medium containing CA or CE (1 µg/ml) and 0.1% DMSO. At the first sign of a reaction to cytochalasin (change in shape of the Spitzenkörper) the preparation was irrigated again with several drops of fixative containing 2% formaldehyde (w/v) and 2% glutaraldehyde (v/v) in 0.1 M sodium cacodylate, pH 7.0. To foster rapid change in the irrigating solutions a fragment of filter paper, approximately 2 cm2, was placed at the near edge of the coverglass. This filter paper provided a sink for excess fluid from under the coverglass when new drops were placed at the upper right edge of the chamber. After all cytoplasmic motion stopped in the tip (20-120 sec) a second photograph was recorded, the slide culture was transferred to a dissecting microscope where the chosen hypha was removed in a small block of agar, placed in a vial of fresh fixative and prepared for electron microscopy by staining with OsO4, acetone dehydration and embedding in Spurr's medium. Serial or partial serial sections were examined after staining with uranyl acetate and lead citrate (5, 12).

Photography

The specimens were observed and photographed on Kodak technical pan film 2415 or Kodak Tri-X with a Zeiss Standard 18 microscope equipped with phase-contrast optics, a transmitted photo-flash system, and a 35 mm camera. Fine structural images were observed and recorded in a JEM 100S electron microscope using DuPont Cronar Ortho Litho film.

Chemicals

Cytochalasins A and E were purchased from Aldrich, Milwaukee, WI., N-ethyl-maleimide (NEM) and p-chloro-mercuribenzoic acid (PCMB) were purchased from Sigma, St. Louis, MO.



RESULTS


Light Microscopic Observations

Apical organization of growing hyphae includes a dynamic Spitzenkörper composed of a phase-bright core surrounded by a region that appears as a dark cloud adjacent to the apical membrane at the tip (Fig. 2). The profile of the bright core is round (0 sec view) except when the cluster of subtending mitochondria partially obscures its subapical perimeter (30 and 60 sec views). A region of phase-bright cytoplasm is visible lateral to the Spitzenkörper and is sometimes penetrated by one or a few mitochondria. Interspersed in the cluster of mitochondria are pleiomorphic phase-bright structures which have smooth linear profiles with occasional enlarged areas. The enlarged or beaded portions seem to migrate along the strand by a peristalsis-like gliding motion in either direction. These bright strands appear to make intermittent contact with or contributions to the subapical perimeter of the core (30 sec view). These bright segments, possibly endoplasmic reticulum (ER), are scattered in the subapical cytoplasm along with elongated mitochondria and numerous very dark, round inclusions, thought to be lipid bodies. Most mitochondria are oriented parallel to the long axis of the hypha with occasional exceptions. In this study control hyphae exhibited apical growth rates ranging from 5-15 µm/min.

The exposure of R. solani hyphal tips to cytochalasin A (CA) or cytochalasin E (CE) yielded a rapid and dramatic effect on hyphal tip organization and growth (Fig. 3) while control hyphae continued growing as usual for 5 min (Fig. 4). The effects of CA and CE were similar except that at equal concentrations the responses to CA were seen approximately one minute faster than those of CE (Fig. 5). We have noted the following reactions to cytochalasin treatment.
  1. Reduction in linear growth rate. Three minutes of treatment with CA or CE rapidly reduced the rate of hyphal tip growth (Fig. 5) from a pretreatment range of 5-15 µm/min down to 25% of the pretreatment rate for CA and to 35% for CE. Control hyphae maintained the pretreatment growth rate for at least 5 minutes.
  2. Disappearance of the phase-contrast dark region of the Spitzenkörper and persistence of the light core region of the Spitzenkörper. The first effect (in 20-50 sec after addition) of CA on hyphal tip organization was a change in shape of the core followed by a dispersion of the dark region of the Spitzenkörper (compare (Fig. 2, Fig. 3 the 15, 30, and 45 sec views, and Fig. 4 with Fig. 3 the 60 - 320 sec views). The phase-contrast light region then retracted (60 sec view) from the hyphal apex and persisted in the apical cytoplasm for several minutes (90 - 320 sec views). It moved in the cytoplasm with no apparent pattern (Fig. 6) and was often in association with mitochondria. We have also noted the persistence of the inner core of the Spitzenkörper in control hyphae that have stopped growing due to manipulation during specimen preparation. The phase-contrast light region in some of these observations persisted for more than twenty minutes (not shown). Control hyphae (undamaged and not treated with cytochalasins) maintained a typical Spitzenkörper (Fig. 2 and Fig. 4 ) for at least five minutes (not shown).
  3. Mitochondrial reorientation. Mitochondria in control hyphae, usually oriented parallel to the long axis of the hypha, did not penetrate the extreme apical dome (the Spitzenkörper region) but remained just behind the Spitzenkörper (Fig. 2). After CA treatment, usually within a minute after disappearance of the phase contrast dark region of the Spitzenkörper, mitochondria were seen in the extreme apical dome and exhibited an altered orientation in the hypha (Fig. 3 the 60 - 320 sec views). This phenomenon started in the apical cytoplasm and gradually spread to the subapical cytoplasm.
  4. Hyphal tip swelling. Changes in the Spitzenkörper were accompanied by changes in the profiles of CA-treated hyphae which soon developed swollen tips (Fig. 3 the 60 - 320 sec views, Fig. 6 the 60 - 240 second views). This nonpolar growth continued during the observation period. Control hyphae (Fig. 4) maintained normal apical shape and polar growth for at least 5 minutes (not shown).

Since our working hypothesis was that cytochalasins act primarily as anti-microfilament agents, we examined other compounds that might mimic other potential effects of the cytochalasins. NEM and PCMB are general sulfhydryl reagents and were used as controls for the sulfhydryl reactivity that has been demonstrated for CA (24). At 100 times the concentration of CA, NEM and PCMB had less effect on linear growth rate than CA (Fig. 7). Moreover, this slowing of growth was not accompanied by the hyphal tip swelling that is typical of cytochalasin treatment (Fig. 3 & Fig. 6).

Electron Microscopic Observations

In near-median sections of control hyphae the region corresponding to the Spitzenkörper core of a fixed hypha viewed with phase contrast optics (Fig. 8a, 8b), a small core area was devoid of vesicles (Fig. 8c, 8d) but containing granular or filamentous matrix and small elements of tubular smooth membrane ( Fig. 9a - 9c). The characteristic tight cluster of large and small vesicles surrounded this core and some of the vesicles seemed to be embedded in the outer part of the core matrix. The tubular smooth membrane and small vesicular profiles (Fig. 9a - 9c) seemed much more frequent in the lateral and subapical portions of the Spitzenkörper region. They were present here in frequencies similar to those found in the subapical cytoplasm. Microfilaments are visible in the core and extend out between the vesicles (Fig. 9a &9c). Mitochondria were absent in the apical dome, but ribosomes and some vesicles were seen in apical cytoplasm surrounding the Spitzenkörper (Fig. 8c, 8d).

In specimens treated with cytochalasin E for one min or less and then fixed in situ, the persistent Spitzenkörper core seen in a fixed hypha using phase contrast optics (Fig. 10a & 10b) corresponded to a region in electron micrographs which was granular, free of microfibrils, and was usually off center in a more rounded or blunt apical dome (Fig. 10c). The apical cytoplasm in these hyphae contained a few scattered apical vesicles. Mitochondria and other cytoplasmic components where dispersed in the area usually occupied by an organized cluster of vesicles (Fig. 10c). The location corresponding to the core (see Fig. 10c enlargement) lacked apical vesicles, mitochondria, and other organelles except for a few ribosomes. Many of the mitochondrial profiles were in cross section and some were located at the apical, lateral and subapical peripheries of the core. The cytoplasm surrounding the core also contained microvesicles, small tubular membrane profiles, and some ribosomes. In addition, the apical cytoplasm contained prominant clusters of tubular ER (Fig. 10c) not seen in controls (Fig. 8c, 8d). The subapical cytoplasm within 1.5 hyphal diameters of the apex in the treated tips also contained similar accumulations of tubular ER (Fig. 10c) which were not present in controls (Fig. 8c, 8d). The ER in the subapical cytoplasm appeared as long, rough cisternae with limited smooth regions and very few of the unusual tubular clusters (not shown).

DISCUSSION


Responses to cytochalasins

Our primary purpose in using CA and CE was to investigate the role of microfilaments in hyphal tip growth. We previously presented evidence supporting CA as a microfilament inhibitor in fungi (12). Here we have provided additional indirect support for this suggestion since two general sulfhydral reagents fail to mimic the effect of CA, thereby making unlikely a potential mechanism of action for CA (24) in our study. Though we are unable to prove that cytochalasins are acting by inhibiting actin polymerization, accepting this hypothesis yields the most straightforward interpretation of our observations and is consistent with the observations of others.

Support for a significant role for actin in hyphal tip growth is increasing. Highly polar F-actin networks have been demonstrated in a variety of fungi (reviewed by Heath in 19) and ultrastructural observations reveal a microfilament network in the hyphal apex (see review by Heath in 19, 10). The microfilaments were found to be most abundant in the core region (2, 25) and were identified in the core as actin via ultrastructural immunolocalization (21). Howard (2) suggested that this microfilamentous network serves as a mechanism to control the localized secretion of growth materials in the tip. Actin microfilaments might be involved in movement of vesicles from subapical regions to the hyphal tip. They have also been found in association with mitochondria and other organelles in a variety of organisms (26, 27, 28, 29) and may help determine the distribution and orientation of these organelles. If CA and CE are inhibiting actin polymerization then our observations support all of these roles for microfilaments. Cytochalasin treatment leads to rapid depolarization of growth, an observation consistent with a role for microfilaments in supporting localized secretion. Cytochalasin treatment also leads to dispersal of the cluster of vesicles and to a switch from polar to nonpolar growth. This observation is consistent with a role for microfilaments in the movement of vesicles to the hyphal apex and in organizing the vesicle cluster. Finally, cytochalasin treatment leads to a reorientation of mitochondria suggesting that together with microtubules (23, 30), microfilaments help determine the position of these organelles.

Bartnicki-Gracia, et al (4) proposed a mathematical model to explain hyphal tip growth. The model hypothesizes that a vesicle supply center (VSC) controls hyphal morphogenesis. The model predicts that the position of VSC determines the polarity of growth and that as the VSC moves away from the apex, growth becomes nonpolar. Cytochalasin treatment of growing hyphal tips leads to retraction of the phase contrast light portion of the Spitzenkörper from the hyphal tip and a change from polar to nonpolar growth. This observation suggests that the phase contrast light core of the Spitzenkörper is the natural equivalent of the proposed VSC.

The persistent inner core of the Spitzenkörper:

Our novel observation that the core of the Spitzenkörper is a stable component of the cytoplasm also suggests that the core is the equivalent of the VSC. Earlier the Spitzenkörper was found to accompany tip growth in septate fungi (31, 32, 5) but its nature and content have not been adequately defined. Apical vesicles are clearly present and are assumed to have a secretory function. But controversy exists over whether the cluster of vesicles constitutes the whole of the structure or if other components contribute (32, 5, 2). Evidence for cytoskeletal material within the apical cytoplasm has been found using fluorochromes to label specific materials (16, 18, 15, 17) and in freeze-substituted specimens observed with electron microscopy (33, 9, 2, 25, 10). This study indicates that the phase contrast dark portion of the visible Spitzenkörper may include only those vesicles tightly clustered around the periphery of a more complex phase contrast bright core. This central core includes microfilaments (10, 25, 21) and smooth membrane profiles (10, 25). This core is retained and can be identified even after the usual structure and position are disrupted by anti-microfilament agents as shown in the present study.

Little attention has been given to the potential contribution of endoplasmic reticulum to tip growth although small tubules or cisternae were seen in and around the Spitzenkörper (11, 25). In Sclerotium rolfsii after 30 min of treatment with 0.1 µg/ml of the demethylase inhibitor, cyproconazole, the Spitzenkörper was still present but a proliferation of smooth ER had occurred (10). The present study finds a rapid proliferation of tubular ER in response to applied inhibitor and suggests that much shorter times (<1 min) are needed to detect direct responses of growing hyphal tips to environmental stresses. It is interesting that during growth of control hyphae intermittent interactions are observed via phase-contrast microscopy between ER components and the core of the Spitzenkörper (personal observations not shown). It is significant that similar interactions were recently video recorded by Rosa Maria Lopez-Franko (34 and personal communication). Could these interactions represent the contribution of membrane and luminal material from the ER directly to the core? If so, does the rapid proliferation of tubular ER in treated tips indicate a buildup of material due to blocked transfer caused by the disintegration of core structure and function? Does this reveal an alternate to transport by apical vesicles, possibly a system for transferring a subset of products from ER directly to the apex?

To better illuminate the nature of the Spitzenkörper or proposed VSC (4) a method is needed to allow direct observation by video-enhanced microscopy of responses to applied stresses followed shortly by rapid preservation and study of the same hypha by electron microscopy. Until then the video-enhanced observations can be profitably coupled with standard chemical fixation of high quality to extend our understanding of the growing hyphal apex. In the study of such delicate and complex phenomena the value of correlations made on the same specimen with different appropriate techniques should not be underestimated.




ACKNOWLEDGEMENTS

This research was supported in part by grants from the Faculty Research Fund at Goshen College, The William and Flora Hewlett Foundation via Research Corporation, and the NIH (AI 14678).


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