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Cell biology/ultrastructure

Functional necessity of the cytoskeleton during cleavage membrane development and zoosporogenesis in Allomyces macrogynus

     School of Life Sciences, Box 874501, Arizona State University, Tempe, Arizona 85287-4501

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

Cleavage membrane development and cytokinesis were examined in zoosporangia of Allomyces macrogynus treated with cytoskeletal inhibitors and compared to zoosporogenesis under control conditions. Developing membranes were visualized in living zoosporangia with laser-scanning confocal microscopy using the lipophilic membrane dye FM4-64. Under control conditions, cleavage membranes developed in four discrete stages, ultimately interconnecting to delimit the cytoplasm into polygonal uninucleate domains of near uniform size. Disruption of microtubules did not impede the normal four-stage development of cleavage membranes, and cytokinesis occurred with only minor detectable anomalies, although zoospores lacked flagella. Disruption of actin microfilaments did not inhibit membrane formation but blocked nuclear migration and significantly disrupted membrane alignment and cytoplasmic delimitation. This resulted in masses of membrane that remained primarily in cortical regions of the zoosporangia, as did nuclei, throughout zoosporogenesis. Zoospores formed in the absence of microtubules had only a slightly larger mean diameter than control zoospores, although nearly 50% of spores contained two or more nuclei. Microfilament inhibitor treatments produced spores with substantially larger mean diameters and correspondingly larger numbers of nuclei per spore, with greater than 85% containing three or more nuclei. These results showed that a functional actin microfilament cytoskeleton was required for proper alignment of cleavage elements and cytokinesis in Allomyces zoosporangia while microtubules played a less significant role.

Key words: cytoskeleton inhibition, FM 4-64, live cell imaging, zoospore formation

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Various cellular factors have been implicated as essential to cytoplasmic cleavage in the zoosporic fungi. While previous studies (Olson and Lange 1983a, Oertel and Jelke 1986, Heath and Harold 1992, Hyde and Hardham 1993) have reported that disruption of actin microfilaments or microtubules results in severe abnormalities in cleavage products, the influence of cytoskeletal elements on the dynamics of cleavage membrane development remains largely undetermined. General studies of zoosporogenesis at times have produced conflicting results with respect to membrane genesis, points of origin or mode of assembly (Gay and Greenwood 1966, Hohl and Hamamoto 1967, Moore 1968, Lessie and Lovett 1968, Williams and Webster 1970, Hoch and Mitchell 1972, Barron and Hill 1974, Lunney and Bland 1976, Olson and Lange 1983b, Hyde et al 1991a, Beaks et al 1992, Beaks et al 1993, Fisher et al 2000). This is due in part to variations in membrane development among organisms believed to have diverse evolutionary affinities but also possibly to the fact that development of cleavage membranes within multinucleate sporangia presents a substantially more intricate example of cytokinesis relative to simpler uninucleate systems. Furthermore, analyses of zoospore development traditionally have employed transmission electron microscopy (TEM) of samples prepared for study using standard chemical fixation methods. Unfortunately, such methods do not faithfully preserve cytoplasmic organization. This has led to misinterpretation of data and incompatible conclusions regarding fundamental aspects of membrane development in zoosporic fungi. More recent fine structure analyses of Phytophthora (Oomycota; Hyde et al 1991b) and Allomyces (Chytridiomycota; Fisher et al 2000) prepared by high-pressure freezing-freeze substitution (HPF-FS), in addition to live-cell imaging (Fisher et al 2000) using epifluorescence dyes such as FM4-64 (Vida and Emr 1995) that target preferentially the plasma membrane and endocytotic membranes, have shown that cytokinesis in these organisms takes place via the progressive extension and ultimate interconnection of multiple membranous sheets. These results have served to displace an earlier model that cited membrane-vesicle alignment and coalescence in zoospore cleavage (Barron and Hill 1974, Hyde et al 1991a).

The purpose of this study was to determine the effects of cytoskeleton disruption on the development of cleavage membranes and cytokinesis during zoospore formation in Allomyces macrogynus. In an earlier report (Fisher et al 2000), we studied living cells labeled with FM4-64 to examine previously unresolved spatial and temporal questions of cleavage membrane development during zoosporogenesis in A. macrogynus and concluded that four distinct stages of membrane development occurred. In the present study, we have applied similar techniques and extended upon our earlier work by focusing on the effects of cytoskeleton disruption on membrane development leading to cytoplasmic cleavage during zoosporogenesis in A. macrogynus. Results indicate that the microtubule cytoskeleton played a less significant role in membrane development and cytoplasmic cleavage than previously reported for A. macrogynus (Olson and Lange 1983a), whereas the actin microfilament cytoskeleton was mandatory for proper membrane elongation, ramification and interconnection. Neither the microtubule or actinmicrofilament cytoskeletons were required for membrane initiation or cortical extension or the production of cleavage membrane material.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Culture conditions and cytoskeletal inhibitors. – Cultures of A. macrogynus (strain Burma 3-35; ATCC No. 38327) were grown and induced to sporulate as described by Lowry and Roberson (1997). Nocodazole (Sigma Chemical Co., St. Louis, Missouri) and cytochalasin D (Sigma) were prepared in dimethyl sulfoxide (DMSO) and administered under controlled conditions as previously described (Lowry et al 1998).

Video-enhanced light microscopy. – Three–four d old cultures were rinsed in a dilute salts (DS, Sistrom and Machlis 1955) solution and transferred to a Petri dish containing 10 ml of DS amended with appropriate volumes of DMSO for control observations or cytoskeletal inhibitors. Colonies were selected and gently transferred into a drop of the appropriate DS solutions on a glass slide and overlaid with a cover slip. DS solution was perfused under the cover slip at approximately 5 min intervals to maintain aerobic conditions. Video-enhanced light microscopy (VELM) was performed according to Fisher et al (2000).

Zoospore diameters were obtained by incubating colonies under control or experimental conditions for 60–75 min to allow an adequate period for zoospore formation and release. Suspensions of zoospores were added to a microfuge tube and vortexed at full speed for approximately 20 s, which caused spores to assume a rounded shape. A drop of suspension was placed immediately on a glass slide and spore diameters were determined with standard light microscope equipped with an ocular micrometer.

Epifluorescence microscopy. – Living zoosporangia labeled with FM4-64 (N-(3-triethylammoniumpropyl)-4-(6-(4-(dieth-ylamino)phenyl)hexatrienyl)pyridinium dibromide; Molecular Probes Inc., Eugene, Oregon) were visualized and images captured according to Fisher et al (2000). Sporangia briefly were induced to sporulate as described above, except colonies selected for observation were placed in drops of DS amended with FM4-64 at a concentration of 32 µM. FM4-64/DS solution was perfused under the cover slip at 5–10 min intervals. For experimental studies, inhibitors first were diluted into DS and FM4-64 was added. This FM4-64/DS/inhibitor solution then was perfused under the cover slip at regular intervals, as in control treatments. Living ZS labeled with FM4-64 were visualized and images digitized using a Leica TCS NT (Leica Imaging System, Exton, Pennsylvania) LSCM with either a Planapo 63x /1.2 water immersion objective or Planapo 100x/1.4 oil immersion objective. An argon laser supplied illumination with appropriate filters (Ex 488, Em 500–560). Sporangia were optically scanned longitudinally from top to bottom as a series of 32 optical sections and viewed as complete stacks.

Nuclei per zoospore counts were accomplished by incubating colonies in DS under control or experimental conditions 60–75 min to permit zoospore release. Zoospores were fixed in freshly made 4% formaldehyde in PIPES buffer (100 mM, pH 6.8) for 10 min, washed twice in distilled H₂O, and stained 5–10 min with the DNA fluorophore 4,6-diamidino-2-phenylindole (DAPI) at a concentration of 0.1 µg/ml in H₂O for 5–10 min. After a rinse in H₂O, cells were mounted in 90% glycerol/10% 0.1 M phosphate buffered saline amended with 1 mg/ml N-propyl gallate (Sigma). Images of nuclei were obtained using standard epifluorescence microscopy as described by Lowry et al (1998).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Events of zoospore formation in control zoosporangia have been described (Lowry and Roberson 1997, Fisher et al 2000) and briefly are reviewed here. During the initial 15–20 min of zoosporogenesis, sporangia contained many homogeneously distributed cytoplasmic inclusions of various sizes with nuclei exclusively located at the zoosporangia cortex (FIG. 1). FM4-64 labeling was observed at the plasma membrane of zoosporangia during the initial 15–20 min of zoospore formation (FIG. 2). Also, during these early stages of zoosporogenesis cleavage, element formation was evident within the cortical cytoplasm (FIG. 2). Between 20–30 min post-induction, cytoplasmic reorganization was evident with nuclei and other cellular contents becoming progressively partitioned into incipient zoospore domains by advancing cleavage elements labeled with FM4-64 (FIGS. 3–6). Cleavage into uniformly sized individual polyhedral-shaped domains was completed by approximately 40–50 min post-induction (FIGS. 7, 8), and zoospore release ensued (FIG. 7). The majority of zoospores from control treatments possessed a single nucleus (97%), with the remaining 3% possessing two nuclei (n = 200). Zoospore diameter with standard deviation (SD), viewed as an indicator of cleavage accuracy, had a mean of 9.87 ± 0.69 µm (n = 50).

View larger version (146K): [in this window] [in a new window]   FIGS. 1–8. Control treatments of zoospore formation in living sporangia of Allomyces macrogynus observed with video-enhanced DIC optics (FIGS. 1, 3, 5, 7) and LSCF microscopy after FM4-64 staining (FIGS. 2, 4, 6, 8). Minutes and seconds in the upper left corners indicate post induction times. Scale bars = 10 µm. FIGS. 1, 3, 5, 7. Nuclei were located in the cortical cytoplasm during early stages of zoospore formation (arrow, FIG. 1) and by 20–30 min post induction were positioned through out the cytoplasm (arrows, FIG. 3). Cytoplasmic domains became distinct (FIG. 5) and eventually the papillum (asterisk, FIG. 1) deliquesces and mature zoospores (arrowhead, FIG. 7) maneuver out of the sporangium into the surrounding medium. FIGS. 2, 4, 6, 8. By 12–20 min post induction, areas of increased fluorescence (arrows, FIG. 2) were observed along regions of the plasma membrane. Cleavage elements initially extended from these regions within the sporangial cortex (arrows, FIG. 4) followed by a rapid elongation inward toward the center of the sporangium (FIGS. 6). By 40–50 min post induction zoospore initials were delimited into polyhedral-shaped cells (asterisk, FIG. 8).

View larger version (135K): [in this window] [in a new window]   FIGS. 9–16. Nocodazole treatments of zoospore formation in living sporangia of Allomyces macrogynus observed with video-enhanced DIC optics (FIGS. 9, 11, 13, 15) and LSCF microscopy after FM4-64 staining (FIGS. 10, 12, 14, 16). Minutes and seconds in the upper left corners indicate post induction times. Scale bars = 10 µm. FIGS. 9, 11, 13, 15. Nuclear position (arrows, FIGS. 9, 11), cytoplasmic compartmentalization (FIG. 13), and spore discharge (FIG. 15) appeared mostly undisrupted in the presence of nocodazole, except for zoospores with fused nuclear caps (asterisk, FIG. 13) and zoospores lacking flagella (not shown). FIGS. 10, 12, 14, 16. Cleavage element formation and development (arrows) in the presences of nocodazole was similar in many respects to that observed in control sporangia though alignment of cleavage elements were sometimes slightly altered.

View larger version (163K): [in this window] [in a new window]   FIGS. 17–24. Cytochalasin D treatments of zoospore formation in living sporangia of Allomyces macrogynus observed with video-enhanced DIC optics (FIGS. 17, 19, 21, 23) and LSCF microscopy after FM4-64 staining (FIGS. 18, 20, 22, 24). Minutes and seconds in the upper left corners indicate post induction times. Scale bars = 10 µm. FIGS. 17, 19, 21, 23. Nuclei remained in peripheral regions of the cytoplasm as a result of cytochalasin treatment (arrows, FIGS. 17, 19). By 30–40 min, zoosporangia exhibited reorganization of cytoplasmic contents and fused nuclear caps (asterisks, FIGS. 21, 23). Cytochalasin treatment resulted in the release of 2–4 large multinucleate and mutlflagellate (arrowhead, FIG. 23) zoospores. FIGS. 18, 20, 22, 24. Cytochalasin D did not appear to influence the early stages of membrane development. Sites of increased FM4-64 staining were observed in cortical cytoplasm (white arrowheads, FIGS. 18, 20). Elongation of cleavage membranes into the sporangial cytoplasm was inhibited due to cytochalasin action. Membranous material continued to accumulate in the cortical region (black arrowheads, FIGS. 22, 24) and appeared interconnected in this region.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

A primary aspect of cytokinesis in many eukaryotic coenocytic cells is believed to be the formation of cytoplasmic domains, or cytoplasts, before cleavage (see review by Pickett-Heaps et al 1999). According to this concept, microtubules play a central role in establishing spatially organized units within the mother cell and define the plane(s) of cleavage. For multinucleate zoosporangia, this concept was suggested after the observation of microtubule asters associated with each nucleus (Heath and Greenwood 1971) and later elaborated upon (Hyde and Hardham 1992). Microtubules were envisioned to stabilize or condense the cytoplasm surrounding each nucleus, thus forming discrete, multiple blocks of cytoplasm within the uncleaved sporangia. This allowed cleavage membranes to develop through less dense cytoplasmic regions between the nuclei. Microtubules also have been hypothesized to play a critical role in delivering membrane precursors to the cleavage planes, where assembly occurs incrementally (Heath and Greenwood 1971, Olson and Lange 1983a).

It has been shown that administration of nocodazole at 0.33 µM was the minimal concentration that resulted in the loss of the microtubule cytoskeleton within 10 min in zoosporangia of A. macrogynus (Lowry et al 1998), well before cleavage membrane initiation begins. In the present study, experiments using nocodazole at this concentration, while visualizing cleavage membrane development in living sporangia, revealed a moderate disruption of cleavage-plane alignment but the four stages of membrane development and near normal cleavage occurred. This suggests an intact microtubule cytoskeleton and its associated motors did not play an obligatory role in cytoplasmic cleavage in Allomyces. In addition, zoospore diameters for nocodazole treatments were only slightly larger than those of control spores, which was likely the result of the relatively minor disruption of cleavage plane alignment. Deviations between the numbers of nuclei in control and nocodazole-treated zoospore might have been due to the misalignment of cleavage membranes observed in nocodazole treated zoosporangia. However, differences in cleavage-plane alignment between control and nocodazole treatments were not extreme and the extent to which this might have contributed to multinucleate spores is not clear. Irregular spacing of nuclei also was observed in zoosporangia of A. macrogynus treated with nocodazole (Lowry et al 1998), and therefore disrupted nuclear positioning also might have been of consequence in the production of the multinucleate spores under these conditions.

In screening tests for inhibitor thresholds with A. macrogynus, application of nocodazole at concentrations above 1 µM, as well as treatment with the nocodazole analogue methyl benzimidazole-2-ylcarbamate or griseofulvin at 10 µM, not only caused microtubule loss but produced significantly greater disruption of cleavage products than was observed under our experimental conditions at 0.33 µM (Lowry and Roberson unpubl). With this in mind, we think it is possible that earlier reports of gross cleavage disruption resulting from microtubule-disrupting treatments in Allomyces (Olson and Lange 1983a) and Phytophthora (Hyde and Hardham 1993) could be attributable to secondary cellular effects from excessive levels of chemical inhibitors.

The involvement and importance of actin microfilaments in the process of cytokinesis has been well established in eukaryotic organisms. Studies of zoosporic fungi using different cytochalasins each have demonstrated that large multinucleate masses resulting from incomplete or noncleavage of the cytoplasm was a consequence of actin microfilament disruption (Oertel and Jelke 1986, Heath and Harold 1992, Hyde and Hardham 1993). Our results of living cells labeled with FM4-64 indicated that in Allomyces, cytochalasin D did not block the origination and cortical extension of cleavage membranes but significantly inhibited the cytoplasmic extension, ramification and interconnection required for normal zoospore delimitation. Predischarge zoosporangia typically displayed large aggregates of cleavage membrane primarily in the cortex of the cytoplasm in FM4-64 stained cells. It was clear from the epifluorescence data that membrane synthesis was not inhibited by the disruption of actin microfilaments in the presence of cytochalasin D. Furthermore, in certain instances cleavage elements in fact did delimit portions of the cytoplasm although the process undoubtedly was random in the presence of cytochalasin D and zoospores that are discharged display a much larger standard deviation than under either control or microtubule-disrupting conditions. Immunofluorescence observations of actin labeling in Allomyces indicate actin was localized as a diffuse layer along the plasma membrane and nuclear surfaces in early stages of zoospore formation and outlines zoospore domains in late stages (Lowry and Roberson unpubl). Similar observations of actin localization have been reported from microinjection studies of living zoosporangia of Phytophthora ( Jackson and Hardham 1998) and in fixed cells of Saprolegnia and Achlya (Heath and Harold 1992). However, due to our inability to adequately and consistently label actin for epifluorescence observations under control and experimental conditions, we can suggest only that actin, like microtubules, does not play a role in membrane biogenesis; however, it is possible with application of 5 µM cytochalasin D that small amounts of the actin cytoskeleton remained intact and functional in the critical regions near the nucleus/plasma membrane interface where cleavage membranes were initiated. Similar concentrations of cytochalasin applied to oomycete fungi have been shown with rhodamine phalloidin (RP) staining to substantially remove actin plaques but small "rods" of actin remain (Heath and Harold 1992). RP does not stain actin in Allomyces. Based on earlier studies of cleavage irregularities and our results, however, it is reaffirmed that actinmicrofilaments are of primary importance in the orderly progression and cytoplasmic extension/interconnection of membrane sheets.

To summarize, our results support the contention that removal of microtubule arrays does not cause severe disruption of cleavage products in A. macrogynus, aside from the absence of flagella and a minor increased incidence of multinucleate spores. The actinmicrofilament cytoskeleton is mandator y for proper membrane elongation, ramification and interconnection. Neither the microtubule or actinmicrofilament cytoskeletons are required for membrane initiation or cortical extension or the production of cleavage membrane material.

	ACKNOWLEDGMENTS

 
This research was supported by the Plant Biology Department, ASU. LSCM was performed in the W.M. Keck Bioimaging Laboratory (ASU). Electron microscopy was performed in the Life Sciences Electron Microscope Facility (ASU). FIGURES 2, 4, 6 and 8 were published originally by Fisher et al (2000).

	FOOTNOTES

 
Accepted for publication July 30, 2003.

¹ Corresponding author. E-mail: Robert.Roberson{at}asu.edu

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