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Mapping Woronin body position in Aspergillus nidulans

     Department of Botany, University of Georgia, Athens, Georgia 30602

Gregory Jedd

     Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, New York 10021-6399

	ABSTRACT

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The positions of all Woronin bodies in five germlings of Aspergillus nidulans prepared by plunge freezing and freeze substitution were determined by transmission electron microscopy. As expected, Woronin bodies were found near septa. High numbers of morphologically identical organelles were also found in apical regions. To verify that these organelles were authentic Woronin bodies, we used antibodies raised against the Neurospora crassa Woronin body matrix protein Hex1. Anti-Hex1 antibodies labeled Woronin bodies at septa and in apical regions of A. nidulans. In germlings that had not yet formed septa, at least fifty percent of Woronin bodies were found within 2.5 µm of the tip. In germ tubes that had formed septa, the total number of Woronin bodies remained the same, but only twenty percent were near the tip. Our results clearly establish that Woronin bodies are found in apical regions of Aspergillus germ tubes and suggest that Woronin bodies are transported from the apex to the more basal regions of the cell immediately before or during septation.

Key words: organelle movement, peroxisome, septum

	INTRODUCTION

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Woronin bodies have been reported near the septal pore in mycelial ascomycetes and deuteromycetes, including Aspergillus nidulans, Aspergillus gigantaceae, Fusarium oxysporum, and Neurospora crassa (Collinge and Markham 1982 , Richle and Alexander 1965 , Wergin 1973 ) and have been used taxonomically (Alexopoulus et al 1996 , Markham and Collinge 1987 ). Elegant work by Trinci and Collinge (1974) showed that Woronin bodies rapidly plug septal pores in N. crassa hyphae damaged by a razor blade or flooding with water. Later work demonstrated that Woronin bodies also plug Penicillium chrysogenum septal pores after damage (Collinge and Markham, 1985 ). These experiments firmly established that a major role of Woronin bodies is to plug septal pores to prevent the escape of cytoplasm and cell death.

Woronin bodies were first described based on light microscope observations as refractive particles associated with septa (Woronin, 1864 ). This early description made it easy to confuse Woronin bodies with other organelles (Markham and Collinge, 1987 ). With transmission electron microscope (TEM) observations it is clear that these electron-opaque bodies are either spherical or hexagonal, surrounded by a single unit membrane, and range from 150–500 nm in size. While the TEM description is more exact than the one furnished by light microscopy, there is still some debate about exactly how to identify Woronin bodies, especially in those instances where they have been reported distant from septa.

Recently, the HEX1 gene encoding the N. crassa Woronin body matrix protein was cloned (Jedd and Chua 2000 , Tenney et al 2000 ). Antibodies raised against recombinant Hex1 protein labeled Woronin bodies exclusively. Deletion of HEX1 resulted in hyphae that could not seal the septal pore and leaked cytoplasm upon lysis. Expression of HEX1 in S. cerevisiae directed the synthesis of vesicles with hexagonal morphology similar to N. crassa Woronin bodies. The sequence of HEX1 contains a peroxisome-targeting signal that was shown to correctly direct localization of a Hex1-GFP fusion protein to peroxisomes in S. cerevisiae. Individual peroxisomes are surrounded by a single membrane and contain diverse enzymes specialized for functions ranging from peroxide metabolism to biosynthesis of lipids (reviewed by Tabak et al 1999 ). The Woronin body clearly represents a new class of fungal peroxisome (Jedd and Chua 2000 ).

Despite this new knowledge of their composition, Woronin body distribution within hyphae remains unclear. Previous work has reported the presence of apical Woronin bodies in several fungi and their absence in other fungi, including A. nidulans (McClure et al 1968 , Collinge and Markham 1982 , reviewed by Markham and Collinge 1987 ). Because Woronin bodies have classically been defined based on morphology coupled with location near the septum, there has been debate on whether these apical organelles were authentic Woronin bodies. In this study we examined A. nidulans germlings grown to the time when the first septa are forming and prepared for TEM by plunge freezing and freeze substitution. Serial sections through five germ tubes were examined and the positions of all Woronin bodies and morphologically identical organelles were mapped. To verify that all of these organelles were authentic Woronin bodies, we used antibodies raised against the N. crassa Woronin body matrix protein Hex1. Anti-Hex1 antibodies labeled all Woronin bodies at septa and morphologically identical organelles throughout the cytoplasm of A. nidulans cells. Our results clearly demonstrate that Woronin bodies are found in the apical cytoplasm of germ tubes in A. nidulans, as well as near septa. Further, our results show that while the total number of Woronin bodies remains constant, the number at the tip decreases after septation, suggesting transport away from the tip.

	MATERIALS AND METHODS

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Fungal growth – Conidia of A. nidulans wildtype strain A28 (Fungal Genetics Stock Center, Department of Microbiology, University of Kansas Medical Center, Kansas City, Kansas) were prepared for study with TEM using cryofixation and freeze-substitution methods. A single layer of moist, sterile dialysis membrane precut into approximately 3mm square pieces was placed on solid, complete medium (1% glucose, 0.2% peptone, 0.1% yeast extract, 0.1% casamino acids, nitrate salts, trace elements, and 0.1% vitamins, 1.5% agar, pH 6.5). Trace elements, nitrate salts, vitamins, and amino acid supplements are described in the appendix to Kafer (1977) . Conidia were transferred onto the dialysis membrane and incubated at 30 C for 12 h.

Fixation and electron microscopy – The dialysis membranes with adherent actively growing cells were rapidly plunged into liquid propane according to the procedure of Hoch (1986) . The samples were transferred to vials of substitution fluid consisting of 2% osmium tetroxide and 0.05% uranyl acetate in anhydrous acetone as described by Mims et al (1988) . After 4 d at -80 C, the vials were warmed slowly to room temperature (-20 C for 4 h, 4 C for 2 h, room temperature for 45 min). The substitution fluid was removed and the samples rinsed in 3 changes of HPLC grade acetone for 15 min each. Samples were infiltrated at room temperature using Embed 812/Araldite resin (Electron Microscopy Sciences, Ft. Washington, Pennsyvania), flat embedded between Permanox slides (Electron Microscopy Sciences), and polymerized for 48 h at 60 C. Well-preserved cells were selected according to Howard and O'Donnell (1987) and sectioned using an RMC 6000XL ultramicrotome. Serial sections were picked up on gold-gilded slot grids (Electron Microscopy Sciences) and allowed to dry on Formvar-coated aluminum racks (Rowley and Moran 1975 ). Sections were post-stained for 3 min in aqueous uranyl acetate followed by lead citrate (Reynolds 1963 ). Grids were examined in a Zeiss EM 902A TEM operated at 80 kV.

Mapping Woronin body position – Serial sections spanning the entire cell were examined and Woronin bodies were identified based on being spherical, electron-opaque, roughly 120 nm in diameter, and enclosed by a single unit membrane. The position of each Woronin body was recorded within cytoplasmic regions defined as follows: (1) basal—in the conidium; (2) septal—2.5 µm on either side of the septum; (3) subapical—2.5 µm above the septum to 2.5 µm behind the tip; (4) apical—final 2.5 µm encompassing the tip.

Immunocytochemistry – The cloning of the N. crassa HEX-1 gene and production and affinity purification of rabbit polyclonal antibodies against recombinant Hex1 have been previously described (Jedd and Chua 2000 ). The purified antibodies were incubated with A. nidulans thin sections prepared exactly as described above except that LR White resin was used instead of epoxy resin and samples were polymerized for 24 h at 50 C. For immunolabeling, each gold-gilded slot grid containing serial sections of A. nidulans was hydrated for 5 min on a 10 µL drop of 0.01 M potassium phosphate buffer (pH 7.2) containing 0.15 M NaCl (KPBS) then transferred to a drop of 3% w/v nonfat dried milk in KPBS for 1 h. Grids were transferred to a drop of KPBS for 5 min and incubated for 1 h on a drop of undiluted affinity-purified antibody against Hex1. Grids were rinsed for 30 s with a steady stream of KPBS and incubated for 1 h on a drop of 10 nm-gold-conjugated goat F(ab) anti-rabbit IgG secondary antibody (Vector Laboratories, Inc., Burlingame, California) diluted 1:10 v/v in KPBS. After incubation, grids were rinsed for 30 s with KPBS and for 30 s with distilled water. Grids were post-stained and imaged as described above. No label was seen in negative controls that omitted incubation with antibodies against Hex1.

	RESULTS

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Woronin bodies were identified based on four morphological characteristics: spherical shape, high electron opacity, size of approximately 120 nm, and enclosure within a single unit membrane. As expected, Woronin bodies were observed near septa (Fig. 1 ). We sometimes observed a ribosome-free zone surrounding Woronin bodies along with apparent fibrous connections to the septum (Fig. 1 ). Morphologically identical organelles were also found in the apical region, often subtending the Spitzenkörper (Figs. 3, 4 ). These organelles were also occasionally seen associated with microbodies in the apical region of the cell (data not shown). To provide biochemical support that these apical bodies were authentic Woronin bodies, sections were incubated with polyclonal antibodies against the N. crassa Woronin body matrix protein Hex1. Anti-Hex1 antibody labeled both Woronin bodies at the septum (Fig. 2 ) and presumed Woronin bodies in the apical cytoplasm (Fig. 5 ); however, the label pattern differed significantly between the organelles. In septal Woronin bodies, the Hex1 protein was distributed throughout the organelle interior, while in apically positioned organelles the localization was restricted to the peripheral regions of the Woronin body (Fig. 5 ). Colloidal gold markers were also observed in narrow regions of cytoplasm immediately adjacent to apically positioned Woronin bodies (Fig. 5 ). Occasionally, label was seen in apical regions where no Woronin body was obvious (Fig. 5 ). However, examination of adjacent serial sections revealed the presence of Woronin bodies in these regions (data not shown) that resulted in Hex1 labeling on adjacent sections.

View larger version (83K): [in this window] [in a new window]    FIGS. 1–2. Transmission electron micrographs of longitudinal sections through Aspergillus nidulans septa. 1. Mature septum with five Woronin bodies (arrowheads) near central pore. Arrow indicates possible fibrous connection between Woronin body and septum. Asterisk indicates ribosome-free area surrounding Woronin body. Bar = 0.25 µm. 2. Septal Woronin body labeled with affinity-purified antibodies against Hex1 and secondary anti-rabbit antibody coupled to 10 nm gold particles. Note membrane surrounding Woronin body (small arrow). Inset: Same image overexposed to better show gold particles. Bar = 0.125 µm

View larger version (193K): [in this window] [in a new window]    FIGS. 3–5. Transmission electron micrographs of near median sections through Aspergillus nidulans germ tube apical regions. Arrowheads indicate Woronin bodies. Asterisks indicate Spitzenkörper vesicles. 3. Mitochondria (M) and Golgi body equivalents (GB) are visible in the cytoplasm. Bar = 0.25 µm. 4. Woronin bodies are visible just behind the Spitzenkörper. Arrow denotes Woronin body enlarged in inset. Bar = 0.25 µm. Inset: Enlargement of Woronin body marked with arrow. Note surrounding membrane. Bar = 0.125 µm. 5. Apical Woronin bodies labeled with affinity-purified polyclonal antibodies against Hex1 and secondary anti-rabbit antibody coupled to 10 nm gold particles. Bar = 0.25 µm. Inset: Uppermost Woronin body enlarged and overexposed to better show gold particles. Bar = 0.125 µm

View larger version (18K): [in this window] [in a new window]    FIG. 6. Diagram of Woronin body position. Graphic representation of data in Table I . Serial sections spanning the entire cell were examined and Woronin bodies were identified based on morphology. The position of each Woronin body was recorded as apical, subapical, septal, or basal. All Woronin bodies are shown disproportionately large and in a single plane for clarity. In actuality, only a few Woronin bodies were visible in each thin section. Cells drawn roughly to scale

	DISCUSSION

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Our results show a change in Woronin body distribution with septation. In cells that had not yet formed septa, at least half of the Woronin bodies were found clustered in the apical cytoplasm. However, in cells that had undergone septation, only one fifth of Woronin bodies were found in the apical cytoplasm. When conidia of A. nidulans are inoculated to rich medium they break dormancy and remain roughly synchronous until well after the first septum is formed (Momany and Taylor 2000 ). Though the entire population will eventually develop septa, the germ tubes examined in this study were fixed at the time when septa are typically just beginning to appear (Harris et al 1994 , Momany and Taylor 2000 ). Hence we expected that cells would be observed in various stages of septum development. Since the number of Woronin bodies observed was similar in cells that had not yet septated versus those that had just septated, the difference in their distribution suggests that Woronin bodies are transported from the tip basally during the time just prior to or early in septum formation.

Other investigators have reported Woronin bodies associated with membranous sacs in apical compartments and have postulated that Woronin bodies are made in these sacs (Brenner and Carroll 1968 , Wergin 1973 ). We also occasionally observed Woronin bodies that appeared to be associated with membranous sacs in the apical region of the cell. If they are indeed synthesized in the apical region, there must be a mechanism for moving mature Woronin bodies subapically into areas where new septa will form. Such a mechanism most likely involves travel along microtubules and/or filamentous actin. Indeed, there is considerable evidence for regulated, microtubule-mediated movement of peroxisomes in animal systems (Rapp et al 1996 ,Wiemer et al 1997 , Huber et al 2000 , Schrader et al 2000 ).

While our mapping results suggest transport of Woronin bodies away from the cell apex, they do not address how Woronin bodies maintain positions near septa, nor how they plug the septal pore. Berns et al (1992) observed that Woronin bodies snapped back into position near the septum after being displaced by a laser trap, prompting them to suggest that Woronin bodies are tethered to specific sites on the septum. We sometimes observed possible connections to the septum along with a ribosome-free zone surrounding Woronin bodies, as though they might be embedded in a filamentous meshwork. Several other researchers have reported similar structures associated with Woronin bodies at septa (McKeen 1971 , Collinge and Markham 1985 , Markham and Collinge 1987 ). It is possible that these structures capture and/or tether Woronin bodies near septa.

Woronin bodies are thought to move from their positions near the septum into the septal pore by bulk diffusion as cytoplasm flows toward a wound. Neither transport from the apex nor tethering near septa precludes bulk flow as a plugging mechanism. However, as pointed out by Markham and Collinge (1987) , in cases of damage, a single Woronin body is usually found plugging the septal pore. If movement to the pore were strictly by bulk flow, one might expect multiple Woronin bodies to be swept into the pore as cytoplasm rushed out.

It is possible that Woronin body movement employs both active and passive mechanisms. Perhaps active microtubule-based movement is responsible for the long-distance relocation of these fungal peroxisomes from apical to septal regions. The molecular motor dynein has been implicated in moving animal peroxisomes along microtubules (Schrader et al 2000 ). After relocation to the septal region one could envision either active or passive mechanisms for Woronin body capture/tethering and plugging. Future experiments will address the mechanisms of long-range and short-range Woronin body movement.

	ACKNOWLEDGMENTS

 
This research was funded by DOE Biosciences grant DE-FG02-97ER20275 and NSF grant MCB9904629 to MM. CV was supported by an NSF REU supplement. GJ is supported by NSF grant MCB0090908. We thank Charles Mims (UGA) for many useful discussions.

	FOOTNOTES

 
¹ Corresponding Author: Email: momany{at}dogwood.botany.uga.edu

Accepted for publication July 25, 2001.

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