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Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, SK S7N 5E2, Canada
Correspondence
Susan G. W. Kaminskyj
Susan.Kaminskyj{at}usask.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-2,6-sialyltransferase, tagged with red fluorescent protein (ST–RFP). CopA–GFP and ST–RFP distributions responded similarly to brefeldin A, which impairs Golgi/GE trafficking. We used a CopA–GFP, hypA1 strain to study GE distribution and behaviour in growing A. nidulans hyphae. This strain has a wild-type phenotype at 28 °C, can be manipulated by changing growth temperature or by use of cytoskeleton inhibitors, and its GE behaviour is consistent with that in a wild-type-morphology strain. A. nidulans GEs were more abundant at hyphal tips than subapically, and showed saltatory motility in all directions. Anterograde GE movements predominated. These were positively correlated with, but at least 10-fold faster than, hyphal growth rate, under all growth and experimental conditions investigated. The actin inhibitor latrunculin B reduced both anterograde GE movement and hyphal growth rate, whereas the microtubule (MT) depolymerizer benomyl increased anterograde GE movement and decreased hyphal growth rate. The MT stabilizer taxol increased A. nidulans GE movement but not hyphal growth rate. A. nidulans GE motility appears to have a complex dependence on both actin and MTs. We present a model for apical delivery of growth materials in which A. nidulans GEs play a role in long-distance transport.
-2,6-sialyltransferase; TEM, transmission electron microscopyA supplementary figure (video clip) showing Golgi equivalent motility in Aspergillus nidulans strain AAB1 is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Heath, 1990, 1995). Golgi bodies process and sort materials (Farquhar & Palade, 1981, 1998; Matheson et al., 2006; Mogelsvang & Howell, 2006), including those destined for secretion. Fungal Golgi equivalents (GEs) differ morphologically from Golgi bodies in animals and plants but perform similar functions (Beckett et al., 1974; Bentivoglio & Mazzarello, 1998; Cole et al., 2000), making them central players in the tip growth process. As in most fungi, Aspergillus nidulans GEs imaged with transmission electron microscopy (TEM) have single pleiomorphic cisternae (Beckett et al., 1974; Kaminskyj & Boire, 2004; Kurtz et al., 1994).
The distribution of organelles (Bartnicki-Garcia, 2002) and proteins (McGoldrick et al., 1995; Sharpless & Harris, 2002) can be used to infer their function. Evidence from A. nidulans (Breakspear et al., 2007), Aspergillus oryzae (Akao et al., 2006), Candida albicans (Rida et al., 2006) and Saccharomyces cerevisiae (Matsuura-Tokita et al., 2006) suggests that GE distribution in fungi is related to tip growth.
The behaviour of organelles in living A. nidulans hyphae has been revolutionized by fluorescent protein tagging (e.g. Suelmann & Fischer, 2000a, b). Such studies depend on verified localizations, using comparisons with independent localization methods. In A. nidulans, sodVIC (stabilization of disomy in chromosome VI) has been shown to encode a protein with high similarity to Saccharomyces cerevisiae COPI (Whittaker et al., 1999). The COPI coat is required for one type of GE-derived vesicle formation (Bentivoglio & Mazzarello, 1998; Boevink et al., 1998; Hawes & Satiat-Jeunemaitre, 2005). Breakspear et al. (2007), who renamed sodVIC as copA, showed that CopA–GFP localized to punctate arrays in A. nidulans hyphae. These arrays were adversely affected by brefeldin A (BFA) treatment, suggesting that they were A. nidulans GEs. Rat -2,6-sialyltransferase (ST) contains a transmembrane domain that has been shown to be important for Golgi body retention in animals (Munro, 1991) and plants (Wee et al., 1998), and for GE localization in yeast (Schwientek et al., 1995), suggesting that it is a reliable marker with which to confirm the localization of CopA–GFP.
A. Breakspear and S. S. Assinder (University of Bangor, UK) provided us with a CopA–GFP strain in a hypA1 temperature-sensitive background. The hypA gene encodes an orthologue of Saccharomyces cerevisiae Trs120p, a regulatory subunit in the COPII secretory pathway implicated in fungal GE transit (Shi et al., 2004). When grown at 28 °C, hypA1 strains have a wild-type phenotype (Kaminskyj & Hamer, 1998; Kaminskyj & Boire, 2004; Shi et al., 2004). When grown at 42 °C, hypA1 strains have reduced cell polarity, swollen GEs and thick cell walls, but these restrictive-phenotype cells establish wild-type-morphology hyphal branches within an hour of being shifted to 28 °C (Kaminskyj & Hamer, 1998; Kaminskyj & Boire, 2004; Shi et al., 2004).
Here, we confirm the localization of CopA–GFP at GEs in growing A. nidulans hyphae by co-localization with the ST transmembrane domain tagged with red fluorescent protein (RFP). A. nidulans GEs showed saltatory motility, with movements being predominantly anterograde. We explored the relationship between the distribution and movement of A. nidulans GEs with respect to cell polarity and tip growth using the hypA1 temperature-sensitive morphogenesis allele and cytoskeleton-targeting drugs. Our results suggest that long-distance delivery of growth-related materials to the hyphal tip may employ anterograde GE movements.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Media and culture methods are described in Hubbard & Kaminskyj (2007) and Kaminskyj (2001). Unless stated otherwise, all cells for all experiments were grown at 28 °C, and had a wild-type phenotype (Kaminskyj & Hamer, 1998). Most experiments used strain AAB1 with GFP under the control of the alcA promoter (Felenbok, 1991). GFP expression was induced by growth on complete medium (CM) containing 1 % (v/v) glycerol or 1 % ethanol as the sole carbohydrate source, or on Difco nutrient agar supplemented with 0.5 % threonine. Anhydrous ethanol or solid threonine was added to cool, autoclaved media.
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). For drug-treated cells, the dialysis tubing and overlying hyphae were mounted in a microscope slide chamber in 
100 µl liquid medium that contained solvents and cytoskeleton-selective inhibitors as required, and allowed to recover for 30 min before observation, as described in Hubbard & Kaminskyj (2007). Mounting induced transient hyphal tip swelling, which made a convenient marker for identifying vigorously growing cells. Observations were terminated by 120 min. Some hyphae were treated with BFA. In this case, hyphae were mounted in the slide chamber in CM-ethanol or nutrient broth-threonine and given 30 min for recovery, then medium plus BFA was added by rinsing through the slide chamber. Observations began as soon as possible following addition of BFA and were terminated after 60 min.
Inhibitors.
Benomyl, paclitaxel (trade name, Taxol) and anhydrous DMSO were from Sigma. Latrunculin B (hereafter, latrunculin) was from Molecular Probes. All other chemicals were from VWR. Inhibitors were diluted from stock solutions with room-temperature liquid medium immediately before use.
Benomyl was stored at 4 °C as a 10 mg ml–1 stock in 100 % ethanol, and used at 1 µg ml–1 in 0.01 % ethanol. Latrunculin was stored as a 25 mg ml–1 stock in 100 % ethanol at –20 °C, and used at 5 µg ml–1 in 0.2 % ethanol. Taxol was stored at –20 °C as a 2 mM stock in 100 % DMSO and used at 50 µM in 0.25 % DMSO. DMSO was purchased as 1 ml ampoules of dry solvent, and the stock was stored over desiccant. BFA was stored at –20 °C as a 5 mg ml–1 stock in 100 % methanol, and used at 10 µg ml–1 in 0.5 % methanol. The inhibitor concentrations were similar to or lower than those typically used (benomyl, BFA and latrunculin), or were the lowest for which a response was detected (taxol) as described in Hubbard & Kaminskyj (2007).
Confocal microscopy.
A. nidulans hyphae were imaged with a Zeiss META 510 laser scanning confocal microscope with a Plan Apochromat x63, numerical aperture 1.2 multi-immersion objective equipped with phase-contrast optics. CopA–GFP imaging used 488 nm excitation, 5–10 % power from an argon multispectral laser operated at 5.9 A, with emission controlled by a BP505-530 filter. ST–RFP imaging used 543 nm excitation, 5–10 % power from a 25 mW HeNe1 laser, with emission controlled by an LP585 filter. For each type of imaging, eight or 16 scans at 0.6–2.5 µs per pixel were used to improve signal to noise ratio. Optical sections were 1.2 µm thick, and chosen to be near-median focal level, as judged by cell profile. Observations were based on single optical sections taken from time-lapse series, 5–20 images collected over 60–300 s. Fluorescence images for studying GE movement and transmitted images for hyphal growth rate were collected at the same time.
Hyphae were chosen for analysis if they were located at the colony margin, had an even width profile, had a smoothly tapered tip and had grown out from the characteristic mounting-induced morphology, that is, a swollen tip or an abrupt change in growth direction. Hyphae that had not responded in this way to mounting were assumed to be non-growing and were not selected for analysis. However, whether a particular hypha was actually growing, and at what rate, was not determined until after the data were collected.
A. nidulans containing RFP-tagged ST.
A 52 aa transmembrane domain from rat ST (Munro, 1991), tagged at the C terminus with monomeric red fluorescent protein (mRFP, hereafter RFP) under the control of the cauliflower mosaic virus (CaMV) 35S promoter was provided by F. Brandizzi (formerly University of Saskatchewan) and C. Hawes (Oxford Brookes University). A. nidulans strain AMH1 was transformed using 4 µg ST–RFP DNA plus 1 µg ARp1-pyr4 DNA, which contains Neurospora crassa pyr4+ as a selectable marker, following the procedure described in Shi et al. (2004). Transformants were grown at 28 °C for 72 h before colonies were tested for expression of RFP and GFP fluorescence. Strain AMH2 expressed RFP on all growth media, and expressed GFP on CM-ethanol or nutrient broth-threonine. GFP and RFP expression were probed on Western blots of AAB1 and AMH2 grown under AlcA-inducing and -suppressing conditions, respectively, and using a polyclonal anti-GFP that can recognize both GFP and RFP [Santa Cruz Biotechnology Inc. (SCBI) Technical Support, personal communication]. Both constructs had bands with the expected mobility, and in addition CopA–GFP had some free GFP (see figure 3-3 in Hubbard, 2007) that likely contributed to background fluorescence.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, b). These were roughly 0.5–1 µm in diameter, and co-localized extensively (Fig. 1c). The CopA–GFP pattern had a somewhat coarse cytoplasmic background, consistent with COPI-coated vesicles that cannot be resolved by confocal fluorescence. Comparing fluorescence and transmitted images (Fig. 1c, d) at least seven of 26 large fluorescent structures appeared to co-localize with cytoplasmic structures of a similar size that were visualized with transmitted light (arrows), although many others did not (arrowheads). Epifluorescence images viewed with confocal optics have a shallower depth of focus than their associated transmitted light images, which makes precise spatial correlations difficult. Nevertheless, this is intriguing, since GEs have not, to our knowledge, been imaged previously without staining in living fungal cells.
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) which is consistent with the anticipated BFA effect. After 60 min of BFA treatment there were preliminary indications of a reticulate network (arrows in Fig. 2e) that was entirely unlike the punctate arrays seen in untreated cells.
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A. nidulans GEs are mobile, independent of tip growth
A. nidulans GEs were mobile within the near-apical cytoplasm, showing randomly directed saltatory movements. Some individual GEs could be tracked between frames of time-lapse series of single near-median optical sections, and were found to move independently in all directions (see Supplementary Fig. S1). We defined GE movements as anterograde, retrograde or lateral (Fig. 3a
), by comparing their positions between sequential frames. Transmitted light images were used to measure growth rate after the series were collected. GE motion was faster in growing hyphae, and tended to be anterograde (Fig. 3c). Rates were similar in strains with wild-type (AMH1, AMH2) and hypA1 (AAB1) backgrounds grown at 28 °C. GE movements were examined in repolarizing hypA1 cells, which had been grown for 14 h at 42 °C (at which temperature they form viable but poorly polarized cells) and then shifted to 28 °C. Under these conditions, new branches visible with transmitted light formed within 1 h (Fig. 3b). These would later grow into wild-type-morphology hyphae.
In repolarizing hypA1 phenotype cells, anterograde GE movements were about twice as fast (Fig. 3c) and twice as frequent (Fig. 3c) as those in other directions. Rates of anterograde GE movement were most different between poorly (grown at 42 °C) and highly polarized (grown at 28 °C) cells. There was a clear trend towards an increasing rate of anterograde movement during repolarization (Fig. 3c), but not towards retrograde and lateral movements (Fig. 3b) over the same time-course. Since anterograde GE movements seem intuitively to be the most immediately relevant to tip growth, the remaining data will focus on these.
Fig. 3(d) compares tip growth rates and anterograde GE movement rates in hypA1 cells growing at permissive and restrictive temperatures, and during cell repolarization, a more detailed analysis of the anterograde data shown in Fig. 3(c). The average anterograde GE movement rate was always at least tenfold faster than growth rate. In other words, there was a net anterograde movement of GEs (Fig. 3d), consistent with delivery of growth-related materials to the cell tip.
Cytoskeletal involvement in A. nidulans GE movements
GE movement will depend on the actin or microtubule (MT) cytoskeletons, or on both. We treated A. nidulans hyphae with actin-depolymerizing (latrunculin), or MT-depolymerizing (benomyl) or -stabilizing (taxol) drugs to study the relative effects on GE movements. The final drug concentrations were prepared from concentrated stock solutions in solvent, so we compared drug effects with those for solvent-treated control cells. Preliminary studies comparing GE movement and hyphal growth rates for strain AAB1 growing on CM-ethanol, CM-glycerol and nutrient broth-threonine showed no significant difference in GE movement or hyphal growth rate between different growth media (Fig. 4a; data not shown).
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), consistent with the importance of an intact actin cytoskeleton for many cellular processes. Anterograde GE movement in latrunculin-treated cells was significantly reduced compared with controls, but was not abolished (Fig. 4b). Hyphae treated with 1 µg benomyl ml–1, which we had shown previously depolymerizes all cytoplasmic MTs within 2 min but does not prevent hyphal tip growth within 1 h (Hubbard & Kaminskyj, 2007), had reduced tip growth rates, but significantly increased anterograde GE movement (Fig. 4b). Hyphae treated with 50 µM taxol, which we had shown previously significantly increases cytoplasmic MT abundance (Hubbard & Kaminskyj, 2007), significantly increased anterograde GE movement rate, but without increasing hyphal growth rate (Fig. 4c). All these drugs caused hyphal morphological abnormalities after several hours; however, we collected growth and GE data only within the first 1 h (benomyl) or 2 h (latrunculin and taxol) of treatment. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-2 in Hubbard, 2007). These were consistent with Aspergillus GEs imaged with TEM, which have single pleiomorphic cisternae (Beckett et al., 1974; Kaminskyj & Boire, 2004; Kurtz et al., 1994), similar to those of Pisolithus (Xu et al., 2004) and Schizophyllum (Rupe
et al., 1995). Fluorescent structures are luminous, which enhances detection and can increase apparent size (Hubbard & Kaminskyj, 2007). The apparent size of A. nidulans GEs is also consistent with results from plants, which have numerous, motile Golgi bodies (Boevink et al., 1998).
The CaMV promoter has been shown to induce gene expression in plants (Jefferson et al., 1987; Odell & Nagy, 1985; Wee et al., 1998) and in fungal species, including S. cerevisiae (Hirt et al., 1990), Uromyces appendiculatus (Li et al., 1993), Ganoderma lucidum and Pleurotus citrinopileatus (Sun et al., 2002), and Pleurotus ostreatus (Xu et al., 2004). This is, to our knowledge, the first demonstration of its efficacy in A. nidulans.
The transmembrane domain of ST has been shown to be associated with Golgi bodies in animal (Munro, 1991), plant (Wee et al., 1998) and fungal (Schwientek et al., 1995) systems. This is, to our knowledge, the first demonstration of its localization in A. nidulans, in which the ST–RFP fluorescence labelling pattern was clearly consistent with that of CopA–GFP labelling. Thus, ST–RFP is likely to be useful for tracking A. nidulans GEs in order to examine their behaviour in living cells without the pharmacological effects noted previously with fluorescent BFA labelling in Pisolithus (Cole et al., 2000), Pleurotus (Xu et al., 2004) and Schizophyllum (Rupe et al., 1995).
The effect of BFA on CopA–GFP fluorescence distribution in living hyphae was consistent with that shown in Breakspear et al. (2007), and with changes seen with ST–RFP. BFA blocks the function of a Sec7 guanine nucleotide exchange factor (Donaldson et al., 1992), which is required early in COPI coat formation (Helms & Rothman, 1992). Within 25 min of applying BFA, both the ST–RFP and CopA–GFP patterns became less distinct, as expected, since BFA dissociates COPI from vesicles and later affects GE morphology by perturbing vesicle trafficking (Jackson & Casanova, 2000). Khalaj et al. (2001) showed that 100 µg BFA ml–1 induces perinuclear endomembrane redistributions in Aspergillus niger after 2 h. Our milder and shorter BFA treatments had less extreme effects, but were consistent with more dramatic redistributions over longer timescales.
Distribution and motility of A. nidulans GEs
Most cellular constituents (turgor being a notable exception) show a tip-high gradient of abundance in growing hyphae, although the length and steepness of the individual gradients vary (Heath & Kaminskyj, 1989). Organelle distributions can be altered by cytoplasmic contractions during chemical fixation (Kaminskyj et al., 1992), so we examined GE distributions in living A. nidulans hyphae. In near-median optical sections, fluorescence-localized GE populations were similar to those shown elsewhere with freeze-substitution TEM (Kurtz et al., 1994). In addition, Breakspear et al. (2007) showed a similar GE abundance pattern in fixed hyphae. A. nidulans GEs are appropriately distributed to contribute to apical growth, being relatively more abundant in the near-apical cytoplasm.
In growing hyphae, the near-apical cytoplasm appears to move as a unit (sometimes called bulk flow) with respect to the lateral hyphal walls, using actin-dependent processes (Heath, 1990, 1995; Kaminskyj & Heath, 1996). Within the cytoplasm, independent nuclear (Morris et al., 1995; Suelmann & Fischer, 2000b) and mitochondrial (Suelmann & Fischer, 2000a) movements are generated by cytoskeleton-dependent processes. When cytoskeletal function was perturbed by benomyl, latrunculin or taxol, there was a trend for GEs to become relatively more concentrated near hyphal tips (data not shown), also seen in Breakspear et al. (2007) following nocodazole treatment. This is consistent with a tendency for cytoplasm to contract toward the apex following cytoplasm perturbation (Kaminskyj et al., 1992; Kaminskyj & Heath, 1996).
The GE distribution pattern in Aspergillus correlates with that of cytoplasmic MTs, which is circumstantial evidence suggesting that longitudinal (but perhaps not lateral) GE motility could use MT-dependent motors. However, unexpectedly, both cytoplasmic MT depolymerization with benomyl and MT polymerization with taxol increased anterograde GE motility. Breakspear et al. (2007) showed that the nudA1ts cytoplasmic dynein heavy chain mutation does not affect GE positioning at restrictive temperature, despite substantial effects on nuclear positioning, suggesting that nuclear and GE motility are differently regulated.
In contrast to their lack of dependence on MTs, Aspergillus GE movement and tip growth are exquisitely dependent on the actin cytoskeleton. Actin-dependent motility has also been shown for plant Golgi bodies (Boevink et al., 1998). A. nidulans hyphae treated for more than 2 h with any of these cytoskeleton-targeting drugs began to develop morphological abnormalities (Hubbard, 2007; Hubbard & Kaminskyj, 2007), suggesting that wall deposition and cell extension can continue despite sublethal cytoskeletal impairment, albeit with aberrant apical targeting. Clearly, the mechanisms underlying GE motility will prove to be complex.
Anterograde transport of tip growth-related materials
Given the size of an A. nidulans wall vesicle (50 nm diameter) and the area of cell membrane required for a growth rate of about 0.5 µm min–1 (Hubbard & Kaminskyj, 2007) of a hypha about 3 µm in diameter (Kaminskyj & Hamer, 1998), at least 600 vesicles per minute must fuse at the hyphal tip. If long-distance anterograde transport of growth-related materials in Aspergillus was in vesicles, then vesicle abundance should be relatively consistent in near-apical cytoplasm. However, TEM shows that Aspergillus hyphae have a substantially larger vesicle population in the apical 2–3 µm compared with even a few micrometres slightly further back (see figure 4a in Kurtz et al., 1994). Thus, long-distance wall material transport in vesicles is not consistent with vesicle distribution patterns in Aspergillus hyphae. This is unlike Saprolegnia ferax, where individual wall vesicles produced by Golgi bodies located in the central cytoplasm appear to migrate to and then be transported along the cell periphery (Heath & Kaminskyj, 1989). Fungal and oomycete hyphae have similar shapes, and the organisms have comparable lifestyles, but they are phylogenetically distant.
There are about 15 cytoplasmic MTs or MT bundles in the apical 20 µm of an A. nidulans hypha (Hubbard & Kaminskyj, 2007). If A. nidulans growth materials were transported in vesicles, along MTs, vesicle distributions visualized with electron tomography should be spatially correlated with cytoplasmic MTs. However, figure 2 in Hohmann-Marriott et al. (2006) does not provide support for this relationship. In addition, the average separation between vesicles and MTs is about 50 nm, twice the length expected for cytoplasmic dynein (R. Roberson, personal communication). Vesicle distribution in Aspergillus hyphae does not appear to be spatially correlated with cytoplasmic MTs, arguing against a direct role for MTs in vesicle motility.
It has generally been assumed that wall-forming materials in filamentous fungi are transported to hyphal tips in vesicles, by means of cytoplasmic MT-dependent motors (Bartnicki-Garcia, 2002; Breakspear et al., 2007; Fischer & Veith, 2007). However, direct evidence to support this model is sparse, or argues to the contrary (Hohmann-Marriott et al., 2006). Results described in Hubbard & Kaminskyj (2007) suggest that tip growth rates in A. nidulans are not quantitatively related to cytoplasmic MT number. Vesicle transport could also be mediated by actin, which has multiple roles in tip growth (Bartnicki-Garcia, 2002; Harris et al., 1994; Heath, 1990, 1995; Kaminskyj & Heath, 1996). The mechanisms underlying organelle movement in A. nidulans have not been explored, apart from those of MTs in nuclear (Morris et al., 1995; Suelmann & Fischer, 2000b) and actin in mitochondrial motility (Suelmann & Fischer, 2000a). Our data showing that A. nidulans GEs have predominant anterograde motility at rates far exceeding the rate of tip growth in the same cells suggest that growth-related materials might be transported to the tip packaged in GEs.
In contrast to a model proposing long-distance transport of wall vesicles in A. nidulans, the pattern of tip-localized vesicle abundance in Aspergillus hyphal tips is complementary to that of Aspergillus GEs (Kurtz et al., 1994; Breakspear et al., 2007; this paper). This then is consistent with the notion that GEs offload their contents in the vicinity of the Spitzenkörper, presumably as vesicles, prior to changes that permit them to fuse at the hyphal apex. In A. nidulans hyphae examined following diverse treatments, anterograde GE movement was consistently at least 10-fold faster than tip growth rates. This suggests that GEs may be delivering wall-building materials to near-apical regions of the hypha, potentially supplementing (or supplanting?) bulk delivery by individual vesicles.
A model for the role of GE movement in A. nidulans tip growth
The model shown in Fig. 5 concerns the role of GE motility in A. nidulans hyphal growth. As discussed earlier, fungal GEs have a function analogous to that of Golgi bodies in animals and plants. Endoplasmic reticulum (ER) to GE transport in fungi (Fig. 5a) might occur at specialized sites, as has been proposed for yeasts (Bevis et al., 2002; Mogelsvang et al., 2003) and plants (daSilva et al., 2004; Matheson et al., 2006). Both the ER (Fernández-Ábalos et al., 1998; Maruyama & Kitamoto, 2007) and GEs (this paper; Breakspear et al., 2007) are widely distributed in the apical cytoplasm of A. nidulans hyphae. Anterograde GE transport may be accompanied by maturation from cis-like to trans-like GE elements (Fig. 5b), a functional analogy to cisternal maturation in Golgi bodies, consistent with changes recently documented in yeast (Losev et al., 2006; Matsuura-Tokita et al., 2006). Despite predominantly anterograde GE movement, GEs do not accumulate at the tips of A. nidulans hyphae. Indeed, GEs were notably less abundant within 5 µm of the tip, suggesting that they offload their cargo (Fig. 5c), presumably as vesicles destined for the Spitzenkörper (vesicle supply centre; VSC). VSC- and exocytosis-related events (Fig. 5d) are shown for completeness, but are not directly addressed by this model. Following unloading, GEs might be transported subapically to acquire more growth-related materials (Fig. 5e), which could account for at least some retrograde and lateral movements. Studies to investigate the events involved in the loading, transport and unloading of GEs are under way.
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ACKNOWLEDGEMENTS |
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, and anonymous reviewers for useful comments. Edited by: S. D. Harris
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Received 7 November 2007;
revised 19 February 2008;
accepted 20 February 2008.
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