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Differential distribution of the endoplasmic reticulum network in filamentous fungi

Jun-ichi Maruyama, Katsuhiko Kitamoto
DOI: http://dx.doi.org/10.1111/j.1574-6968.2007.00758.x 1-7 First published online: 1 July 2007

Abstract

Filamentous fungi are composed of hyphal compartments divided by septa, which communicate via septal pores. Apical compartments can elongate to over 100 µm without septum formation and possess a polarized distribution of organelles. In Aspergillus, subapical compartments are arrested in interphase but can reinitiate mitosis and growth by branching. Recent reports using green fluorescent protein (GFP) technology have demonstrated the highly differentiated localization of the endoplasmic reticulum (ER) network in various regions of the hyphae: the gradient distribution from the apical region, the localization along the septum, differential distributions in adjacent compartments, and the dynamic morphological change during septum formation. In this review the spatial regulation of the ER network in multicellular filamentous fungi is discussed.

Keywords
  • filamentous fungi
  • endoplasmic reticulum
  • multicellular microorganism
  • septum

Introduction

Filamentous fungi grow as straight hyphae and sometimes branch to create a new growing axis. In this highly polarized morphology, organelles are positioned in the appropriate regions of the hyphae; the Spitzenkörper, containing many vesicles, is found at the apical tip (Harris et al., 2005) and the vacuoles are largely developed in the basal regions of the hyphae (Hyde et al., 2002). Dynein, a minus-end-directed motor dependent on microtubules, transports vacuolar precursors towards the basal end of the hyphae (Seiler et al., 1999; Lee et al., 2001; Maruyama et al., 2002, 2003). This raises a question ‘Where are these vacuolar precursors transported to?’

The hyphae of the filamentous fungi are composed of compartments divided by septa. In Aspergillus the subapical compartments are arrested in interphase while the apical ones are mitotically active (Fiddy & Trinci, 1976). When the subapical compartments branch, the nuclei resume mitosis and migrate into the new branch (Fiddy & Trinci, 1976). Although adjacent compartments show different cellular states, they are considered to communicate with each other via the septal pores. The Woronin body, a wound-healing organelle, plugs the septal pore to prevent an excessive loss of the cytoplasm from the flanking compartment in injured hyphae (Jedd & Chua, 2000; Maruyama et al., 2005).

The endoplasmic reticulum (ER) consists of a polygonal network of tubules connected with the nuclear envelope. Green fluorescent protein (GFP) technology has allowed the visualization of the ER network distribution in filamentous fungi. Wedlich-Söldner (2002) demonstrated the polarized distribution of the ER network in the apical compartment. Moreover, diverse patterns of the ER network distribution, characteristic of multicellular filamentous fungi was recently observed (Maruyama et al., 2006). In this study, the spatial differences of the ER network distribution in various regions of the hyphae such as the apical and subapical compartments were reviewed.

Visualization of the ER network in filamentous fungi

In filamentous fungi, the localization and dynamics of the ER network have been studied by expression of the GFP-fused proteins (Fernandez-Abalos et al., 1998; Wedlich-Söldner et al., 2002; Maruyama et al., 2006). The fusion construct consists of GFP with the signal sequence and the ER retention signal attached at its amino and carboxyl termini, respectively. Expression of the construct in the yeast, Saccharomyces cerevisiae, results in GFP fluorescence along the nuclear envelopes and beneath the plasma membranes, which is typical of the yeast ER structure (Prinz et al., 2000; Maruyama et al., 2006). In filamentous fungi, the ER network is observed as a tubular network elongated along the hyphae that shows motility (Fernandez-Abalos et al., 1998; Wedlich-Söldner et al., 2002; Maruyama et al., 2006). In dikaryotic hyphae of Ustilago maydis, the nuclear envelope can be clearly visualized (Wedlich-Söldner et al., 2002). In Aspergillus oryzae, however, the ER network visualized by the BipA–GFP fusion protein only partially surrounds the nuclei, although the heterologous expression of the same construct in S. cerevisiae labeled complete ring structures showing the nuclear envelope (Fig. 1a) (Maruyama et al., 2006). The localization patterns of the GFP-fused ER membrane proteins (i.e., the ER SNAREs–AoSec20, AoUse1) coincide with the nuclear envelope structures in A. oryzae (Kuratsu et al., submitted for publication). These observations allow one to infer that the organization of the ER domains around the nuclei in A. oryzae differs from that in S. cerevisiae. Sec12, a transitional ER (tER) protein, shows dot-like localization in Pichia pastoris, but localizes throughout the ER, including the nuclear envelope, in S. cerevisiae, indicating a different spatial organization of tER between the two yeasts (Rossanese et al., 1999). These reports raise the possibility that the spatial organization of ER domains is governed by some mechanism specific to A. oryzae, which is not present in S. cerevisiae.

Figure 1

ER network in Aspergillus oryzae (Maruyama et al., 2006). (a) Confocal image of the ER localization in A. oryzae hypha and Saccharomyces cerevisiae. Cells expressing the BipA-GFP fusion protein were observed. Bars: 5 µm. (b) Gradient localization of the ER network from the apical region of straight growing hypha. The arrowhead indicates the hyphal tip. Bar: 5 µm. (c) Three-dimensional structure of the ER network along the first septum (from two different angles). Bar: 2 µm. (d) Confocal image of differential distribution of the ER network in adjacent compartments divided by the first septum. The arrow and ‘V’ indicate the first septum and the vacuoles, respectively. Bar: 5 µm.

Gradient distribution of the ER network in the apical compartment

In filamentous fungi, the apical compartments sustain polarity during hyphal growth and can elongate over 100 µm without septum formation. The ER network shows a gradient distribution with increasing content towards the hyphal tip in A. oryzae (Fig. 1b) and the dikaryotic hyphae of U. maydis (Wedlich-Söldner et al., 2002; Maruyama et al., 2006). During apical branching in A. oryzae, the ER network is concentrated at the newly formed tip and displays a new gradient distribution towards the basal region (Maruyama et al., 2006). This observation suggests that the orientation of the ER network gradient is altered when a hypha establishes a new axis of growth polarity. Similarly, a new Spitzenkörper emerges de novo at the new site of branch initiation (Girbardt, 1957; Riquelme & Bartnicki-Garcia, 2004; Harris et al., 2005). The apical gradient of ER in the dikaryotic hyphae of U. maydis seems to consist of small dots and disappears upon exposure to brefeldin A, an inhibitor of anterograde transport from the ER to the Golgi (Wedlich-Söldner et al., 2002). It is suggested that the dot structures found in the gradient are ER–Golgi cycling vesicles. Considering these microscopic observations together, it is proposed that the gradient distribution of the ER network supports a continuous bulk flow for polarized secretion and hyphal growth.

Distribution of the ER network along the septum

In filamentous fungi, the ER distribution has been investigated mainly in the apical regions of hyphae (Fernandez-Abalos et al., 1998; Wedlich-Söldner et al., 2002). Recent study of the authors (Maruyama et al., 2006) revealed unexpected distributions of the ER network in various parts of the hyphae. One of the findings was the ER network distribution along the septa (Fig. 1c). The ER-resident SNARE proteins fused with GFP were also found to distribute along the septa in A. oryzae (Kuratsu et al., submitted for publication). A three-dimensional reconstruction of confocal images revealed that the ER is able to form a tubular network along the septum but is excluded from the septal pores (Fig. 1c). In basidiomycetes the ER locates parallel to the septal pore caps (Müller et al., 1998, 2000). Although A. oryzae does not have a similar structure at the septa (Maruyama et al., 2005) as other filamentous ascomycetes, it can be hypothesized that the ER network surrounding the septal pores might regulate intercellular communication such as transport of cytoplasmic constituents through the septal pores.

Differential distribution of the ER network in adjacent compartments

It is generally assumed that the subapical compartments are arrested in interphase while the apical one remains mitotically active (Fiddy & Trinci, 1976). The ER network distribution was analyzed in the adjacent compartments flanking the first septum (Maruyama et al., 2006). In most cases the ER network proliferated more extensively in the second compartment, whereas its distribution gradient in the apical compartment had been completed up to the first septum (Fig. 1d), resulting in a discontinuity in the distribution of the ER network across the first septum. In the apical compartment, the vacuoles accumulated near the first septum, which showed the opposite gradient to the ER (Fig. 1d). This suggests that the final destination of the vacuolar precursors may be the apical side of the first septum. A gradient distribution of the ER network was also found during branching at the newly formed tip from the second compartment, in which the vacuoles were sequestered to the distal end of the compartment (Fig. 2). This revealed that some of the second compartments were differentiated to behave independently from the flanking apical and distal ones for branching and had established their own gradient unit of organellar distribution as the apical compartment does. Although hypercellular mutants with unusual numbers of nuclei and abnormal enlargement in the subapical compartments have been isolated from Aspergillus nidulans (Kaminskyj & Hamer, 1998), the independent behavior of the subapical compartment is poorly understood at the molecular level.

Figure 2

Schematic model of the differential distribution of the ER network in adjacent compartments (Blue, ER; Green, nucleus; Yellow, vacuole; Red, Woronin body). Discontinuity of the ER network distribution in the second compartment is shown. Branching from the second compartment (Left) causes an independent gradient unit of the ER network distribution at the newly formed tip. Vacuoles are developed and localize near the distal septum. After a hypotonic shock (Right), the ER network distribution is retained due to plugging of the Woronin body at the septal pore. The second compartment restarts growth and forms an intrahyphal hypha, resulting in proliferation of the ER network for polar hyphal extension.

As suggested above, septum formation independently regulates the ER network distribution in adjacent compartments in A. oryzae. However, this notion conflicts with the evidence in some of the filamentous fungi that adjacent compartments communicate through the septal pore and that intracellular structures (nuclei, tubular endomembranes, microtubules) pass through the septal pores (Shatkin & Tatum, 1959; Burnett, 1968; Hunsley & Gooday, 1974; Shepherd et al., 1993; Markham, 1994; Freitag et al., 2004). In the basidiomycete Pisolithus tinctorius, the tubular endomembranes crossing the septal pore have been suggested to exchange materials between the vacuole systems in adjacent compartments (Shepherd et al., 1993). In Neurospora crassa, with a wider diameter of hyphae, cytoplasmic constituents are apparently streamed through the septal pores (Tey et al., 2005). That obvious cytoplasmic streaming is not observed in A. oryzae, is possibly due to the narrower size of its septal pore, and its slower growth rate, compared with N. crassa (Freitag et al., 2004). Electron microscopic images capturing septal pores demonstrate that some are plugged by an electron dense structure that is not the Woronin body (Trinci & Collinge, 1973; Maruyama et al., 2005). More recently, Fleissner & Glass (2007) have reported that N. crassa SO protein is localized at the septal plugs during hyphal damage and aging. Since the Woronin body does not seem to plug the septal pores in undamaged living vegetative hyphae (Maruyama et al., 2005), other structures and/or molecules might plug the septal pores to induce independent behavior of the ER networks in adjacent compartments.

Dynamics of the ER network during septum formation

Formation of a new septum involves the supply of new cell wall materials at a perpendicular axis to hyphal growth. In A. nidulans some of the chitin synthases and chitinases localize at the septum formation site (Ichinomiya et al., 2005; Takeshita et al., 2005, 2006). In Aspergillus niger and A. oryzae, secretory proteins are in part targeted to the septa (Gordon et al., 2000; Masai et al., 2003; Maruyama et al., 2005), implying that the secretory pathway may play a role in septum formation. However, little is known about organellar distribution during septum formation, in filamentous fungi. Time-lapse analysis in our study using the BipA–GFP fusion protein (Maruyama et al., 2006) provided novel information about the ER network distribution during the formation of a new septum (Fig. 3). Before septum formation the ER network is continuously distributed across the site to be septated later. Formation of a new septum is initiated from the cortical side (Harris, 2001). Coinciding with this event, the ER is found as a line at the septum formation site. When septum formation has just been completed, approximately 10 min after its initiation, the ER seems to concentrate near the center of the new septum. Subsequently, this ER spot disappears and then the neighboring region of the new septum temporarily lacks an ER distribution compared with the other hyphal regions. These ER dynamics are in accord with that of formin SepA during septum formation in A. nidulans (Sharpless & Harris, 2002). The formins are involved in nucleation of the actin cytoskeleton during cytokinesis (Glotzer, 2003). It might be that the dynamics of the ER network during septum formation are regulated by the formins and actin. Although other marker proteins for the ER and Golgi should be carefully investigated for their localizations during septum formation, our observation supports the idea that the secretory pathway components, such as the ER and Golgi could supply additional membranes and wall materials at septum formation sites to complete cytokinesis.

Figure 3

Schematic representation of the ER network distribution during formation of the new first septum (Blue, ER; Green, nucleus; Red, Woronin body). Before septum formation no discontinuity of the ER network distribution is found. When septum formation is initiated from the cortical region, the ER appears as a line at the location of septum formation. During ongoing septation the line structure of the ER is shortened into a dot at the septal center and then disappears. Soon after completion of septum formation, the ER network proliferates on the side of the new first septum away from the hyphal tip while it is poorly developed on the other side of the septum. Consequently, discontinuity of the ER network distribution is generated across the septum.

Soon after septum formation, the ER network in the second compartment proliferates towards the septum and reaches along the side of the new septum that is away from the hyphal tip (Fig. 3). In contrast, the ER network is poorly developed on the side of the new septum that is the closest to the hyphal tip, resulting in discontinuity of the ER network distribution across the septum soon after completion of septum formation. Westfall & Momany (2002) reported that, after the completion of septum formation, the A. nidulans septin AspB resides at the apical side, which is opposite to the side of the proliferated ER network in this study. In S. cerevisiae septins compartmentalize the ER during polarized growth (budding) (Luedeke et al., 2005). This evidence leads to the suggestion that the septins, such as AspB, may generate a discontinuity of the ER network distribution across the septum and induce independent polarities to adjacent compartments in the filamentous fungi.

The ER network distribution during hyphal tip bursting and intrahyphal hyphae formation

The authors had previously established a method for visualizing hyphal tip bursting when they are subjected to hypotonic shock. In this experimental system, the cytoplasm of many hyphal tips at the margin of the colonies grown on agar medium burst out within several minutes after the addition of water (Maruyama et al., 2005). Extensive loss of the cytoplasm from the second compartment adjacent to the burst hyphal tip is prevented by the Woronin body, which plugs the septal pore (Maruyama et al., 2005; unpublished data). When hyphal tip bursting is induced by hypotonic shock, the ER in the apical cell is dispersed and fragmented into small dots. In the second compartment, however, the ER network is still intact and shows motility (Fig. 2). Moreover, the ER network distribution along the septa is retained normally, whereas the septum flanking the burst apical compartment is bent. These results suggest that the Woronin body plugs the septal pore immediately after hyphal tip bursting without disturbing the second compartment.

The compartment adjacent to the damaged cell consolidates the plugged position by new wall synthesis and can form new hyphae, such as branching and intrahyphal hyphae (Trinci & Collinge, 1974; Jedd & Chua, 2000). This study (Maruyama et al., 2006) investigated the ER network distribution during regrowth after hyphal tip bursting. At 1–2 h after hypotonic shock, some of the burst hyphae resumed growth by perforating the plugged septa, resulting in the formation of intrahyphal hyphae into the lysed apical compartments (Fig. 2). The ER network at the regrowing tip proliferated more intensively than that in the second compartment immediately after hypotonic shock (Fig. 2). These observations reveal that the second compartment adjacent to the burst hyphal tip is released from an arrested state, and then the ER network distribution is reorganized for polarized growth of the intrahyphal hypha. Intrahyphal hyphae are seen in mature regions of colonies in filamentous fungi (Buller, 1958; May, 1992), and disruption of the A. nidulans csmA and csmB genes encoding class V chitin synthases with a myosin motor-like domain are known to cause intrahyphal hyphae formation (Horiuchi et al., 1999; Takeshita et al., 2006). However, it has not been shown how the subapical compartment releases the arrested state for regrowth.

conclusions

Filamentous fungi are multicellular microorganisms with intercellular communication through septal pores. This characteristic is also found in higher eukaryotes as gap junctions and plasmodesmata in animal and plant cells, respectively. Although this interesting feature has not been studied well, the highly differentiated distributions of the ER network allowed the focus on compartmental regulation in filamentous fungi. Studies of the ER network distribution should aid the understanding of the maintenance of homeostasis in eukaryotic multicellular microorganisms.

Acknowledgement

This study was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (Japan).

Footnotes

  • Editor: Richard Staples

References

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