Cell-to-cell transport via motile tubules in growing hyphae of a fungus

Basidiomycete fungal hyphae grow continuously by divisions of a tip cell. This results in an extensive mycelium, of which the tip cell is the final outpost. Growth of this cell requires continuous synthesis of cell wall, as well as migration of cytoplasm and organelles. Cell division produces a file of cells that, in contrast to the tip cell, grow only by branching. The result is an extensive, branched mycelium, which in some fungi may culminate in a mycorrhizal association with roots. Transport of nutrients is through the mycelium and is likely to have both symplastic and apoplastic components (Cairney, 1992). Symplastic transport is reported to occur along hyphae by diffusion, cytoplasmic streaming or osmotically generated mass flow through the fungal cytoplasm (Jennings, 1987, 1989; Thompson et al., 1987). In basidiomycetes, this symplastic continuity depends on transport through the dolipores in the septa and it is usually assumed that transport across the dolipores occurs exclusively in the cytoplasmic compartment.

Recently we have shown that the fluorochrome 6-carboxyfluorescein (CF) is accumulated by a pleiomorphic system of motile tubules and vacuoles in the tip cells of the fungus Pisolithus tinctorius (Pers.) Coker and Couch (Shepherd et al., 1993). The tubules of this system, which bear a close resemblance to tubular endosomal networks of cultured cancer cells, or lysosomal networks of macrophages, can extend and retract across large intracellular distances. They can move fluorescent material by peristaltic motion between clusters of vacuoles situated at intervals along the terminal and penultimate cells. Although their movement may similarly depend on the cytoskeleton, it is independent of both the rate and direction of cytoplasmic streaming. During these observations we noted that tubules intermittently crossed the dolipore septum separating the tip and penultimate cells, indicating that this pleiomorphic tubule and vacuole system plays a role in cell-to-cell transport as well as intracellular transport. This would imply that there is a compartment additional to the cytoplasm that can, at least transiently, act as a conduit across the connecting bridges of the symplast in fungi. This contrasts with the prevailing view that transport between walled cells (higher plants and most basidiomycete fungi) occurs primarily via cytoplasm in continuities between the adjacent cells, and that any substructures in such cytoplasmic bridges are nonconducting (see Robards and Lucas, 1990).

In this paper, we provide evidence that tubular elements of the tubule and vacuole system pass through the septum and transfer material between the vacuoles in adjacent cells. This occurs at a specific stage in the cell division cycle of the tip cell in Pisolithus tinctorius. The feasibility of cell-to-cell transport across completed dolipores via tubular elements is further indicated by electron micrographs of freeze-substituted hyphae, which show that smooth tubular cisternae, of similar appearance and dimensions to the tubules that interconnect vacuoles, pass through the pores in the parenthesomes and into the entrance of the septal pore.

Mycelium of Pisolithus tinctorius (Pers.) Coker and Couch, strain DI 15, isolated by Grenville et al. (1986), was grown on Modified Melin-Norkrans agar medium (Marx, 1969) at 22°C in the dark. Samples of the peripheral growth zone of 1-to 3-week-old cultures were treated with 6-carboxyfluorescein diacetate (20 µg/ml) at pH 4.8 and prepared for microscopy as described by Shepherd et al. (1993). Cells loaded with CF were observed at different stages of the cell cycle including clamp formation, cell division and septum formation. Material was viewed with a Zeiss Axiophot microscope with the filter combination BP 450-490, FT 510 and LP 520, photographed on Kodak Technical Pan film, rated at 400 ISO and developed in Technidol. Sequences of tubule movements, lasting between 35 and 45 min, were photographed at 4 s intervals for analysis. Correlated images using Nomarski differential interference contrast (DIC) optics indicated the completeness and position of the septum. The tubule system was also seen in untreated cells with DIC optics (Orlovich and Ashford, 1993), indicating that it is not induced by CF loading.

A Leica Confocal Laser Scanning Microscope was also used to record sequences of movements of CF-labelled tubules in the vicinity of the septum during clamp formation and cell division. Cells were scanned in a single optical plane at half-second intervals and images were captured with 512 × 256 pixels resolution. One representative sequence of movements in a single plane, lasting for a total of 15 s, is presented here. The combination of filters was 488 excitation, 515 dichroic beam splitter and 515 nm barrier.

Mycelium from the peripheral growth zone was freeze-substituted and prepared for electron microscopy exactly as described by Shepherd et al. (1993). Sections were examined and photographed with an Hitachi H-7000 transmission electron microscope at 100 kV.

The mycelium of this strain of Pisolithus tinctorius is a heterokaryon with two nuclei per cell. The process of nuclear division and cytokinesis of the tip cell is complex, involving development of a clamp connection, formation of two septa, and fusion of the clamp with the penultimate cell, so that a nucleus of each type is compartmentalized into a new tip cell and the penultimate cell, respectively. This process has been recorded in detail in Pisolithus tinctorius by Orlovich and Ashford (1993) and follows the usual sequence for dikaryotic basidiomycetes. The tubular reticulum in the tip cell is extensive in the nuclear region throughout interphase (Fig. 1), is maintained during mitosis, and is distributed between the daughter cells as the septa form between them. Following mitosis, septa form synchronously across the base of the clamp and across the main hypha, separating the dikaryon in the tip cell from the other daughter nuclei of each kind (one in the clamp and the other in the new penultimate cell). Septal development takes about 5 min from initiation to completion, under our conditions.

Before and during the formation of the main septum, many tubules and some vacuoles traversed the incipient septal region in both directions, passing across the narrowing gap in the furrowing membrane. At the stage when the two septa were just completed, as determined under DIC optics, this tubule traffic temporarily ceased. This stage with complete dolipore septa is shown in Fig. 2A. Shortly afterwards, tubule movement resumed in this region, but was restricted to single tubules, which could originate in either the penultimate or the terminal cell (Figs 2B-G, 3A-J). Individual tubules transiently penetrated the neighbouring cell, and made contact with vacuoles or dilated tubules in that cell (Figs 2E, 3H). As far as they could be traced the tubules were continuous with an extensive tubular reticulum located in the nuclear regions of either cell. Fluorescent material was sometimes transferred across the septum within the tubule by peristaltic motion. One of these tubule dilations on one side of the septum is seen in Fig. 2D. Tubule movements across the main septum continued for at least 45 min and included both extensions (Fig. 2B-G) and retractions (Fig. 3A-J), with tubules from adjacent cells moving in opposite directions. For example, in Fig. 3A-D the tubule that entered the terminal cell from the penultimate cell has retracted as a tubule approaches from the opposite direction in the terminal cell. This implies that the movements of tubules originating in different cells are coordinated.

Movement across the main septum continued during the period when the clamp cell wall was being dissolved, preparatory to the movement of the nucleus to restore the binucleate condition of the penultimate cell. For example, in the sequence shown in Fig. 3 a branch has penetrated into the clamp cell (Fig. 3D), indicating that some dissolution of the clamp cell wall must already have occurred.

Tubule movements were sporadic but all extensions and retractions within several micrometres of the septum took place in a single plane. This suggests that tubule movements follow pre-determined tracks in this region. The tubule branch, which passed through the dissolving clamp cell wall, also occupied this plane, suggesting that tubules of adjoining cells share an underlying mechanism that coordinates their movements intercellularly. Most of the tubule movements in the terminal and penultimate cells described above, including those of the tubule branch that entered the clamp cell, took place in the cellular region between and including the nuclei in these three cells.

To confirm that tubules could move across intact dolipore septa, hyphal tips were freeze-substituted at a stage when the dolipore apparatus was expected to be complete (Fig. 4A,B). Both micrographs show typical dolipores with perforated parenthesomes and associated sheets of ER, lying parallel to the septum. In Fig. 4A many tubular elements are captured near and in some cases through the pores in the parenthesome, which are approximately 70 nm in diameter. These tubules were of similar dimensions to many of the tubular bridges that connected vacuoles (compare Fig. 4 with Figs 5 and 6 of Shepherd et al., 1993). They were invariably aligned perpendicular to the parenthesome, to focus on the septal pore entrance, in a plane similar to the fluorescent tubules during their movements in vivo apparently along specific tracks. They were wider than and somewhat different in appearance from ER that lies parallel with the septum and they disappeared in serial sections, indicating that they are tubules, not sheets. In Fig. 4B two tubules that had penetrated the parenthesome and passed at least as far as the entrance of the septal pore are shown. Profiles of tubules and small vacuoles were the only membrane-enclosed structures that we found between the parenthesome and the dolipore entrance in these freeze-substituted hyphae. The diameter of the septal pore was such as to allow two tubules to pass side by side in one plane of the pore.

The results show that tubular elements of the motile pleiomorphic vacuole system, which are known to move material across long intracellular distances (Shepherd et al., 1993), can also transport material from cell to cell across the dolipore septum. The data indicate that this occurs when the septa are complete, with intact dolipores. Septa are laid down synchronously across the base of the clamp and main hypha at the stage when the clamp has formed and the daughter nucleus has migrated into it, but before the clamp tip has fused with the main hypha. Septum formation is known to take about 5 min and the parenthesome is assembled very late in this process, but both septa have fully organised dolipores well before dissolution of the main hyphal wall and clamp (Orlovich and Ashford, 1993, unpublished data). Tubules extend and withdraw across the septum for most of this period; they have been recorded doing so for at least for 45 min, much longer than it takes to complete the septum. This eliminates the possibility that a tubule could have crossed the septum before its completion, and subsequently become trapped. The capacity of the dolipore for passage of tubules of the appropriate size is confirmed at the EM level by the finding that tubular cisternae, of similar dimensions and appearance to the tubules connecting vacuoles, pass through the parenthesome and into the mouth of the septal pore, and there is ample space for them to pass through the pore. The CF-loaded tubules ranged from the limit of resolution up to about 1 µm in diameter. At the EM level the cisternae radiating towards the pore were somewhat wider and more irregular than typical ER profiles in the vicinity, and the system as a whole is most like the endosomal or lysosomal networks of mammalian tissue culture cells (Swanson et al., 1987; Hopkins et al., 1990; Tooze and Hollinshead, 1992a,b). The connections of the tubule system with vacuoles that contain hydrolytic enzymes, and which have long been thought of as the lysosomes in fungal cells (Klionsky et al., 1990), also support this view. Endosomal and lysosomal networks are known to be involved in intracellular transport in cultured mammalian cells (Hopkins et al., 1990), but have not been shown to be involved in cell-to-cell transport. Identification of the compartment will require the use of specific markers to label it (Tooze and Hollinshead, 1992a,b). However, whatever the identity of the tubules in the fungal cells, they represent a distinct sub-compartment of the cytoplasm in which trans-dolipore transfer of material can occur and this has implications for long distance transport in the vacuole compartment of basidiomycete fungi.

The findings are relevant to observations on other symplastic continuities such as the plasmodesmata of higher plant cells, where an axial structure, the desmotubule, thought to be modified ER, passes through the channel (Robards and Lucas, 1990). This appears to be permanently located in the channel, whereas the motile tubular system in this fungus makes intermittent trans-dolipore movements. It is controversial whether the desmotubule is closed, or is an open tubule that operates as a transport channel through the plasmodesmata. This controversy is based on the apparent connection of the desmotubule to ER cisternae that have open lumina, whilst the tubule in most micrographs appears so constricted that its dimensions should allow space for only a few water molecules (Gunning and Overall, 1983). While the tubule and pore in this fungus are very different from those of plant plasmodesmata, the observations in this paper do show that cell-to-cell transport via a membrane-bound subcellular compartment can occur.

The work was supported by an Australian Research Council grant awarded to A.E.A.; D.A.O. was in receipt of an Australian Postgraduate Research Award. The authors are grateful to Suzanne Bullock for printing the plates and Lydia Kupsky for photographic assistance. We also thank Bill Allaway, Brian Gunning and Fred Rost for comments and criticism of the manuscript; Leica Instruments Australia for the use of the Confocal microscope; Frank Lie for his technical assistance; and Carl Zeiss Pty Ltd. for the loan of an Axiophot microscope.