Journal of Cell Science 105, 1173-1178(1993)
Printed in Great Britain © The Company of Biologists Limited 1993
1173
Cell-to-cell transport via motile tubules in growing hyphae of a fungus
V. A. Shepherd*, D. A. Orlovich and A. E. Ashford
School of Biological Science, University of New South Wales, PO Box 1, Kensington, NSW 2033, Australia
*Author for correspondence
SUMMARY
The system of pleiomorphic, motile tubules and vacuoles
in growing hyphal tips of Pisolithus tinctorius has been
shown to play a role in intracellular transport. Here we
show that the same system also exchanges material
between adjacent cells. This exchange is most obvious
between terminal and penultimate cells following
nuclear division in the tip cell and just before dissolution of the cell wall between the clamp connection and
penultimate cell. At this stage the two new dolipore septa
are complete. The process was studied in living hyphae
using confocal and conventional fluorescence
microscopy. Tubules could move in either direction
across the septum and often extended and retracted several times and penetrated for some distance (e.g. 40 m)
into the receiving cell. Movements appeared co-ordinated and during the exchange tubules transiently interconnected vacuoles in adjacent cells and by peristaltic
movements appeared to transfer material between them.
INTRODUCTION
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 (Shep-
The fluorescent tubules occupied a specific plane in the
vicinity of the septum and remained in this plane for
the duration of their movement, suggesting that their
orientation and direction of movement is controlled. In
freeze-substituted hyphae, tubular cisternae of similar
dimensions to fluorescent tubules passed through the
parenthesome pores perpendicular to the septum and in
some cases entered the mouth of the septal pore. This
indicates that the septal pore is of an appropriate dimension to accommodate the tubules and that they can cross
the septal pore to exchange material between vacuole
systems of adjacent cells. This is the first direct demonstration of such intercellular transport via a sub-cellular compartment.
Key words: pleiomorphic vacuolar system, Pisolithus tinctorius,
cell-to-cell transport, 6-carboxyfluorescein, freeze-substitution,
confocal microscopy
herd 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
1174 V. A. Shepherd, D. A. Orlovich and A. E. Ashford
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 cellto-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.
MATERIALS AND METHODS
Fungal material, loading of fluorochromes, and
fluorescence microscopy
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.
Laser scanning confocal microscopy
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.
Freeze-substitution and electron microscopy
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.
RESULTS
Intercellular transport via motile tubules in living
cells
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, 3AJ). Individual tubules transiently penetrated the neighbour-
Fig. 1. Accumulation of fluorescent material in motile tubules (t) and vacuole (v) clusters in the terminal cell of Pisolithus tinctorius, after
loading with CF. Both tubules and vacuoles accumulate CF. The position of the hyphal tip is indicated by arrowheads.Bar, 10 µm.
Cell-to-cell transport via motile tubules 1175
ing 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
Fig. 2. Sequence of intercellular movements of a motile tubule as shown by fluorescence microscopy. (A) Following nuclear division in
the terminal cell a septum forms across the main hypha and at the base of the clamp to isolate the migratory nucleus (n) in the clamp. This
process takes about 5 min; the clamp septum lags very slightly behind the main septum but both are complete here. The clamp has not yet
fused with the penultimate cell to produce the heterokaryon and some time may elapse before it does so. The clamp septum shows a
typical dolipore, while the main septum is sectioned above the plane of the pore. Bar, 1 µm. (B-G) A CF-loaded tubule originating in the
penultimate cell (P) is shown crossing the septum of the main hypha, to communicate between the penultimate and terminal cell. The
clamp cell is out of focus below the main hypha. In (B) initially tubules are present in both the terminal (T) and penultimate (P) cells, but
at this stage there is no tubule across the septum (arrowheads), the precise position of which (arrowheads) is shown in (C) by DIC optics.
The septum appears complete and its position is indicated relative to the vacuole (v) in the penultimate cell which is both fluorescent and
visible with DIC optics. In (D) 4 s later a tubule (p) originating in the penultimate cell has passed vacuole (v) and crossed the septum
(position arrowed). On the other side of the septum the tubule is dilated. In (E) 8 s later than (C), the tubule (p) has fused with a small
vacuole in the terminal cell. The septum and tubule are seen together by simultaneous use of epifluorescence and DIC optics. (F) After a
further 8 s connection between vacuoles on either side of the septum may be seen; and (G) ultimately the tubule (p) fragments into shorter
lengths. Bar, 20 µm.
1176 V. A. Shepherd, D. A. Orlovich and A. E. Ashford
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.
Electron microscopy
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 membraneenclosed 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.
DISCUSSION
10 µm
10 µm
The results show that tubular elements of the motile
pleiomorphic vacuole system, which are known to move
Fig. 3. Confocal laser scanning microscopy shows tubule
movements across the septum, here captured in stills from a
sequence scanned at 0.5 s intervals in a single optical plane over a
period of 15 s. The tubule (t) originates in the terminal cell, and
that labelled p in the penultimate cell. The clamp (c) is partially
out of focus, but may be used as a reference point, and the
position of the septum across the main hypha is indicated in each
micrograph by an arrowhead. (A) Tubule (p), which is continuous
with vacuoles (v) in the penultimate cell, passes through the centre
of the septum, and for about 2 µm into the terminal cell. Tubule t
is also seen in the terminal cell, passing below the vacuole
(B) Tubule p has changed position and orientation and has begun
to withdraw, while tubule t has extended. (C) Tubule p has
withdrawn from the septum, whilst tubule t continues to extend.
(D) Tubule p has withdrawn while tubule t has approached closer
to the septum. Tubule p now branches into the clamp (small
arrowhead), indicating that the cell wall has begun to dissolve and
that cell fusion has commenced. (E) Tubule p has extended further
and has reached the septum. Tubule t also continues to extend, and
in (F) penetrates the penultimate cell, sliding more or less in
parallel past tubule p. (G) Tubule p remains still at the septum,
while tubule t continues to extend parallel to it, further into the
penultimate cell. (H) Tubule p remains at the septum, whilst
tubule t extends further and fuses with vacuoles in the penultimate
cell. Tubule t can be traced for at least 20 µm from the terminal
cell into the penultimate cell. (I) Tubule p now retracts 1-2 µm
from the septum, but its tip is still aligned parallel to tubule t
(J) Tubule p retracts further and is now separated from tubule t.
Thus during the sequence both tubules have crossed the septum, in
opposite directions, and have been transiently linked to a vacuole
cluster. Bar, 10 µm.
Cell-to-cell transport via motile tubules 1177
Fig. 4. Two electron micrographs from a series through the septum between terminal and penultimate cells of the main hypha, showing
smooth membrane cisternae in the dolipore region. (A) Several cisternae radiate towards the parenthesome and three pass through the
parenthesome pores. The cisternal profiles appear precisely aligned so that they pass through the pores more or less perpendicular to the
parenthesome, towards the mouth of the septal pore. (B) Some cisternal profiles can be traced to the dolipore entrance, where they
partially occlude the septal pore and displace the electron-opaque material in the pore. The diameter of the parenthesome pore (about 70
nm), is similar to that at the septal pore entrance. Two cisternal profiles occur at the septal pore mouth (arrows), although more than this
number penetrate the parenthesome. A vacuole profile (v) occurs close to the septal pore and these were the only organelles apart from
cisternae found in the sub-parenthesome region. er, endoplasmic reticulum; tc, tubular cisternae. Bar, 0.5 µm.
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.,
1178 V. A. Shepherd, D. A. Orlovich and A. E. Ashford
1990), but have not been shown to be involved in cellto-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 transdolipore 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.
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(Received 30 March 1993 - Accepted 23 April 1993)