Journal of Cell Science 104, 495-507 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
495
A dynamic continuum of pleiomorphic tubules and vacuoles 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 vacuole system in growing hyphal tips of Pisolithus
tinctorius is a dynamic continuum of vacuoles and extensible tubular elements. The system varies from a tubular reticulum with few vacuoles across a spectrum of
intermediate forms to clusters of vacuoles with few
tubules. Spherical vacuoles interconnected in clusters
are situated at intervals along the hyphal tip and are
transiently linked by tubules that extend from a vacuole
in one cluster and fuse with that of another. Extension
and retraction of the tubules is independent of cytoplasmic streaming, can occur in either direction, and
covers distances as great as 60 µm. The tubules pulsate
and peristalsis-like movements transfer globules of
material along them between the vacuoles in different
clusters. The tubules also generate vacuoles. The tubular system has the potential for intracellular transport
of solutes in the hyphal tips without concomitant transfer of large amounts of membrane. This contrasts with
models of intracellular transport via vesicles, where the
ratio of membrane transferred to internal content is
very much higher. The system has many features in
common with tubular endosomal and lysosomal systems
in cultured animal cells.
INTRODUCTION
network, endosomes and some lysosomes that were formerly thought to be discrete to form extensive tubular networks throughout the cell (see Tooze and Hollinshead,
1992). In fungi the vacuole system has been examined
using fluorescent markers in living cells of only a few
species, mostly yeasts (Makarow, 1985; Preston et al.,
1987; Roos and Slavik 1987; Weisman et al., 1987; Banta
et al., 1988; Weisman and Wickner, 1988; Basrai et al.,
1990). These studies demonstrate that, at least in yeast,
intervacuolar transport occurs at a particular stage in the
life cycle and is a precisely controlled event (Weisman and
Wickner, 1988), but the mechanism for transfer is far from
clear. Fluorescent trails connecting vacuoles have been variously interpreted as chains of small vesicles moving along
predetermined tracks in the cytoplasm (Weisman and
Wickner, 1988), or tubular connections (Klionsky et al.,
1990). In higher plants a dynamic association between
tubular elements and vacuoles has been shown in developing Allium guard cells (Palevitz and O’Kane, 1981; Palevitz et al., 1981). It is also becoming clear that the “large
central vacuole” of higher plant cells can be accompanied
by other vacuole systems of different morphology and
motility, some of which are tubular; these include the
pleiomorphic canalicular system of tomato hairs (McCully
and Canny, 1985) and the tubular system in cultured carrot
cells (Hillmer et al., 1989). Although neglected, such
motile vacuolar systems appear to be widespread amongst
higher plant cells of different types (McCully and Canny,
Fungal vacuoles, like higher plant vacuoles, play an important role in controlling the composition of the cytoplasm,
storage and lytic activity (Boller and Wiemken, 1986), and
are generally depicted in the literature as separate, more or
less spherical, bodies enclosed by a single membrane. They
are usually considered to act as a repository for material
and an end-point for intracellular transport, in contrast to
the endoplasmic reticulum (ER), other reticula and various
types of vesicles, which play an intermediary role in transport. This rather static view of vacuoles is understandable,
since most of our knowledge of the structure and functions
of fungal vacuoles is based on electron microscopy of thin
sections of chemically fixed material or biochemical analysis involving cell fractionation (Klionsky et al., 1990). The
tendency of both these approaches to fragment organelle
systems that form a reticulum and represent tubules or reticula as discrete spherical structures has been widely discussed (Mersey and McCully, 1978; Hopkins et al. 1990;
Wilson et al., 1990; Tooze and Hollinshead, 1992).
Recent methods used to investigate organelles using fluorescent markers in living cells, freeze substitution, and
electron-opaque markers of physiological significance
avoid some of these problems. Data from these techniques,
mostly from animal cells, have emphasised: firstly, the
dynamic nature of organelle interactions and; secondly, the
capacity of a number of organelles, such as the trans-Golgi
Key words: tubules, vacuoles, endosomes, Pisolithus tinctorius,
intracellular transport, 6-carboxyfluorescein, freeze-substitution
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V. A. Shepherd, D. A. Orlovich and A. E. Ashford
1985). These observations contrast with a prevailing view
of the static nature of vacuoles and the view that transfer
of materials between cellular compartments, including vacuoles, is predominently or indeed almost exclusively, via
vesicles.
We have loaded the vacuoles of young living hyphae of
Pisolithus tinctorius, a mycorrhizal fungus, with 6-carboxyfluorescein (CF) and monitored the changes in form
and interactions of the vacuoles using fluorescence
microscopy. CF has been used as a symplastic tracer in
higher plants (Oparka, 1991) and is known to be compartmented by vacuoles of Egeria densa (Goodwin et al., 1990).
It is considered to be membrane impermeant in dissociated
state, but can be loaded into cells as the non-fluorescent
diacetate, which is cleaved by intracellular esterases to form
the highly fluorescent CF (Goodall and Johnson, 1982). The
images from fluorescence microscopy are compared with
electron micrographs of freeze-substituted cells. Freeze substitution has well-known advantages over chemical fixation
in capturing the morphology of dynamic vacuole and tubule
systems in plants and fungi (McCully and Canny, 1985;
Howard and O’Donnell, 1987). Together, these methods
reveal a motile, pleiomorphic and interactive vacuole and
tubule system, which has characteristic behaviour and transports the fluorochrome between vacuole clusters situated at
intervals along the growing hyphal tips. The smaller tubules
are morphologically indistinguishable from tubular smooth
ER (see Hepler et al., 1990). However, their accumulation
of fluorochrome, their patterns of motility and behaviour
and their connection with vacuoles show similarities with
the endosomal and lysosomal networks recently described
in animal tissue culture cells.
MATERIALS AND METHODS
Loading cells with 6-carboxyfluorescein
Pisolithus tinctorius (Pers.) Coker and Couch, strain DI-15, isolated by Grenville et al. (1986), was cultured on modified MelinNorkrans (MMN) agar medium (Marx, 1969) at 22°C in the dark.
Actively growing hyphae from cultures 1 to 3 weeks old were
treated with 6-carboxyfluorescein diacetate (CFDA). A 20 µg/ml
solution of CFDA (Molecular Probes, OR, USA) was made up by
diluting a 1 mg/ml stock solution in acetone with reverse osmosis water (final pH 4.8). The solution was not buffered; both phosphate and zwitterionic buffers diminished cell viability, as indicated by cessation of cytoplasmic streaming. Solutions retained
low fluorescence for at least 24 hours, indicating a low CFDA
hydrolysis rate in the external medium. A large segment (about 4
mm × 4 mm) was cut from the growing edge of the fungal colony
and floated upside-down in each solution to submerge the fungus
at the agar-air interface. Optimal staining of the vacuole system
was obtained with a 10-min pulse of CFDA followed by 30 min
in medium without CFDA. Each mycelial segment was then
placed agar-side-up on a slide, a coverslip was pressed gently on
to it, and it was viewed by fluorescence microscopy. Observations
were confined to terminal and penultimate cells of actively growing submerged hyphae. Any aerial hyphae and hyphae submerged
more than 1 mm below the agar surface were disregarded. Cytoplasmic streaming continued and the vacuole system maintained
its characteristic behaviour in the 6-carboxyfluorescein (CF)loaded cells for a minimum of 1 h and up to 4 h during exposure
to blue excitation.
Test for purity of the fluorochrome and its identity
after compartmentation
Whole fungal colonies (4 replicates), cut from agar plates and
floated in the CFDA solution as described above, were macerated
with a mortar and pestle. Control colonies floated in distilled
water, and agar soaked in either CFDA or water were similarly
macerated. CF was obtained by hydrolysis of the diacetate solution. Samples of each were loaded on to thin-layer chromatography plates (Kieselgel 60; Merck, Darmstadt, GDR) and developed
in n-butanol:acetic acid:pyridine:water, 15:3:10:12, by volume.
The Rf values of fluorescent components in each solution were
calculated following examination under UV light.
Light microscopy
Fluorescence micrographs were taken on a Zeiss Axiophot microscope equipped with an HBO 50 mercury arc lamp, using filter
combination BP450-490, FT 510 and LP 520. Photomicrographs
were taken on Kodak Technical Pan film rated at 400 ISO and
processed in either Technidol or HC 110 developer. To check
whether CF-loading plus irradiation modified vacuole morphology or movement, CF-loaded and control (water-soaked) hyphae
were compared using Nomarski differential interference contrast
(DIC) optics.
Freeze-substitution and electron microscopy
The mycelium was allowed to grow over 5 mm discs of autoclaved Nuclepore brand ‘Membra-fil’ gridded membrane filter (8
µm pore size) placed on the agar surface just ahead of the growing front of the colony. The membrane filters were cut from the
agar surface before the hyphal tips had reached the other side.
Each filter was placed on a disc of aluminium foil attached to the
end of a plunging rod, which was then slammed on to the polished surface of a liquid nitrogen-cooled copper block. The frozen
hyphae were transferred to vials containing 4 ml of 2% osmium
tetroxide in acetone with 3 Å molecular sieve, and substituted at
−70°C for six days. Vials were returned to room temperature in
stages (1 hour at −20°C, 1 hour at 4°C and 1 hour at room temperature), rinsed three times (3 × 10 min) in fresh dry acetone and
infiltrated with epoxy resin (Spurr, 1969). Sections were collected
on Formvar/carbon-coated copper slot grids and stained with
methanolic uranyl acetate (10 min) followed by undiluted lead citrate for 20 min (Reynolds, 1963). Electron micrographs were
taken with an Hitachi H-7000 transmission electron microscope
at 100 kV.
RESULTS
Purity of fluorochrome and identity after
compartmentation
Chromatograms of the CFDA-treated fungal macerate gave
one yellow fluorescent spot (Rf 0.77) identical to the single
spot obtained from a hydrolysed CFDA solution, indicating that the CFDA was pure and that the compartmented
fluorochrome was the single compound, CF. Two pale blue
fluorescent spots, with Rf values of 0.85 and 0.72, were
present in both CFDA-treated and control fungal macerates,
but the only fluorescence detected by microscopy of whole
live cells under blue excitation was a faint orange autofluorescence in the hyphal walls. Freshly prepared CFDA solution also gave a faint spot of Rf 0.77, showing that the solution contained a small amount of hydrolysis product. The
agar contained no fluorescent compounds and agar soaked
in CFDA gave only the usual spot of Rf 0.77.
Continuum of tubules and vacuoles in hyphae
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Fig. 1. Fluorescence micrographs (except B) showing variation in form of the vacuole and tubule system in various terminal cells of
actively growing hyphal tips, after CF loading. Arrowheads indicate the position of the hyphal tip in each case. Bar, 20 µm.(A) Clusters
of vacuoles are situated at intervals along the terminal cell with tubules in the intervening regions. The cytoplasm at the tip is relatively
free of vacuoles and tubules. (B) Phase-contrast micrograph showing the same cell as (A) to demonstrate the position of the hyphal tip.
(C) Terminal cell containing mostly vacuoles, larger than those in A. Single tubules (t) span the nuclear zone, which is free of vacuoles.
(D) Terminal cell containing mostly a tubular reticulum, which interconnects along a considerable portion of the cell, with a few small
vacuoles. Some tubules have dilated tips (small arrowhead) and others are confluent with vacuoles (larger arrowheads). (E) Tubules
commonly round up to form long strings of small, similar-sized vacuoles (v). These are seen to be part of the tubular reticulum, which
includes tubular parts (t). A tubule extends into the apical zone at the extreme tip. (F) The strings of vacuoles (arrowheads), connected by
fine fluorescent bridges, can span long distances and are continuous with tubules. The basal zone contains larger vacuoles (v) and tubules.
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Accumulation of 6-carboxyfluorescein
CF was rapidly accumulated by many of the hyphal tips.
A low level of fluorescence was transiently present in the
cytoplasm, and intense fluorescence rapidly appeared in an
interconnected tubule and vacuolar network (Fig. 1A). This
was only seen in growing hyphal tips (Fig. 1B). More
mature cells of each hypha, usually including the penultimate cell, contained fewer, larger, more or less elliptical or
rounded vacuoles. These were also interconnected and
accumulated fluorochrome. There was no apparent effect of
accumulated CF plus irradiation on either cytoplasmic
streaming or the behaviour of the vacuole system in the
short term, as demonstrated by comparison of CF-loaded
and control hyphae under DIC optics. However, after about
3 h under blue excitation the tubules broadened and coalesced, and their movements slowed.
General morphology and movements of the
vacuole system in terminal cells
The labelled vacuole and tubule system consisted of clusters of more or less spherical vacuoles located at intervals
along the terminal cell with an interconnecting system of
tubules (Fig. 1A,B). The variation in form in different
hyphal tips at the growing edge of the same colony is shown
in Fig. 1; the two extremes were either clusters of vacuoles
with very few associated tubules (Fig. 1A,C), or a tubular
reticulum with a few small vacuoles (Fig. 1D). Most tips
contained a combination of both. Tubules were of various
lengths and could be either single (Fig. 1C) or branched
(Fig. 1D,E). Their diameter ranged from the limit of resolution to about 0.6 µm, and they were up to 60 µm long.
They were invariably oriented approximately parallel or at
a small angle to the long axis of the hypha (Fig. 1C,D,E,F)
and, in some cells, they formed a continuous reticulum,
which was interconnected for the full length of the cell.
The terminal cell could be subdivided into zones (apical,
subapical, nuclear and basal) on the basis of the distribution and behaviour of the vacuole and tubule system. There
was usually at least one vacuole cluster in each zone, with
the exception of the zone containing the two nuclei, which
was generally free of vacuoles, but often contained single
tubules (Fig. 1C) or a tubular reticulum. Vacuoles were
absent from the extreme tip of the cell and those in zones
anterior to the nucleus were usually small (Fig. 1A,E),
though not always (Fig. 1C). The basal zone, between the
nuclei and septum, was the most obviously variable, being
occupied either almost exclusively by tubules, or by several large vacuoles interconnected by bridges (Fig. 1F), or
occasionally both.
During the observation period vacuole clusters tended to
remain in the same location although they underwent saltatory movements, changed shape and showed transient
fusions within the cluster (Fig. 2A-D). This contrasted with
the movement of the tubular elements, which could rapidly
extend and retract over long cellular distances. Individual
tubules could extend from a vacuole in one cluster (Fig.
2E,F) into an adjacent zone and fuse with another vacuole
or tubule. Tubule movement was intermittent, unpredictable
and multidirectional. Some tubules underwent and completed their movements within a second or two, while others
remained extended in a single plane for at least one minute,
Fig. 2. Time-lapse sequences illustrating characteristic
movements of the vacuoles and tubules. Bars, 10 µm. (A-D)
Sequence from a series of fluorescence micrographs taken at 4-s
intervals to show the saltatory movements of interconnected
vacuoles. (A) Several vacuole groups, one of which initially
contains 3 vacuoles (a). (B) Another vacuole has been added to
group (a), which remains strung out. In (C) the group (a) now
shows more irregular arrangement of the 4 vacuoles. In (D) the
group has rounded up to form a small vacuole cluster. Other
vacuole groups show similar types of movements, and are
interconnected by just perceptible fluorescent bridges.
(E,F) Sequence illustrating the extension of a tubule from a large
vacuole. In (E) the tubule extends from a large vacuole (v) for a
short distance and shows a dilated tip (arrowhead). (F) 4 s later the
tubule has extended towards the adjacent vacuole (v*) and a
narrow dilation (arrowhead) marks the original position of the
dilated tip, suggesting that material has flowed forward from it.
(G,H) Sequence showing retraction and change in position of a
non-dilated tubule tip. In (G) the tubule tip (arrowhead) is
extended beyond the small vacuole (v) and curves around it. In
(H) 4 s later the small vacuole (v) has remained in the same
position, but the tubule has retracted relative to it and its tip is
pointing away from the vacuole. (I) A very fine tubule with a
vacuole-like dilation (d) close to its tip, and a narrower dilation
behind. These dilations moved along the tubule and generated
vacuoles. (J-L) This sequence in the same plane of focus captures
movement and transfer of material between a series of vacuoles
along a tubule. (J) shows three small vacuoles (1,2,3) along the
tubules between the two larger vacuoles (v) and a further three
(4,5,6) to the right. In (K) there are only two small vacuoles left
(2,3)between the two large vacuoles, while to the right 4and 5
have lost material and are much smaller while 6is larger. In (L)
there is now only one vacuole (3) between the two large vacuoles
and on the right only number 6 remains.
usually until the fluorochrome had faded (Fig. 2G,H).
Although the direction and distance varied, it appeared that
the tubules were always moving along specific pathways.
The nuclear zone occupied a central position; tubules traversed it in either direction, or originated from it. They
moved acropetally from vacuoles in the basal zone and transiently formed connections with subapical zone vacuoles at
least 40 µm away, or they moved basipetally from the subapical zone into the nuclear and basal zones. Frequently a
single tubule, originating from subapical zone vacuoles or
the reticulum in the nuclear zone, projected into the apical
zone to within a micrometre or two of the hyphal tip (Fig.
1E).
In addition to their extension and retraction the tubules
also dilated and contracted by peristaltic movement and, in
this way, transported globules of fluorescent material along
the tubule (Fig. 2I-L). Fluorescent content from vacuoles
of the subapical zone moved along the tubular element as
dilations, giving the impression that the vacuole itself was
moving along the tube. Extending tubules often had dilations at their tips and these were commonly observed to
separate and produce a vacuole (Figs 1D, 2A-D). Dilations
travelling along a tubule either produced a vacuole when
they arrived at the tip, or their content was incorporated
into an existing vacuole. A pulsating tubule often suddenly
underwent transformation into a string of small vacuoles
(about 1 µm diam.), connected by fine fluorescent bridges
(Fig. 1E,F). These were commonly strung out and moved
Continuum of tubules and vacuoles in hyphae
in unison along the same tracks, in continuity with the
tubules, and they ultimately grouped into vacuole clusters
(Fig. 2A-D).
Tubule movement was independent of cytoplasmic
streaming. The cytoplasmic streams contained very small
499
fluorescent particles (≤0.1 µm in diameter), presumed to be
vesicles that accumulate fluorochrome, moving in them.
There were usually several streams. The tubules moved in
various directions relative to these cytoplasmic streams and
at different rates from them. Fig. 2G-H shows changes in
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Fig. 3. Time-lapse photomicrographs taken at 4-s intervals (except E, where the interval is greater) in the same focal plane showing
movements of the tubular reticulum. Bar, 20 µm. A vacuole (v) remains connected to the reticulum via a narrow fluorescent bridge
throughout. The sequence shows consecutive frames of the retraction of a tubule (t1) that constitutes a branch of the reticulum. Initially
(A) the tubule is quite long. In (B) it is now only about one third of its original length and in (C) it is about half the length it was in (B). In
(D) it has retracted further, so that it now is represented only by a small dilation at the branch point. Other less dramatic changes can be
followed in other parts of the system. (E) taken 60 s later than (D) shows what are interpreted to be changes resulting from the damaging
effects of irradiating CF-loaded cells for long periods. The vacuole (v) serves as a reference point and indicates that the plane of focus is
the same as in the previous figures of the sequence. Most tubules are now much thicker and the configuration of the reticulum has
changed. At some branch points (arrowheads) a plaque-like ring structure surrounding a non-fluorescent area occurs. The tubule (t2),
which remained very fine initially in (A-D), has become obliterated by a much broader tubule, which is confluent with the fluorescence of
the vacuole cluster.
position and retraction of the tip of a tubule that branches
from a reticulum that is connected to a vacuole. Fig. 3AD shows the sequence of changes over a short period of
time in a reticulum and its continued attachment to individual vacuoles throughout. In particular, one of the tubules
retracts back to the branch point. A vacuole is attached to
Continuum of tubules and vacuoles in hyphae
501
Fig. 4. Consecutive sections from a series through a vacuole cluster showing that all vacuoles are interconnected in some plane or other
by narrow bridges and the vacuole cluster is a continuum. The position of individual vacuoles (labelled v1 etc) can be determined relative
to the other organelles such as the adjacent Golgi reticulum (g). The bridges (arrowheads) between some vacuoles are as narrow as
adjacent rough ER (rer) cisternae. Microtubules (mt) running parallel to the long axis of the hypha may be traced in several micrographs.
Bar, 1 µm.
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V. A. Shepherd, D. A. Orlovich and A. E. Ashford
a tubule via a narrow bridge that contains fluorescent material. From fluorescence micrographs the vacuoles (dilated
region) had a diameter of roughly 1.3 µm while the bridge
(narrower region) varied from about 0.7 µm in diameter to
the limit of resolution. Finally, the reticulum (Fig. 3E)
shows changes characteristic of long irradiations and interpreted as damage, where the tubules thicken and form new
fusions as their motility ceases.
Fine structure of freeze-substituted hyphae
Interconnections between tubules and vacuoles were confirmed at the ultrastructural level. Serial sections through
vacuole clusters showed that each vacuole was connected
via narrow membrane-enclosed bridges to usually more
than one adjacent vacuole, indicating that the clusters are
a complex fully interconnected system (Fig. 4). The interconnecting bridges were often wider, but could be as narrow
as cisternae of adjacent rough ER. Vacuoles contained a
dispersed electron-opaque material, were mostly 0.4 - 0.8
µm in diameter (n=39) and ranged from more or less circular to ovate, pyriform, irregular or elongate in profile.
Two adjacent vacuole clusters in the subapical zone are
shown in Fig. 5A,B. An elongate vacuole has extended from
one of the clusters and lies parallel to the long axis of the
hypha (Fig. 5A). In another section through the same region
(Fig. 6A,B) an elongate vacuole lies parallel to the hyphal
long axis adjacent to both these vacuole clusters, without
apparently connecting with them. This elongate vacuole
shows an undulating profile and some constricted regions.
In other cases vacuoles and constrictions alternate (Fig. 7B)
to produce structures identical in appearance to the long
strings of connected vacuoles seen by fluorescence
microscopy (Fig. 1F). The dilations were 0.3 - 0.8 µm in
diameter and the constrictions were 40 - 150 nm (n=4) (Fig.
7B). The narrowest constrictions were of similar diameter
to the lumen (39 nm, n=4) of rough ER cisternae, which
also lay more or less parallel to the hyphal long axis (Fig.
6). Rough ER was most obvious in intervacuolar regions
(Figs 5, 6), where it was continuous over long distances
with occasional breaks in individual sections, indicating that
it occurred mostly as perforate sheets (Figs 5A, 6A).
Smooth tubular cisternae were widespread. They occurred
in peripheral and central parts of the terminal cells in areas
both with and without vacuole clusters (Figs 5, 6, 7A), in
cytoplasm around the nucleus (Fig. 7C), and associated with
the dolipore septum at the base of the cell. Tubular cisternae were often found in characteristic ring-like structures
(Fig. 7A,C) and they frequently extended into the extreme
hyphal tips, which did not contain vacuoles (Fig. 7A). The
lumen was variable in diameter, from totally constricted at
some points, to 50 - 73 nm (n=14) in dilated regions. Profiles of smooth tubular cisternae were often seen adjacent
and parallel to elements of the cytoskeleton, oriented
approximately parallel to the long axis of the hypha.
Cytoskeletal elements were also located close and parallel
to the elongate vacuoles (Figs 5, 6) and were identified as
both microtubules (Fig. 7B) and microfilaments (Fig.
6A,B). Groups of microtubules (Fig. 7D,E) and bundles of
microfilaments (Fig. 7E,F) were seen in cross-section in
transverse sections of the hyphae. Golgi bodies were seen
as clusters of vesicle-like profiles, short tubules with dilated
Fig. 5. Adjacent areas of the sub-apical zone of a freezesubstituted terminal cell, in longitudinal section, from a section
cut parallel to the long axis of the hypha. Bar, 1 µm. The large
arrow in (A) indicates the direction of the hyphal tip and the
bottom of A is continuous with the top of B. There are two
clusters of vacuoles. The one smaller in vacuole number and size
(v1), is nearer the tip than the other (v2). Several of the vacuoles in
each cluster are connected. The intervening space between
vacuole clusters contains many profiles of rough ER cisternae
(rer) oriented more or less parallel to the long axis of the hypha
and with sparse ribosomes. Clusters of short tubules and vesiclelike profiles, identified as Golgi bodies (g1, g2 and g3) occur at
intervals along the zone. Several sectioned cytoskeletal elements
are identified as partially sectioned microfilaments (mf) and run
more or less parallel to the long axis of the hypha. Mitochondrial
profiles (m) are of moderate electron opacity. Bar, 1 µm.
ends, and occasionally a reticulum, with contents of moderate to high electron opacity (Figs 5, 6, 7A).
DISCUSSION
Significance and potential role in transport
The system demonstrated here in the actively growing
hyphal tips differs from any previously reported vacuole
system in fungi in its motility, interconnectedness and
pleiomorphism. It is clearly not induced by CF-loading and
UV irradiation (cf. Lee and Chen, 1988), since it can also
be shown in untreated, live cells by DIC microscopy and
by electron microscopy after freeze-substitution. This constantly changing continuum of tubules and vacuoles must
play an important role in intracellular transport in living
hyphae. It provides an alternative transport pathway to
either the cytoplasmic streams or endomembrane vesicles.
Indeed, the tubules appear to be ferrying material specifically between the clusters of vacuoles stationed at intervals
along the hyphal tip. Fluorescent globules are moved along
the tubules and transferred between these vacuoles by peristalic movements and new vacuoles are produced by pinching off such globules from the tubule tip. Tubule movement is multidirectional and so the system can
accommodate the bidirectional transport observed in hyphal
tips. Transfer via tubules rather than vesicles allows larger
volumes to be moved at one go and, if peristalsis is
involved, without a concomitant transfer of membrane. It
can be predicted that the ratio of membrane to fluid transfer along the pathway will be lower than for transfer of a
similar volume via a collection of vesicles, where the ratio
of membrane to internal content depends on the size of the
vesicle. An involvement of the cytoskeleton in tubule movement is indicated by the constant close association of
cytoskeletal elements.
Comparison with other systems
Although this system has not previously been demonstrated
in living hyphae, circumstantial evidence indicates that it
may be widespread in fungi. The fluorescent threads or
“trails” observed connecting vacuoles in yeast (Weisman
and Wickner, 1988) bear a strong resemblance to the tubular elements seen in Pisolithus, and the view that these are
Continuum of tubules and vacuoles in hyphae
“tracks” of vesicles, manifest as fluorescent threads, may
need re-interpretation. Failure to appreciate the tubular connections from electron microscopy probably arises from the
use of thin sections of chemically fixed material. However,
there is some evidence for the existence of tubular systems
503
in fungi from electron microscopy. A tubular vacuole
system with only a few small associated spherical vacuoles
is shown in freeze-substituted hyphal tips of the basidiomycete, Sclerotium rolfsii (Robertson and Fuller, 1988).
Tubular vesicular complexes (TVC 1) are described by
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Knauf et al. (1989) in the cytoplasm of Uromyces appen diculatus, another basidiomycete, and consist of cisternae
with an irregular luminar width of 30 - 70 nm. They were
considered to be a specialised portion of rough ER, but most
are smooth cisternae associated with larger structures that
are circular in profile (see, for example, their figure 5). A
Fig. 6. See p. 506 for legend
Continuum of tubules and vacuoles in hyphae
similar system is reported in Erisyphe graminis (Dahmen
and Hobot, 1986). Tubules in Pisolithus hyphae, identical
in appearance and behaviour to those that accumulate CF,
505
also accumulate the fluorochrome DiOC6(3), whilst the vacuoles do not. DiOC6(3) was widely considered to be a
specific marker for the ER (Terasaki et al., 1984; Quader
Fig. 7. See p. 506 for legend
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Fig. 6. Another section from the same series as Fig. 5, showing
the same vacuole clusters (v1 and v2). In this section there are
fewer vacuole profiles in v1 than shown in Fig. 5A. An elongated
vacuole (v), showing typical dilated and narrow regions, is
oriented parallel to the long axis and passes adjacent to vacuole
clusters v1 and v2, but does not connect up with them. There are
many rough ER profiles (rer), and also profiles of smooth tubular
cisternae (tc). They are wider and more irregular in diameter than
the rough ER cisternae, and show distinct constrictions. Like
many of the rough ER cisternae, they are often longitudinally
oriented, although they are not always parallel to the long axis.
Microfilaments (mf) run for long distances parallel to the hyphal
long axis. Individual Golgi bodies (g1, g2 and g3) can be traced
through the series. The large arrow again indicates the direction of
the hyphal tip and the bottom of A is continuous with the top of B.
Mitochondrial profiles (m) are continuous through the series of
sections, indicating a mitochondrial reticulum. Bar, 1 µm.
Fig. 7. Electron micrographs of freeze-substituted hyphae. (A)
Apical zone at the extreme hyphal tip, indicated by the cluster of
electron-opaque vesicles (ve) that surround the Spitzenkörper.
Behind this region are many smooth tubular cisternae (tc) oriented
in various planes, including two rings of tubules (arrows), but no
vacuoles. Posterior to this are several Golgi (g), mitochondrial
(m), and ER profiles, and then a vacuole (v) cluster, and rough ER
cisternae. Bar, 1 µm. (B) An elongate vacuole (v) with alternating
dilated and constricted regions and characteristic content runs
longitudinally for a distance of at least 8 µm. The section also
shows short sections of several microtubules (mt), all oriented
parallel to the hyphal long axis. Bar, 1 µm. (C) Transverse section
from a series through the nuclear zone (n, nucleus) in one hypha
and a vacuole (v) cluster in another. Many circular profiles of
organelles with smooth membranes and of different dimensions
are obvious in both hyphae. The larger profiles (v) can be
identified by their size and content as dilated regions of the
interconnected vacuole system, while the smaller profiles (tc) are
of tubules of the dimensions of smooth ER. A ring of tubules
(arrow) similar to those in A is present in one of the hyphae.
Single microtubules and groups of two or more, all in crosssection, occur throughout both hyphae. Bar, 1 µm. (D, E, F)
Enlargement of three areas from (C) showing the microtubules
(mt) and microfilaments (mf) in more detail. All are sectioned
transversely. Bar, 0.5 µm.
and Schnepf, 1986), but recently it has been reported to be
non-specific and to stain other intracellular membranes,
including Golgi and endosomes (Terasaki and Reese, 1992).
Furthermore, there are other vacuolar systems with tubular
elements and properties very similar to those in Pisolithus
that are not considered to be synonymous with smooth ER;
for example, the tubular vacuolar network that develops in
Allium guard cells during differentiation (Palevitz and
O’Kane, 1981; Palevitz et al., 1981). This tubular network
initially arises from large spherical vacuoles, exhibits complex movements and shape changes, and is pleiomorphic.
The elements are of similar dimensions to those of
Pisolithus, and they enlarge to form dilations. The reticulate form is transient and as differentiation proceeds the network is transformed into larger cisternae, which eventually
coalesce into a single large vacuole. Tubular networks
sometimes continuous with vacuoles have also been found
in cultured plant and animal cells (see for example, Hillmer
et al., 1989; Cole et al., 1990; Tooze and Hollinshead, 1992).
Such networks would be almost impossible to distinguish
from smooth ER cisternae in electron micrographs on morphology alone. However, several tubule systems that form
a reticulum in animal cells have been identified as physiologically distinct from the ER compartment by use of
specific markers. Among these are the trans-Golgi reticulum, tubular endosomes and tubular lysosomes (Swanson
et al., 1987; Hopkins et al., 1990; Luo and Robinson, 1992;
and see Tooze and Hollinshead, 1992) The Golgi tubules
can be recognised by their electron-opaque content in
freeze-substituted Pisolithus hyphae, while the tubule
system is electron lucent or contains electron-opaque dispersed material. It bears a strong resemblance in appearance and behaviour under the fluorescence microscope to
the tubular endosomes described by Hopkins et al. (1990),
and to the same system viewed in the electron microscope
(Tooze and Hollinshead, 1992). Its characterisation in
fungal hyphae will depend upon the use of specific electron-opaque markers, as with animal cells. Vacuoles in
fungi are considered analogous to animal cell lysosomes
because of their low pH and hydrolytic enzyme content
(Klionsky et al., 1990) and there are many reports of the
apparent endocytosis of fluorescent probes in fungi (for
example, Makarow, 1985; Preston et a1., 1987; Basrai et
al., 1990). The tips of growing hyphae are a very likely site
for endocytosis, since fungi are heterotrophic and the highest rates of uptake occur at their tips. However, endocytosis remains unproven in fungi, because the cell wall precludes the use of large molecules as tracers and the
mechanism of uptake of the fluorescent probes used is controversial (Oparka, 1991; Wright et al., 1992). It is probable that movement of the tubules is under control of the
cytoskeleton. We have noted the close association between
both the tubules and elongated vacuoles, and microtubules
and microfilaments. The functional relationship remains to
be determined, but the mechanism of transport by peristalsis may be widespread in other reticular systems in plant
and animal cells, as an alternative to transfer via vesicles.
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, Bill Allaway for comments on the
manuscript, Lydia Kupsky for photographic assistance, Renate
Sandeman for photographic advice, and Carl Zeiss Pty Ltd. for
the loan of an Axiophot microscope.
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(Received 31 August 1992 - Accepted 5 November 1992)