Journal of Cell Science 106, 847-858 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
847
Ultrastructure of the endocytotic pathway in glutaraldehyde-fixed and
high-pressure frozen/freeze-substituted protoplasts of white spruce (Picea
glauca)
M. E. Galway*, P. J. Rennie and L. C. Fowke†
Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 0W0, Canada
*Present address: Department of Biology, University of Michigan, Ann Arbor, Michigan 48109-1048, USA
for correspondence
†Author
SUMMARY
An ultrastructural study of endocytosis has been made
for the first time in protoplasts of a gymnosperm, white
spruce (Picea glauca), fixed by high-pressure freezing
and freeze substitution. Protoplasts derived from the
WS1 line of suspension-cultured embryogenic white
spruce were labelled with cationized ferritin, a nonspecific marker of the plasma membrane. The timing of
cationized ferritin uptake and its subcellular distribution were determined by fixing protoplasts at various
intervals after labelling. To address concerns about
using chemical fixation to study the membrane-bound
transport of cationized ferritin, protoplasts were fixed
both by conventional glutaraldehyde fixation and by
rapid freezing in a Balzers high-pressure freezing apparatus (followed by freeze substitution). Cationized ferritin appeared rapidly in coated pits and coated vesicles
after labelling. Later it was present in uncoated vesicles,
and in Golgi bodies, trans-Golgi membranes and partially coated reticula, then subsequently in multivesicu-
lar bodies, which may ultimately fuse with and deliver
their contents to lytic vacuoles. The results show that
the time course and pathway of cationized ferritin
uptake in the gymnosperm white spruce is very similar
to the time course and pathway elucidated for cationized ferritin uptake in the angiosperm soybean. Highpressure freezing yielded much better preservation of
intracellular membranes and organelles, although
plasma membranes appeared ruffled. Protoplasts fixed
by both methods possessed numerous smooth vesicles in
the cortex and smooth invaginations of the plasma membrane. These became labelled with cationized ferritin,
but apparently did not contribute directly to the internalization of cationized ferritin, except via the formation of coated pits and vesicles from their surfaces.
INTRODUCTION
Recently, a number of ultrastructural studies using membrane-impermeant, electron-opaque tracers have confirmed
that endocytosis via coated pits and coated vesicles is possible in plant cells and protoplasts (reviewed by Fowke et
al., 1991; see also Samuels and Bisalputra, 1990; Owen et
al., 1991; Lazzaro and Thomson, 1992). The subsequent
appearance of tracers in various organelles, vesicles and
vacuoles has provided valuable information about organelle
functions and the pathways of intracellular transport in plant
cells. Two types of markers have been employed in ultrastructural studies: soluble tracers of fluid-phase uptake such
as heavy metal salts and lucifer yellow (Hübner et al., 1985;
Samuels and Bisalputra, 1990; Owen et al., 1991; Lazzaro
and Thomson, 1992) and the insoluble plasma membranebound tracers, cationized ferritin (CF) and lectin-gold conjugates (Tanchak et al., 1984; Joachim and Robinson, 1984;
Hillmer et al., 1986; Domozych and Nimmons, 1992). Since
the soluble fluid-phase markers can penetrate cell walls,
Endocytosis via clathrin-coated pits and vesicles, and the
subsequent fate of endocytosed proteins and other substances, has been extensively studied in animal cells. In
plant cells until recently, research on endocytosis was hindered by (a) the supposition that endocytosis was not energetically possible in turgid plant cells, and (b) the presence
of cell walls, which limit access to plant plasma membranes
(Fowke et al., 1991).
Clathrin-coated pits on the plasma membrane and
clathrin-coated vesicles in the cytoplasm of plant cells are
believed to function mainly in the uptake and recycling of
plasma membrane and plasma membrane components
(Coleman et al., 1988). However, many studies have indicated that plant cells can internalize a variety of large,
exogenously supplied molecules (Nishizawa and Mori,
1977; Coleman et al., 1988, and references therein).
Key words: cationized ferritin, endocytosis, freeze substitution,
high-pressure freezing, Picea glauca, protoplasts
848
M. E. Galway, P. J. Rennie and L. C. Fowke
they can be used to study endocytosis in intact cells and
tissues at normal turgor pressure. However, they must be
precipitated in situ during fixation to visualize them for
electron microscopy. Drawbacks are that lucifer yellow may
not be completely membrane-impermeant (Robinson and
Hedrich, 1991), while the heavy metals are toxic and can
have deleterious effects on cell structure and function
(Wheeler et al., 1972; Romanenko et al., 1986; Lazzaro and
Thomson, 1992).
Although endocytosis of insoluble membrane-bound
tracers should reflect the normal processes of vesicle-mediated turnover of plasma membrane (Fowke et al., 1991),
these tracers can’t penetrate the cell walls of higher plants
and therefore have only been used to study endocytosis in
protoplasts. Nevertheless, the most detailed information on
the time course and pathway of tracer uptake has been
obtained from studying the endocytosis of CF in protoplasts
of suspension-cultured soybean cells (Tanchak et al., 1984;
Tanchak, 1987; Tanchak and Fowke, 1987; Tanchak et al.,
1988; Fowke et al., 1989). This is because the plasma membranes of many protoplasts can be rapidly and uniformly
labelled at once, resulting in the endocytosis of large quantities of cationized ferritin and subsequent labelling of relatively large numbers of organelles.
We wished to determine whether the time course and
pathway of CF endocytosis as elucidated in soybean and
also in bean leaf protoplasts (Joachim and Robinson, 1984)
differs in protoplasts of more distantly related species. For
this purpose, we chose meristematic protoplasts prepared
from embryogenic suspension cultures of the gymnosperm
Picea glauca, or white spruce (Attree et al., 1989a,b; Attree
and Fowke, 1993).
Another concern to us was that all previous ultrastructural studies of endocytosis (apart from one study of heavymetal uptake) have employed slow-acting aldehyde fixatives, which can disrupt labile cellular membranes and
membrane-bound compartments (Mersey and McCully,
1978; Wilson et al., 1990) and may therefore be particularly unsuitable for studying the ultrastructural cytology of
endocytosis. To address the possibility of fixation artefacts,
we employed both glutaraldehyde fixation and high-pressure freezing in a prototype Balzers HPM 010 high-pressure freezing apparatus (see Gilkey and Staehelin, 1986 for
details), followed by freeze substitution.
MATERIALS AND METHODS
WS1 embryogenic suspension culture
The WS1 line of suspension-cultured embryogenic white spruce,
originally established by Hakman and Fowke (1987), was maintained as previously described (Hakman and Fowke 1987), except
that subculturing was carried out every 7 days, by mixing 50 ml
of fresh LP medium with each 50 ml of WS1 culture, and decanting half of the mixture into a new 250 ml flask.
Protoplast isolation
The optimized method for a rapid, high yield of WS1 protoplasts
was modified from Attree et al. (1989a) by substituting pectolyase
(Sigma Chemical Company, St. Louis, MO, USA) for the mixture of rhozyme HP-150, pectinase and driselase. WS1 suspen-
sion-cultured cells (6-7 days old) were collected on Miracloth
(Chicopee Mills, New York, USA), drained of excess liquid and
weighed. They were then preplasmolyzed for 1 hour on a gyratory shaker at 50-75 rpm in a protoplast isolation buffer consisting of 10 mM 2[N-morpholino]ethanesulfonic acid (MES) at pH
5.6, 6 mM CaCl2.2H2O, 0.7 mM NaH2PO4 and 0.44 M sorbitol.
The plasmolyzed cells were collected again by filtration and transferred to isolation buffer containing 1% (w/v) Onozuka R-10 cellulase (Yakult Pharmaceutical Industry, Nishinomiya, Japan) and
0.2% (w/v) pectolyase. This enzyme solution was centrifuged
before use for 5-10 minutes at 2500 g to precipitate insoluble
solids. The preplasmolyzed cells were added to the enzyme solution at a ratio ranging between 0.2 and 0.6 g wet weight of WS1
cells per ml of solution, and incubated on a gyratory shaker at
100 rpm for 2-2.5 hours. The resultant protoplasts were collected
by filtration through an 80 µm mesh nylon filter and collected in
conical centrifuge tubes by centrifugation for 4 minutes at 100 g.
Protoplasts were resuspended and washed in enzyme-free isolation buffer and then in an incubation buffer containing 10 mM N[2-hydroxyethyl]piperazine-N′-2-ethanesulfonic acid (HEPES)
(tissue culture grade) at pH 7, in place of MES. Protoplasts were
allowed to recover for at least 1 hour prior to subsequent labelling
with CF in the incubation buffer.
Cationized ferritin incubation, glutaraldehyde
fixation and resin embedding
Incubation of protoplasts in CF was carried out as described by
Tanchak et al. (1988). Protoplasts in incubation buffer were collected by centrifugation in 1.5 ml polypropylene microcentrifuge
tubes to yield pellets of approximately 0.15 ml volume. Pellets
were resuspended to 0.4 ml, then diluted to 0.8 ml in incubation
buffer containing 1 mg/ml CF (stock contained 10 mg/ml in 0.125
M NaCl, 2 mM azide; Molecular Probes, Eugene, OR, USA), to
give a final concentration of 0.5 mg/ml CF. Control protoplasts
were incubated in CF-free incubation buffer containing 6.3 mM
NaCl, 0.1 mM NaN3 (approx. 0.006%). The small amounts of
NaCl and NaN3 had no detectable effects on protoplasts. Protoplasts incubated for 30 minutes or less in CF were fixed by the
addition of 0.4 ml of ice-chilled 3% w/v glutaraldehyde (in incubation buffer) to give a final concentration of 1% glutaraldehyde.
For incubations of 60 minutes or more, protoplasts were first
washed several times in succession by centrifugation and resuspension in CF-free incubation buffer before fixation in ice-chilled
1% glutaraldehyde. All protoplasts were fixed in 1% glutaraldehyde on ice for 1-2 hours, followed by fixation in 3% glutaraldehyde in incubation buffer for 2-3 hours at room temperature. Fixed
protoplasts were washed in 10 mM HEPES, then postfixed in 1%
osmium tetroxide in 10 mM HEPES, dehydrated first to 100%
ethanol, then to propylene oxide, followed by infiltration and
embedding in Araldite resin for electron microscopy.
High-pressure freezing, freeze substitution and
resin embedding
Protoplasts isolated as described were washed and resuspended in
incubation buffer containing 0.5% agarose (Sigma Type IX, ultralow gelling temperature, Sigma Chemical Co., St. Louis, MO,
USA) to cushion protoplasts during freezing and hold them
together during subsequent processing. Agarose increased the viscosity of the incubation medium, but did not interfere with coating of protoplasts with CF since protoplasts with or without
agarose in the incubation medium became rapidly coloured as the
orange-brown CF adsorbed to their surfaces. Electron microscopy
of freeze-fixed protoplasts incubated in CF with agarose revealed
that the plasma membranes were extensively coated with CF, similar to protoplasts not exposed to agarose. The protoplasts were
incubated in CF as described above, except that agarose was added
Endocytosis in freeze-fixed protoplasts
and the final ratio of total volume to protoplast pellet volume
during CF incubation was decreased from 5.3:1 to 2:1, so that the
final concentration of CF used ranged from 0.5 to 1 mg/ml. This
increase in protoplast density ensured that protoplast samples were
sufficiently concentrated for subsequent sectioning and electron
microscopy, since pelleting the CF-labelled protoplasts before
freezing was not possible for short incubations in CF. Droplets of
the protoplast suspensions were frozen in liquid nitrogen under
high pressure in a prototype Balzers HPM 010 high-pressure freezing apparatus using the specimen cups and holder designed by
Craig et al. (1987). Just before use, the upper part of each specimen cup was coated in a freshly prepared solution of vegetable
lecithin in chloroform (100 mg/ml) to assist in separating the cups
after freezing. Freezing took place within 30 seconds of loading
the samples into the cups.
After freezing, the cups were transferred immediately to liquid
nitrogen, the two halves were separated, and the specimens were
transferred to vials containing a liquid nitrogen-cooled solution of
2% osmium tetroxide in acetone. Substitution was carried out in
this solution at approximately −79°C in a dry ice/acetone bath for
a minimum of 3 days. The samples were gradually warmed to
room temperature over a 6 hour period, rinsed in acetone, and
infiltrated with Spurr’s resin (which infiltrates freeze-substituted
tissue better than Araldite) over 3 days before polymerization.
Electron microscopy
Ultra thin sections were cut on a diamond knife. Sections were
mounted on uncoated or formvar-coated copper mesh grids, and
were observed either unstained or briefly stained in lead citrate
(for visualizing CF) or stained fully in uranyl acetate and lead citrate, and viewed with a 420 model Philips electron microscope.
RESULTS
Protoplast production
The WS1 line of suspension-cultured, embryogenic white
spruce consists of small clumps of undifferentiated cells
and somatic embryos at various stages of development.
These embryos, like zygotic embryos, are a mass of small
densely cytoplasmic meristematic cells from which extend
a ‘tail’ of larger, elongated and vacuolate suspensor cells
(Fig. 1; see also Attree et al., 1991). Suspensor cells seldom
released intact protoplasts. Nearly all protoplasts were small
and cytoplasmic, like the meristematic cells of the embryos.
Since the cells in the meristematic region are interconnected
by numerous plasmodesmata (Hakman et al., 1987), the best
yields were obtained by vigorous shaking during the
enzyme treatment in order to separate the protoplasts, which
otherwise remained connected by their plasmodesmata
and/or fused to yield multinucleate protoplasts. Fluorescein
diacetate staining confirmed that 80 to 90% of newly isolated protoplasts were viable (not shown).
Protoplast preservation
Thin sections of single well-preserved, uninucleate protoplasts were used to establish the time course of CF uptake,
and to study protoplast ultrastructure (Figs 2 and 3). Much
of the volume of each protoplast was occupied by the
nucleus, which was surrounded by cytoplasm, organelles
and large vacuolar profiles. Membranous material was
usually attached to the outer surface of the plasma membranes in glutaraldehyde-fixed protoplasts (Fig. 3), and may
849
have been extruded from the protoplast during fixation
and/or released from damaged protoplasts during protoplast
preparation.
In contrast, freeze-fixed protoplasts had little or no extraneous membrane at their surfaces, although the plasma
membranes were quite ruffled (Fig. 2). The nuclear chromatin in some freeze-fixed protoplasts had a holey appearance (Fig. 2), indicating ice damage within the nucleus.
However, the cytoplasm of these protoplasts was not
excluded from observation if it was free of detectable ice
damage.
More glutaraldehyde-fixed than freeze-fixed protoplasts
were examined in this study because the small size of the
specimen cups limited the number of protoplasts that could
be frozen, and because only a subpopulation of protoplasts
were well-preserved by high-pressure freezing. A survey of
protoplasts fixed in five different freezing runs showed that
there were between 2 and 6% uninucleate, well-preserved
protoplasts (for 128 to 413 protoplasts sampled per run).
Protoplast ultrastructure
The ultrastructure of CF-labelled and unlabelled WS1 protoplasts was similar. However, significant differences were
noted in the ultrastructure of freeze-fixed compared to glutaraldehyde-fixed protoplasts. In general, the membranes of
all organelles and vacuoles were smoother and the
organelles appeared turgid in freeze-fixed protoplasts as
compared to glutaraldehyde-fixed protoplasts (Figs 2-4).
Nuclear profiles were full and round after freeze fixation,
but convoluted after glutaraldehyde fixation (compare Figs
2 and 3). Membrane contrast was generally poorer after
freeze fixation regardless of staining method employed.
Both freeze-fixed and glutaraldehyde-fixed WS1 protoplasts contained smooth invaginations of the plasma membrane, which were 4 times larger than the invaginations of
coated pits. In the cytoplasm, smooth vesicles similar in
size and appearance to these large smooth invaginations
were only observed near the plasma membrane (Figs 2 and
3; see also below). These large smooth vesicles should not
be confused with the small smooth or uncoated vesicles,
similar in size to coated vesicles, which were distributed
throughout the cytoplasm.
All glutaraldehyde-fixed protoplasts contained amorphous dark deposits scattered over protoplast membranes,
particularly plasma membrane, the bounding membranes of
plastids, mitochondria, multivesicular bodies (MVBs) as
well as Golgi bodies (e.g. Figs 3, 8, 10, 12, 24, 28) where
they hindered observation of CF particles within the cisternae. These osmiophilic deposits were visible in both
unstained and stained sections, and in unstained sections a
linear/punctate substructure was detected, suggesting that
the material consisted of closely packed rod-shaped elements (Fig. 5). These deposits were absent from high-pressure frozen material and from intact WS1 embryos (not
shown).
Golgi body ultrastructure differed somewhat between
embryonic cells of the somatic embryos and the protoplasts
derived from them (Figs 6-11). In the Golgi bodies of
embryonic cells (studied by Hakman et al., 1987) coated or
smooth vesicles were associated with the trans-Golgi faces
850
M. E. Galway, P. J. Rennie and L. C. Fowke
(Fig. 6); reticulate networks of tubular membranes bearing
clathrin-coated regions, the partially coated reticula (PCR),
were often near by, although not detectably connected to
the Golgi bodies (Fig. 6, see also Fig. 13 Hakman et al.,
1987). In the protoplasts, similar Golgi bodies with adjacent PCR were observed (Fig. 7), but in most Golgi bodies
the structure of the trans faces was more complex (Figs 811). The ends of the transmost Golgi cisternae were often
Figs 1-8. For legends see p. 852.
Endocytosis in freeze-fixed protoplasts
curled inward and associated with clusters of coated or
smooth vesicles (Figs 8, 9, 11). Profiles of closed or almost
closed membrane rings were also found adjacent to trans-
851
Golgi cisternae (Figs 9, 10, 11). Some of these rings also
enclosed coated and smooth vesicles (Fig. 10). Like embryonic cells, PCR was often located near Golgi bodies in the
Figs 9-15. For legends see p. 852
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M. E. Galway, P. J. Rennie and L. C. Fowke
protoplasts, typically covering an area about two to three
times that occupied by the Golgi bodies (Figs 4, 7, 12, 23).
Microtubules lay just beneath the plasma membrane in
Figs 1-8
Fig. 1. Light micrograph of somatic embryo from WS1
suspension culture. Note small meristematic cells (single
arrowhead) and larger vacuolate suspensor cells (double
arrowhead). Bar, 0.2 mm.
Fig. 2. Portion of freeze-fixed WS1 protoplast containing nucleus
(N), mitochondria (M), plastids (P), vacuoles (V). Note wavy
plasma membrane; also peripheral vesicles (single arrowhead).
Holes in one region of chromatin (double arrowhead) indicate ice
damage. Lead citrate staining. Bar, 2 µm.
Fig. 3. Portion of glutaraldehyde-fixed WS1 protoplast with
organelles labelled as in Fig. 2. Note also membrane material at
outer surface of plasma membrane (arrowheads). Unstained. Bar,
2 µm.
Fig. 4. Organelle morphology in freeze-fixed WS1 protoplast.
Golgi body (G), vacuole (V), PCR (single arrowhead) and two
MVB profiles (double arrowhead) are visible. Lead citrate
staining. Bar, 300 nm.
Fig. 5. Punctate and linear substructure is apparent in these
osmiophilic deposits lying along the inner side of the vacuolar
membrane in a glutaraldehyde-fixed WS1 protoplast. Note also
darker particulate matter in one deposit. Unstained. Bar, 100 nm.
Fig. 6. Golgi body (G) and PCR in meristematic cell of a WS1
somatic embryo. Note coated regions of the PCR (arrowheads).
Uranyl acetate and lead citrate staining. Bar, 300 nm.
Fig. 7. Golgi body (G), and PCR with abundant coated regions
(arrowheads) near vacuole (V) in WS1 protoplast. Uranyl acetate
and lead citrate staining. Bar, 300 nm.
Fig. 8. Golgi bodies with curled cisternae at the trans (T) face in
glutaraldehyde-fixed WS1 protoplast. Amorphous deposits
(tannin?) cover portions of the Golgi bodies. Note CF in vesicles
(arrowheads) at trans face of Golgi body after 5 minutes exposure
to CF. Lead citrate staining. Bar, 200 nm.
Figs 9-15
Fig. 9. Golgi bodies in freeze-fixed WS1 protoplast. Note curled
cisternae at trans (T) face of one Golgi. Note also membrane ring
near curled cisterna (arrowhead). Uranyl acetate and lead citrate
staining. Bar, 300 nm.
Fig. 10. Membrane ring (single arrowhead) enclosing coated and
uncoated vesicles is positioned between two Golgi bodies (G) and
adjacent to PCR (double arrowhead) in glutaraldehyde-fixed WS1
protoplast. Lead citrate staining. Bar, 200 nm.
Fig. 11. Membrane ring (single arrowhead) close to curled
cisterna at trans face of Golgi body (G) in freeze-fixed WS1
protoplast. Note also profiles of two MVBs (double arrowheads).
Lead citrate staining. Bar, 200 nm.
Fig. 12. Glutaraldehyde-fixed WS1 protoplast 15 minutes after
exposure to CF showing CF particles (single arrowhead) in PCR
adjacent to unlabelled Golgi body (G), mitochondrion (M) and
vacuole (V). Note also unlabelled dilation in PCR (double
arrowhead). Lead citrate staining. Bar, 200 nm.
Fig. 13. Transverse and grazing sections of microtubules (single
arrowheads) are visible adjacent to plasma membrane labelled
with CF (double arrowheads) in WS1 protoplast freeze-fixed 60
minutes after exposure to CF. Uranyl acetate and lead citrate
staining. Bar, 200 nm.
Fig. 14. Grazing section of plasma membrane from glutaraldehydefixed WS1 protoplast 30 minutes after exposure to CF contains
cortical microtubules (single arrowheads), and large smooth
vesicles or invaginations of the plasma membrane bearing coated
pits (double arrowheads). Lead citrate staining. Bar, 300 nm.
Fig. 15. Bundle of microfilaments in glutaraldehyde-fixed WS1
protoplast. Lead citrate staining. Bar, 200 nm.
both freeze-fixed and glutaraldehyde-fixed protoplasts (Figs
13 and 14). All microfilament bundles observed in glutaraldehyde-fixed protoplasts were straight and compact
(Fig. 15), but one bundle consisting of wavy microfilaments
was found in an otherwise well-preserved protoplast after
freeze-fixation (Fig. 16).
Endocytosis of cationized ferritin
Protoplasts were labelled continuously in CF at room temperature and not by pulse-labelling chilled protoplasts, then
rewarming them as described by Joachim and Robinson
(1984) and Hillmer et al. (1986) because fewer coated pits
were found on the plasma membranes of rewarmed protoplasts than on the membranes of comparable unchilled protoplasts, and CF formed discontinuous clumps at the plasma
membranes of rewarmed protoplasts (unpublished results).
These factors reduced CF uptake in rewarmed protoplasts
compared to protoplasts held at room temperature, which
in turn reduced CF labelling of organelles and made it more
difficult to determine the timing of CF transit into different organelles.
No differences were detected in the time course and pathway of CF endocytosis in glutaraldehyde-fixed compared
to freeze-fixed protoplasts, so examples have been selected
from protoplasts fixed by both methods (Figs 17-29). Ultrastructural observations of CF uptake were made either from
unstained thin sections of protoplasts or after 5 minutes
staining in lead citrate only, since combined uranyl
Figs 16-24
Fig. 16. Bundles of wavy and straight microfilaments in freezefixed WS1 protoplast. Uranyl acetate and lead citrate staining.
Bar, 200 nm.
Fig. 17. CF-filled coated pit (single arrowhead) 30 minutes after
exposure to CF in glutaraldehyde-fixed WS1 protoplast. Note two
CF-filled uncoated vesicles (double arrowheads). Unstained. Bar,
200 nm.
Fig. 18. CF-filled coated pit (arrowhead) on grazed portion of
plasma membrane of WS1 protoplast freeze-fixed 10 minutes after
exposure to CF. Lead citrate staining. Bar, 200 nm.
Fig. 19. A CF-filled coated pit (single arrowhead) budding from a
CF-labelled smooth invagination of the plasma membrane in WS1
protoplast freeze-fixed 10 minutes after exposure to CF. Note CFfilled uncoated vesicle (double arrowhead). Lead citrate staining.
Bar, 200 nm.
Fig. 20. CF-filled coated vesicle (arrowhead) near plasma
membrane of glutaraldehyde-fixed WS1 protoplast after two
minutes exposure to CF. Unstained. Bar, 200 nm.
Fig. 21. CF-filled coated vesicle (arrowhead) near plasma
membrane in WS1 protoplast freeze-fixed 10 minutes after
exposure to CF. Lead citrate staining. Bar, 200 nm.
Fig. 22. CF-labelled large smooth vesicle (single arrowhead) near
plasma membrane in protoplast freeze-fixed 10 minutes after
exposure to CF. Note CF-filled coated vesicle or pit (double
arrowhead) between plasma membrane and smooth vesicle. Lead
citrate staining. Bar, 200 nm.
Fig. 23. Numerous CF particles (arrowheads) in PCR and vesicles
adjacent to a group of Golgi bodies (G) in WS1 protoplast freezefixed 10 minutes after exposure to CF. Unstained. Bar, 200 nm.
Fig. 24. Tangential and transverse sections of Golgi bodies (G) in
glutaraldehyde-fixed WS1 protoplast 60 minutes after exposure to
CF. CF particles are located in cisternae at the periphery of one
Golgi body (arrowheads). Note amorphous deposits in Golgi
bodies. Lead citrate staining. Bar, 200 nm.
Endocytosis in freeze-fixed protoplasts
acetate/lead citrate staining obscured the less electronopaque CF. However, this meant that the clathrin coats of
coated pits, vesicles and membranes were often poorly
stained, although still identifiable by a ribosome-free zone
of similar thickness to clathrin coats (Figs 17-22).
853
The time course of endocytosis
Within 10 seconds of labelling, and thereafter, protoplasts
were coated with a layer of CF, which was also present in
coated pits (Figs 17 and 18), large smooth invaginations of
the plasma membrane (Fig. 19), coated vesicles (Figs 20-
854
M. E. Galway, P. J. Rennie and L. C. Fowke
22) and large smooth vesicles lying close to the plasma
membrane (Fig. 22). The large smooth invaginations were
connected by openings of various sizes to the surface of the
protoplasts, and serial sectioning showed that some of the
large smooth vesicles were also connected by narrow chan-
nels to the surface (not shown). Coated pits appeared on
both the large smooth invaginations and vesicles just as on
the plasma membrane proper, and coated vesicles were
observed in their vicinity (Figs 14 and 19). When the membranes of these invaginations/vesicles were CF-labelled, the
Endocytosis in freeze-fixed protoplasts
associated coated pits and vesicles were often labelled also
(Fig. 19). CF in uncoated vesicles (Figs 17 and 19) first
appeared in samples fixed 2 minutes after exposure to CF.
Some CF was observed in Golgi bodies, trans-Golgi
membranes (Fig. 8), and PCR after only 5 minutes of
labelling in both freeze-fixed and glutaraldehyde-fixed
samples, whereas in MVBs the first CF was not detected
until 10 minutes after labelling. The number of CF-labelled
coated and smooth vesicles, Golgi bodies, PCR (Fig. 23)
and MVBs in the protoplasts increased rapidly within the
first 30 minutes of labelling. CF within Golgi bodies was
located principally at the periphery of medial and trans cisternae (Fig. 24). CF was also observed in clathrin-coated
and smooth vesicles in the vicinity of Golgi cisternae, and
in the trans-Golgi membranes and associated vesicles (Fig.
8). However, more CF appeared in PCR and MVBs than
in Golgi bodies in protoplasts incubated in CF for 30 minutes to 2 hours. In PCR, CF occurred throughout the tubular membranes and their coated buds (Figs 12, 23, 25).
Although some CF particles were observed at the internal surface of the bounding membranes of MVBs, most CF
inside these organelles was located at the surfaces of the
clustered small internal vesicles (Figs 25-27). Vesicles,
sometimes containing CF, were occasionally seen fused to
the outer surface of CF-labelled MVBs (Fig. 25). After
incubation in CF for 1-2 hours, some MVBs contained a
large number of CF particles (Fig. 26). In a few cases
MVBs lay immediately adjacent to vacuoles (Figs 25, 27).
Clusters of vesicles similar in size to those present in MVBs
were also noted within some smaller vacuolar profiles (Fig.
28), and in one instance these vesicles were labelled with
CF (Fig. 29).
DISCUSSION
Freeze fixation and freeze substitution
To the best of our knowledge, this is the first ultrastructural
Figs 25-29
Fig. 25. Region of CF-labelled PCR (single arrowheads) lies
adjacent to an MVB. An uncoated vesicle containing CF appears
fused to the surface of the MVB (double arrowhead) in this
glutaraldhyde-fixed protoplast 30 minutes after exposure to CF. V,
vacuole. Unstained. Bar, 200 nm.
Fig. 26. In WS1 protoplast freeze-fixed 60 minutes after exposure
to CF, an MVB (arrowhead) containing indistinct internal
vesicles, but numerous CF particles, lies between nucleus (N) and
plastid (P). Unstained. Bar, 200 nm.
Fig. 27. Glutaraldehyde-fixed WS1 protoplast after 60 minutes of
exposure to CF showing MVB protruding into large vacuole (V).
Note CF (arrowheads) at surface of vesicles within MVB. Lead
citrate staining. Bar, 200 nm.
Fig. 28. Glutaraldehyde-fixed WS1 protoplast showing Golgi
body lying between two vacuoles (V), one of which contains a
group of vesicles (arrowhead) similar to those inside MVBs.
Uranyl acetate and lead citrate staining. Bar, 200 nm.
Fig. 29. Two Golgi bodies and mitochondrion (M) lie adjacent to
a large vacuole (V) containing CF-labelled vesicles (arrowhead)
similar to those present in MVBs, in WS1 protoplast fixed in
glutaraldehyde 60 minutes after exposure to CF. Lead citrate
staining. Bar, 300 nm. Inset: detail of CF at surface of vesicles in
vacuole. Bar, 100 nm.
855
study of rapidly frozen and freeze-substituted protoplasts.
Freeze fixation provided excellent preservation of a small
number of protoplasts. It should be possible to increase the
proportion of protoplasts preserved free from detectable ice
damage by modifying the freezing or freeze substitution
method used here. The lack of direct contact between the
20 to 50 µm diameter protoplasts and the specimen cups,
which act as heat sinks for larger specimens such as root
tips (Moor, 1987) may have contributed to ice crystal formation in protoplasts during the initial freezing by slowing
heat loss from the cells. The 0.44 M (8% w/v) sorbitol in
the CF incubation buffer should have provided some cryoprotection for the protoplasts during the initial freezing,
by reducing extracellular ice crystal formation and therefore reducing the release of heat, which accompanies this
crystallization (Gilkey and Staehelin, 1986; Moor, 1987).
However, cryoprotection could be increased further by
adding inert non-penetrating cryoprotectants such as dextran or 1-hexadecene (Kiss et al., 1990) to the protoplasts
during incubation. Large ice crystals can also form by secondary growth during specimen rewarming if ice removal
during substitution is incomplete. Although the substitution
and rewarming method used here has been successfully
used to preserve seedling root tips after high-pressure freezing (Kiss et al., 1990), and the 3-day substitution period at
−79°C was considered adequate due to the small sample
volumes imposed by specimen cup sizes, it may be necessary to increase this substitution time to ensure complete
removal of ice from protoplasts and the surrounding
medium.
Ruffled plasma membranes have not previously been
reported in well-preserved cells after high-pressure freezing, although rippled plasma membranes were reported in
ice-damaged walled cells of seedling root tips (Craig and
Staehelin, 1988). It is possible that the ruffling is an artefact of exposure to high pressure. Although samples are
pressurized for only about 0.5 seconds, a number of such
artefacts have been reported, including the bursting or
breakage of large vesicles in root cap cells of Arabidopsis
(Kiss et al., 1990) and large peripheral vesicles in fungal
sporangia (Hyde et al., 1991a).
The appearance of loose wavy bundles of microfilaments
in cells after high-pressure freezing/freeze substitution may
be another artefact of pressurization, since Ding et al.
(1992) noted such microfilament bundles in Nicotiana leaf
tissue after high pressure freezing, but found only compact
bundles of straight, regularly spaced microfilaments in wellpreserved cells after chemical or freeze fixation at normal
atmospheric pressure.
Protoplast ultrastructure
The ultrastructure of protoplasts freshly prepared from WS1
embryos has not previously been examined in detail. WS1
protoplasts evidently differ from the meristematic cells of
the embryos since protoplasts but not embryo cells have
large smooth invaginations of the plasma membranes and
similar smooth vesicles, amorphous dark deposits associated with various intracellular membranes, and different
arrangements of membranes at the trans face of Golgi
bodies.
The large smooth plasma membrane invaginations and
856
M. E. Galway, P. J. Rennie and L. C. Fowke
vesicles present in protoplasts may reflect disturbances of
the plasma membranes during protoplast isolation, when,
for example, the numerous plasmodesmata between the
meristematic cells of the embryos become severed. It is not
clear whether the large smooth vesicles near the plasma
membranes of protoplasts are formed by internalization of
large smooth plasma membrane invaginations. However,
since these vesicles remained in the peripheral cytoplasm
and did not fuse with other organelles or vacuoles, there is
no evidence that they delivered CF directly to the endocytotic pathway. Indeed, the occurrence of CF-labelled coated
pits on such smooth invaginations and vesicles and of CFlabelled coated vesicles nearby suggest that these structures
continued to behave as plasma membrane.
The membrane-associated amorphous deposits detected
in glutaraldehyde-fixed protoplasts are absent from intact
somatic embryos and older white spruce protoplasts in culture (Hakman et al., 1987, and unpublished results). They
may have been induced by stress, caused for example by
exposure to cell wall-degrading enzymes, or water loss
upon plasmolysis, since very similar ‘membraglobuli’ form
in Arabidopsis leaves exposed to low temperatures (Ristic
and Ashworth, 1993). The absence of deposits in freezesubstituted protoplasts suggests that they were retained by
glutaraldehyde and osmium tetroxide fixation but not by
exposure to osmium tetroxide and acetone during freeze
substitution. These deposits could be tannins, since tannin
synthesis is induced in suspension-cultured white spruce by
mild environmental changes (Durzan et al., 1973) and
tannin synthesis is associated with the formation of amorphous dark deposits in the white spruce cells (Chafe and
Durzan, 1973). Soluble phenols, including some tannins,
are known to diffuse throughout the cytoplasm during fixation (McClure, 1979), perhaps accounting for the odd association of the amorphous deposits with cellular membranes.
The elaboration of membranes at the trans face of Golgi
bodies in WS1 protoplasts marks another difference
between protoplasts and their source embryonic cells. The
curled trans cisternae and associated vesicles observed in
the protoplasts resemble the trans-Golgi membranes identified as trans-Golgi network (TGN) in root cap cells (Staehelin et al., 1990 Fig. 6; Hillmer et al., 1988 Fig. 1b). We
distinguished between PCR and TGN on the basis that both
WS1 protoplasts and embryonic cells contained partially
coated reticula, but only in protoplasts were TGN-like
curled membranes and membrane rings associated with
trans faces of Golgi bodies. The terms TGN and PCR have
at times been used interchangeably to describe all partially
coated membranes in plant cells (e.g. see Hillmer et al.,
1988; Staehelin et al., 1990). Some of this uncertainty over
the identity of PCR and TGN has been due to our incomplete knowledge of the biogenesis and functions of these
membranes (Griffing, 1991), and to variations in the prominence of TGN and PCR in different cell types (for examples see Pesacreta and Lucas, 1985). Fixation can also result
in differential preservation of TGN and PCR. For example,
Samuels and Bisalputra (1990) did not find any PCR in glutaraldehyde-fixed root cells of Lobelia, but they found that
rapid freezing and freeze substitution preserved this
organelle and revealed that it accumulated a fluid-phase
tracer of endocytosis. Griffing (1991) has summarized the
physical and cytochemical evidence suggesting that PCR
and TGN are indeed distinct membrane-bound compartments with different functions in plant cells. At present,
PCR is believed to be an intermediate organelle of the lytic
pathway, capable of fusing with endocytotic vesicles from
the plasma membrane and perhaps (by analogy with similar structures in animal cells) sorting the contents of these
vesicles for recycling to the cell surface or delivery to Golgi
bodies or MVBs (Tanchak et al., 1988; Fowke et al., 1991).
The TGN membranes are thought to be sites for sorting
vacuolar proteins from proteins and polysaccharides destined for secretion (Staehelin et al., 1990; Moore et al.,
1991). The development of TGN-like membranes at the
Golgi bodies of WS1 protoplasts therefore suggests there
is an increase in the secretory activity of the protoplasts
compared to embryonic cells, perhaps related to the
initiation of cell wall regeneration in the protoplasts.
Time course and pathway of endocytosed
cationized ferritin
We were unable to detect any differences between the time
course and pathway of internalized CF in WS1 protoplasts
and that detailed for protoplasts from suspension-cultured
soybean (Tanchak et al., 1984; Tanchak and Fowke, 1987;
Tanchak et al., 1988). The presence of exogenously supplied CF in coated vesicles and in coated pits at all stages
of invagination shows that CF enters the protoplasts via this
pathway as in soybean protoplasts (Tanchak et al., 1984),
bean leaf protoplasts (Joachim and Robinson, 1984) and
wound-induced protoplasts of the green alga Boergesenia
(O’Neil and La Claire, 1988). All coated pits are assumed
to be invaginating, not fusing with the plasma membrane,
because of evidence that the clathrin coat must disassemble before membrane fusion can occur (Altstiel and Branton, 1983). Serial sectioning of similar coated vesicles in
soybean protoplasts has revealed that coated vesicles
located in the cytoplasm beyond the depth of coated pit
invaginations on the plasma membrane are indeed free vesicles (Fowke et al., 1989).
The intracellular distribution of endocytosed CF changed
and increased during labelling, allowing different stages in
the uptake of this marker to be identified. Thus CF first
appeared rapidly in coated and smooth vesicles near the
plasma membrane then deeper in the cytoplasm. As in soybean protoplasts, Golgi bodies and PCR became CFlabelled within the first 5 minutes of exposure to CF (Tanchak et al., 1984). Likewise, lectin-gold conjugates were
endocytosed and transported to the Golgi bodies of carrot
protoplasts within 7 minutes of labelling (Hillmer et al.,
1986). These results and others confirm that both fluidphase and membrane-bound tracers of endocytosis are
delivered after uptake to PCR in protoplasts and walled
cells of higher plants (Hübner et al., 1985; Hillmer et al.,
1986; Tanchak et al., 1988; Samuels and Bisalputra, 1990;
Owen et al., 1991). However, access of tracers to Golgi
bodies differs between fluid-phase and insoluble membrane-bound tracers, suggesting that while tracers are transported directly in plasma membrane-derived coated vesicles to PCR, transport to Golgi bodies is secondary or
indirect (Fowke et al., 1991). Our results agree with previous studies that insoluble membrane-bound tracers pass into
Endocytosis in freeze-fixed protoplasts
the peripheries of both cis or trans Golgi cisternae after
endocytosis (Hillmer et al., 1986; Tanchak et al., 1988).
The distribution of endocytosed lead nitrate in Golgi cisternae of seedling root cells was similar (Hübner et al.,
1985), but lanthanum nitrate did not enter Golgi bodies at
all in epidermal cells of Lobelia roots (Samuels and
Bisalputra, 1990), while lucifer yellow was distributed
throughout Golgi cisternae after endocytosis in absorptive
trichomes of Brocchinia (Owen et al., 1991). The efficiency
of transport of endocytosed tracers into Golgi bodies (from
PCR?) probably varies according to tracer characteristics
such as charge and solubility (Samuels and Bisalputra,
1990; Owen et al., 1991).
In WS1 protoplasts, the first lightly labelled MVB was
observed after 10 minutes; MVB labelling clearly lagged
behind that of the Golgi and PCR, a lag that was also noted
in CF-labelled soybean protoplasts (Tanchak et al., 1984).
This lag suggests that endocytosed CF is transported either
from PCR or Golgi bodies to MVBs (Fowke et al., 1991).
The fusion of CF-labelled smooth vesicles with MVBs as
observed in WS1 protoplasts is consistent with a vesiclemediated delivery of CF to the MVBs. Such fusion events
would deliver CF to the interior surface of the bounding
membrane; subsequent internal invagination of this membrane could account for the occurrence of internal vesicles
with surface-bound CF, as proposed by Tanchak and Fowke
(1987). MVBs with labelled internal vesicles were also
observed in protoplasts exposed to gold-labelled lectins
(Hillmer et al., 1986), and in lanthanum-labelled root epidermal cells (Samuels and Bisalputra, 1990).
The detection of MVB-type vesicle clusters, in one case
CF-labelled, in vacuoles of the WS1 protoplasts suggests
that CF in MVBs may ultimately be delivered to the vacuoles for degradation. Similar evidence for MVB fusion
with vacuoles was obtained in soybean protoplasts (Tanchak and Fowke, 1987; Record and Griffing, 1988). The
co-localization of CF and acid phosphatases (enzymes associated with the lysosomal or degradative pathway in cells)
in some MVBs and the vacuoles of soybean protoplasts provides further evidence that internalized CF is ultimately
degraded (Record and Griffing, 1988).
In conclusion, our results confirm that there is a common
time course and pathway for the uptake of CF by endocytosis in protoplasts derived from taxonomically distant
species of vascular plants. This endocytotic pathway is
probably similar to that in intact higher plant cells, judging
by the available data on endocytosis of fluid-phase tracers
in seedling roots and absorptive trichomes. However, physiologically-based differences in the fate of endocytosed
tracers are evident in studies of CF taken up via contractile vacuoles in fungal zoospores and flagellated green algae
(Cerenius et al., 1988; Domozych and Nimmons, 1992) and
in CF taken up via coated pits of wound-induced protoplasts of the coenocytic green alga Boergesenia (O’Neil and
LaClaire, 1988).
In contrast to Samuels and Bisalputra (1990) and Hyde
et al. (1991b) we have found no significant differences in
the ultrastructure of endomembranes after freeze fixation as
compared to glutaraldehyde fixation, although high-pressure freezing and freeze substitution greatly improved the
857
preservation of cell membranes, organelle structure and
probably cytoplasmic organization. These results confirm
the view of CF transport and distribution within the
endomembrane system previously established from study
of aldehyde-fixed protoplasts. Improvements to the freezefixation methods used here should enable larger samples of
well-preserved protoplasts to be examined in future, which
will assist in cytological and developmental studies of these
fragile cells.
We thank Dr Andrew Staehelin for use of his Balzers highpressure freezing apparatus, Dr Tom Giddings for his assistance
and much helpful advice in the operation of the high-pressure
freezer and in freeze substitution of the samples and Dr Stephen
Attree for advice on culturing and preparing protoplasts from the
WS1 culture. This research was supported by the Natural Sciences
and Engineering Research Council of Canada, including an
NSERC postdoctoral award to M.E.G.
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(Received 6 July 1993 - Accepted 13 August 1993)