Development 109, 753-764 (1990)
Printed in Great Britain © The Company of Biologists Limited 1990
753
Visualization of the endoplasmic reticulum in living buds and branches of
the moss Funaria hygrometrica by confocal laser scanning microscopy
M. MICHELE McCAULEY* and PETER K. HEPLER
Department of Botany, University of Massachusetts, Amhersl, Massachusetts 01003, U.S.A.
•Author for reprint requests
Summary
Caulonemata of the moss Funaria hygrometrica were
vitally stained with the fluorescent, lipophilic carbocyanine dye DiOC6(3) and examined via confocal laser
scanning microscopy. Although DiOCfi(3) stained nearly
all of the organdies, cortical endoplasmic reticulum
(ER) could be resolved under favorable conditions and
appeared as a network of irregular polygons, interspersed with lamellar cisternae in some cell types. The
pattern of cortical ER was examined first during side
initial formation and then in young branches and buds.
The ER network extends into the outgrowth of a
developing side initial, keeping pace with elongation of
the outgrowth. Prior to the cell division that cuts off the
outgrowth from the underlying cell, the network in the
outgrowth becomes tighter, i.e. the polygons become
Introduction
Bud formation in the moss Funaria hygrometrica (L.)
Sibth, and the subsequent development of the buds into
upright, 'leafy' gametophytes, represents a switch from
two-dimensional, filamentous growth to three-dimensional growth. Cytokinins, e.g. benzyl adenine (BA),
play a role in this developmental switch as well as the
earlier filamentous growth. Low [BA] ( 1 0 ~ 8 - 1 0 ~ I 4 M )
promotes branching of filaments by stimulating caulonemal cells to divide asymmetrically, forming small side
initials (Bopp and Jacob, 1986). The developmental
fate of such side initials is determined by the concentration of BA to which they are exposed. Side initials
cultured under continued low [BA] develop into
branches (Bopp and Jacob, 1986). In contrast, side
initials exposed to high [BA] (10~6 M) develop into buds
(Brandes and Kende, 1968; Bopp, 1984), the first
morphological indications of bud formation being cessation of tip growth and apical swelling. Under continued high [BA], the dome- or club-shaped bud initials
divide in a highly regular pattern to form murticellular
buds.
Previous studies have demonstrated that the internal
membranes of Funaria change qualitatively and quanti-
smaller. If the side initial develops as a branch, this
somewhat tighter ER network is maintained in the tipgrowing side branch. If the side initial develops as a bud,
dramatic changes in both the configuration and the
quantity of the ER network occur. Coincident with the
apical swelling that marks the first visible sign of bud
formation, the network becomes increasingly tighter
until eventually the polygonal configuration is barely
discernible. The increased coverage of the bud cortex by
the ER network demonstrates that a significant increase
in the quantity of membranes also takes place during
bud formation in Funaria.
Key words: Funaria, endoplasmic reticulum, DiOQ(3),
confocal laser scanning microscopy.
tatively during bud formation. A fluorescence microscopy study (Saunders and Hepler, 1981) used chlortetracycline (CTC) to show that the relative amount of
membrane-bound Ca2+ increases in the bud initial and
that an increased level is maintained throughout the
early stages of bud development. Membrane density,
which was measured with the general membrane
marker /V-phenyl-l-naphthylamine (NPN), also increases during bud formation although not as much as
membrane-associated Ca 2+ . The increase in membrane
density measured by NPN fluorescence was corroborated by an ultrastructural study of bud formation in
which morphometric analysis showed a 3-fold increase
in the relative volume of ER between the protuberance
and 3-cell stages of bud formation (Conrad et al. 1986).
We decided to further investigate membrane distribution and quantity in Funaria using DiOC6(3) (3,3'dihexyloxacarbocyanine iodide) staining and confocal
laser scanning microscopy (CLSM). Our goals were to
delineate the pattern of endoplasmic reticulum (ER)
during side initial formation and then to compare the
patterns of ER of buds and branches. Such a comparison should enable us to understand more fully how
changes in membrane organization correlate to the
switch to three-dimensional growth. The lipophilic,
754
M. M. McCauley and P. K. Hepler
fluorescent dye DiOC6(3) was first shown to stain ER in
living and glutaraldehyde-fixed animal cells (Terasaki et
al. 1984). Subsequently, other workers have used
DiOC6(3) to visualize ER in plant cells, specifically
onion epidermal cells (Quader and Schnepf, 1986;
Lichtscheidl and Url, 1987), and to study the relationships of the ER to microtubules and microfilaments in
these same cells (Quader et al. 1987, 1989). The ability
to resolve fluorescing structures within a cell is greatly
enhanced by using CLSM. Confocal microscopy provides fluorescent images in which out-of-focus blur is
virtually eliminated and enables one to optically section
fairly thick specimens (White and Amos, 1987; Shotton,
1988). Whereas onion epidermal cells are flat and
relatively thin, Funaria branches and buds are cylindrical or spherical and relatively thick. Thus we felt that
CLSM would be a necessary adjunct to staining with
DiOC6(3) if we were to resolve the ER in these densely
cytoplasmic cells.
In this paper, we demonstrate the presence of an
irregular, polygonal network of cortical ER in Funaria
hygrometrica using DiOC6(3) staining and confocal
microscopy. Furthermore, using these procedures, we
compare the patterns of cortical ER in developing buds
and branches and show that a significant increase in the
quantity of ER, as indicated by a dramatic decrease in
the openness of the network, occurs during bud formation.
Confocal Imaging System which uses an argon ion laser
(10 mW at 488 nm) and a Nikon Optiphot microscope.
Funaria cells were illuminated with the intensity filter wheel
set at its lowest position (neutral density filter no. 2; 1%
transmission) and with the pinhole generally at its smallest
diameter. Images were obtained using a Nikon PlanApo 60x
oil objective (N. A. 1.40) and the Kalman integration algorithm. Photographs were taken of the monitor with a 35 mm
Nikon camera using Kodak T-Max 100 film. Minor image
processing (scaling to enhance brightness of the image followed by basing to darken the background) was performed on
—one-third of the images prior to photographing them.
Slightly more than half of the images shown in this paper (1 A,
1C, 2A, 2B, 3B, 5A, SB, 5C, 6C, 6D, 7A, and 7C) were
processed in this manner. Estimates of ER density were made
from selected prints using Marsh's modification (Tennant,
1975) of the line intersect method of length estimation
(Newman, 1966).
Results
Confocal microscopy of DiOC6(3)-stained Funaria cells
provides striking images of the ER and enables one to
compare the patterns of ER in different cell types.
However, DiOC6(3) stains not only ER, but also
mitochondria, plastids, nuclei, vacuoles, etc. and thus
various dye concentrations were tested in an attempt to
maximize staining of the ER. DiOC6(3) concentrations
<1.0^gml~ 1 do not stain any cellular structure,
whereas a l.OjigmP 1 concentration stained the ER and
other organelles lightly. Optimal staining of Funaria is
obtained at 2.5-5.0 fig ml"1 concentrations of
Materials and methods
DiOC6(3). At these concentrations, up to 80% of the
Funaria hygrometrica spores were sown on liquid Laetsch's cells in a given preparation are well-stained. No dye
concentration tested produced staining of only the ER:
medium. 7- to 20-day-old sporelings were then transferred to
1 HM naphthalene acetic acid (NAA)/-PO4 Laetsch's medium in a cell with well-stained ER, all the organelles are
solidified with 1% agar and overlaid with cellophane. The
well-stained. In fact, the ER is often the most lightly
transferred sporelings were allowed to grow for an additional
stained of all the cellular components. Frequently,
5-9 days before use in experiments. Buds were induced by
optimal confocal images are obtained from cells that are
transferring the cellophane and attached protonematal colonslightly overstained as judged by conventional epifluories to 1;UM BA/-PO4 Laetsch's medium. Side branches were
escent illumination.
either produced spontaneously on the NAA/-PO4 Laetsch's
medium or induced on 100pM BA/-PO4 Laetsch's medium.
The best observations of DiOC6(3)-stained ER are
All Funaria material was propagated in a growth chamber
made in living Funaria cells. The ER was first observed
with a 16 h photoperiod and constant temperature of 20°C.
in Funaria caulonemata that had been lightly fixed with
The light intensity was 100^einsteinsm~2sec~' at the level of
glutaraldehyde prior to staining, but the collapsed
the colonies.
appearance of the ER tubules in these cells indicated
The ER and other membrane-bound organelles of Funaria
fixation damage. Thereafter, cells were stained without
cells were vitally stained with the fluorescent dye DiOC6(3)
prior fixation; all the figures in this report show vitally
and observed with confocal laser scanning microscopy. A
Funaria cells.
stained
stock solution of DiOQ(3) (Eastman Kodak, Rochester, NY)
Cytotoxicity
of the DiOCg(3) was tested by staining
in ethanol (0.5 mg ml~') was diluted in either distilled H2O or
Funaria as if for confocal microscopy and then observliquid Laetsch's medium, with dilution just prior to use giving
the best staining. Final concentrations ranging from
ing individual cells with DIC optics over a few days.
0.5-5.0/igmr 1 DiOQ(3) were tested. Protonemata were
(Cytoplasmic streaming cannot be used as an indicator
transferred to a pool of DiOC^S) solution on a microscope
of dye effect since this phenomenon does not occur in
slide, stained for 10-20 min, and then rinsed with the approFunaria.) Although some of the cells observed died
priate liquid Laetsch's medium for 5-10 min. The protonemafter ~12h, many of the cells remained alive up to 2
ata were mounted in liquid Laetsch's medium, covered with a
days after application of the dye. The DiOC6(3) seemed
cover slip and excess medium wicked away. The coverslip was
to arrest further development of some cells, but others
not sealed because all sealants tried leached into the Laetsch's
continued to elongate and divide, and buds were
solution and damaged the cells. Wicking away the medium to
formed in 1 ftM-BA-treated preparations. Occasionally,
a minimum is sufficient to prevent specimen drift under the
DiOC6(3) staining caused rapid plasmolysis and cytocoverslip.
plasmic disorganization, but cells exhibiting these efStained cells were observed with the Bio-Rad MRC-500
Endoplasmic reticulum in Funaria
fects were easily discerned under bright-field optics and
not examined via confocal microscopy.
Laser illumination of cells was limited to short
intervals (30-70 s) at the lowest intensity setting to
minimize cell damage and photobleaching of the
DiOC6(3). Occasionally, the duration of irradiation was
70-180 s. Irradiated cells appeared undamaged afterwards as judged by their bright-field images. In a few
cases, the same cell was reexamined about 1 h after the
first examination and the 2 images compared. The cells
had apparently suffered no ill effects nor was there a
noticeable degradation of the images except for some
fading of the dye. Photobleaching, when it occurred,
seemed to affect the ER image more than the image of
the other organelles.
The ER was first observed in caulonemal cells from
the bases of filaments, i.e. older caulonemata. Caulonema cells have a large central vacuole surrounded by a
thin layer of parietal cytoplasm and older caulonemata
755
tend to be especially vacuolate. In these cells, the ER
forms a continuous network of tubules interspersed
with lamellar cisternae (Fig. 1A). The network is
located in the cell cortex close to the plasma membrane
(PM) along the entire length of a cell. The tubules of
the network delimit irregular polygons of quite variable
sizes. This variation in polygon size can result in large
differences in the openness of the network within a
single cell. The flattened sac-like portions of ER, when
present, occur at the intersections of some of the
polygons (Fig. 1A) and are also quite variable in size.
Occasionally, one observes tubules of ER that branch
off the network and then end blindly (Fig. IB). The
junctions of the network are commonly formed from
the intersection of 3 tubules; organelles are sometimes
observed to underlie the junctions.
In these highly vacuolate caulonemata, confocal
microscopy allows visualization of the ER network in
the cortical regions of both the upper and lower surfaces
Fig. 1. (A) Older caulonemal cell of Funaria showing various organelles stained by DiOC6 (3), including the tubular ER
network (arrows), interspersed with cisternae of lamellar ER (asterisks), m, mitochondrion; c, chloroplast. (B) Younger
caulonema with polygonal ER network, but no lamellar ER cisternae. Arrows point to blindly ending ER tubules.
(C) Caulonemal tip cell with ER network tighter than that of caulonemata in A and B. X3000. Bar, 5 f/m. (All figures in
manuscript are at same magnification except Figs 6B, 7B, and 7D.)
756
M. M. McCauley and P. K. Hepler
of the cells. (Large expanses of the upper and lower
surfaces of the cells can sometimes be viewed owing to
vertical compression of the cells under the coverslip.)
Even with conventional fluorescent microscopy, one
can often see the ER network in the upper cortical
region of these same cells, especially at their proximal
ends where the cytoplasm tends to be sparse in organelles. The network does not appear to move except for
some oscillations characteristic of Brownian movement.
In all other cell types examined - younger caulonemal cells, filament tips, branches and buds - ease of
ER imaging is variable, even with confocal microscopy.
All of these cell types are more cytoplasmically dense
and less vacuolate than older caulonemata. Staining
dense cells with DiOC6(3) produces very bright images
in which many of the organelles are clearly delineated.
About half of these cells also reveal spectacular ER
profiles. Optical sectioning shows that the ER occurs
just beneath the upper plasma membrane in an approximately 1-2 ^m thick region of the cortex. ER is never
visualized in the corresponding area of the lower
surface of the cell nor in the more interior regions of the
cell. Only rarely can the ER in these cells be observed
with conventional fluorescence microscopy: the optical
advantages of confocal microscopy and integration of
the resulting image over several seconds are required to
see the ER in these dense cells.
In the same preparation, however, the ER of about
half of the well-stained, densely cytoplasmic cells is not
observed. Even optical sectioning through these cells
does not reveal ER profiles. In the cortical region,
where the ER should be found, one obtains an image of
diffuse fluorescence that is apparently not associated
with any particular organelle.
The ER of the younger caulonemata is similar to that
of the older cells except that often the patches of
lamellar cisternae are smaller and fewer or even absent
(Fig. IB). Major discontinuities in the ER network,
with variable-length segments of completely unconnected tubules, are also seen in some of these caulonemal cells. The ER of filament tip cells was also
examined for comparative purposes. The pattern of the
ER network in tip cells is similar to that of caulonemal
cells, although the network tends to be slightly tighter in
the tip cells (Fig. 1C). (To allow direct comparison of
the spacing of the ER network, all figures in the
manuscript are at the same magnification except
Figs6B, 7B, and 7D.) Lamellar cisternae were never
observed in tip cells.
The ER configuration of caulonemal cells was examined during the formation of side initials. The changes
described below hold whether the side initial was
induced under high or low concentrations of BA, i.e.
whether the side initial was to become a branch or a
bud. A slight swelling at the distal end of the caulonemal cell marks the first stage in side initial production. Cortical ER occurs in the region of swelling
(Fig. 2A-C); this ER is in continuity with the surrounding ER and exhibits the same polygonal pattern. As the
swelling begins to elongate, the ER network develops
apace, ensheathing the outgrowth while maintaining its
reticulate configuration. When the outgrowth has
elongated to a length of ~15-20^m, but usually prior to
the division that separates the initial cell from the
underlying caulonemal cell, a change in the ER becomes noticeable (Fig. 3A-C). The network of cortical
ER throughout the elongating outgrowth is less open
than that in the rest of the cell. The degree to which the
network of the outgrowth becomes more closely spaced
varies from cell to cell and the tightness is always
relative to the ER spacing in the underlying caulonema
cell.
The pattern of ER seen subsequent to the one
described above is dependent upon the developmental
fate of the newly formed side initial cell. Under
conditions promoting branch development, the side
initial will continue elongating and in some cases divide
to form a multicellular branch. The closer spacing of
ER that is observed in the outgrowth prior to division
continues to be seen in the developing branch: the
branch ER is always somewhat tighter than the underlying caulonemal cell ER (Fig. 4A,B). Besides this difference, the pattern of ER in the two cells is similar. The
branch's network of tubular ER lacks lamellar cisternae
and extends along the length of the cell. In a 2-cell
branch, both cells have a polygonal network of ER.
Under conditions promoting bud formation, the side
initial will begin to swell (Figs 3A, 5A) and at about the
same time, the side initial's ER will undergo further
changes in its configuration. The polygonal network
remains, but the spacing of the meshwork, first seen to
decrease in the outgrowth, continues to tighten in the
swelling bud initial (Figs 5A-C, 6A). The increased
tightness of bud ER is associated with apical swelling of
bud initials and not dependent on mitosis and cytokinesis since in a few cases in bud-inducing medium,
swollen outgrowths that had not yet been separated by
cell division were observed to have the closely spaced
ER of bud initials (Fig. 5A). As the bud develops, the
network becomes so closely spaced that individual
tubules become difficult to distinguish and are just
barely resolved out of a diffuse fluorescent haze
(Figs 5B,C, 6A). At this stage, the polygonal pattern of
the cortical ER is not readily apparent. In some 1-celled
bud initials, a somewhat circular area of the cortex
lacking any ER was observed within the midst of
tightening ER near the cell apices. In other buds, the
tightening ER network was not evenly distributed but
displayed foci of densely aggregated or radially
an-anged tubules (Fig. 6C, asterisks); these foci were
sometimes associated with underlying organelles.
The tightness of the network forms a gradient within
the developing bud as a stalk region becomes delineated (Fig. 6B-D). This region is found at the base of
an elongated 1-celled bud (Fig. 5C, asterisk), and after
division is composed of the basal cell of the 2-celled bud
(Fig. 6B). As it develops, the stalk becomes highly
vacuolate and simultaneously its ER network becomes
more open (Fig. 6B, D). As one approaches the apex of
Endoplasmic reticulum in Funaria
757
Fig. 2. Successive (1, 2 and 4)
1 fun sections through distal end
of Funaria caulonema during early
swelling stage of side initial
formation. (A) Cortical ER
(arrows) forms polygonal pattern.
(B) ER network extends into
region of swelling (arrows).
(C) Optical section deeper into
cell showing staining of other
organelles and ER network in
cortex (arrows), c, chloroplast;m,
mitochondrion; v, vacuole. X3000.
Bar, 5/OTI.
the 1- or 2-celled bud, the tightness of the network
gradually increases so that the most closely packed ER
is found at the very apex of the bud (Fig. 6C). Note
that, again, the ER configuration is observed to change
within a singe cell. The ER continues to be closely
spaced in the apical regions of buds as they develop into
multicellular structures (Fig. 7A-D) and form tetrahedral apical cells. Tightly packed ER characteristic of
bud initials and buds was never observed in branch
initials or branches.
It seems apparent by inspection that the tightening of
the polygonal network during budding represents an
increase in the quantity of ER and thus an increase in
the percentage of cytoplasm overlaid with cortical ER.
In addition, an estimate of ER tubule length per area
was made for various cell types in an attempt to
quantify the change in ER density during budding.
These estimates are presented in Table 1 and indicate
that older, presumably more metabolically inactive cells
have the least dense cortical ER while younger, more
active cells have a denser network. As expected, both
branch and bud initials (meristematic cells) have high
ER densities, with the bud initials having the highest
values.
Table 1. ER density in various cell types o/Funaria
hygrometrica
Cell type
ER density
(
/an ER tubule I
//nr' cytoplasm /
Range
Older caulonema
Younger caulonema
Caulonemal tip cell
0 .72-0.93
1 .05-1.38
1 .15-1.59
Caulonema
A
1,33
B
1.11
C
1.38
Branch initial
1.88
1.70
1.57
B
Caulonema
A
1,28.
C
1.05
Bud initial
1.98
2.01
1.96
Discussion
The confocal micrographs of Funaria demonstrate that
a dramatic change in the pattern of the cortical ER
network occurs during budding and, furthermore, that
this reorganization of membranes represents a significant increase in the quantity of cortical ER. Rearrange-
758
M. M. McCauley and P. K. Hepler
Fig. 3. Two examples of a later stage in Funaria side initial formation, but prior to cell division. Note that the ER network
in the outgrowths is somewhat tighter than that in the underlying caulonemata. (A) ER of side initial in bud-inducing
medium, showing apical swelling characteristic of bud initials. (B) ER in outgrowth developing in branch medium. (C) More
median section of outgrowth in B and underlying caulonema with ER network on left (arrows). Rim of brightness at apex of
outgrowth (arrowheads) represents pieces of ER network in cross-section. X3000. Bar, 5 fan.
Endoplasmic reticulum in Funaria
759
Fig. 4. Cortical ER in two Funaria caulonemata with side branches. Note that the ER in the branches is slightly tighter than
the ER in the caulonemata. (A) Medium length 1-cell branch. (B) Longer 1-cell branch. X3000. Bar, 5/.cm.
ment of the cortical ER coincides with the apical
swelling that marks the first morphological sign of
budding and thus may be related to the switch from
two-dimensional to three-dimensional growth. In general, the pattern of cortical ER correlates with both cell
type and the developmental status of the cell. Older,
presumably more quiescent cells have a more widely
spaced ER network while younger, more meristematic
cells have a tighter network. Thus older caulonemata
have a more open network than younger caulonemata,
and tip caulonemata have a tighter network than either
of the intercalary caulonemal cells. Some tightening of
the ER network occurs in the outgrowth during side
initial formation whether the side initial will go on to
form a branch or a bud. Thus initiation of a new growth
point correlates with a change in the pattern of cortical
ER. However, it is during bud formation that the
greatest changes in cortical ER are observed.
The evidence of an increase in cortical ER during
budding presented in this study supports and extends
earlier work-with Funaria. In the membrane study of
Funaria using fluorescent dyes, membrane density as
measured by NPN fluorescence increased 1.5-fold in
1-cell buds and 2-fold in 6-cell buds compared to
caulonemal cells (Saunders and Hepler, 1981). Similarly, the TEM study of Funaria showed a 3-fold
increase in the relative volume of ER between the
protuberance and 1- and 3-cell stages of bud formation
(Conrad et al. 1986). This study demonstrates that the
increases in membrane density and ER volume correlate with the increase in the quantity of the cortical ER
network via the tightening of the polygonal network.
760
M. M. McCauley and P. K. Hepler
Fig. 5. (A) Caulonema with swollen bud initial demonstrating increased tightness of cortical ER in initial. Initial not yet
separated by cell division. (B) Cortial ER network in upper half of 1-cell bud. Note tightness of network at this stage.
(C) More median section of bud in B, showing developing stalk region (asterisk). X3000. Bar, 5 jan.
Indeed, Conrad and coworkers (1986) note that in a
1-cell bud, the ER becomes concentrated in the cortical
region of the cell.
The functional significance of the increase in cortical
ER may be found in the CTC study of Funaria
(Saunders and Hepler, 1981). CTC fluorescence
increased 4-fold in 1-cell buds and about 6-fold in 3- and
6-cell buds compared to caulonemal cells (Saunders and
Hepler, 1981). These results coupled with the NPN
results show that the relative amount of membranebound Ca 2 + increases during budding. The authors
suggest that the pattern of CTC-staining may be indica-
tive of Ca 2 + associated with the ER, but were not
actually able to resolve which intracellular compartments were stained. It is tempting to speculate that the
increase in the quantity of the cortical ER network
accounts for the increase in the membrane-bound Ca 2 +
shown to occur during budding. Interestingly, Drawert
and Ruffer-Bock (1964) showed that the cortical ER
network of onion cells fluoresces after tetracycline
treatment, while Allen and Brown (1988) obtained
similar results after CTC staining and propose that the
ER sequesters calcium. It would be informative to
combine CTC staining of Funaria with confocal mi-
Endoplasmic reticulum in Funaria
761
Fig. 6. (A) 1-cell bud and underlying caulonema. Compare tightness of ER in two cells. X3000. (B) Non-cortical section of
2-cell bud at lower magnification. x%0. (C) Apical cell of bud in B. Note gradient of ER density with tightest ER at very
apex and foci of aggregated ER tubules (asterisks). X3000. (D) Stalk of bud in B with fairly open ER network. X3000. Bar,
5 jim.
762
M. M. McCauley and P. K. Hepler
Fig. 7. (A) Tight cortical ER of apical cell of 2-cell bud. Note that individual tubules are difficult to discriminate. X3000.
(B) Lower magnification of section of bud in A. X1800. (C) ER of 4-cell bud. X3000. (D) Low magnification of non-cortical
section of 4-cell bud in C. x640. Bar, 5/an.
Endoplasmic reticulum in Funaria
croscopy, but this is not possible at the present time
because of the lack of the proper excitation wavelength
of the confocal system.
Other lines of evidence suggest that the change in
cortical ER quantity during budding may represent
changes in Funaria'?, ability to sequester Ca2+ and/or
changes in the level of intracellular Ca 2+ . The ER has
been implicated as an intracellular storage pool of Ca2+
in a variety of systems (Hepler e/a/. 1990). For instance,
Luttmer and Longo (1985) present morphometric evidence that eggs (sea urchin, mouse) whose development is activated by intracellular Ca 2+ release have
significantly more cortical ER than eggs (surf clam) not
so activated. A correlated CTC staining and ultrastructural study of dividing eggs of the sea urchin Lytechinus
variegatus demonstrated that endomembranes, including the ER, are the Ca2+-sequestering organfelles indicated by CTC (Hinkley and Newman, 1988). Recent
work with a calsequestrin-like protein (CSL) purified
from eggs of the sea urchin Strongylocentrotus purpuratus provides further evidence for a calcium-sequestering role for the cortical ER (Henson et al. 1989).
(Calsequestrin is the Ca2+ storage protein in the sarcoplasmic reticulum of muscle cells.) Staining of isolated
egg cortices with CSL antibodies revealed the presence
of a submembranous polygonal, tubular network similar to the ER network, while ultrastructural analysis
localized CSL to the ER lumen. The authors postulate
that, in general, the ER of nonmuscle cells may serve a
Ca2+ storage role during the regulation of intracellular
Ca 2+ .
Certainly the CTC results with Funaria (Saunders
and Hepler, 1981) plus later work with calcium antagonists (Saunders and Hepler, 1983; Markmann-Mulisch
and Bopp, 1987) indicate that a long-term change in
Ca2+ is associated with bud formation. It is known, for
example, that Ca2+ in the external medium is required
for buds to develop beyond the swollen, 1-cell stage
(Markmann-Mulisch and Bopp, 1987), precisely the
stage during which the changes in the ER network
occur. Perhaps the cytokinin signal for further bud
development is mediated through release of Ca2+ from
the cortical ER into the swollen bud initial and this
intracellular Ca2+ mobilization is coupled to Ca2+ entry
from the external medium. This idea is based on the
hypothesis that the ER of nonexcitable cells transduces
extracellular signals via a release of Ca2+ and Putney's
(1986) 'capacitative Ca2+ entry' model for receptorregulated calcium movement into cells. Putney's model
postulates that initial Ca2+ release from the ER is
coupled to entry of Ca2+ from extracellular sources via
the second messenger inositol (1,4,5) trisphosphate and
close apposition of the ER and PM. It is not known
whether such close appositions of the ER and PM occur
in Funaria, but have been postulated to occur in onion
epidermal cells in which the peripheral network is not
dislodged by centrifugation (Quader et al. 1987).
The network of cortical ER dramatically visualized in
living Funaria cells is localized only to the cortex. This is
clearly demonstrated by serial optical sectioning: the
network was never observed in deeper regions of the
763
cell but was restricted to the first micron or so beneath
the plasmalemma. Funaria's network, with its frequent
tripartite junctions, irregular polygons and occasional
lamellar cisternae, is similar to an ER configuration
described for both animal (Terasaki et al. 1986; Lee and
Chen, 1988; Sanger et al. 1989) and plant (Drawert and
Ruffer-Bock, 1964; Quader and Schnepf, 1986; Lichtscheidl and Url, 1987; Lichtscheidl and Weiss, 1988;
Allen and Brown, 1988) cells. The cortical ER in
Funaria corresponds to the relatively stationary ER
network found in close proximity to the plasmalemma
of onion epidermal cells (Quader et al. 1987, 1989;
Lichtscheidl and Weiss, 1988; Allen and Brown, 1988).
The longer ER tubules that occur deeper in the cytoplasm of the onion cells, often in parallel bundles, and
that are highly dynamic (Drawert and Ruffer-Bock,
1964; Lichtscheidl and Weiss, 1988; Allen and Brown,
1988) are not observed in Funaria. This is not surprising
since these dynamic tubules are associated with cytoplasmic streaming in onion cells and Funaria lacks
cytoplasmic streaming.
It is not clear how the change in cortical ER density
during budding is accomplished. Images of 1-celled
buds lacking cortical ER in small, localized areas
indicate that ER formation may not always keep pace
with cell expansion, but the manner in which such an
area becomes associated with ER has not been discovered. Foci of aggregated or radial tubules observed
in buds are reminiscent of the 'focal aggregates' of
lettuce root tip cells stained by osmium tetroxidepotassium ferricyanide (Hepler, 1981). It is possible
that the foci seen in the cortex of Funaria buds may also
represent localized areas of membrane growth and
proliferation.
A number of workers have reported rearrangements
within the polygonal network of animal cells or animal
cell extracts (Lee and Chen, 1988; Dabora and Sheetz,
1988; Sanger et al. 1989). Lee and Chen (1988) documented highly localized movements of ER that are
capable of generating the basic elements of the
network. These movements, characterized as tubule
branching, ring closure and sliding, are generally completed in 10-20 s. Sanger and coworkers (1989) report a
continual shifting of the ER network over 1-2 min
periods including changes in the size and position of
indivdual polygons and reversible divisions of polygons.
Presumably these types of rearrangements could
generate the tightening of the network associated with
budding in Funaria. In general, movements of the ER
were not detected in our system, but comparison of 2
images of the same Funaria cell sometimes showed
minor changes in the ER pattern. Usually, however,
these seemed to be lateral displacements and distortions of the polygons rather than wholesale rearrangements. The acquisition of crisp images of the ER
network over integration times of 30 s or more argues
against the occurrence of movements of 10-20 s duration, but it may be that ER rearrangements take much
longer in Funaria as budding occurs over the period of a
couple of days.
The difficulty of resolving the ER in many cases is
764
M. M. McCauley and P. K. Hepler
somewhat puzzling. Lack of ER staining in the more
internal regions of the cells and in cortical regions
adjacent to the slide is most likely explained by a falloff
of signal intensity (White etal. 1987; Amos, 1988). Such
falloff is presumably due to attenuation of incident
exciting and emerging fluorescent beams by intervening
structures. This could be especially true for the organelle-rich buds and branches of Funaria in which the
DiOC 6 (3) stains a multitude of cellular structures.
Inability to resolve the ER out of a fluorescent haze
from the upper cortical regions of otherwise wellstained cells is more problematic. Interference from
fluorescence of the nearby PM or nonspecific staining of
the cell wall are possibilities as is lack of uptake of the
dye by the ER in certain cells. Rearrangements of the
ER network during integration of the image may
explain some of the hazy images. If multiple layers of
ER occur in the cortices of Funaria buds, DiOC6(3)
staining might produce a general fluorescent haze
rather than distinct staining of individual tubules. Multiple layers were not resolved but are a possibility.
We have shown that the pattern of cortical ER in
Funaria correlates with both cell type and developmental status of the cell. The most dramatic example of this
is seen during bud formation when the spacing of the
polygonal ER network tightens and thus the quantity of
the cortical ER greatly increases.
This work was supported in part by an NSF Postdoctoral
Research Fellowship in Plant Biology to M.M.M. an NSF
grant no. DCB87-02057 to P.K.H. and an NSF grant no.
BBS-8714235 to the Univ. of Massachusetts Microscopy
Center. The authors would like to thank E. Sheldon for
teaching M.M.M. the confocal microscope system.
References
ALLEN, N. S. AND BROWN, D. T. (1988). Dynamics of the
of the sea urchin: localization and dynamics in the egg and first
cell cycle embryo. J. Cell Biol. 109, 149-161.
HEPLER, P. K. (1981). The structure of the endoplasmic reticulum
revealed by osmium tetroxide-potassium ferricyanide staining.
Eur. J. Cell Biol. 26, 102-110.
HEPLER, P. K., PALEVTTZ, B. A., LANCELLE, S. A., MCCAULEY, M.
M. AND LICHTSCHEIDL, I. (1990). Cortical endoplasmic reticulum
in plants: a review. J. Cell Sci. In press.
HINKLEY, R. E. JR AND NEWMAN, A. N. (1988). Changes in the
distribution of calcium-sequestering membranes during the first
cell cycle of the sea urchin, Lytechinus variegatus. Develop.
Growth. Differ. 30, 219-228.
LEE, C. AND CHEN, L. B. (1988). Dynamic behavior of endoplasmic
reticulum in living cells. Cell 54, 37-46.
LICHTSCHEIDL, I. K. AND URL, W. G. (1987). Investigation of the
protoplasm of Allium cepa inner epidermal cells using ultraviolet
microscopy. Eur. J. Cell Biol. 43, 93-97.
LICHTSCHEIDL, I. K. AND WEISS, D. G. (1988). Visualization of
submicroscopic structures in the cytoplasm of Allium cepa inner
epidermal cells by video-enhanced contrast light microscopy.
Eur. J. Cell Biol. 46, 376-382.
LUTTMER, S. AND LONGO, F. L. (1985). Ultrastructural and
morphometric observations of cortical endoplasmic reticulum in
Arbacia, Spisula and mouse eggs. Develop. Growth Differ. 27,
349-359.
MABKMANN-MUUSCH, U. AND BOPP, M. (1987). The hormonal
regulation of protonema development in mosses. IV. The Role
of Ca 2 + as cytokinin effector. J. Plant Physiol. 129, 155-168.
NEWMAN, E. I. (1966). A method of estimating the total length of
root in a sample. J. Appl. Ecol. 3, 139-145.
PUTNEY, J. W. JR (1986). A model for receptor-regulated calcium
entry. Cell Calcium 7, 1-12.
QUADER, H. AND SCHNEPF, E. (1986). Endoplasmic reticulum and
cytoplasmic streaming: fluorescence microscopical observations in
adaxial epidermis cells of onion bulb scales. Protoplasma 131,
250-252.
QUADER, H., HOFMANN, A. AND SCHNEPF, E. (1987). Shape and
movement of the endoplasmic reticulum in onion bulb epidermis
cells: possible involvement of actin. Eur. J. Cell Biol. 44, 17-26.
QUADER, H., HOFMANN, A. AND SCHNEPF, E. (1989).
Reorganization of the endoplasmic reticulum in epidermis cells
of onion bulb scales after cold stress: involvement of cytoskeletal
elements. Planta 177, 273-280.
SANGER, J. M., DOME, J. S., MITTAL, B., SOMLYO, A. V. AND
SANGER, J. W. (1989). Dynamics of the endoplasmic reticulum in
living non-muscle and muscle cells. Cell Motil. Cytoskel. 13,
301-319.
endoplasmic reticulum in living onion epidermal cells in relation
to microtubules, microfilaments, and intracellular particle
movement. Cell Motil. Cytoskel. 10, 153-163.
AMOS, W. B. (1988). Results obtained with a sensitive confocal
scanning system designed for epifluorescence. Cell Motil.
Cytoskel. 10, 54-61.
BOPP, M. (1984). The hormonal regulation of protonema
development in mosses. II. The first steps of cytokinin action. Z.
Pflanzenphysiol. Bd. 113, 435-444.
BOPP, M. AND JACOB, H. J. (1986). Cytokinin effect on branching
and bud formation in Funaria. Planta 169, 462-464.
BRANDES, H. AND KENDE, H. (1968). Studies on cytokinincontrolled bud formation in moss protonemata. Plant Physiol.
43, 827-837.
membrane-associated calcium following cytokinin treatment in
Funaria using chlorotetracycline. Planta 152, 272-281.
SAUNDERS, M. J. AND HEPLER, P. K. (1983). Calcium antagonists
and calmodulin inhibitors block cytokinin-induced bud formation
in Funaria. Devi Biol. 99, 41-49.
SHOTTON, D. (1988). The current renaissance in light microscopy.
II. Blur-free optical sectioning of biological specimens by
confocal scanning fluorescence microscopy. Proc. Roy. Micros.
Soc. 23, 289-297.
TENNANT, D. (1975). A test of a modified line intersect method of
estimating root length. J. Ecol. 63, 995-1001.
CONRAD, P. A., STEUCEK, G. L. AND HEPLER, P. K. (1986). Bud
TERASAKI, M., CHEN, L. B. AND FUJIWARA, K. (1986). Microtubules
formation in Funaria: organelle redistribution following cytokinin
treatment. Protoplasma 131, 211-223.
DABORA, S. L. AND SHEETZ, M. P. (1988). The microtubule-
dependent formation of a tubulovesicular network with
characteristics of the ER from cultured cell extracts. Cell 54,
27-35.
DRAWERT, H. AND ROFFER-BOCK, U. (1964). Fluorochromierung
von Endoplasmatischem Reticulum, Dictyosomen und
Chondriosomen mit Tetracychn. Ber. Dtsch. Bot. Ges. 77,
440-449.
HENSON, J. H., BEGG, D. A., BEAUUEU, S. M., FISHKIND, D. J.,
BONDER, E. M., TERASAKI, M., LEBECHE, D. AND KAMINER, B.
(1989). A calsequestrin-like protein in the endoplasmic reticulum
SAUNDERS, M. J. AND HEPLER, P. K. (1981). Localization of
and the endoplasmic reticulum are highly interdependent
structures. J. Cell Biol. 103, 1557-1568.
TERASAKI, M., SONG, J., WONG, J. R., WEISS, M. J. AND CHEN, L.
B. (1984). Localization of endoplasmic reticulum in living and
glutaraldehyde-fixed cells with fluorescent dyes. Cell 38, 101-108.
WHITE, J. G. AND AMOS, W. B. (1987). Confocal microscopy comes
of age. Nature 328, 183-184.
WHITE, J. G., AMOS, W. B. AND FORDHAM, M. (1987). An
evaluation of confocal versus conventional imaging of biological
structures by fluorescence light microscopy. J. Cell Biol. 105,
41-48.
{Accepted 3 May 1990)