J. Cell Sci. 68, 83-94 (1984)
83
Printed in Great Britain © The Company of Biologists Limited 1984
ALTERED LABELLING OF THE CELL SURFACE AND
INTRACELLULAR ORGANELLES WITH
[3H]MANNOSE IN ENUCLEATED AMOEBAE
CHARLES J. FLICK1NGER
Department of Anatomy, University of Virginia School of Medicine, Charlottesville,
Virginia, U.SA.
SUMMARY
The production, transport, and disposition of material labelled with pHJmannose were studied
in microsurgically enucleated and control amoebae. Cells were injected with the precursor and
samples were prepared for electron-microscope radioautography at intervals, up to 24 h later.
Control cells showed heavy labelling of the rough endoplasmic reticulum and the Golgi apparatus
at early intervals after injection. Later, labelling of groups of small vesicles increased, and the
percentage of grains over the cell surface peaked 12 h after administration of the precursor. Two
major changes were detected in enucleate amoebae. First, the kinetics of labelling of cell organelles
with [3H]mannose were altered in the absence of the nucleus. The Golgi apparatus and the cell
surface both displayed maximal labelling at later intervals in enucleates, and the percentage of grains
over the rough endoplasmic reticulum varied less with time in enucleated than in control cells.
Second, the distribution of radioactivity was altered. A greater percentage of grains was associated
with lysosomes in enucleates than in control cells. The change in the kinetics of labelling of the
endoplasmic reticulum, Golgi apparatus and cell surface indicates that intracellular transport of
surface material was slower in the absence of the nucleus. It is suggested that this is related to the
decreased motility of enucleate cells.
INTRODUCTION
In previous work, the synthesis and intracellular transport of cell surface components were studied in amoebae by radioautography after microinjection of individual cells with [3H]mannose (Flickinger, 1975, 1982; Read & Flickinger,
1980a,6). This precursor was used because mannose is the major sugar of the
amoeba's cell surface (O'Neill, 1964; Allen, Ault, Winzler & Danielli, 1974). It is
known to be incorporated into the carbohydrate portion of glycoproteins in the
endoplasmic reticulum in other cells (Struck & Lennarz, 1980), and thus is expected
to label these substances at the beginning of their pathway of intracellular transport.
Heavy labelling of the endoplasmic reticulum and the Golgi apparatus of amoebae was
observed at the earliest intervals after injection of [3H]mannose, followed by labelling
of groups of small vesicles and, later, the cell surface. An interpretation of these
observations, consistent with studies on other cells, is that [3H]mannose is incorporated into glycoprotein in the rough endoplasmic reticulum and the labelled
material is rapidly transported to the Golgi apparatus, where it is modified and
packaged into small vesicles for transport to the cell surface (Flickinger, 1975, 1982).
The nucleus of amoeba is necessary for normal motility and attachment to the
84
C. J. Flickinger
substrate (Lorch&Danielli, 1953a,6; Prescott & Carrier, 1964;Jeon, 1968); removal
of the nucleus resulting in cessation of organized movement within a few minutes
(Goldstein & Jelinek, 1966; Jeon, 1968). Our hypothesis, therefore, is that the
nucleus might also be necessary for intracellular movement of cell products such as
surface coat material. The hypothesis can be tested in amoebae because they are easily
enucleated, and enucleate cells continue to synthesize protein (Mazia & Prescott,
1955). Therefore, amoebae were enucleated microsurgically, injected with [3H]mannose, and prepared for electron-microscope radioautography. The results show that
production of material labelled with [3H]mannose continued, but transport to the cell
surface was slower than normal in the absence of the nucleus. In addition, some of the
label was redistributed to lysosomes.
MATERIALS
AND
METHODS
Cultures of Amoeba proteus were maintained in Prescott's amoeba medium with daily feedings of
washed Tetrahymena (Prescott & Carrier, 1964). The cells were not fed for the 24 h preceding an
experiment.
Amoebae were placed on an agar-coated slide (Jeon, 1970) and enucleated by pushing the nucleus
from the cell with a glass probe controlled with a deFonbrune micromanipulator. Within lOmin
each cell was injected with a solution of [l- 3 H]mannose (Amersham, Arlington Heights, II: sp. act.
2-7-5 1 Ci/mmol) as previously described (Flickinger, 1974). Control cells were normal nucleated
amoebae, which were injected in a similar manner. After injection, the amoebae were placed in
amoeba medium at 21 °C. Samples were fixed at intervals of 30min, and 1, 2, 6, 12 and 24 h, after
administration of the precursor. An earlier sample was not prepared because we observed previously
that incorporation of precursor 10— 15min after injection was too low for electron-microscope
radioautography to be practical (Flickinger, 1974), perhaps because some time is required for the
cells to recover from the direct injection. Aspects of the use of this precursor, including the extent
to which it may be metabolized to other sugars, have been discussed previously (Flickinger, 1975,
1982). The amoebae were immersed for 1 h in Karnovsky's (1965) fixative, rinsed overnight in
0-05 M-cacodylate buffer, and embedded in a small cube of agar to prevent their dispersion (Flickinger, 1969). The blocks were postfixed in 1 % OsO4 in 0-1 M-cacodylate buffer (pH 7-3) dehydrated
in a graded series of ethanols followed by propylene oxide, and embedded in Araldite. Sections for
light and electron microscopy were cut on a Porter-Blum MT-2 ultramicrotome with glass or
diamond knives, respectively.
Light-microscope radioautographs were obtained by dipping slides in Kodak NTB-2 emulsion
(Prescott, 1964). Grids for electron-microscope radioautography were coated individually with
Ilford L-4 emulsion by the loop method (Stevens, 1966). The preparations were stored for 8—12
months in light-tight boxes, developed in Microdol-X, fixed, washed and stained with lead citrate.
The radioautographs were studied with a Philips EM-300 electron microscope. Additional features
of the radioautographic methods can be found in previous publications (Flickinger, 1975, 1982).
The distribution of silver grains at intervals after injection was assessed by determining the
percentage of grains overlying different cell organelles in electron-microscope radioautographs. To
take into account the scatter of radiation from a source in assigning grains to cellular structures, a
circle of 2720 A radius was drawn around each silver grain in the electron-microscope
radioautographs. The probability is approximately 50% that the radioactive structure responsible
for the grain lies within this circle under the conditions of emulsion and section thickness used in
the present study (Salpeter & McHenry, 1973). Grains were scored as 'exclusive gTains' when only
one structure lay within the circle, and as 'shared grains' if more than one structure lay within the
circle. Fractions of shared grains were apportioned equally to the structures within the circle, i.e.
half a grain to each of two structures, etc. (Weinstock & Leblond, 1974). For each sample ~40
micrographs representing portions of approximately 20 cells were obtained at random at an original
magnification of X7800. Approximately 1000 grains were counted for each interval. Silver grains
were assigned to the following compartments: rough endoplasmic reticulum, Golgi apparatus, cell
Labelling of enucleated amoebae
85
surface, mitochondria, nucleus, small vesicles, cytoplasmic matrix, lipid droplets or one of the types
of vacuoles described in Results. Since the cell surface included both the plasma membrane and the
cell coat, it constituted a band (not a line), which occupied a definite area in radioautographs.
Therefore, with the use of the 'probability circle' method of analysis it was possible to assign grains
to the 'surface'. The cytoplasmic matrix was scored as a separate compartment only when a circle
contained matrix alone, because it was present within almost all circles except those over the interior
of the nucleus, and counting all these as 'shared' grains would have complicated the analysis greatly.
The percentage of grains overlying each structure was calculated by dividing the number of grains
over the structure by the total number of grains counted at that interval (X100).
The radioautographic background, determined by counting areas of the section lacking cells, was
less than 0-5 grain/1000/im 2 . This was considered negligible, so all the grain counts shown exceed
background. The difference in distribution of actual numbers of grains between the experimental
and the control samples was tested for significance at each interval by chi-square analysis of a
contingency table. The differences between treated and control values for individual organelles were
also analysed for significance by subdividing the contingency tables and conducting chi-square tests
(Zar, 1974).
RESULTS
Fine structure
The ultrastructure of Amoeba proteus has been described on numerous occasions
(e.g. see Daniels, 1973; Flickinger, 1973, 1975, 1982). Therefore, only a few features
pertinent to the radioautographic analysis will be reviewed.
The rough endoplasmic reticulum was represented by irregular cisternae and
tubules bounded by ribosome-studded membranes (Figs 1, 5). The Golgi apparatus
comprised multiple stacks of smooth curved cisternae and associated vesicles (Fig. 1).
At the concave pole, vesicles and expanded cisternae had afilamentouslining resembling the cell surface coat. The cell surface (Fig. 1) included both the plasma membrane and the cell coat, which was 200—250 nm thick and was composed in turn of an
inner amorphous layer and an external filamentous layer. Mitochondria had tubular
cristae and variable matrix density.
Amoebae contain many vesicles and vacuoles. The following different types were
distinguished in the analysis of radioautographs. Small vesicles and tubular structures, 35—50 nm wide, were present in conspicuous groups and scattered singly in the
cytoplasm. Polymorphous vacuoles (Fig. 4) had a variable content of amorphous
material, vesicles and debris; cytochemical studies indicate that they are a form of
lysosome (Stockem, 1969; Flickinger, 1977, 1982). Fringed vacuoles were so-called
because they had a membrane and filamentous lining similar to the plasma membrane
and cell coat. The descriptively termed empty vacuoles were those lacking obvious
content. Other vacuoles included those not assignable to one of the preceding
categories.
The nucleus of control amoebae was bounded by the usual two membranes and a
prominent honeycomb-like fibrous lamina. The nuclear interior contained nucleoli,
chromatin and helices.
Although enucleated amoebae undergo ultrastructural changes several days after
removal of the nucleus (Flickinger, 1968), few alterations occurred during the period of
the present study, and then only after 24-h. In a detailed ultrastructural examination of
C. J. Flickinger
•
• 'f
"•
.
w * *
' - « • • • »
^
•
Fig. 1. Radioautograph of a portion of a control amoeba 30min after injection of
[3H]mannose. Silver grains are associated with the Golgi apparatus (G). er, rough
endoplasmic reticulum; s, cell surface. X23 000.
Fig. 2. Control amoeba 12 h after administration of precursor. The cell surface is heavily
labelled. X17 000.
Labelling of enucleated amoebae
87
enucleated amoebae, decreased size of the Golgi apparatus and alterations in the morphology of the endoplasmic reticulum were observed at this time (Flickinger, 1968).
Radioautography
Sample electron-microscope radioautographs, which also serve to illustrate some
of the ultrastructural features of the cells, are depicted in Figs 1—6. The quantitative
analysis of the distribution of silver grains over parts of enucleate and control cells is
presented in Table 1, and is summarized graphically for selected organelles in Fig. 7.
The data shown in Table 1 indicate that there were significant differences between
enucleates and controls in the percentage of grains over the Golgi apparatus, the cell
surface, and polymorphous vacuoles at multiple intervals. In addition, among the
membranous organelles, the proportion of grains over the endoplasmic reticulum and
other vacuoles differed significantly between enucleates and controls at one interval.
The percentage of grains over the rough endoplasmic reticulum of control cells was
Fig. 3. Radioautograph of an enucleate amoeba 1 h after injection of [3H]mannose. Silver
grains overlie a portion of the Golgi apparatus (C). X26000.
Fig. 4. A portion of a polymorphous vacuole in an enucleate amoeba 12 h after injection
of the precursor. Silver grains are associated with some of the dense amorphous contents
of the vacuole. X 17 000.
C. J. Flickinger
Figs 5 and 6
Labelling of enucleated amoebae
89
Table 1. Percentage of silver grains associated with organelles of amoebae at intervals
after injection of pHJmannose
Time after injection (h)
r
30min
Organelle
A
1
2
6
12
24
Endoplasmic reticulum
Control
Enucleates
16-8
6-8*
12-4
8-5
121
8-8
7-6
6-4
8-2
7-4
4-9
4-9
Golgi apparatus
Control
Enucleates
27-1
4-4*
11-8
21-6*
4-6
3-4
1-7
1-2
1-4
0-7
11
1-7
Cell surface
Control
Enucleates
2-4
4-6
1-4
2-5*
3-8
7-1
23-5
7-7*
39-2
21-0*
12-7
30-9*
Small vesicles
Control
Enucleates
5-3
0-5*
9-1
1-7
8-6
l-7(*
6-1
1-4*
2-6
11
3-9
1-9
Fringed vacuoles
Control
Enucleates
'Polymorphous' vacuoles Control
Enucleates
11-3
8-2
8-7
7-0
1-9
10-0*
13-9
17-3
12-7
141
6-9
14-7*
17-6
17-7
60
15-0*
6-0
13-2"*
3-6
21-4*
2-0
19-4*
10-3
10-4
0-5
3-5*
1-2
1-2
2-3
1-6
'Empty' vacuoles
Control
Enucleates
2-4
1-7
2-2
3-2
2-3
1-2
'Other' vacuoles
Control
Enucleates
13-6
17-0
13-4
14-6
19-0
15-4
19-6
16-9
14-6
13-2
25-4
12-9*
Mitochondria
Control
Enucleates
31
4-4
4-6
4-1
5-1
6-5
2-7
6-9
5-1
3-8
51
5-8
Lipid droplets
Control
Enucleates
0-7
1-5
0-5
0-5
0-1
21
1-3
1-7
2-5
0-4
0-3
0-4
Nucleus
Control
Enucleates
0-7
0
0-5
0
1-5
0
2-4
0
2-6
0
2-7
0
Cytoplasmic matrix
Control
Enucleates
8-7
37-1*
21-3
19-3
17-1
20-4
15-2
17-2
110
15-3
11-5
10-5
Other
Control
Enucleates
6-2
3-7
6-0
3-0
3-0
1-5
2-8
1-8
2-2
1-4
Total grains counted
Control
Enucleates
1048
1010
1000
1016
1115
1025
1049
1021
1223
993
8-1
1-9*
1147
890
The significance of the difference between the number of grains over a given organelle in experimental and control cells is indicated as follows: *P< 0 0 0 1 ; no symbol, not significant. The total
distributions of grains over experimental and control samples were significantly different
(P< 0-001) at all intervals.
Fig. 5. Enucleate cell 12 h after administration of [3H]mannose. A small polymorphous
vacuole (p) is labelled, and a few silver grains are associated with the rough endoplasmic
reticulum (er). The cell surface (s) contains a smaller proportion of grains than in controls
at this interval (see Table 1) (cf. Fig. 2). X23 000.
Fig. 6. Enucleate amoeba 24 h after injection. The cell surface is conspicuously labelled
at this time. X20 000.
90
C.J.Flickinger
highest at the earliest interval and decreased with time. However, in enucleated cells
it reached its highest values at 1 and 2 h, before declining slightly. Similarly, the Golgi
apparatus of control amoebae was heavily labelled in the initial sample (Fig. 1) and
its radioactivity then declined precipitously, but in enucleates (Fig. 3) the peak
percentage of grains associated with the Golgi apparatus was not reached until 1 h
after injection of precursor. In the case of the small vesicles, groups were most heavily
labelled in controls between 1 and 6 h, while in enucleates they never acquired more
than 1-2% of the grains.
40
30
w
c.
$
2 0
10
1
2
12
24
Time (h)
30r
20
10
o
1 2
12
24
Time (h)
Fig. 7. The percentages of silver grains associated with organelles at intervals after injection of amoebae with [3H]mannose. A. Control cells; B, enucleate amoebae. ER, rough
endoplasmic reticulum; G, Golgi apparatus; CS, cell surface; SV, small vesicles.
Labelling of enucleated amoebae
91
An especially prominent feature of the labelling pattern of normal amoebae was a
rise in the percentage of grains associated with the cell surface (Fig. 2) to a peak of
almost 40 % of the grains 12 h after administration of precursor. This pattern was also
modified in enucleated cells. Although the cell surface acquired a substantial proportion (30-9%) of the grains, this level was not reached until 24 h in enucleates (Figs
5,6).
The only remaining organelles that showed significant differences in labelling
between enucleates and controls at multiple times were the polymorphous vacuoles
(Fig. 4). At each interval except the last they were associated with a significantly
higher proportion of grains in enucleated than in control amoebae.
Scattered differences in labelling of compartments at a single interval were recorded
in the fringed and empty vacuoles, the 'other' category, and the cytoplasmic matrix.
No significant differences were observed in labelling of nuclei, mitochondria, and the
remaining compartments.
DISCUSSION
As discussed in detail previously (Flickinger, 1975, 1982), the observation that the
cell surface of normal amoebae becomes heavily labelled with [3H]mannose indicates
that a substantial proportion of this precursor is incorporated into cell surface components, most probably glycoproteins. In addition, the sequence and pattern of labelling of cell organelles suggest that the assembly of cell surface coat material takes place
in the rough endoplasmic reticulum and the Golgi apparatus.
In enucleated cells a substantial proportion of the incorporated [3H]mannose also
appeared in cell surface material, almost a third of the grains being associated with the
surface at the time of its maximum labelling. This is in accord with the observation
that protein synthesis continues in enucleated amoebae, although incorporation of
amino acid is reduced to about one-third that of normal nucleated cells (Mazia &
Prescott, 1955).
Although material labelled with [3H]mannose continued to be produced and to
reach the surface in enucleates, the kinetics and distribution of labelling of cellular
components were both altered in the absence of the nucleus. The first major difference
was that the intracellular transport of material labelled with [3H]mannose was slower
in enucleates than in control cells. This was indicated by the following observations:
(1) the Golgi apparatus was maximally labelled at 30 min in controls but not until 1 h
in enucleates; (2) the cell surface was maximally labelled at 12h in controls but its
percentage of grains was highest at 24 h in enucleates; and (3) the percentage of grains
associated with the endoplasmic reticulum did not fall as rapidly with time in
enucleates as in controls. Since enucleated cells were studied only at short intervals
after enucleation, it is not known if these changes become more pronounced with
time. However, slower renewal of cell surface material could account at least in part
for the progressively increasing fragility of enucleated amoebae (e.g. see Wise &
Flickinger, 1971).
The nucleus is known to be important for normal motility, which ceases almost
92
C. J. Flickinger
immediately upon enucleation of amoebae (Goldstein & Jelinek, 1966; Jeon, 1968).
Such an influence on motility of the cell as a whole may be accompanied by impaired
movement of organelles intracellularly, thus accounting for slower transport of
material to the surface. It should be noted, however, that while motility and attachment of the cells to the substrate are virtually absent in enucleate amoebae, the
transport of-labelled material to the cell surface was not abolished by the removal of
the nucleus. This is consistent with Jeon's (1968) observation that some internal
cytoplasmic movements continue in enucleated amoebae. The mechanism of the
changes in cell motility in the absence of the nucleus is uncertain. The immediacy of
the effect appears to preclude its mediation through changes in RNA and protein
synthesis, and has led to speculation that changes in intracellular ion distribution
and/or cell membrane or intracellular electrical potentials may be involved (Bingley,
Bell & Jeon, 1962; Jeon, 1968). In any case, the effect of the nucleus on motility is
presumably mediated through the cytoplasmic filaments that are involved in motility
in amoebae and other cells (Allen, 1981; Pollard, 1981).
Since the level of protein synthesis is diminished in enucleate amoebae (Mazia &
Prescott, 1955), the influence of this change on the results must also be considered.
It should be emphasized, however, that the kinetic data (Table 1, Fig. 7) are expressed in terms of the percentages of grains over various compartments. These are
measures of the relative amounts of radioactivity in different organelles, not absolute
levels of radioactivity. Therefore, a decrease in total incorporation of precursor alone
would not alter the curves for the percentages of grains versus time, if the kinetics of
transport were unchanged. Furthermore, Jamieson & Palade (1968) showed that in
the exocrine pancreas virtually complete inhibition of protein synthesis with
cycloheximide did not block subsequent intracellular transport of secretory product.
A slight retardation of transport in the pancreas was attributed to an effect of the drug
on respiration (Jamieson & Palade, 1968), but altered energy metabolism does not
appear to be a factor in the present study because ATP levels in nucleate and enucleate
amoebae are reported to be similar (Brachet, 1955). On the other hand, if the
[3H]mannose were available to the cells for a longer time as a result of its being
consumed more slowly, changes in the kinetic data might be observed. We have no
direct information on the utilization of precursor in normal or enucleate cells, but the
sharp peak in radioactivity in the Golgi apparatus in enucleates argues against prolonged availability of precursor.
The second major change observed in enucleates was an increase in the proportion
of radioactivity associated with polymorphous vacuoles, which have been identified
cytochemically as lysosomes (Stockem, 1969; Flickinger, 1977, 1982). Since the
lysosomal hydrolases themselves are glycoproteins (Riordan & Fustner, 1978), labelling of polymorphous vacuoles with [3H]mannose may reflect radioactivity in the
enzymes themselves. Although the significance of increased lysosomal labelling is
unclear, it may represent preparation for increased digestion of the cell's own
components, since enucleate amoebae are no longer able to capture food organisms.
Another possibility is that it simply reflects a general response to deleterious
conditions, because similar changes were observed in amoebae exposed to several
Labelling of enucleated amoebae
93
other unusual conditions (Read & Flickinger, l980a,b; Flickinger, 1982).
Differences between enucleate and control cells in labelling of a given compartment
at a single interval are more difficult to interpret. An increased percentage of grains
over fringed vacuoles at 12 h and 'other' vacuoles at 24 h in enucleates could be related
to the increased proportion of radioactivity associated with their congeners the
polymorphous vacuoles. The relatively heavy labelling of the cytoplasmic matrix of
enucleates at 30min is more puzzling. This might be attributed to a shift in relative
incorporation of precursor from membrane-associated components destined for the
cell surface to other molecules retained within the cytoplasm. If this were the case,
however, the percentage of radioactivity associated with the matrix would be expected
to remain fairly constant throughout the experiment, but it subsequently fell to
control levels. In any event, it is very unlikely that the matrix labelling at the 30-min
interval represented unincorporated precursor, because the small [3H]mannose
molecules are expected to be washed out of the cells during the lengthy exposure to
fixatives, rinses, dehydration fluids, etc.
In summary, components labelled with [3H]mannose continued to be synthesized
and to reach the cell surface after enucleation, but the kinetics of intracellular transport were slowed. In addition, a larger proportion of the radioactivity was associated
with lysosomes in the absence of the nucleus.
The author is indebted for technical assistance to Ms Ellen Parker, Ms Kathleen Glasheen and
Ms Helen Gray. This research was supported by a grant from the American Cancer Society'(CD58E).
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{Received 13 October 1983-Accepted 20 December 1983)