J. Cell Set. 58, 79~93 (198a)
Printed in Great Britain © Company of Biologists Limited 198 a
REDISTRIBUTION OF MATERIAL LABELLED
WITH [3H1MANNOSE IN AMOEBAE INDUCED
TO UNDERGO PINOCYTOSIS
CHARLES J. FLICKINGER
Department of Anatomy, School of Medicine, University of Virginia,
Charlottesville, Virginia, 22908, U.S.A.
SUMMARY
The synthesis, transport, and disposition of material labelled with PH]mannose were studied
by electron microscopic radioautography in normal amoebae and in cells that had internalized
cell surface as a result of being induced to undergo pinocytosis. Control amoebae were injected
with the precursor and placed in normal medium. The Golgi apparatus and rough endoplasmic
reticulum were heavily labelled at the earliest intervals, while radioactivity of the cell surface
peaked 12 h after injection of precursor. The experimental cells were injected, placed in bovine
Berum albumin solution from 15 to 60 min after injection, and then removed to normnl medium
until fixation. Incorporation of the precursor into the rough endoplasmic reticulum was near
normal, but the proportions of grains associated with the Golgi apparatus and the cell surface
were greatly reduced. The percentage of grains overlying vacuoles increased 12 h after injection,
notably in the case of polymorphous vacuoles and dense vacuoles, both of which were identified
as lysosomes with the acid phosphatase reaction. The results suggest that addition to the surface
of components labelled with [lH]mannose was diminished following induction of pinocytosis.
Incorporation of the precursor appeared to be shifted from cell surface material to lysosomal
contents, possibly lysosomal enzymes. It is thought that this shift occurred in response to the
need for the cell to digest unusually large amounts of endocytosed protein. Recycling of cell
surface under these conditions is considered possible.
INTRODUCTION
A biogenetic relationship between intracellular membranous organelles, such as the
rough endoplasmic reticulum and the Golgi apparatus, and the plasma membrane
has been demonstrated radioautographically in a variety of cells by labelling glycoproteins with radioactive sugars (Ito, 1969; Bennett, Leblond, & Haddad, 1974;
Sturgess, Moscarello, & Schachter, 1978). Recently, steps in the synthesis and transport of a membrane protein, the G glycoprotein of vesicular stomatitis virus, have
been detailed by biochemical analysis. Synthesis of this molecule is begun in the
rough endoplasmic reticulum, and it is subsequently transported through the Golgi
apparatus to the cell surface (Bergmann, Tokuyasu & Singer, 1981; Rothman &
Fries, 1981).
The membranous organelles of amoebae appear to be related similarly. Radioactively labelled sugar is incorporated into the rough endoplasmic reticulum and the
Golgi apparatus, and labelled material subsequently appears at the cell surface
(Flickinger, 1975). Small vesicles have been implicated in transport of material from
the Golgi apparatus to the plasma membrane (Flickinger, 1975), as in intestinal
80
C. J. Flickinger
epithelial cells (Michaels & Leblond, 1976). The glycoprotein surface coat of amoebae
is remarkably thick, and cytoplasmic vacuoles that have a similar lining are believed to
be endocytic in nature (Wise & Flickinger, 1974 a; Flickinger, 1975).
While the participation of intracellular organelles in renewal of the cell surface coat
has been studied, there is little information on environmental conditions that affect
this renewal and how it is regulated by the cell. In the present study, we have investigated the influence of inducing endocytosis on synthesis, intracellular transport and
turnover of components of the cell surface. We reasoned that increased internalization
of the plasma membrane and cell coat might lead to accelerated renewal of the
surface, if its components were sequestered or degraded intracellularly. On the other
hand, amoebae might be able to accommodate increased internalization of membrane
without acceleration of the rate of surface production and transport if the plasma
membrane and its coat were recycled, as apparently occurs in other systems (see
Discussion).
Amoebae were injected with [:lH]mannose, which is the major sugar in the amoeba's
cell surface (Allen & Winzler, 1973), and immersed in a solution of bovine serum
albumin, which induces vigorous pinocytosis (Chapman-Andresen, 1962). Electron
microscopic radioautographs were prepared at intervals thereafter and compared with
those of control amoebae. Contrary to expectations, production of the surface coat
was neither increased nor did it remain constant. Instead, labelling of the cell surface
was greatly diminished, while relatively more radioactivity appeared in cytoplasmic
vacuoles, including those identified as lysosomes.
MATERIALS AND METHODS
Cultures of Amoeba proteus were maintained in Prescott's amoeba medium (Prescott &
Carrier, 1964). They were fed daily with washed Tetrahymena. The cells were starved for 2
days prior to an experiment.
Amoebae were placed on an agar-coated slide (Jeon, 1970) and injected individually with a
solution of [i-3H]mannose (Amersham-Searle, Arlington Heights, IL; sp. act. 5 Ci/mmol) as
previously described (Flickinger, 1974a). Mannose was used as a precursor because it constitutes two-thirds of the neutral sugars in the cell surface of amoebae (Allen & Winzler, 1973).
This form of radioactive mannose was used, to be consistent with earlier work (Flickinger,
1974a, 1975; Read & Flickinger, 1980). Its initial use in amoebae (Flickinger, 1975) was in
turn modelled after the successful employment of this compound for radioautographic studies
of glycoprotein synthesis and secretion in another system (Whur, Herscovics & Leblond, 1969).
After the injection, the cells were permitted to recover for 15 min in Prescott's medium and
were placed in a 1 % solution of bovine serum albumin (BSA) (Nutritional Biochemicals,
Cleveland, OH), pH 44, in amoeba medium for 45 min. Vigorous pinocytosis occurs under
these conditions (Chapman-Andresen, 1962). The amoebae were then rinsed and placed in
amoeba medium for the remainder of the experiment. Control cells were maintained in normal
medium throughout. The experimental cells were injected before being placed in BSA, rather
than vice versa, because they were more fragile than normal while in BSA, which made them
difficult to manipulate.
Samples were removed and fixed at intervals of 30 min, and 1, 2, 6, 12 and 24 h after injection
of precursor. An earlier sample was not prepared because previous work had shown that
incorporation of precursor 10-15 m ' n after injection was too low for electron microscopic
radioautography to be practical (Flickinger, 1974a); this may be so because some time is
required for the cells to recover from the direct injection. For fixation, the amoebae were
immersed for 1 h in Karnovsky's glutaraldehyde/formaldehyde mixture (Karnovsky, 1965),
Pinocytosis in amoebae
81
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 o-i M-cacodylate
buffer (pH 7-3), dehydrated in a graded series of ethanols, and embedded in Araldite. Sections
~ 1 /tm thick for light microscopy were cut with glass knives and mounted on slides. Light
microscopic radioautographs were prepared by dipping slides in Kodak NTB-2 emulsion
(Prescott, 1964). Preparations were developed at intervals of 2, 4 and 6 weeks and stained with
Azure II.
Thin sections for electron microscopy were cut with a diamond knife on a Porter-Blum
MT-i or MT-2B ultramicrotome. For electron microscopic radioautography gold sections
were mounted on copper grids and coated with Ilford L-4 emulsion using a loop (Stevens,
1966). Undeveloped sample grids from each coating operation were viewed in the electron
microscope to ensure that a uniform monolayer of silver halide crystals had been applied. After
8 months in light-tight boxes, the grids were developed in Microdol-X, fixed, washed and
stained with lead citrate. The radioautographs were studied with a Philips EM-300 or AEI
EM-8B electron microscope. Additional features of the radioautographic methods can be
found in a previous publication (Flickinger, 19746).
The distribution of silver grains at intervals after injection was studied by determining the
percentage of grains overlying different cell organelles in electron microscopic radioautographs,
according to the procedure of Weinstock & Leblond (1974). 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 microscopic radioautographs. For the emulsion
and section thickness used in the present study, the probability is approximately 50 % that the
radioactive structure responsible for the grain lies within this circle (Salpeter & McHenry,
1973). Determination of the structures within circles resulted in scoring of 'exclusive grains'
(one structure within the circle) and 'shared grains' (more than one structure within the circle).
Shared grains were apportioned according to the number of structures within the circle; e.g.
half a grain to each of two structures, etc. (Weinstock & Leblond, 1974). For each sample
approximately 30 micrographs were obtained at random at an original magnification of 7800 x
(Philips) or 6300 x (AEI). Silver grains were assigned to the following compartments: rough
endoplasmic reticulum, Golgi apparatus, cell surface, mitochondria, nucleus, small vesicles,
cytoplasmic matrix, or one of the several types of vacuoles described in Results. It should be
noted that 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. This fact,
coupled with the use of the 'probability circle' method of analysis made it possible to assign
grains to the 'surface1. Approximately 1000 grains were counted for each interval. 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 ( x 100).
In the present electron microscopic radioautographs the background was less than 0-5 grain/
1000 /1m1, which was considered negligible, so all the grain counts shown exceed background.
At each interval, the difference in distribution of actual numbers of grains between the experimental and the control samples was tested for significance by chi-squared analysis of a contingency table. The differences between treated and control values for individual organelles were
then analysed for significance by subdividing the contingency tables and conducting chisquared tests (Zar, 1974).
Analysis of electron microscopic radioautographs indicated that structures morphologically
resembling lysosomes were labelled in amoebae in which pinocytosis had been induced.
Therefore, to aid in identification of these structures, acid phosphatase activity was localized
cytochemically. Amoebae were immersed in BSA solution for 1 h, transferred to amoeba
medium for 1 h and then fixed for 30 min in Karnovsky's fluid. After rinsing overnight in
distilled water, they were embedded in agar and incubated for 40 min at 37 °C in a medium
consisting of 1 vol. of 1-25% sodium /?-glycerolphosphate (Sigma), 1 vol. of Tris-maleate
buffer (pH 5'o), 2 vol. of 0-2% lead nitrate, and 1 vol. of distilled water (Barka & Anderson,
1962; Wise & Flickinger, 19706). Control cells were incubated in medium lacking the substrate.
After incubation, the samples were rinsed successively in distilled water, 1 % acetic acid and
distilled water (5 min each). The cells were then post-fixed and embedded in Araldite as
usual.
82
C.J. Flickinger
RESULTS
Morphology
Normal fasted amoebae had an irregular shape due to their possession of several
pseudopods. They attached to the substrate and were actively motile. Experimental
cells that had been exposed to BSA solution for 45 min were rounder, had many short
pseudopods, did not attach to the substrate and were not motile, as described in
detail by Chapman-Andresen (1962). The experimental amoebae gradually resumed a
normal configuration when placed in amoeba medium for the remainder of the
experiment.
The ultrastructure of A. proteus has been described and reviewed (Daniels, 1973;
Flickinger, 1973). Therefore, only the major pertinent features are summarized here.
Many of these are illustrated in the radioautographs (Figs. 1-6, which are arranged
in a timed sequence).
The rough endoplasmic reticulum was composed of cisternae, tubules and vesicles of
ribosome-studded membranes (Figs. 1, 3). The Golgi apparatus consisted of multiple
stacks of curved, smooth-surfaced cisternae with expanded ends (Fig. 1). Cisternae
toward the concave pole, especially at their margins, contained a filamentous material
closely resembling the cell surface coat in morphology and staining properties
(Stockem, 1969; Wise & Flickinger, 1970a; Flickinger, 1981). Groups of small
vesicles and tubules (Fig. 6) have been shown to contain a similar filamentous material
(Flickinger, 1981). The cell surface of A. proteus comprised the plasma membrane and
its external coat, which in turn included a conspicuous layer of filaments that extended
up to 150-200 nm from the membrane (Fig. 5). Mitochondria with tubular cristae
were scattered through the cytoplasm. The nucleus was bounded by the usual two
membranes with pores and an elaborate internal honeycomb-like fibrous lamina. The
interior of the nucleus contained chromatin, nucleoli and helices.
For analysis of radioautographs, cytoplasmic vacuoles were classified as follows.
Fringed vacuoles had a lining of filamentous material resembling the cell coat (Fig. 6).
Polymorphous vacuoles (Fig. 4) had a variable content including granules, membranous
material or an amorphous substance, and were previously identified as lysosomes
(Read & Flickinger, 1980). Some vacuoles (mainly in experimental cells) had an
electron-dense content and were therefore termed dense vacuoles (Fig. 6). Empty
vacuoles were devoid of obvious contents (Fig. 5). Vacuoles not readily assignable to
one of the above categories were designated other vacuoles.
Fig. 1. Control amoeba 30 min after injection of [3H]mannose. Numerous silver grains
overlie the stacks of curved cisternae of the Golgi apparatus (g). Some grains are also
associated with the rough endoplasmic reticulum (er). x 32000.
Fig. 2. Periphery of a control cell 12 h after injection of precursor. Silver grains are
concentrated over the cell surface. The plasma membrane (m) is clearly defined, but
the external filamentous coat is barely visible because the section is lightly stained (cf.
Fig. 5). x 19000.
Pinocytosis in amoebae
-£
*
C.J.FUcUngtr
Fig. 3. Experimental cell, induced to undergo pinocytosis, 30 min after injection of
[*H]mannose. Grains overlie the rough endoplasmic reticulum (er), but few are
associated with the Golgi apparatus (g). x 26 500.
Fig. 4. Experimental cell 6 h after administration of precursor. A polymorphous
vacuole, containing amorphous or granular material, pieces of membrane, and clear
areas, is heavily labelled, x 22000.
Pinocytosis in amoebae
85
The fine structure of the experimental cells generally resembled that of normal
amoebae, but in addition many pinocytic channels and vacuoles were present. The
multiple channels were sinuous membrane-bounded structures of variable length
with a moderately dense homogeneous or variegated content (Figs. 6, 7). Vacuoles
with a similar content, probably protein ingested by pinocytosis, were numerous in
experimental cells (Figs. 6-8), and as noted above, they were descriptively termed
dense vacuoles. Because of their similarity in content, the channels were included
with the dense vacuoles when scoring the radioautographs.
Table 1. Percentage of silver grains associated with organelles of amoebae at intervals
after injection of \?H]mannose
Time after injection (h)
A
Organelle
30 min
Endoplasmic reticulum
Control
Expl
Golgi
Control
Expl
Cell surface
Control
Expl
Small vesicles
Control
Expl
Fringed vacuoles
Control
Expl
' Polymorphous' vacuoles Control
Expl
'Empty' vacuoles
Control
Expl
Dense vacuoles
Control
Expl
'Other' vacuoles
Control
Expl
Mitochondria
Control
Expl
Lipid droplets
Control
Expl
Control
Nucleus
Cytoplasmic matrix
Other
Expl
Control
Expl
Control
Expl
Total no. grains counted Control
EXDI
168
191
27-1
6-9*
2-4
os»
53
10-4*
ii-3
34'
19
49'
24
46"
o-o
2- 4 »
136
133
I
124
10-9
n-8
I2-I
II-2
76
n-4 b
46
17
1-7
2- 9 »
32
1-4
23
91
38
i-9 b
8-6
6-6c
87
6
2
235
8-2
49
11-5°
45
i-4
i-5
392
o-8
i-i
127
5'2»
6-i
2-6
127
37
69
39
2-5
176
5-i
10-9*
2-O
103
7-1
49'
60
60
58
5-7
4-2"
36
6-7b
57'
24
4-7'
81
139
12
7-7'
3-o"
2-2
2-3
05
1-2
23
49'
7o»
3 -6»
42'
15-9'
o-o
55'
o-o
6-8*
19-0
16s
o-o
o-o
o-i
13-2*
34'
134
19-6
53"
146
172
25 41
146
5'i
4i
51
2-9
34
95b
46
46
0-7
05
o-i
1-3
25
0-3
i-8"
o-o
o-6
o-s
o-o
°-5
i-5
4-5'
2-6
7-i»
2-7
49'
24
19
15-2
2I-8 1
ii-o
26l»
11-5
30
28
28
3-i
o-ob
0-7
2-oc
87
213
24-2»
30-7
62
81
49
44'
1048
1000
1147
1122
171
51
2-7
6-6
8-4'
171
194
6-o
3-4b
1000
1006
3-7
1115
1049
1000
IOI4
3-8
257'
2-2
3O
1223
498
The significance of the difference between the number of grains over a given organelle in
experimental and control cells is indicated as follows: • P < o-ooi; b P < o-oi; c P < 0 0 5 ;
no symbol, not significant. The total distributions of grains over experimental and control
samples were significantly different (P < o-ooi) at all intervals.
C. Jf. FUckinger
Pinocytosis in amoebae
d
c
a
A
8
Fig. 7. Lightly stained section of an amoeba induced to undergo pinocytosis and
incubated for demonstration of acid phosphatasc activity. Copious reaction product
is present in a vacuole with a moderately dense content (d). Small amounts of reaction
product appear in a channel-like structure (c). x 30000.
Fig. 8. Acid phosphatase control. There is no precipitate in vacuoles (d). x 33000,
Electron microscopic radioautography
The electron microscopic radioautographs shown in Figs. 1-6 serve as samples of
the preparations that were analysed and qualitative illustrations of variations in
labelling at different intervals. To follow changes in labelling patterns at different
intervals, attention is directed toward the quantitative analysis of the radioautographs
shown in Table 1 and summarized graphically for some organelles in Fig. 9.
Fig. 5. Amoeba induced to undergo pinocytosis, 12 h after injection of [3H]mannose.
Silver grains overlie the endoplasmic reticulum (er), empty vacuoles (w), and some
vesicles, but very few are associated with the cell surface (s) compared to control
cells at this interval (cf. Fig. 2). The filamentous cell coat (s) is conspicuous in this
micrograph, x 19000.
Fig. 6. Experimental cell at the 12 h interval. Dense vacuoles (d), which have a
content that probably represents endocytosed protein, are accompanied by fringed
vacuoles (/), and a group of small vesicles (a), x 27000.
C. J. FHcfanger
40
30
a
20
W
Tl
)
I
2
12
24
Time (h)
40 r—
30
c
a
20
10
Time (h)
Fig. 9. Labelling of selected compartments of amoebae at intervals after injection of
pH]mannose. Abcissa: time after injection; ordinate: percentage of silver grains.
A, control; 8, experimental celJs induced to undergo pinocytosis. g, golgi apparatus;
gr, rough endoplasmic reticulum; ct, cell surface; sv, small vesicles; dv, dense vacuoles.
Values for other organelles are included in Table r.
Pinocytosis in amoebae
89
Controls. At the first interval, 30 min after injection of precursor, the most heavily
labelled organelles were the Golgi apparatus and the rough endoplasmic reticulum
(Figs. 1, 9). The proportion of grains associated with these organelles then declined
with time. The percentage of grains over the cell surface was low to begin with but
rose to a conspicuous peak of almost 40 % grains 12 h after administration of precursor
(Figs. 2, 9). Labelling of groups of small vesicles reached its maximum at 1 h. Some
grains appeared over the various types of vacuoles at all intervals, but the highest
percentages for fringed, polymorphous and other vacuoles were reached in the later
samples (Table 1).
Labelling of other organelles, such as mitochondria, nuclei, and lipid droplets,
tended to increase with time but were not otherwise remarkable. Approximately 1020 % grains were attributed to the cytoplasmic matrix throughout the period of study
However, since the matrix represents about 50% cell volume (Flickinger, 1975) the
concentration of label in this compartment remained much lower than in the major
membranous organelles.
Experimental cells. In amoebae that were induced to undergo pinocytosis, as in the
controls, the rough endoplasmic reticulum was conspicuously labelled at the earliest
interval (Figs. 3, 9). In striking contrast to the controls, however, the proportion of
radioactivity associated with the Golgi apparatus in the pinocytosing cells was greatly
diminished, being reduced from 27% to only 6 9 % (Figs. 3, 9; Table 1). Although
the percentage of grains associated with these organelles dropped at subsequent
intervals, the proportion over the rough endoplasmic reticulum did not decrease as
much as in the normal amoebae.
At later intervals, the most obvious difference between the experimental and
control cells was a large decrease in the proportion of grains associated with the cell
surface in those induced to undergo pinocytosis (Figs. 5, 9). At 6 h and later this
difference was highly significant, the percentage of grains being reduced from 39-2 to
4-5 at 12 h, which is the time of the normal peak in labelling of the surface. On the
other hand, in several instances larger proportions of grains were associated with
vesicles and vacuoles in the experimental amoebae (Table 1). Most obvious was the
labelling of polymorphous vacuoles (Fig. 4) and dense vacuoles (Fig. 6), the latter
rising from low levels to a peak 6 h after administration of precursor (Fig. 9). Since
dense vacuoles appeared to contain endocytosed protein, morphologically similar
vacuoles were rare in control cells. Thus, to some extent the labelling scored for dense
vacuoles simply reflects the reclassification of other types, such as fringed vacuoles,
as they acquired an electron-dense content. It should be emphasized, however, that
this is only a partial explanation of the pattern of vacuole labelling in experimental cells
because, when the percentage of grains associated with all types of vesicles and vacuoles
was summed, it was seen that at the 12 h interval (when the surface label normally
peaks) the proportion of radioactivity over all the vacuoles increased from 27% in
controls to 41 % in cells induced to pinocytose. A higher percentage of grains was also
associated with the cytoplasmic matrix in experimental cells at all intervals.
The overall pattern of distribution of silver grains over the experimental group of
cells was significantly different from that over the controls at all intervals (P < 0001).
4
CEL58
90
C. J. Flickinger
The number of grains associated with individual organelles also differed significantly
between experimental and control samples in many instances, as indicated in Table i.
Cytochemistry
Amoebae that were incubated in medium for acid phosphatase displayed electrondense reaction products in cisternae and vesicles at the convex pole of the Golgi
apparatus and in a variety of forms of vacuoles, including polymorphous vacuoles and
those with a dense content thought to represent endocytosed protein (Fig. 7). Some
dense vacuoles and channels (Fig. 7) contained small amounts of reaction product or
lacked itJentirely; these may represent pinocytic channels or vacuoles early in the
acquisition of hydrolytic activity by fusion with lysosomes. Cytochemical controls
(Fig. 8) did not contain electron-dense deposits of reaction product, the only staining
being a fine stippling that seemed randomly distributed through some of the sections.
DISCUSSION
The sequence of labelling of normal amoebae after injection of [•1H]mannose most
probably reflects the incorporation of sugar into glycoprotein in the rough endoplasmic reticulum and Golgi apparatus, followed by transfer of much of the labelled
material to the cell surface, as has been described in more detail (Flickinger, 1975).
The labelling pattern of normal cells also suggests that small vesicles may function in
transport of coat material, because their peak labelling occurred between that of the
rough endoplasmic reticulum and Golgi apparatus, and the cell surface. The appearance of maxima in vacuoles, especially those of the fringed variety, after peak labelling
of the cell surface has been interpreted as reflecting internalization of surface by endocytosis (Flickinger, 1975). Some earlier vacuolar labelling, particularly in the case of
polymorphous vacuoles, was attributed to radioactivity of lysosomal erwymes (Read &
Flickinger, 1980).
Although incorporated into the rough endoplasmic reticulum at approximately
normal proportions, a much lower percentage of the material labelled with fH]mannose subsequently reached the surface in cells induced to undergo pinocytosis.
This change was especially dramatic at the normal peak interval of 12 h when the
fraction of radioactivity associated with the surface was reduced to less than 14% of
its normal value. Instead, at this interval more labelled material appeared in vacuoles,
including the polymorphous and dense varieties, which were identified as lysosomes
on the basis of their morphology and acid phosphatase activity. The simplest interpretation of this shift in distribution of labelled material is that the induction of pinocytosis
by protein introduces into the cell much material that requires digestion. In response
to this demand, the manufacture of components of the digestive apparatus, including
lysosomal enzymes that are glycoproteins (Riordan & Fustner, 1978), may take
precedence even over renewal of the cell surface. Thus, the radioactive precursor
would be diverted from pathways leading to the surface into lysosomal vacuoles. It is
very unlikely that the vacuoles could have obtained their label as a result of endocytosis of radioactive cell surface during the course of the experiment, because
Pinocytosis in amoebae
91
labelling of the surface was never as high as that of the vacuoles and a peak in the
percentage of grains over the surface did not precede those over the vacuoles. In fact,
the normally conspicuous peak in labelling of the surface at 12 h was abolished.
If the production of surface components is decreased under these conditions, how
then is the surface maintained ? The large amounts of surface internalized by pinocytosis in various cell types (Bowers & Olszewski, 1972; Stockem, 1973; Steinman,
Brodie& Cohn, 1976; Ryter & de Chastellier, 1977; Bowers, Olszewski & Hyde, 1981)
have suggested that recycling occurs. For example, it has been estimated that in amoebae
50% of the cell surface is ingested within 15-30 min when vigorous pinocytosis is
induced (Chapman-Andresen, 1963; Holter, 1965). It has been argued that the
synthesis of new components cannot account for the necessary replacement of
surface internalized by pinocytosis, because reported turnover rates of the plasma
membrane are too low (Steinman et al. 1976). In the most direct demonstration of
recycling, recent studies of the fate of enzymically iodinated membranes have shown
that plasma membrane polypeptides enter phagolysosomes and return from phagolysosomes to the cell surface (Muller, Steinman & Cohn, 1980a, b). While the present
observations do not indicate directly whether recycling of surface components has
occurred, recycling of pre-existing (unlabelled) surface is consistent with the data.
Such a mechanism, whereby vesicles deliver protein to lysosomes and then return
membrane to the surface, would permit maintenance of the cell surface even in the
face of intense pinocytic activity.
While initial incorporation of radioactivity into the rough endoplasmic reticulum
after administration of prTJmannose was normal, labelling of the Golgi apparatus was
greatly reduced in cells induced to undergo pinocytosis. This may be related to the
observation that the number of Golgi bodies decreased in pinocytosing amoebae
(Stockem & Korohoda, 1975). In addition, a higher proportion of labelled material
may have been retained in the endoplasmic reticulum instead of being transported to
the Golgi apparatus, as suggested by the failure of the percentage of grains over the
endoplasmic reticulum to fall to normal levels (Fig. 9). Furthermore, radioactively
labelled material could have been transferred directly from the endoplasmic reticulum
to vacuoles without passing through the Golgi apparatus, if primary lysosomes were
formed directly from the endoplasmic reticulum. The pathway for primary lysosome
formation in amoebae has not been definitely established, and acid phosphatase
activity is present in some Golgi cisternae and vesicles (Wise & Flickinger, 19706),
but formation of lysosomes from the endoplasmic reticulum has been postulated in a
variety of cells (Novikoff, 1976).
The extent to which [3H]mannose may be metabolized to other molecules in
amoebae is not known directly. In rat thyroid glands incubated under controlled
conditions in vitro, however, approximately 50-75 % of the radioactivity from ["C]mannose in protein fractions is recovered as mannose, the remainder being in galactose
and a fraction that contains mainly amino sugars along with some amino acids
(Herscovics, 1969). Thus, if the situation is at all similar in amoebae, the majority of
the label after injection of [3H]mannose probably remained in mannose. Furthermore,
any label in the radioautographs attributable to other sugars or amino acids was
4-2
92
C.J.Flickinger
probably in protein or glycoprotein (as is the case also for mannose), because most
free small molecules would have been washed from the cells by the fixatives and
dehydration fluids.
Although addition to the cell surface of material labelled by injection of pHjmannose
was diminished in response to endocytosis in the present study, induction of pinocytosis was previously reported to stimulate incorporation of pHJglucosamine into the
cell surface of A. proteus (Allen, Winzler & Danielli, 1976). This difference is unexplained, unless components labelled by the two different precursors are handled
differently by the cells.
REFERENCES
ALLEN, H. J. & WINZLER, R. J. (1973). The chemistry of amoeba surface. In Biology of Amoeba
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{Received 3 March 1982)