Published June 1, 1972
PINOCYTOSIS IN ACANTHAMOEBA
CA S TELLANII
Kinetics and Morphology
BLAIR
BOWERS
and T H O M A S
E. OLSZEWSKI
]~'rom the National Heart and Lung Institute, Laboratory of Biochemistry, Section on Cellular
Biochemistry and Ultrastructure, Bethesda, Maryland ~0014
A]3STRACT
INTRODUCTION
Weisman and K o r n (29), while studying phagocytosis by the small soil ameba, Acanthamoeba
castdland, found evidence for a low rate of uptake
of soluble molecules from the incubation m e d i u m
T h e uptake occurred in the presence or absence
of particle uptake. W e have now examined this
slow uptake of solutes more closely to determine
its mechanism. W e present here biochemical and
morphological evidence that the uptake of five
quite different soluble molecules occurs by pinocytosis in Acanthamoeba. Pinocytosis goes on continuously and appears to be the feeding mechanism in a culture m e d i u m containing no particles.
T h e results further show that although the total
volume of m e d i u m ingested by pinocytosis is not
large, the surface to volume ratio of most pinosomes is high. As a consequence, pinocytosis
results in a high rate of surface internalizaUon in
Acanthamoeba.
MAT]~RIALS
AND
METHODS
Cell Incubations
Acanthamoeba castellam~ (Neff strain) was cultured
axenically in a medmm containing 1.5% proteose
peptone and 1.5% glucose as described by Korn
(12). After 5-7 days of growth the cells weie harvested
by low speed centnfugation and either resuspended in
TH~ ffO~;R~L O~ CELI~ BIoLoGY • VOLUME 58, 197~2 - pages 681-694
681
Downloaded from on June 16, 2017
T h e uptake of radioactively labeled albumin, inulin, leucine, and glucose by Acanthamoeba
castdlanii (Neff strain) was measured. The uptake is linear with time and appears to be
continuous under the conditions of these experiments. Uptake is abolished at 0°C. No
evidence for saturation of the uptake mechanism was obtained with either albumin or
leucine. Each of the four tracer molecules enters the ameba at a similar rate when the
uptake is calculated as volume of fluid ingested per unit time. T h e data suggest that each
of these molecules enters the cell by pinocytosis. The highest rate of uptake was obtained
with celIs in their usual culture m e d i u m containing proteose peptone, glucose, and salts
but pinocytosis also continued at a reduced rate in a simple salt solution. The calculated
volume of fluid taken in during pinocytosis in culture m e d i u m was about 2 /A,/hr per 106
cells. T h e route of uptake was examined m the electron microscope using horseradish peroxidase (HRP) as a tracer. H R P activity was found exclusively within m e m b r a n e profiles
within the cytoplasm, confirming the pinocytotic mode of uptake. An estimate of the rate
of surface m e m b r a n e turnover due to pinocytosis was made using the biochemical and
morphological data obtained. This estimate suggests that the plasma m e m b r a n e turnover
of one cell is on the order of several times an hour.
Published June 1, 1972
Tracer ~golecules
Tracer molecules were. human serum albumin-13~I
(Abbott Laboratories, North Chicago, Ill ), inulinmethoxy-3H, uniformly labeled D-glucose-14C, uniformly iabeled L-leueineJ4C, and z-leueine-4,5-aH
(aIl from New England Nuclear Corp., Boston,
Mass.). The ablumln was filtered on a Sephadcx G-25
column (Pharmacia Fine Chemicals I n c , Piscataway,
N. J.) before use to remove unincorporated 131I.
682
T~n JOUI~AL oF CELL BIOLOGY • Vo-b~E
53,
About 95~0 of the counts were recovered in the void
volume, and this fraction was used for the experiments. The conceatration of albumin in the column
fraction was determined from a standard curve by
reading the absorption at 280 nm. Inulin counts
ehromatographed in a single broad peak on Sephadex
G-50 and were all in the void volume after filtration
on Sephadex G-15; therefore, the inulin was used
without purification. Glucose and lcueine were also
used without further purification.
Electron Microscopy
For morphological studms on the uptake of horseradish peroxidase (HRP), amebas were incubated in
medium containing 1 mg/ml of H R P (type HPOD,
Worthington Biochemical Corp, Freehold, N. J.).
After suitable intervals, the ceils were fixed for 1 hr by
the addition of 2 vol of 3% glutaraldehyde in 0.1 ~z
phosphate buffer, pH 6 8 For the demonstration of
peroxidase activity the fixed cells were rinsed and
incubated for 15 rain in the medium described by
Graham and Karnovsky (10), postfixed in 1% OsO4,
dehydrated in ethanol, and embedded in Epon 812
(14). As controls, cells exposed to exogenous H R P
were incubated in the Graham-Karnovsky medium
containing 0.0I ~ NaCN, and cells not exposed to
exogenous H R P were incubated in complete mediuin.
In neither case was activity observed.
RESULTS
Albumin Uptake
Pinocytosis was initially tested by using isotopically labeled h u m a n serum albumin as a
marker for fluid uptake. Cells at a concentration
of 10s/nil were incubated in culture m e d i u m containing about 0.02 m g / m l of a l b u m i n J a I plus
nonradioactive albumin to make a total concentration of I m g / m l . The uptake was examined as a
function of time and temperature (Fig 1). T h e
uptake at 30°C is linear for about 15 rain and
begins to plateau after 30 rain There is no uptake
at 0°C, indicating that the uptake process is not
simply a physical binding of albumin to the surface.
T h e plateauing of the uptake curve after 30
rain m i g h t be due to cessation of uptake or to loss
of label due to excretion of l a I after metabolism of
the ingested protein T o distinguish between
these alternatives a second experiment was performed with the results shown in Table I. I n
Experiment B, cells were preincubated with 4
m g / m l of nonradioactive albumin for 15, 30, or
45 rain and then a trace a m o u n t of a l b u m i n - l a I
197~
Downloaded from on June 16, 2017
culture medium at a cell concentration of 1 2 X 106
cells/ml, or washed twice and resuspended in replacement medium at a concentration of 1.2 X l0 s ceils/
ml. Cell concentrations were determined by light
scattering (28). 10 ml of the ceil suspension, in a
total volume of 12 ml, were used in each experimental
flask, giving a final cell concentration of l0 s cells/ml
Cell protein content as determined by the Lowry
method (13), using bovine serum albumin as a
standard, was 0.53 mg/106 ceils. Osmolalities of
suspending media were determined by freezingpoint depression with an Advanced Instruments
Osmometer (Advanced Instruments Inc., Newton
Highlands, Mass.).
The amebas were equilibrated for 15 rain on a
reciprocal shaker (80 strokes/rain) at 30°(3, then the
radioactive tracer was added and the incubation
continued for the required length of time Duplicate
2 ml samples from each flask were pipetted into 10
ml of ice-cold wash solution (0.1 ~ NaH2PO4,
Na2HPO4 buffer, p H 6.8) to terminate the incubation. For the albumin uptake studies, the wash soIution contained I mg/rrfl nonradioactive human
serum albumin (Sigma Chemical Co, St. Louis,
Mo ). The presence or absence of nonradioactive
inulin or leucine in the wash solutions had no effect on
the measurement of the uptake of those two molecules
The cells were washed three times with 5 ml of
buffer in a refrigerated centrifuge and pelleted. The
wash solution was removed and the pellet was &ssolved in 0.5 nfl NCS solubilizer (Amersham/Searle
Corp., Arlington Heights, Ill.). The sample was then
quantitatively transferred to scintillation vials and
counted in 0.4% 2,5-diphenyloxazole in toluene.
In the experinaents with albumin-lalI, the
counts were corrected for the half-life of 131I.
For collection of 1 4 C O 2 , a center well containing
0.5 mI NCS was suspended from the stopper of the
incubation flask. The incubation medium was
acidified with HCI at the end of the incubation period
to release CO2, and the NCS solution was quantitatively transferred to a counting vial.
For "zero time" samples, the tracer was mixed
with the contents of the incubation flask, and duplicate samples were taken immediately. This procedure
required about 60 sec. Zero time values for uptake
were subtracted from the experimental values
Published June 1, 1972
TABLE I
Albumm-IalI Uptake by Aeanthamoeba
l J p t a k e p e r 10 ~ cells p e r 15
rain
Premcubatton
m i l e (rmn)
cpm
/~g p r o t e i n
Experimen~A*
None
15
30
45
432
461
439
476
0.04
0 04
0.04
0.05
Experiment B$
None
15
30
45
242
258
263
281
0 57
0.60
0.61
0.66
was added a n d the u p t a k e measured over a n
interval of I5 min. I n a parallel experiment,
E x p e r i m e n t A, the cells were p r e i n c u b a t e d in
culture m e d i u m w i t h o u t the addition of nonradmactive albumin. I n b o t h these experiments,
the uptake measured over 15-rain intervals is
constant for at least 60 min. T h e flattening of the
curve in Fig 1 t h e n can be a t t r i b u t e d to loss of
l a I from the cells r a t h e r t h a n to a d i m i n u t i o n of
a l b u m i n u p t a k e with time This experiment also
shows t h a t our i n c u b a t i o n conditions do n o t result
m any decrease in celi activity over a period of an
hour
T h e loss of isotope from cells was investigated
directly after preloading with a l b u m i n - l a I or
mulin-3H for 15 rain. T h e ceils were washed free
of external isotope in the cold a n d r e m c u b a t e d in
culture m e d i u m a t 30°C. T h e results of this experiment, shown in Fig. 2, support the idea
t h a t the protein m a y be rapidly degraded after
uptake, with the consequent loss of I a I from the
cells. O v e r 60,07o of the counts were lost from alb u m i n - l o a d e d cells after 30 rain of remcubation,
as c o m p a r e d with only a b o u t 15% from inulinloaded cells.
T h e u p t a k e of a l b u m i n is nearly linear with
Inulin Uptake
Inulin, a polysaccharide of i n t e r m e d i a t e molecular weight (5000-5500), is in general n o t enzymatically degraded or actively transported b y
cells I t therefore m i g h t be expected to provide a
more convenient m a r k e r t h a n a l b u m i n for measuring u p t a k e processes in Acanthamoeba
Experiments similar to those w i t h a l b u m i n were
performed using inulin-3H as a tracer molecule.
Fig. 4 shows t h a t inulin, like a l b u m i n , is r e m o v e d
fi'om the culture m e d i u m at a constant rate.
~¥e were not a b l e to do a meaningful test of
u p t a k e as a function of c o n c e n t r a t i o n with inulin
because of its limited solubility.
U p t a k e measurements were routinely carried
out with amebas in their culture medium. T h e
culture m e d i u m contains 1.5~o proteose-peptone
a n d 1 5~vv
0 glucose as m a j o r components. T h e effect
of proteins a n d a m i n o acids on stimulating pinocytosis in the large amebas has been well docum e n t e d (5), a n d there is evidence in a n o t h e r
protozoan, Tetrahymena, t h a t proteose-peptone
solution stimulates vacuole formation a n d fluid
u p t a k e (24) In order to rule out t h a t the proteosep e p t o n e solution m i g h t be i n d u c i n g pinocytosis
in Acanthamoeba, inulin uptake was m e a s u r e d
B~Am BOWERS A~D Tno~IAs E. OLSZEWSKI P, nocygosi~ in Acanthamoebacastellan~i
683
Downloaded from on June 16, 2017
~0 17 m g / m l albumin-l~lI (1.8 X 106 c p m / m l )
was added after p r e i n c u b a t i o n in culture m e d i u m
for the indicated times a n d the uptake was det e r m i n e d after 15 rain.
:~ I d e n t m a i to E x p e r i m e n t A, except t h a t a l b u m i n laiI was added after p r e i n c u b a t i o n in culture
m e d i u m containing 4 m g / m l of n o n r a d i o a c t i v e
albumin. Both Experiments A a n d B were performed with the same cell b a t c h
c o n c e n t r a n o n , at least to concentrations as h i g h
as 10 m g / m i (Fig. 3). ~¥e interpret t h e b r e a k in
the curve at a b o u t 4 m g / m l to signify a general
inhtbition of cellular activity by high concentrations of a l b u m i n r a t h e r t h a n a n y saturation of
a l b u m i n uptake. This inhibition of u p t a k e in the
presence of a l b u m i n is also shown in T a b l e I
Experiments A a n d B were performed with tile
same b a t c h of ceils a n d the m e d i u m c o n t a i n e d
identical a m o u n t s of radloactimty, b u t m this
experiment, in the presence of 4 m g / m l of album i n total, uptake is depressed to a b o u t half t h a t
with trace amounts of albumin. Decrease in uptake of other solute molecules in the presence of
a l b u m i n has also been observed, so this c a n n o t be
interpreted as overload of some transport mechanism specific for a l b u m i n , b u t is related to a n
unidentified effect of a l b u m i n on cellular acuwty.
Active transport processes, w h i c h are m e d i a t e d b y
enzymes, characteristically show a l i m k m g rate,
or saturation, of t r a n s p o r t wtth increasing concentrations of substrate T h e inability to o b t a i n
saturation of the u p t a k e m e c h a n i s m in this
instance is therefore consistent with a m e c h a n i s m
of bulk t r a n s p o r t for this molecule.
Published June 1, 1972
150
m
0
100
hi
I.-
H
-
50
I
rn
-.1
z
!
0
0oc
I
I
5
15
INCUBATION
IO0~
ti
t
30
50
TIME
(rnm)
._t
d
F- 5 O
Z
b_
0
I
I
I
I
30
60
90
120
INCUBATION
TIME
(mm)
lqkGURE 1 Uptake of albumin as a function of incubation time and temperature. Cells were incubated as
described under Methods with human se~arm albumin-t51I (1.9 X 105 cpm/ml). Nom'adioaetive albumin
was added to obtain a concentration of 1 mg/mI.
FmVRE ~ Loss of label fi.ona cells preloaded with albumln-lZlI (circles) or inulin-SH (triangles). Cells
were prelabeled for 15 mln, washed free of external isotope, and re-incubated in culture medium. Each
time point is per cent of initial cellular radioactivity. For albumin-loaded cells, initial radioactivity was
1400 cpm/106 cells, and for inulin-loaded cells, 975 epm/106 cells.
after washing the cells free from culture m e d i u m
w i t h 0.02 M N a C I containing 0.1 M glucose, or
0.1 M glucose plus 0 001 ~ MgC12, or w i t h 0 02 M
N a C I alone (Fig 4). I n e a c h case u p t a k e continued. T h e rate of u p t a k e found in the glucosesalt solution was within the r a n g e of values we
o b t a i n e d w i t h ceils in culture m e d i u m , so that,
for short-term experiments at least, the rate is
n o t significantly altered from t h a t found in culture
m e d i u m . I n the salt solution alone, the rate was
684
a b o u t one-half t h a t found with the same b a t c h of
cells in the salt solution plus glucose T h e presence
or absence of small amounts of M g ++ in the inc u b a t i n g solution h a d no effect. T h e reasons for
the reduction in rate in the 0.02 ~ NaCI solution
have n o t been further explored, b u t two obvious
variables are the m a r k e d change in osmolality,
from 200 mosmols for the culture m e d i u m to 40
mosmols for the salt solution, a n d the lack of a n
energy source. I n any event, these results show the
TH~ SOCIAL OF CELL BIO~OGr • VOLV~ 53, 197~
Downloaded from on June 16, 2017
®
-
d
i,1
rn
Published June 1, 1972
.50--
o
o_
"- 2 0
=L
LO
<[
bn
I0
I
Z
{23
d
<
~
I
I
t
I
I
I
2
4
6
10
ALBUMIN CONCENTRATION (mg/ml)
5000
-
Downloaded from on June 16, 2017
®
o
0.9_
/,.,,/
Medtum
o_
2000
{3
LIJ
<
&
[3-
~
K
,ooo
j
m
I
5
j
15
x
~
50
INCUBATION TIME (mm)
91y~lu~° s~_
50
FlelYaE 8 Uptake of h u m a n serum albumin-131I as a function of concentration in the medium. Results
from three experiments are plotted. Uptake was measured for 15 rain with total concentrations of
albumin of 0.08, 0.16, 0.~5, 0.50, 1~ 2, ~t, 6, and 10 mg/ml. A constant amount of radioactive albumin
was used (about 18 Izg/mI), and the final concentration achieved with nom.adioactive human serum
albumin I n the experiments described by the open symbols the medium contained 1.9 X 105 cpm/mL
I n the experiment described b y the closed triangles, the medium contained ¢.4 X 10 5 cpm/ml. Protein
taken up was calculated from the specific activity of the protein and the cpm found in the cells.
I~OV~E 4 Uptake of inulin-3I-I. Three experiments are plotted: (a) the uptake of inulin by cells in
culture medlttm (circles) ; (b) uptake in 0.02 ~ NaC1, O 1 g glucose, 0.001 M 2~gClz (open triangles) ; and
(c) a comparison with the same cell batch of uptake in 0.0~ M NaCI, 0.1 5I glucose (closed triangles) with
uptake in 0.0~ M NaC1 (squares). The concentration of imdin in these experiments was 0.9 X 10- ~ M;
1 X 10 Gcpm/ml.
BI~AIn Bownns a~p TRO~IAs E, OLSZEWSKI Pinocytos~, in Acanthamoeba castellanii
685
Published June 1, 1972
uptake process to be a continuous function that
proceeds readily in a simple salt solution with an
energy source The experiment shown in Fig. 2
shows that the rate of loss of counts from cells
which were preloaded with inulin-3H is only
about 1/( 2 the rate of accumulation. This result
contrasts with that found for albuminAaq and is
consistent with the presumed inability of amebas
to metabolize inulin. The experiment also indicates that uptake as measured in the experiment
m Fig. 4 is in fact a summation of the uptake and
toss of label from the amebas. In the case of inulin, the rate of loss is too low to affect the constancy of uptake over a period of 1 hr.
Leucine and Glucose Uptake
685
THE JOURNALO~ C~LL BIOLOGr • V O ~ E
Morphology of Pinoeytosis
In order to visualize the uptake process of
Acanthamoeba in the electron microscope, we have
used horseradish peroxidase (HRP) as the tracer
molecule. Incubations were performed exactly
as for the biochemical experiments, except that
H R P was added instead of a radioactive solute
molecule and the incubations were terminated by
the addition of fi afive. We used a very short
incubation time (20 see) to attempt to visualize
the primary events of transport, but have also
used a variety of longer incubation times up to 1
hr. As expected, all H R P found within the cells
was enclosed within membrane profiles, confirming the pinocytotic nature of the uptake process.
Figs. 7 and 8 illustrate some of the variety of
labeled profiles observed in cells after a very
brief exposure to tracer. ~ After longer incubation
1 The H R P activity of the pinosomes most often is
localized along the membrane of the vesicles rather
than being uniformly distributed within them. Although this might appear contradictory to the conclusion reached from the biochemical data that up-
53, 197~
Downloaded from on June 16, 2017
By analogy with other ceils, small molecules such
as amino acids and sugars might be expected
to be taken up by active-transport mechanisms.
We therefore examined the uptake of L-leucineAaC
and D-ghicose-l~C by Azantharnoeba.
T h e kinetics of leucine uptake are shown in
Fig. 5 to be the same as for the uptake of albumin
and inulin. Leucine is removed from the medium
in a temperature-dependent process that is constant for at least an hour. Counts from leucine1~C were rapidly incorporated into trichloroacedc
acid-precipitable material and thus loss of counts
through metabolism was not observed during
these short experiments. The rate of leucine ingestion is proportional to its concentration in the
m e d i u m up to a concentration of 0 01 M (Fig. 6).
A t 0.1 ~ leucine, uptake is slightly depressed. This
is presumably due to the high osmolality of the
solution (300 mosmols), rather than to any saturation of an uptake mechanism. Osmolalities in
this range have been shown to inhibit particle
uptake in Acanthamoeba (18, 29), and it is likely
that they wouId affect the uptake of soluble molecules also. T h e absence of saturation kinetics
with leucine, as with albumin, is evidence for
bulk transport rather than active transport of
this amino acid.
The data for uptake of albumin, inulin, and
leucine have been. given in terms of the amount of
radioactivity taken in per 10 6 cells per unit time
These tracer molecules were of different specific
activities and were present in the medium in
different concentrations. I f it is assumed that
each o[ them goes into the cell by a mechanism of
bulk transport, then their uptake rates may be
more directly compared by calculating the rate
of uptake as the volume of medium ingested per
106 cells per unit time. Values calculated in this
way are identical for replicate flasks within a
given experiment. For example, four replicate
flasks in an albuminA~tI experiment gave 0.42 ±
0 01 ~1/106 cells per 15 min There was, of course,
considerably greater variation between uptakes
with different batches of cells. T h e average uptake expressed m/~1/106 cells per 15 min for four
different experiments with albumin-18q was 0 39
4- 0.08; for seven experiments with inulin-SH
was 0 59 4- 0.15; and for two experiments with
teucineA4C was 0 65 (0.55 and 0.75) In a single
experiment with glucose-I4C, uptake for 15 min
was equivalent to a rate of 0 47 /~1/108 cells per
15 min. Radioactive CO2 collected during the
incubation accounted for an addiuonal 10%
uptake. This value of 0 5 #I/106 ceils per 15 rain is
virtually identical with those found for the other
molecules tested and indicates that glucose is
ingested in the same manner.
Thus, the rate of uptake, when expressed as
volume of medium ingested per unit time, is
the same regardless of the tracer used or its concentration in the medium This finding points to a
mechanism of uptake that does not involve selective binding of the substance transported to the
cell surface. In other words, the uptake mechanism
appears to be nonconcentrafive, and a true,
nonspecific "gulping" of the medium.
Published June 1, 1972
7000
t9
O
-
@
o
5000
~ 50~c
~D
u.l
x~
In
D
~D
3000
0
I
ILl
z
1000
(D
ILl
M
5
15
50
50
70
INCUBATION TIME (mm)
A
E IO -I - =
®
O
10-2-
Downloaded from on June 16, 2017
¢0
o
10-3
10-4
~_ 1 0 - 5
-1mI
10-6
hi
Z
,,,
_1
10-7
10-6
10-5
10-4
10-3
10-2
I0-1
I0 0
LEUCINE CONCENTRATION(mM/ml)
FXOVl~E 5 Uptake of Ieueine as a function of time a n d temperature. Cells were incubated in culture
m e d i u m containing 3 8 X 10- 6 ~I leueine-14C (9 4 X 106 c p m / m l ) .
I~GURE 6 Uptake of leucine as a function of concentration in t h e medium. Replicate flasks were incubated for 15 m i n with total concentration of 10- 1 to 10- 6 ~ leueine. :Each flask contained t h e same a m o u n t
of leucine-~H (~.1 M 106 e p m / m l ; 0 4 N 10_6 ~) with nonradloaetive leueine added to m a k e t h e required
concentration.
periods the images are similar but more numerous,
a n d t h e r e is a relative i n c r e a s e in t h e n u m b e r of
take of soluble molecules is nonconcentrative, it
should be r e m e m b e r e d t h a t the n e w l y f o r m e d pinosome contains a dilute solution with too few large
molecules to f o r m a gel on fixation. T h u s , it is
reasonable to expect t h a t the reaction p r o d u c t will
n o t r e m a i n in sztu t h r o u g h o u t the fixation, d e h y d r a tion, a n d e m b e d d i n g process, b u t will only be
visualized w h e r e it ]s s u b s e q u e n t l y b o u n d to a surface
i n t e r m e d i a t e size ( 0 . 3 - 0 7 # m d i a m e t e r ) p r o files I n i n c u b a t i o n p e m o d s e x t e n d i n g for s e v e r a l
m i n u t e s it is clear t h a t s m a l l p i n o s o m e s fuse
w i t h larger, u n l a b e l e d vesicles, c a u s i n g s o m e
a m b i g u i t y as to w h m h a r e t h e initial u p t a k e
vesicles. F o r this r e a s o n w e h a v e l o o k e d m o s t
c a r e f u l l y a t cells e x p o s e d to t r a c e r for o n l y 20
see, t h e m i n i m u m t i m e for a r e a s o n a b l e a m o u n t of
l a b e l to go into t h e ceils, I n o r d e r to i l l u s t r a t e t h e
r a n g e of profile sizes o b s e r v e d a n d t h e w f r e q u e n c y ,
BLAIR BOWERS _t~n TttO~AS E. OLSZEWSKI Pinoeytosi8 in Aeanthamoeba eastellanil
687
Published June 1, 1972
Downloaded from on June 16, 2017
FIGVR~ 7 Section of Acanthamoebacytoplasm showing small HR1)-containing vesicles (arrows). The cell
was exposed to H R P for $0 sec before fixation. Section is stained with lead citrate and tu'anyl acetate.
PM, plasma membrane; DV, digestive vacuole. X 81,500.
688
Published June 1, 1972
Downloaded from on June 16, 2017
:FI~vRn 8 a-c Shows the range of I-IRP-labeled profiles found in cells exposed ¢o Ht~P for g0 sec. Sections
are unstained. PM~ plasma membrane; DV, digestive vacuole. X ~9~000.
689
Published June 1, 1972
)z
w
o
Lo
r~
;:&
O-
.08
.16
.24
.32
.40
,48
.56
.64
.7'2
.80
.86
.5 2.5
DIAMETER iN MICRONS
FmcRE 9 The frequency of the sizes of profiles containing H R P activity measured in thin sections d
cells t h a t were exposed to H R P for ~0 see a t 30°C. I n order to compare HRP-labeled profiles of irregular
shape and to simplify plotting, the average of the major axis (e a) and the minor axis (~ b) was used for
nonch-cular profiles when ~ a was not greater than twice ~ b. :For more eccent14c profiles, the diameter was
calculated as the diameter of a ch'cle of equivalent area (~ ~¢Zab).
690
sites on the surface are p r o b a b l y engaged in
pinocytosis within a n y given dine interval.
T h e image seen in Fig. 8 c has some special
characteristics. I t is found close to the edge of
the cell a n d has the appearance of a narrow,
highly convoluted invagination, often with cytoplasmic protrusions in the center. W e have included these images in the frequency chart, b u t
feel t h a t they p r o b a b l y do not contribute significantly to uptake. I n unpublished observations of
cells i n c u b a t e d at 0°C a n d exposed to H R P for
15 min, we found a n u m b e r of similar channels
labeled with H R P , b u t in ,similar experiments
we find n o measurable uptake of radioactive
molecules at 0°C. T h e volumes of such invaginations m a y be very small or they m a y represent
incompletely closed-off channels in the surface
from which t r a p p e d molecules can be removed
by extensive washing (as in the experiments with
radioactive tracers).
DISCUSSION
W e have examined the uptake of four quite
different kinds of molecules by Acanthamoeba.
S e r u m a l b u m i n is a large protein with a molecular
weight of 65,000 and a net negative charge at
the p H of these experiments. I n u l i n is a neutral
sugar, Intermediate in molecular weight (50005500). Glucose (mol we 180) a n d ]eucine (mol
wt 131) are small molecules t h a t enter readily
THE JOURNAL OF CELL BIOLOGY " VOLVME 5S, 197~
Downloaded from on June 16, 2017
we m e a s u r e d all the labeled profiles in a n u m b e r
of m i c r o g r a p h s from three different 20-see uptake experiments. T h e results are plotted as a
h i s t o g r a m i n Fig. 9. T h i s is not a statistically
valid, r a n d o m sample because the n u m b e r of
labeled vesicles within a n y thin section was so
small t h a t it was n o t feasible to obtain a true
r a n d o m sample. T h e micrographs were obtained
b y s c a n n i n g t h e sections a n d p h o t o g r a p h i n g the
labeled profiles observed. Vesicles of large size
m i g h t b e overrepresented, if anything, because
of t h e i r greater visibility as c o m p a r e d with vesicles
of 120 n m diameter.
T h e histogram indicates t h a t the most frequently
observed profile is a r o u n d 120 n m in diameter,
with a whole s p e c t r u m of sizes extending up
to a b o u t 2.5 # m in d i a m e t e r occurring m u c h less
frequently. T h e smaller vesicles were usually
spherical or cucumber-shaped, while the larger
profiles were often more irregular in shape.
Some of t h e m a p p e a r to be profiles from fingerlike invaginafions of the surface (Fig. 8 c), others
from flattened spheroids (Fig. 8 b). M o s t of the
labeled profiles were shapes of high surface to
volume ratio, t h a t is, quite small, or, if large,
flattened, or spindle-shaped.
L a b e l e d vesicles often occurred in clusters
(Fig. 7), a n d single vesicles or clusters were freq u e n t l y seen at one or two widely separated sites
a r o u n d the p e r i p h e r y of a single cell. This app e a r a n c e suggests t h a t one or more r a n d o m
Published June 1, 1972
vestigations on pinocytosis have been carried out
with two large amebas, Chaos chaos and Amoeba
proteus (11) Those amebas have in common a
prominent mucoid surface coating (15, 17). They
can be induced to pinocytose intensively for
short periods of time by certain cations and
other positively charged molecules (5) which
appear to initiate pinocytosis by interacting with
the mucoid coat (4, 15, 16) T h e cells are also
capable of concentrative uptake of proteins and
other materials that bind to the mucoid coat
(5, 15, 23). In induced pinocytosis the amebas
characteristically cease locomotion and a n u m b e r
of protuberances are formed on the surface. A
channeMike invagination forms in each protuberance and pinches off vesicles at its terminus
that are large enough to see with the light microscope (5) Induced pinocytosls in these amebas
appears to be a highly artificial state (25) Although some spontaneous or " p e r m a n e n t "
pinocytosis may occur in the culture m e d i u m (30)
the amebas cannot be cultured axenically. Thus,
the rate or extent of this permanent pinocytosis is
presumably insufficient to support satisfactory
growth. In Acanthamoeba, on the other hand, we
have not been able to demonstrate a surface
coat in thin-sectioned material (2), nor have we
any evidence that surface binding of molecules
plays a major role in their ingestion Furthermore, pinocytosis appears to be a continuous
process in agitated cultures and is not enhanced
by molecules (e.g., serum albumin) that induce
pinocytosis in Chaos chaos or Amoeba proteus. Finally,
the morphology of uptake appears somewhat
different in Acanthamoeba than in the large amebas
There are obvious limitations to the interpretation
of static images, but our electron microscope
images suggest that the most common mode of
ingesuon is by vesiculations that are too small to
be seen with the light mmroscope
Although there is a plethora of morphological
observations on pinocytosis in a variety- of cell
types, there are relatively little kinetic data Most
of these data come from m a m m a l i a n cells, and
some of the data that are directly comparable
to values we have obtained with Acanthamoeba
are tabulated in Table I I In each instance listed
in the table, the uptake is at least an order of
magnitude lower than that found for A, anthamoeba. This result is perhaps not surprising since
pinocytosls seems to perform no significant nutritional function in m a m m a l i a n cells (9, 21, 27),
BLAIR BOWERS AND TEOMAS E. OLSZnWS~I Pinoeytosis in Acanthamoeba castellanii
691
Downloaded from on June 16, 2017
into the metabolism of the ameba. Each one of
this diverse group of tracers was cleared from the
m e d i u m at a similar rate in a temperaturedependent process. The uptakes do not show
saturation kinetics as would be expected if they
depended upon an active transport process
These data, instead, are consistent with a mechanism of bulk transport that appears to be nonconcentrative in nature. Electron microscope data
support this conclusion and show that the tracer
molecule, horseradish peroxidase, goes into
cells by way of surface invaginations and vesiculations, with no evidence of general surface
binding.
T h e vesicular uptake, or pinocytosis, was
measured in most cases in the regular culture
medium for the amebas U n d e r these conditions
pinocytosis appears to be continuous and the
rate is reproducible from experiment to experiment In a m e d m m containing only salts, there
is a decrease in the rate of uptake, but this may
well be a nonspecific effect such as failure to adjust to changed osmotic conditions, or lack of a
readily available energy source
Pinocytosis
then results in a small but steady accumulation of
material from the exmrnal medium. Using the
values for uptake obtained in this study, the uptake of organic material from the culture medium
by pinocytosis is calculated to be about 60/~g/106
cells per hr This is equivaIent to approximately
7 % of the cellular dry weight (3) per hour and all
the cell weight in about 15 hr. Fluid ingestion is
equivalent to about 21% of a mean cell volume
in an hour. Pinocytotic uptake does not impose
an excessive water load on the ameba since it
possesses a contractile vacuole for water excretmn Pinocvtosls is undoubtedly" the mechanism
by which the cetI normally ingests the culture
medium and is the membrane property that
allows ttus ameba, whmh is highly specialized
for phagocytosis (29), to be cultured axenicalty
on soluble culture medium The slow doubling
time of Acanthamoeba (20-40 hr) is consistent with
this relatively inefficient mechanism of bulk transport.
Pinocytosis has been most extensively studied in
those ceil types that can be obtained in homogeneous populations amebas, polymorphonuclear
leucocytes, macrophages, and cultured m a m malian cells The results obtained with Acantharnoeba differ in several respects from those obtained with other cell types
Some of the earliest and most extensive in-
Published June 1, 1972
'I'A BH.; II
Rales of Pinocytosi.~ in Acanthamoeba and Other Cells
Cell type
Tracer (cone.)
Nh}leculcs/
cell/hr
nl/1 {1~ cells/hr
1600 240{){
Reference
Acanthamoeba
Albumin-lall ({}.4t:~
Inulin-an
t; M 107*
F,hrlich ascites
Albumin-lall (0.5~i
{~ X 104
Ryser, 1963 (19)
Sarcoma 18{1
A l b u m i n - t a q ({}.4'/~,)
I; X 104
Ryser, 1963 (19)
Chinese hamster
cell line (Ale V I I I )
Chondroitin
Sulfatc-aaS
48114
Saito and
1971 (22)
Guinea pig PMN
Inulin-14(1
21 31
Bcrger and K a r n o v sky, 1966 (1)
Uzman,
Abbreviation: PMN, p o l y m o r p h o n u c l e a r leucocytes.
* Value from Fig. 3.
{ Range observed with albumin and inulin.
and indeed its flmction is not well understood
Tile electron microscope images show that a
major p a t h w a y in pinocytotic uptake is by quite
small vesiculations. Some images suggest that
these vesiculations occur directly at the surface
(Fig. 8 a), others, t h a t they m a y form from tim
breakup of surface channels or larger vesiculations
(Fig. 8 b). Perhaps both occur. In any event the
images suggest a high surface to volume ratio in
the pinocytotic uptake event. Tim largest vesicular
profiles with pinocytotic content were found
infrequently a n d we are u n c e r t a i n as to their
significance in the uptake process. Even t h o u g h
they are not numerous in the small sample obtained with the electron microscope, they could
account for the largest part of the volume of
uptake. In spite of these uncertainties, it is possible to make a reasonable estimate of the rate of
internalization of surface m e m b r a n e due to
pinocytosis in Acanthamoeba, using the q u a n t i t a tive data on fluid uptake and knowledge of the
range of vesicle sizes involved in t h a t uptake. We
have previously (3) obtained an estimate of a b o u t
2200 # m 2 for the surface area of the average
ameba.
A m i n i m u m value for surface t u r n o v e r is obtained by assuming t h a t the entire uptake occurs
by spherical vesicles of a d i a m e t e r equivalent to
the largest observed ( ~ 2 # m ) . This assumption
results in a value for turnover of a b o u t 2.7 times
an hour. A t the other extreme, if it is assumed
692
that the 120 n m d i a m e t e r vesicles account for
the bulk of tile uptake, then the rate of t u r n o v e r
increases to a b o u t 4{) times an hour. It is p r o b a b l e
that the initial pinosomes are not u n i l b r m in size,
but with the present data we have no accurate
way of assessing their relative contributions.
Nevertheless, a t u r n o v e r rate of 2 10 times an
h o u r seems a reasonable estimate, of the true value.
T h e postulated high rate of smfacc t u r n o v e r is
consistent with one interpretation of the experiments by Ulsamer, Smith, a n d K o r n (26) conccrning the cfl'ect of phagocytosis on m e m b r a n e
synthesis in Acanthamoeba. A m e b a s were i n c u b a t e d
u n d e r conditions in which they took up two to
six times their surface in a b o u t 30 min by phagocytosis, a n d the rate of incorporation of radioactive precursors into cell phospholipids was
c o m p a r e d with the rate in nonphagocytosing
cells. W h e t h e r measured as incorporation into
whole cell lipids (26) or into lipids of isolated
plasma m e m b r a n e s (A. G. Ulsamer a n d E. I).
Korn, personal communication), the rate was not
appreciably affected by phagocytosis. This would
be tile expected result if pinocytosis in n o n p h a g o cytosing cells results in such a high surface turnover rate that it would not be appreciably different
in cells forced to phagocytose. Studies on tile
effect of phagocytosis on pinocytosis (Bowers,
unpublished) show a proportional decrease in
pinocytosis with increasing amounts of phagocytosis, so t h a t tim two events are not additive
with respect to surface turnover.
THE JOURNAL OF CELL BIOLOGY " VOLUME 58, 197~
Downloaded from on June 16, 2017
(20).
Published June 1, 1972
We are grateful to Dr. Edward D. Korn for his
counsel throughout this study, and for a helpful
reading of the manuscript.
Reeewed for publieatwn I December 1971, and in revzsed
form 7 Februmy 1972.
REFERENCES
6.
7.
8.
9.
10.
11.
12.
13.
14
15
t6.
17.
1. BERCER, R. R , and hi. L KARNOVSKY. 1966.
Biochemical basis of phagocymsis. V Effect of
phagocytosis on cellular uptake of extracellular
fluid and on the intraeellular pool of L-c~glycerophosphate. Fed. Proc. 25:840.
2. BOWERS, B., and E. D. Kogvr. 1968. The fine
structure of Acanthamoeba castellam~. I. The
trophozoite. J. Cell Bwl. 39:95.
3. BowERs, B., and E D. ]s2oR~. 1969. The fine
structure of Aeanthamoeba castellam~ (Neff
strain) II Encystment. J. Cell Bwl. 41:786.
4. BRANDr, P. W., and G. D. PAPPAS. 1960. An
Electron microscopic study of plnocytosis in
ameba. I. The surface attachment phase. J.
Biophys. Biochem Cy~ol 8:675.
5. CHAPMAN-ANDRESBN, C 1962. Studies on pino-
18.
19.
20.
21.
22.
cytosis in amoebae. C. R. Tray. Lab. Carlsbe~g.
33:73.
CHAt'MAI'~-AI'~DRES~,C 1965. The induction of
pinocytosis in amoebae. Arch Bml. 76:189.
CHLAPOWSKI, F. J., and X. N. BAND 1971. Assembly of lipids into membranes in Aeanthamoeba palestmens~s. II. The origin and fate of
glycerol-SH-labeled phospholipids of cellular
membranes. J. Cell Bwl 50:634.
COH•, Z. A. 1971. Endocytosls a n d the vacuolar
system. In Cell Membranes. Biological and
Pathological Aspects. Goetz W. Richter and
Dante G Scarpelli, editors. Williams and
Wilklns Co., Baltimore.
EAGLe, H , and K. A Pmz 1960. The utilization
of proteins by cultured h u m a n cells. J. BmL
Chem. 235:1095.
GRAHA~I, RICHARD C., and N~. J KARNOVSKY.
1966. The early stages of absorption of injected horseradish peroxidase in the proximal
tubules of mouse kidney' ultrastructural cymchemistry by a new technique. J H~stochem.
Cytochem. I4:291.
t-tOLTER, H. 1959. Pinocytosis. Int. Rev. Cytol.
8:481.
KORN, E D 1963. Fatty acids of Acanthamoeba
sp. J. Bwl. Chem. 238:3584.
LowRy, O. H , N. J. ROS~BROUOH, A. L. FARR,
and R. J. RANDALL 1951. Protein measurement with the folin phenol reagent. J. BwL
Chem. 193:265.
LUFT, J. It. 1961. Improvements in epoxy resin
embedding methods, or. Bzophys. Bwchem
Cytol. 9:409.
I~IARSHALL, J. ~I , a n d V. T. NAGHMIAS. 1965.
Cell surface and pinocytosls. J. H~stochem.
Cytochem. 13:92.
~iARSHALL, J ~I., V. N. SCHU/~AIKER,and P. W.
BRANDT. 1959. Pinocytosis in amoebae Ann.
N Y. Acad. Sez. 78:515.
PAPPAS, G. D. 1959. Electron microscope studies
on amoebae. Ann. N.Y. Acad. Sc,. 78:448
RABINOVITCH,~{., and hi. J DESTEFANO. 1971.
Phagocytosis of erythrocytes by Acanthamoeba
sp. Exp. Cell Res. 5~:275.
R'zs~R, H. J - P . 1963. The measurement of
IlSl-serum albumin uptake by tumor ceils in
tissue culture. Lab. Invest 12:1009.
RYSER, I~. J.-P 1968 Uptake of protein by
mammalian cells: an underdeveloped area.
Sczence (Washington). 159:390
RYsER, H. J - P . , J C. AuB, a n d J . C. CA~LFIELD
1962. Studies on protein uptake by isolated
tumor celIs II. Quantitadve data on the
adsorption and uptake of I131-serum albumin
by Ehrlich ascltes tumor cells, or. Cell Bwl.
1~:437.
SAITO, H., and B. G UZMA~. 1971. Uptake of
BLAIR I~OWEES AND THOMAS E. OLSZEWSKI Pinocytosis in Acanthamoeba eastellanii
593
Downloaded from on June 16, 2017
T h e rapid deletion of the cell surface suggests
t h a t it is n o t all replaced b y de novo synthesis, b u t
instead points to some recirculation of surface
m e m b r a n e components. T h e n a t u r e of the postulated recirculating u n i t is entirely u n k n o w n
a l t h o u g h Chlapowski a n d Band (7) h a v e suggested, mainiy on morphological grounds, t h a t
"collapsed vesicles" c o m m o n l y seen in the closely
related a m e b a they studied m a y be vehicles for
m e m b r a n e transfer. I n Acanthamoeba, we note t h a t
similar collapsed vesicles (Fig. 8 a) a p p e a r to be
related to pinocytotic u p t a k e b u t we h a v e no
evidence o n the possibility of reinsertion into the
m e m b r a n e as suggested b y Chlapowski a n d Band
T h e estimated rate of ingestion of surface
m e m b r a n e b y Acanthamoeba of several times a n
h o u r is h i g h in comparison w i t h estimates from
some other cells t h a t exhibit pinocytosis. For example, C o h n (8) has calculated t h a t the macrop h a g e m a y interiorize 5 0 % of its surface in 2 - 5
h r d u r i n g active pinocytosis. C h a p m a n - A n d r e s e n
(6) estimates t h a t u p to 5 0 % of the surface of
Amoeba proteus m a y b e ingested during one pinocytotic cycle (15-30 min), b u t there is a required
rest period before a n e w cycle c a n be initiated.
T h u s surface t u r n o v e r in Acanthamoeba is remarkable n o t only for its h i g h rate, b u t also for the
fact t h a t it appears to be continuous u n d e r our
culture conditions
Published June 1, 1972
23.
24.
25.
26.
chondroifin sulfate by mammalian cells in
culture. II. Kinetics of uptake and autoradiography. Exp. Cell Res 66:90.
SCHU~,tAKER,V. N. 1958. Uptake of protein from
solution by Amoeba proteus. Exp. Cell Res. I5:314.
SEAMAN, G. t~. 1961. Some aspects of phagotrophy in Tetrahymena. d. Protozool. 8:204.
STOCI~EM, W. 1966. Pinocytose und Bewegung
von Am/Sben. I. Mitteilung. Die Reaktion yon
Amoeba proteus auf versehiedene Makierungssubstanzen. Z. Zellfo~seh. Mzkrosk. Anat. 74:372
ULSAMER,A. G., F. R. SMITH, and E D. KORN.
1969. Lipids of Aeanthamoeba castellam*. Composition and effects of phagocytosis on incorporation of radioactive precursors, or. Cell
Bwl. 43:105.
27. V~TI, A , a n d V . Bocci. 1970 Minimal catabolism
of "native" l~I-albumin and 131I a-globulin
by polymorphonuclear leucocytes. Exp. Cell
Res. 61:206.
28. WEISMAN,R. A., and E. D. KoR~ 1966. Uptake
of fatty acids by Aeanthamoeba. Bzoch~m. Bzophys. Acta. 116:229.
29. ~EIS~AN, R. A , and E D KORN. 1967. Phagocytosis of latex beads by Aeanthamoeba. I.
Biochemical properties. Bzochemzstry. 6:485.
30. WOHLFART~I-BOTTERMAN, K E , and W. Z.
STOCI~M. 1966. Pinocytose und Bewegung
yon Am6ben II. Mitteilung. Permanente und
induzierte Pmoeytose bei Amoeba proteus. Z
Zellforsch. Mzkrosk. Anat 73:444.
Downloaded from on June 16, 2017
694
T ~ E JOURNAL OF CELL BIOLOGY " VOLUME ~
197~