J. Cell Sd. 83, 119-133 (1986)
119
Printed in Great Britain © The Company of Biologists Limited 1986
CHARACTERIZATION OF EARLY COMPARTMENTS
IN FLUID PHASE PINOCYTOSIS: A CELL
FRACTIONATION STUDY
KIMBERLY A. CASEY, KAREN M. MAUREY AND BRIAN STORRIE*
Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State
University, Blacksburg, VA 24061, USA
SUMMARY
Flotation through a 5-6% Percoll gradient of pinosomes from Chinese hamster ovary (CHO)
cells labelled during a lOmin internalization period with horseradish peroxidase (HRP), a solute,
revealed two pinosomal populations, the expected low-buoyancy population and an unexpected
buoyant population. The buoyant pinosomes that sedimented similarly to plasma membrane were
not an artifact of HRP trapping during homogenization or of cell surface-adherent HRP. No
trapping or cell surface adherence of HRP could be detected by biochemical or cytochemical
assays, even after internalization periods as short as 15 s to 1 min. With short uptake times, the
buoyant pinosome population was the major HRP positive vesicle population, suggesting a
precursor-to-product relationship between the two populations. In pulse-chase experiments, the
buoyant pinosome population was shown to be highly exocytic and the precursor to later
pinosomes. By electron-microscope cytochemistry, rapidly labelled, HRP positive pinosomes (15 s
to 1 min uptake) were typically smooth vesicles with a median diameter of =0 - 30/im and a size
range from =(H0/Um to greater than 1-0/lm in diameter. We suggest that these rapidly labelled
structures are a very early stage in the intracellular processing of pinocytic vesicles.
INTRODUCTION
In recent years, the role of the prelysosomal compartment(s) as a molecular sieve
or filter in endocytic transport has become a key to the understanding of endocytosis.
The prelysosomal compartment(s) has been described by a kinetic approach to
ligand uptake, combined with either microscopy or cell fractionation (for reviews,
see Anderson & Kaplan, 1983; Helenius et al. 1983; Pastan & Willingham, 1985;
Steinman et al. 1983). Prelysosomal vesicles are acid phosphatase negative, large
(^O'Z to 1 fim) in comparison with coated pits and vesicles, and heterogeneous
in appearance (round to tubular). In polarized cells, two classes of prelysosomal
vesicles, peripheral and Golgi-lysosome associated, can be distinguished by location. Collectively, these vesicles have been referred to as pinosomes, endosomes,
intermediate vacuoles, CURL (compartment of uncoupling of receptor and ligand)
or receptosomes. Physiologically, the prelysosomal compartment(s) is the site of
many important processes, including ligand and solute transport, ligand—receptor
dissociation, viral entry, receptor/membrane recycling and membrane-specific
fusion events.
•Author for correspondence.
Key words: pinocytosis, horseradish peroxidase,fibroblasts,endosomes.
120
K. A. Casey, K. M. Maurey and B. Storrie
The heterogeneity of the prelysosomal compartment(s) apparent in many cell
types is probably the consequence of an underlying biochemical separation of
different steps in the endocytic transport pathway. By cell fractionation in commonly
used Percoll gradients, endocytic tracers such as horseradish peroxidase (HRP),
epidermal growth factor, /3-hexosaminidase and low-density lipoprotein are found
first in low-density endosomes/pinosomes and later in higher-density endosomes
(Merion & Sly, 1983; Storrie et al. 1984). Kinetic experiments indicate that the
exocytosis of newly internalized ligand back into the medium occurs early and
hence must occur predominantly from low-density endosomes (Adams et al. 1982;
Besterman et al. 1981; Daukas et al. 1983). Kinetic and inhibitor experiments
indicate that vesicle acidification and accompanying ligand-receptor dissociation
also occur fairly early (Wolkoff et al. 1984). Hence, these biochemical steps should
also occur in low to moderate-density endocytic vesicles. Other steps such as the
segregation of ligand from receptor have been reported to occur later (Wolkoff et al.
1984) and hence should occur in denser endocytic vesicles.
Characterization of individual endocytic compartments by cell fractionation
should make an important contribution to the biochemical dissection of endocytic
pathway(s). In previous work from this (Pool et al. 1983; Storrie et al. 1984) and
other laboratories (Merion & Sly, 1983; Sahagian & Neufeld, 1983), in which the
shortest internalization period was 2min, the earliest endosome identified by cell
fractionation was a vesicle population of distinctly higher density than plasma
membrane, the ultimate origin of any early endosome population. In the present
work, using recently developed cell fractionation procedures (Sahagian & Neufeld,
1983) and uptake periods shortened to as brief as 15 s, we have investigated the
nature of rapidly labelled, early prelysosomal endocytic compartments in fibroblasts.
The chief and unexpected outcome has been to reveal the existence of a buoyant,
very early pinosome population that appears to be a precursor to later pinosome
populations. This work using HRP, an enzyme internalized by fluid-phase pinocytosis in fibroblasts (Adams et al. 1982; Storrie et al. 1984), leads to the conclusion
that early pinosomes fractionate very similarly to plasma membrane.
MATERIALS AND METHODS
Cell culture
Chinese hamster ovary (CHO-S(C2)) cells were grown in suspension culture in Eagle's minimal
essential medium, alpha modification without ribonucleosides (aMEM), supplemented with 10%
heat-inactivated, foetal calf serum (FC10) as described (Pool et al. 1983). Cell number was
quantified with a haemacytometer.
HRP uptake and chase conditions
CHO cells at a final concentration of SXlO5 to 8xlO 5 cells ml" 1 were washed once in aMEM at
24°C and then resuspended at a concentration of 3xlO 7 cells ml" 1 in 37°C a-MEM/FC10. After a
20min preincubation in a shaking water-bath, HRP (type II, Sigma Chemical Co., St Louis, MO)
dissolved in adVIEM/FCIO and brought to 37°C was added to a final concentration of 2mgml~',
unless otherwise stated. Cell viability at the time of HRP uptake was >97 %. After a 15 s to 10 min
Rapidly labelled pinocytic compartments
121
incubation at 37°C, internalization was terminated by pouring the culture onto 0-4 vol. of crushed,
frozen saline (Adams et al. 1982). Cells were washed four times in 4°C NKM/FC10 (NKM is
0-13M-NaCl, SmM-KCl, 5 mM-MgC^; supplemented with 10% foetal calf serum) as described
(Adams et al. 1982). A new centrifuge tube was used for each wash. For chases cells were
resuspended in complete 37°C culture media and incubated for various times. Chases were
terminated by pouring the culture onto crushed, frozen saline. After a chase cells were washed once
in 4°C PBSA (0-37M-NaCl, 2-7mM-KCl, 8-1 mM-Na2PO4, I S mM-KH 2 PO 4 ).
Cell fractionation
Washed cells (Pooled al. 1983) were resuspended at a concentration of =5 XlO7 cells ml" 1 in 1 ml
of 4°C 0-25 M-sucrose, subjected to a pressure of 30 lbf in~ z (1 lbf in~ 2 = 6-9kPa) for 15min with
N 2 and after decompression further disrupted by four gentle strokes of a Potter-Elvehjem homogenizer. Cell breakage was = 5 0 % . Total postnuclear supernatants were prepared and fractionated
by sedimentation in 10 % Percoll gradients (Pool et al. 1983; Storrie et al. 1984) or by flotation in
5'6% Percoll gradients (Sahagian & Neufeld, 1983). For flotation experiments, total postnuclear
supernatants, = 1 ml in 0-25 M-sucrose, were mixed with 4°C 2-2 M-sucrose to give a final sucrose
concentration of 1-85M, overlayered with 4°C 5-6% Percoll in 0-25 M-sucrose, and centrifuged at
14600gaV at 3°C in a DuPont Sorvall SV288 rotor, 25-4mmX89mm tubes (DuPont InstrumentsSorvall Biomedtcal Div., DuPont Co., Wilmington, DE), for 70min. Gradient fractions were
collected by displacement. To determine the density distribution of the gradients, refractive index
readings and direct weighings of 4°C gradient fractions were calibrated against density marker
beads (Pharmacia Fine Chemicals AB, Uppsala, Sweden).
Enzyme assays
HRP, alkaline phosphodiesterase I (plasma membrane marker) and /3-hexosaminidase (lysosomal marker) were assayed as described previously (Pool et al. 1983). Recoveries of enzyme
activities in gradient experiments typically ranged from 80-120%. To determine biochemically
whether HRP was sequestered within cells, peroxidase activity was assayed, before sample storage,
in the absence or presence of 0-1 % Triton X-100 in a buffer consisting of 0-1 M-imidazole (pH7-0)
in 0-25 M-sucrose. CHO cells possess no detectable peroxidase activity. Therefore, intracellular
HRP can be estimated, at a minimum, as latent HRP activity (i.e. HRP activity assayed in the
presence of detergent minus that assayed in the absence of detergent). For cell fractions, organellesequestered HRP can similarly be estimated as latent enzyme activity.
HRP cytochemistry
For cytochemical localization of HRP activity after cell fractionation, gradient fractions were
pooled on the basis of marker enzyme activity and combined with an equal volume of 4°C 5-0%
glutaraldehyde in 0-2M-cacodylate buffer (pH 7-4). After a 15 min fixation, 4°C 0-25 M-sucrose was
added to give a total 10-fold dilution in Percoll concentration. Organelles were then pelleted at
25 2 6 0 ^ for 20 min in a DuPont Sorvall SS-34 rotor. Pellets were resuspended in 10 ml of 4°C
0-25 M-sucrose and washed by centrifugation at 105 000g^ for 1 h to remove residual Percoll. The
pellets were then rinsed three times with 4°C cacodylate buffer, processed for HRP cytochemistry
using diaminobenzidine (DAB) as substrate, and embedded in Epon as previously described
(Storrie et al. 1984).
For localization of HRP activity in intact cells, cells incubated with HRP were washed, fixed, and
processed for HRP cytochemistry using DAB as substrate (Storrie et al. 1984). Sections were
observed with a Zeiss EMI OCA electron microscope at an accelerating voltage of 60 kV.
Morphometry
Vesicle diameters were typically scored from micrographs printed to a final magnification of
between 15 000 and 30 000. The diameter of elongate vesicles was scored as the average of the A" and
Yaxes of an ellipse.
122
K. A. Casey, K. M. Maurey and B. Storrie
RESULTS
Pinosome contents were labelled by incubating CHO cells with HRP for 15 s to
lOmin at 37°C. HRP is ingested by CHO cells through fluid-phase pinocytosis
(Adams et al. 1982); HRP uptake is non-saturable (Adams et al. 1982) and unaffected by yeast mannan (Adams et al. 1982) or by periodate oxidation of the
HRP to open sugar rings (Sullivan & Storrie, unpublished data). Hence HRP
should be present in all pinocytic vesicles, irrespective of the absence or presence of
specific receptors. After various uptake times, CHO cells were either processed for
HRP cytochemistry or homogenized and fractionated by flotation in 5 - 6% Percoll
gradients or by sedimentation in 10% Percoll gradients. In 10% Percoll gradients,
which maximize the separation between pinosomes/endosomes and lysosomes,
HRP internalized by CHO cells during 2-5-10min incubations is present in a
pinosome population that is separated partially from the plasma membrane and
completely from lysosomes (Pool et al. 1983; Storrie et al. 1984). In 5-6% Percoll
gradients, which maximize the separation between plasma membrane and lysosomes,
/3-galactosidase, a ligand internalized by CHO cells by receptor-mediated endocytosis, is present after a 2 min uptake in an endosome/receptosome population that
is separated completely from the plasma membrane and overlaps extensively with
lysosomes (Sahagian & Neufeld, 1983).
Identification of multiple early pinosomal compartments
In the initial experiments, CHO cells were incubated with HRP for a standard
10min uptake period at 37°C and then rapidly chilled to 4°C to stop further
intracellular processing of the labelled pinocytic vesicles (pinosomes). Total postnuclear supernatants were prepared and then fractionated in Percoll gradients (Pool
et al. 1983; Sahagian & Neufeld, 1983; Storrie et al. 1984). In agreement with
previous results (Pool et al. 1983), HRP positive pinosomes behaved as a single
vesicle population after sedimentation in a 10% Percoll gradient (Fig. 1). However,
as shown in Fig. IB, two distinct populations of HRP positive pinosomes were
observed after flotation in a 5 - 6% Percoll gradient. The denser population was
displaced slightly to the light side of the lysosome distribution (/3-hexosaminidase
marker) and exhibited a distribution similar to that of the early endosome population
recently described (Sahagian & Neufeld, 1983) for receptor-mediated endocytosis.
The buoyant, low-density pinosome population (rho = l-037gcm~3) was similar in
distribution to the plasma membrane marker, alkaline phosphodiesterase I.
A series of control experiments was done to test whether the buoyant, plasma
membrane-like, pinosome population might be an artifact of HRP trapping or
membrane adherence as the plasma membrane vesiculated at homogenization. For
cells incubated with HRP for 10 min at 4°C, no HRP positive vesicles were detected
in cell fractionation experiments, indicating that trapping of exogenous HRP did not
occur at homogenization. The inclusion of 10mM-(ethylenedinitrilo)-tetraacetic acid
(EDTA) in the 5-6% Percoll gradient, a treatment that should strip many loosely
adherent proteins from membranes, had no effect on the detection of buoyant pinosomes. The latency of HRP activity and /3-hexosaminidase activity, the lysosomal
Rapidly labelled pinocytic compartments
123
160
24 -
-
100
o
X
-
7
B
min
e
40
D
i
_c
E
j.
OS
< -
0
|
t/1
cd
100
"H
c "
o
ish
SOX
i
u
3C
•a
60
2
u
o
X
20
10
15
20
25
Fraction number
30
35
0
-"0
Fig. 1. Distribution of HRP internalized during a lOmin uptake in a 5-6% (B) or 10%
(C) Percoll gradient. Cells were exposed to 1 mgml" 1 HRP for lOmin in a-MEM/FCIO
and poured onto 0-4 vol. of crushed frozen saline to stop uptake. The total postnuclear
supernatant was divided into two equal portions. One portion was mixed with dense
sucrose and overlaid with 5-6 % Percoll. The other portion was overlaid on 10 % Percoll.
Following centrifugation, fractions were collected and analysed for HRP ( •
• ) , /?hexosaminidase (lysosomal marker, O
O), and alkaline phosphodiesterase I (plasma
membrane marker, A
A) activities. In A density values ( A , 5-6% Percoll; D, 10%
Percoll) were determined by density beads, measurement of refractive index and direct
weighing of fractions from parallel gradients. RFU, relative fluorescence units; A, absorbance.
marker, in the postnuclear supernatant were found to be equivalent (~85 % versus
=80%), indicating that the vast majority of each activity was located inside a
membrane-limited organelle. HRP activity in the buoyant pinosome population was
latent, again consistent with a membrane-delimited location of the activity. No cell
surface HRP activity could be observed by electron-microscope cytochemistry, in
agreement with previous work (Adams et al. 1982; Steinman et al. 1974, 1976). On
124
K. A. Casey, K. M. Maurey and B. Storrie
the basis of these controls and the evidence for fluid-phase pinocytosis of HRP, we
suggest that the buoyant pinosomal population is a very early pinosome population
and not an artifact of HRP trapping or adherence to the cell surface.
Relationship between pinosome populations
The resolution of multiple pinosome populations after an HRP pulse suggests
either a precursor-to-product relationship between the two vesicle populations or
multiple independent pathways for solute uptake. To test for a precursor-to-product
relationship between the two pinosome populations, CHO cells were pulsed with
HRP for decreasing time periods (3min to 15 s) and total postnuclear supernatants
were fractionated in 5 - 6% Percoll gradients. If buoyant pinosomes are the precursor
to later, low-buoyancy pinosomes, then with shorter peroxidase uptake times an
increasing portion of HRP activity should be associated with the upper pinosome
population. As shown in Fig. 2, when the HRP pulse was shortened to 1 min or 15 s,
the buoyant population became the predominant peroxidase-positive compartment.
The HRP activity (1 min pulse) associated with this apparent precursor population
was latent, as expected for peroxidase sequestered within an early pinocytic vesicle
(Fig. 3). The HRP activity that peaked in the bottom portion of the gradient was not
latent (compare Fig. 2B and Fig. 3). Much of this non-latent HRP activity may be
due either to residual exogenous enzyme left after the cell wash steps or to organelle
breakage during cell homogenization.
To test directly for the intracellular localization of HRP after a 1 min uptake,
freshly pulsed cells were assayed for HRP activity in the absence or presence of
detergent. For CHO cells, which have no endogenous peroxidase activity, detergent
disruption of the cell would be required if the majority of enzyme activity after the
1 min, 37°C, internalization period is intracellular. In contrast, if most enzyme
activity consists of HRP that has bound to the cell surface during the 37°C
incubation, then little difference in enzyme activity should be observed after
detergent treatment. For CHO cells incubated with HRP for 1 min at 37°C,
measurement of the vast majority (80 %) of HRP activity required detergent
disruption of the cell, indicating that the enzyme was sequestered within the cell.
By electron-microscope cytochemistry, HRP after a 1 min pulse was located in
intracellular vesicles and no cell surface HRP could be detected (see below).
If buoyant pinosomes are indeed an early precursor population, then in pulsechase experiments HRP ought to chase from light to dense pinosomes. This chase,
however, would not be expected to be quantitative. Earlier work has shown that early
pinosomes/endosomes are exocytic (Adams et al. 1982; Besterman et al. 1981;
Daukas et al. 1983). Hence, even though a precursor-to-product relationship may
exist between the populations, only a fraction of the HRP activity present in buoyant
pinosomes would be expected to be transferred into later pinosomes/endosomes. To
provide evidence for these points, CHO cells were incubated with HRP for 1 min at
37°C, washed, and after various chase times in warm HRP-free media total postnuclear supernatants were prepared. As shown above, after a 1 min HRP pulse
primary pinosomes constituted the major population of peroxidase positive vesicles
Rapidly labelled pinocytic compartments
125
(Fig. 4A). After a 3 min chase period, only a small percentage of the total peroxidase
activity was associated with buoyant pinosomes. This rapid decrease in peroxidase
activity was accompanied by the accumulation of latent HRP activity in the lower
portion of the gradient (Fig. 4D). As shown above, the HRP activity present in
the lower portion of the 5-6% Percoll gradient after a brief pulse was not latent.
During this chase period significant loss of HRP activity from the cell was observed
250
200
150
100
50
o x
E - 300 7_"
E
i
c
1
u.
at
<
200
100
<4
200 12
o
X
150
100
50i
10
15
20
25
Fraction number
35
-'O
Fig. 2. Distribution of HRP activity after short pulses (3min, 1 min or 15s) in 5-6%
Percoll gradients. Cells were exposed to Zmgml" 1 HRP for 3 min (A) or 1 min (B) or to
4 m g m r ' HRP for 15 s (C) in a-MEM/FClO and poured onto 0-4 vol. of crushed frozen
saline to stop uptake. Total postnuclear supernatants were mixed with dense sucrose and
overlaid with 5-6% Percoll. Following centrifugation, fractions were collected and
analysed for HRP ( #
• ) , /3-hexosaminidase (O——O), and alkaline phosphodiesterase I (A
A) activities.
K. A. Casey, K. M. Maurey and B. Storrie
126
1 'c
10
15
20
Fraction number
25
30
35
Fig. 3. Distribution of latent HRP activity after a 1 min pulse in a 5-6 % Percoll gradient.
Cells were exposed to Zmgml" 1 HRP for 1 min and poured onto 0-4vol. of crushed
frozen saline to stop uptake. The total postnuclear supernatant was mixed with dense
sucrose and overlaid with 5-6% Percoll. Following centrifugation, fractions were collected and assayed immediately for HRP activity in the absence and presence of
detergent. The distribution of latent activity (activity in the presence of detergent minus
activity in the absence of detergent) is shown. Overall HRP activity in the gradient
fractions was 75 % latent.
(Table 1). These results suggest both that a precursor-to-product relationship does
exist between the pinosome populations and that rapidly labelled or early pinosomes
are highly exocytic compartments, as expected from previous studies (e.g. see Adams
etal. 1982).
Morphological appearance of rapidly labelled, early pinosomes
To assess the morphology of the buoyant, early pinosome population after fractionation in Percoll gradients, CHO cells were pulsed with HRP for 10 min,
fractionated by centrifugation in a 5-6% Percoll gradient and the upper fractions
were pooled according to the distribution of HRP activity. HRP activity in the
rapidly labelled, early pinosome fraction was localized in round vesicles with the
diaminobenzidine reaction product generally found around the periphery of the
vesicles and facing inward (Fig. 5, arrows). The peroxidase positive vesicles were
typically devoid of any clathrin coat. Within most early pinosomes, smaller vesicles
rimmed with outward-facing reaction product were also observed. Rapidly labelled,
Fig. 4. Distribution of pinocytized HRP activity in 5-6% Percoll gradients after a
pulse (lmin) or a 1-3 min chase. Cells were exposed to 2mgml~' HRP for 1 min
in arMEM/FCIO, poured onto 0-4 vol. of crushed frozen saline, washed and chased in
<*MEM/FC10. Total postnuclear supernatants were mixed with dense sucrose and
overlaid with 5-6% Percoll. Following centrifugation, fractions were collected and
analysed for HRP ( •
• ) , /3-hexosaminidase (O
O), and alkaline phosphodiesterase I (A
A) activities. HRP activity in the bottom portion of the gradient was
non-latent at the end of the pulse and was latent after the chase.
120
90
60
30
0
35
20
2
x
10 _c
I
c
1
25
20 £
o
X
10
0
40
30
20
10 h
10
10
15
20
Fraction number
Fig. 4
25
30
128
K. A. Casey, K. M. Maurey and B. Storrie
Table 1. Effect of chase time on cell-associated HRP activity
Cell-associated HRP activity
Chase time
(min)
Expt 1
(%)
Expt 2
(%)
0
1
2
3
100
48
39
100
67
44
37
45
Cells were incubated with 2mgml ' HRP for 1 min at 37°C, poured onto 0-4vol. of crushed
frozen saline to stop uptake and washed extensively at 4°C. Cells were then chased for various times
at 37°C in complete culture media. The data were derived by correcting the recovery of HRP
activity in postnuclear supernatants for the efficiency of cell homogenization based on the yield
of plasma membrane (alkaline phosphodiesterase I) and lysosomal (j8-hexosaminidase) marker
enzyme activities. The HRP uptake period = 1 min.
early pinosomes were often 0-2—0-3//m in diameter and ranged in size up to about
1 ;Um in diameter. Also present in the early pinosome fraction were various smooth
and rough membranous organelles.
To determine the morphology of rapidly labelled, early pinosomes in situ, CHO
cells were pulsed with peroxidase for between 15 s to 1 min, fixed and HRP activity
r\
<~ x:-*Q
Fig. S. Morphological characterization of rapidly labelled, early pinosomes in Percoll
gradient fractions. Cells were exposed to 2 m g m r ' HRP for lOmin and poured onto
0-4 vol. of crushed frozen saline to stop uptake. The total postnuclear supernatant was
mixed with dense sucrose and overlaid with 5-6% Percoll. The early pinosome-enriched
fractions were pooled on the basis of HRP activity and processed for peroxidase cytochemistry. Black deposits indicate sites of HRP activity (arrows). X22950.
Fig. 6. Morphological characterization of rapidly labelled, early pinosomes in situ. Cells
were exposed to 2 m g m r ' HRP for 15s (A), 30s (B,C) or 60s (D) in tfMEM/FC10,
poured onto 0-4vol. of crushed frozen saline, washed and processed for peroxidase
cytochemistry. Black deposits indicate sites of HRP activity. In B, the arrowhead points
to a pinosome-associated microfilament cluster. In C, the arrowhead points to a microtubule cluster, and the arrow points to a coated pit. A, X28125;B, x59700;C, X28650;
D, X28125.
130
K. A. Casey, K. M. Maurey and B. Storrie
was localized by the DAB procedure. By electron-microscope cytochemistry of intact
cells, HRP activity after a 15 s to 1 min pulse was found in round and elongate
vesicles with the DAB reaction product generally found around the periphery of the
vesicles (Fig. 6). No cell surface reaction product was detected. In some cases,
the reaction product was not continuous about the entire vesicle circumference.
Frequently a small vesicular inclusion surrounded on its outer surface with DAB
reaction product was observed in the peroxidase positive vesicles. The labelled
vesicles were generally 0-1-0-4 jum in diameter, irrespective of whether the uptake
period was 15 s or as long as 1 min. Typically, the diameter of the early pinosomes
was much larger than that of coated pits and the pinosomes were non-coated. For a
30 s uptake period, HRP positive vesicles ranged in size from 0-07-1-1 jUm diameter
with a median diameter of 0-30 fim. At higher magnification, a microfilament cluster
was occasionally observed in association with an early pinosome (Fig. 6B). The
functional significance of this is unknown.
The morphology of the rapidly labelled, early pinosomes both in situ and after
fractionation resembles that reported previously for endosomes/receptosomes (for
review, see Pastan & Willingham, 1985), suggesting that this rapidly labelled
pinosome population is a form of endosome/receptosome.
DISCUSSION
These experiments were done to characterize early vesicles formed during fluidphase pinocytosis in CHO cells, a cell line of fibroblastic origin. Knowledge of the
properties of early pinosomes should provide information on the initial intracellular
steps in the pinocytic pathway. Our results suggest that rapidly labelled, early
pinosomes are buoyant compartments that can be readily resolved from later pinocytic compartments by cell fractionation and imply a unique set of molecular
properties for the compartments.
In these experiments, the pinocytic solute tracer was horseradish peroxidase. For
some cell types such as macrophages (Kaplan & Nielsen, 1978; Stahl et al. 1978;
Sung et al. 1983) and absorptive cells of the neonatal ileum (Gonnella & Neutra,
1984), HRP binds to a mannose/iV-acetylglucosamine receptor. At the HRP concentrations (mg ml" 1 ) used in the present work, such a receptor, if present, would be
saturated and most peroxidase uptake would be by fluid-phase pinocytosis (Sung
et al. 1983). For CHO cells we find no evidence of cell surface binding of HRP
preparations. In the present work surface binding was not detected by either biochemical or cytochemical assays. In previous work we found HRP uptake by CHO
cells to be non-saturable and unaffected by mannose concentrations as high as
150 mM (Adams et al. 1982; Storrie et al. 1984). Recently completed work (Sullivan
& Storrie, unpublished data) indicates that neither, 19mgml~' yeast mannan nor
periodate oxidation of the enzyme to open the ring structure of the sugar side-chains
has any significant effect on HRP uptake by CHO cells.
The identification of rapidly labelled pinosomes as unique pinocytic compartments rests chiefly on cell fractionation data combined with a series of control
Rapidly labelled pinocytic compartments
131
experiments. The first indication of the compartment was the unexpected outcome of
experiments using flotation through a 5-6 % Percoll gradient introduced by Sahagian
& Neufeld (1983) for the separation of endosomes involved in receptor-mediated
endocytosis from plasma membrane. In their work, also with CHO cells, a single
low-buoyancy endosome population that overlapped extensively with lysosomes was
observed after a 2 min period of /3-galactosidase uptake. With this gradient, for CHO
cells pulsed with HRP for 10min, we observed two pinosome populations: the
expected low-buoyancy population and an unexpected buoyant population. The
buoyant population sedimented very similarly to plasma membrane and might have
been an artifact of HRP trapping during homogenization or cell surface adherence of
HRP. However, in control experiments no trapping or cell-surface-adherent HRP
could be detected. The cell-associated HRP was found to be intracellular by direct
biochemical assay, and in the cell fractions it was latent and resistant to removal by
treatment with EDTA.
Because of their similarity in fractionation properties to plasma membrane, the
membrane origin of endocytic vesicles, we postulated that buoyant pinosomes were
early pinocytic compartments. This proved to be the case. In a series of pulseuptake experiments, the buoyant pinosome population was the major rapidly labelled
pinosome population after a 15-60s uptake and in pulse-chase experiments HRP
was rapidly chased from buoyant to low-buoyancy pinosomes. As expected for an
early endocytic compartment, buoyant pinosomes appeared to be a site of reversible
pinocytosis (diacytosis).
The rapidly labelled, early pinosomes were not identified in previous work because
of the gradient systems and uptake protocols used. Sedimentation through 10 % and
20% Percoll gradients, as done earlier in this laboratory (Pool et al. 1983; Storrie
et al. 1984), is incapable of resolving very early pinosomes from other pinosome
populations. The resolution achieved in the 5-6% Percoll gradient is either a
consequence of the very flat density gradient generated or the use of a flotation
versus a sedimentation protocol. Recent work indicates that osmotic contraction in
dense sucrose is not necessary for the separation. Similar resolution occurs with
resuspension of organelles in dense Percoll prepared in 0-25M-sucrose (Wirt &
Storrie, unpublished observations). The preferential labelling of early pinosomes
requires a very brief uptake protocol. With most uptake protocols, early pinosomes
would be only a small portion of the labelled pinosome population and hence would
be easy to miss whatever the resolution of the gradient.
As a fluid-phase marker, HRP should be included in any newly formed endocytic
vesicle, irrespective of the pathway by which the vesicle originated. Marsh &
Helenius (1980), Helenius & Marsh (1982), Ryser et al. (1982), and Pastan &
Willingham (1985) have all suggested that the major site of both fluid-phase and
receptor-mediated endocytosis in fibroblasts is the coated pit. The typically smooth
appearance and large size (mean diameter =0'30^m with vesicles as large as 1 [im
or more) of HRP positive early pinosomes relative to coated pits after even brief
uptake periods (15 s to 1 min) indicates that the kinetics of the precursor-to-product
relationship between these structures must be very rapid. Recent work (Pastan &
132
K. A. Casey, K. M. Maurey and B. Storrie
Willingham, 1985) indicates that the entire endocytic process from open coated pit to
separate endocytic vesicle occurs within a few seconds.
Understanding the exact relationship between prelysosomal endocytic compartments (see also, Besterman et al. 1981; Geuze et al. 1983; Merion & Sly, 1983;
Townsend et al. 1984; Wolkoff et al. 1984) for animal cells will require a more
precise definition of the transport properties, physical properties and composition
of the individual compartments. This goal will be achieved through the direct
characterization of isolated vesicles. Without such information, no clear interpretation of the molecular basis for the separation of vesicle classes can be given.
The physical separation of rapidly labelled, early pinosomes from later pinosomes,
the major contribution of the present work, suggests that molecular differences exist
between these early pinosomes and other pinocytic compartments. Definition of
these differences will have to await the isolation of the individual compartments.
We thank Andrea Ferris for her excellent cell culture work and Ginny Viers for assistance with
electron microscopy. This work was supported by Public Health Service grant GM-28188.
REFERENCES
ADAMS, C. J., MAUREY, K. M. & STORRIE, B. (1982). Exocytosis of pinocytic contents by Chinese
hamster ovary cells. J. Cell Biol. 93, 632-637.
ANDERSON, R. G. W. & KAPLAN, J. (1983). Receptor-mediated endocytosis. Modern Cell Biol. 1,
1-52.
BESTERMAN, J. M., AIRHART, J. A., WOODWORTH, R. C. & Low, R. B. (1981). Exocytosis of
pinocytized fluid in cultured cells: kinetic evidence for rapid turnover and compartmentation.
J. Cell Biol. 91, 716-727.
DAUKAS, G., LAUFFENBURGER, D. A. & ZIGMOND, S. (1983). Reversible pinocytosis in
polymorphonuclear leukocytes. J. Cell Biol. 96, 1642-1650.
GEUZE, H. J., SLOT, J. W., STROUS, G. J. A. M., LODISH, H. F. & SCHWARTZ, A. L. (1983).
Intracellular site of asialoglycoprotein receptor-ligand uncoupling: double-label immunoelectron microscopy during receptor-mediated endocytosis. Cell 32, 277-287.
GONNELLA, P. A. & NEUTRA, M. R. (1984). Membrane-bound and fluid-phase macromolecules
enter separate prelysosomal compartments in absorptive cells of suckling rat ileum. J. Cell Biol.
99, 909-917.
HELENIUS, A. & MARSH, M. (1982). Endocytosis of enveloped animal viruses. Ciba Fdn Symp.
92, 59-69.
HELENIUS, A., MELLMAN, I., WALL, D. A. & HUBBARD, A. (1983). Endosomes. Trends biochem.
Sci. 8, 245-250.
KAPLAN, J. & NIELSEN, M. (1978). Pinocytic activity of rabbit alveolar macrophages in vitro.
J. reticuloendothel. Soc. 24, 673-685.
MARSH, M. & HELENIUS, A. (1980). Adsorptive endocytosis of Semliki Forest virus. J. molec. Biol.
142, 439-454.
MERION, M. & SLY, W. S. (1983). The role of intermediate vesicles in the adsorptive endocytosis
and transport of ligand to lysosomes by human fibroblasts. J. Cell Biol. 96, 644-650.
PASTAN, I. & WILLINGHAM, M. C. (1985). The pathway of endocytosis. In Endocytosis (ed. I.
Pastan & M. C. Willingham), pp. 1-44. New York, London: Plenum Press.
POOL, R. R. JR, MAUREY, K. M. & STORRIE, B. (1983). Characterization of pinocytic vesicles from
CHO cells: resolution of pinosomes from lysosomes by analytical centrifugation. Cell Biol. Int.
Rep. 7, 361-367.
RYSER, H. J., DRUMMOND, I. & SHEN, W. C. (1982). The cellular uptake of horseradish peroxidase
and its poly(lysine) conjugate by cultured fibroblasts is qualitatively similar despite a 900-fold
difference in rate. J . cell. Physiol. 113, 167-178.
Rapidly labelledpinocytic
compartments
133
SAHAGIAN, G. G. & NEUFELD, E. F. (1983). Biosynthesis and turnover of the mannose 6phosphate receptor in cultured Chinese hamster ovary cells. J. biol. Chem. 258, 7121-7128.
STAHL, P. D., RODMAN, J. S., MILLER, J. M. & SCHLESSINGER, P. H. (1978). Evidence for
receptor-mediated binding of glycoproteins, glycoconjugates, and lysosomal glycosidases by
alveolar macrophages. Proc. natn.Acad. Sci. U.SA. 75, 1399-1403.
STEINMAN, R. M., BRODIE, S. E. & COHN, Z. A. (1976). Membrane flow during pinocytosis. A
stereologic analysis. J. Cell Biol. 68, 665-687.
STEINMAN, R. M., MELLMAN, I. S., MULLER, W. A. & COHN, Z. A. (1983). Endocytosis and the
recycling of plasma membrane..?. Cell Biol. 96, 1-27.
STEINMAN, R. M., SILVER, J. M. & COHN, Z. A. (1974). Pinocytosis in fibroblasts: Quantitative
studies in vitro. J. Cell Biol. 63, 949-969.
STORRIE, B., POOL, R. R. JR, SACHDEVA, M., MAUREY, K. M. & OLIVER, C. (1984). Evidence for
both prelysosomal and lysosomal intermediates in endocytic pathways. J. Cell Biol. 98, 108-115.
SUNG, S.-S. J., NELSON, R. S. & SILVERSTEIN, S. C. (1983). The role of the mannose/N-
acetylglucosamine receptor in the pinocytosis of horseradish peroxidase by mouse peritoneal
macrophages. J . cell. Physiol. 116, 21-25.
TOWNSEND, R. R., WALL, D. A., HUBBARD, A. L. & LEE, Y. C. (1984). Rapid release of galactose-
terminated ligands after endocytosis by hepatic parenchymal cells: evidence for a role of
carbohydrate structure in the release of internalized ligand from receptor. Proc. natn. Acad. Sci.
U.SA. 81, 466-470.
WOLKOFF, A. W., KLAUSNER, R. D., ASHWELL, G. & HARFORD, J. (1984). Intracellular
segregation of asialoglycoproteins and their receptor: a prelysosomal event subsequent to
dissociation of the ligand-receptor complex. J. Cell Biol. 98, 375-381.
{Received 9 September 1985 -Accepted, in revised form, 15 January 1986)