characterization of early compartments in fluid … · cell fractionation washed cells (pooled al....

$: CHARACTERIZATION OF EARLY COMPARTMENTS IN FLUID … · Cell fractionation Washed cells (Pooled al. 1983) were resuspended at a concentration of =75 cell XlOs ml" 1 in 1 ml of 4°C$
J. Cell Sd. 83, 119-133 (1986) 119Printed 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 StateUniversity, 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 unexpectedbuoyant population. The buoyant pinosomes that sedimented similarly to plasma membrane werenot an artifact of HRP trapping during homogenization or of cell surface-adherent HRP. Notrapping or cell surface adherence of HRP could be detected by biochemical or cytochemicalassays, even after internalization periods as short as 15 s to 1 min. With short uptake times, thebuoyant pinosome population was the major HRP positive vesicle population, suggesting aprecursor-to-product relationship between the two populations. In pulse-chase experiments, thebuoyant pinosome population was shown to be highly exocytic and the precursor to laterpinosomes. By electron-microscope cytochemistry, rapidly labelled, HRP positive pinosomes (15 sto 1 min uptake) were typically smooth vesicles with a median diameter of =0-30/im and a sizerange from =(H0/Um to greater than 1-0/lm in diameter. We suggest that these rapidly labelledstructures 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 sieveor filter in endocytic transport has become a key to the understanding of endocytosis.The prelysosomal compartment(s) has been described by a kinetic approach toligand 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 heterogeneousin appearance (round to tubular). In polarized cells, two classes of prelysosomalvesicles, peripheral and Golgi-lysosome associated, can be distinguished by lo-cation. 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 ofmany important processes, including ligand and solute transport, ligand—receptordissociation, viral entry, receptor/membrane recycling and membrane-specificfusion 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 celltypes is probably the consequence of an underlying biochemical separation ofdifferent steps in the endocytic transport pathway. By cell fractionation in commonlyused Percoll gradients, endocytic tracers such as horseradish peroxidase (HRP),epidermal growth factor, /3-hexosaminidase and low-density lipoprotein are foundfirst in low-density endosomes/pinosomes and later in higher-density endosomes(Merion & Sly, 1983; Storrie et al. 1984). Kinetic experiments indicate that theexocytosis of newly internalized ligand back into the medium occurs early andhence must occur predominantly from low-density endosomes (Adams et al. 1982;Besterman et al. 1981; Daukas et al. 1983). Kinetic and inhibitor experimentsindicate that vesicle acidification and accompanying ligand-receptor dissociationalso occur fairly early (Wolkoff et al. 1984). Hence, these biochemical steps shouldalso occur in low to moderate-density endocytic vesicles. Other steps such as thesegregation 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 fractionationshould make an important contribution to the biochemical dissection of endocyticpathway(s). In previous work from this (Pool et al. 1983; Storrie et al. 1984) andother laboratories (Merion & Sly, 1983; Sahagian & Neufeld, 1983), in which theshortest internalization period was 2min, the earliest endosome identified by cellfractionation was a vesicle population of distinctly higher density than plasmamembrane, the ultimate origin of any early endosome population. In the presentwork, using recently developed cell fractionation procedures (Sahagian & Neufeld,1983) and uptake periods shortened to as brief as 15 s, we have investigated thenature 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 pinosomepopulations. This work using HRP, an enzyme internalized by fluid-phase pino-cytosis in fibroblasts (Adams et al. 1982; Storrie et al. 1984), leads to the conclusionthat early pinosomes fractionate very similarly to plasma membrane.

MATERIALS AND METHODS

Cell cultureChinese 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 wasquantified with a haemacytometer.

HRP uptake and chase conditionsCHO cells at a final concentration of SXlO5 to 8xlO5 cells ml"1 were washed once in aMEM at

24°C and then resuspended at a concentration of 3xlO7 cells ml"1 in 37°C a-MEM/FC10. After a20min 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 is0-13M-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 wereresuspended in complete 37°C culture media and incubated for various times. Chases wereterminated by pouring the culture onto crushed, frozen saline. After a chase cells were washed oncein 4°C PBSA (0-37M-NaCl, 2-7mM-KCl, 8-1 mM-Na2PO4, I S mM-KH2PO4).

Cell fractionationWashed 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 withN2 and after decompression further disrupted by four gentle strokes of a Potter-Elvehjem homo-genizer. Cell breakage was =50%. Total postnuclear supernatants were prepared and fractionatedby sedimentation in 10 % Percoll gradients (Pool et al. 1983; Storrie et al. 1984) or by flotation in5'6% Percoll gradients (Sahagian & Neufeld, 1983). For flotation experiments, total postnuclearsupernatants, =1 ml in 0-25 M-sucrose, were mixed with 4°C 2-2 M-sucrose to give a final sucroseconcentration of 1-85M, overlayered with 4°C 5-6% Percoll in 0-25 M-sucrose, and centrifuged at14600gaV at 3°C in a DuPont Sorvall SV288 rotor, 25-4mmX89mm tubes (DuPont Instruments-Sorvall Biomedtcal Div., DuPont Co., Wilmington, DE), for 70min. Gradient fractions werecollected by displacement. To determine the density distribution of the gradients, refractive indexreadings and direct weighings of 4°C gradient fractions were calibrated against density markerbeads (Pharmacia Fine Chemicals AB, Uppsala, Sweden).

Enzyme assaysHRP, alkaline phosphodiesterase I (plasma membrane marker) and /3-hexosaminidase (lyso-

somal marker) were assayed as described previously (Pool et al. 1983). Recoveries of enzymeactivities in gradient experiments typically ranged from 80-120%. To determine biochemicallywhether 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, intracellularHRP can be estimated, at a minimum, as latent HRP activity (i.e. HRP activity assayed in thepresence of detergent minus that assayed in the absence of detergent). For cell fractions, organelle-sequestered HRP can similarly be estimated as latent enzyme activity.

HRP cytochemistryFor 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 wasadded to give a total 10-fold dilution in Percoll concentration. Organelles were then pelleted at25 2 6 0 ^ for 20 min in a DuPont Sorvall SS-34 rotor. Pellets were resuspended in 10 ml of 4°C0-25 M-sucrose and washed by centrifugation at 105 000g^ for 1 h to remove residual Percoll. Thepellets were then rinsed three times with 4°C cacodylate buffer, processed for HRP cytochemistryusing 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, andprocessed for HRP cytochemistry using DAB as substrate (Storrie et al. 1984). Sections wereobserved with a Zeiss EMI OCA electron microscope at an accelerating voltage of 60 kV.

MorphometryVesicle 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" andYaxes of an ellipse.


RESULTS

Pinosome contents were labelled by incubating CHO cells with HRP for 15 s tolOmin 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 un-affected by yeast mannan (Adams et al. 1982) or by periodate oxidation of theHRP to open sugar rings (Sullivan & Storrie, unpublished data). Hence HRPshould be present in all pinocytic vesicles, irrespective of the absence or presence ofspecific receptors. After various uptake times, CHO cells were either processed forHRP cytochemistry or homogenized and fractionated by flotation in 5-6% Percollgradients 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 apinosome population that is separated partially from the plasma membrane andcompletely from lysosomes (Pool et al. 1983; Storrie et al. 1984). In 5-6% Percollgradients, which maximize the separation between plasma membrane and lysosomes,/3-galactosidase, a ligand internalized by CHO cells by receptor-mediated endo-cytosis, is present after a 2 min uptake in an endosome/receptosome population thatis separated completely from the plasma membrane and overlaps extensively withlysosomes (Sahagian & Neufeld, 1983).

Identification of multiple early pinosomal compartments

In the initial experiments, CHO cells were incubated with HRP for a standard10min uptake period at 37°C and then rapidly chilled to 4°C to stop furtherintracellular processing of the labelled pinocytic vesicles (pinosomes). Total post-nuclear supernatants were prepared and then fractionated in Percoll gradients (Poolet al. 1983; Sahagian & Neufeld, 1983; Storrie et al. 1984). In agreement withprevious results (Pool et al. 1983), HRP positive pinosomes behaved as a singlevesicle population after sedimentation in a 10% Percoll gradient (Fig. 1). However,as shown in Fig. IB, two distinct populations of HRP positive pinosomes wereobserved after flotation in a 5-6% Percoll gradient. The denser population wasdisplaced slightly to the light side of the lysosome distribution (/3-hexosaminidasemarker) and exhibited a distribution similar to that of the early endosome populationrecently described (Sahagian & Neufeld, 1983) for receptor-mediated endocytosis.The buoyant, low-density pinosome population (rho = l-037gcm~3) was similar indistribution to the plasma membrane marker, alkaline phosphodiesterase I.

A series of control experiments was done to test whether the buoyant, plasmamembrane-like, pinosome population might be an artifact of HRP trapping ormembrane adherence as the plasma membrane vesiculated at homogenization. Forcells incubated with HRP for 10 min at 4°C, no HRP positive vesicles were detectedin cell fractionation experiments, indicating that trapping of exogenous HRP did notoccur at homogenization. The inclusion of 10mM-(ethylenedinitrilo)-tetraacetic acid(EDTA) in the 5-6% Percoll gradient, a treatment that should strip many looselyadherent proteins from membranes, had no effect on the detection of buoyant pino-somes. The latency of HRP activity and /3-hexosaminidase activity, the lysosomal


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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/FCIOand poured onto 0-4 vol. of crushed frozen saline to stop uptake. The total postnuclearsupernatant was divided into two equal portions. One portion was mixed with densesucrose 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 (plasmamembrane 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 directweighing of fractions from parallel gradients. RFU, relative fluorescence units; A, ab-sorbance.

marker, in the postnuclear supernatant were found to be equivalent (~85 % versus=80%), indicating that the vast majority of each activity was located inside amembrane-limited organelle. HRP activity in the buoyant pinosome population waslatent, again consistent with a membrane-delimited location of the activity. No cellsurface HRP activity could be observed by electron-microscope cytochemistry, inagreement with previous work (Adams et al. 1982; Steinman et al. 1974, 1976). On


the basis of these controls and the evidence for fluid-phase pinocytosis of HRP, wesuggest that the buoyant pinosomal population is a very early pinosome populationand 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 suggestseither a precursor-to-product relationship between the two vesicle populations ormultiple independent pathways for solute uptake. To test for a precursor-to-productrelationship between the two pinosome populations, CHO cells were pulsed withHRP for decreasing time periods (3min to 15 s) and total postnuclear supernatantswere fractionated in 5-6% Percoll gradients. If buoyant pinosomes are the precursorto later, low-buoyancy pinosomes, then with shorter peroxidase uptake times anincreasing portion of HRP activity should be associated with the upper pinosomepopulation. 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 populationwas 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 notlatent (compare Fig. 2B and Fig. 3). Much of this non-latent HRP activity may bedue either to residual exogenous enzyme left after the cell wash steps or to organellebreakage 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 ofdetergent. For CHO cells, which have no endogenous peroxidase activity, detergentdisruption of the cell would be required if the majority of enzyme activity after the1 min, 37°C, internalization period is intracellular. In contrast, if most enzymeactivity consists of HRP that has bound to the cell surface during the 37°Cincubation, then little difference in enzyme activity should be observed afterdetergent treatment. For CHO cells incubated with HRP for 1 min at 37°C,measurement of the vast majority (80 %) of HRP activity required detergentdisruption of the cell, indicating that the enzyme was sequestered within the cell.By electron-microscope cytochemistry, HRP after a 1 min pulse was located inintracellular vesicles and no cell surface HRP could be detected (see below).

If buoyant pinosomes are indeed an early precursor population, then in pulse-chase 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 earlypinosomes/endosomes are exocytic (Adams et al. 1982; Besterman et al. 1981;Daukas et al. 1983). Hence, even though a precursor-to-product relationship mayexist between the populations, only a fraction of the HRP activity present in buoyantpinosomes would be expected to be transferred into later pinosomes/endosomes. Toprovide evidence for these points, CHO cells were incubated with HRP for 1 min at37°C, washed, and after various chase times in warm HRP-free media total post-nuclear supernatants were prepared. As shown above, after a 1 min HRP pulseprimary pinosomes constituted the major population of peroxidase positive vesicles


(Fig. 4A). After a 3 min chase period, only a small percentage of the total peroxidaseactivity was associated with buoyant pinosomes. This rapid decrease in peroxidaseactivity was accompanied by the accumulation of latent HRP activity in the lowerportion of the gradient (Fig. 4D). As shown above, the HRP activity present inthe 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

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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 to4 m g m r ' HRP for 15 s (C) in a-MEM/FClO and poured onto 0-4 vol. of crushed frozensaline to stop uptake. Total postnuclear supernatants were mixed with dense sucrose andoverlaid with 5-6% Percoll. Following centrifugation, fractions were collected andanalysed for HRP ( # • ) , /3-hexosaminidase (O——O), and alkaline phospho-diesterase I (A A) activities.


1 'c

10 15 20Fraction 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 crushedfrozen saline to stop uptake. The total postnuclear supernatant was mixed with densesucrose and overlaid with 5-6% Percoll. Following centrifugation, fractions were col-lected and assayed immediately for HRP activity in the absence and presence ofdetergent. The distribution of latent activity (activity in the presence of detergent minusactivity in the absence of detergent) is shown. Overall HRP activity in the gradientfractions was 75 % latent.

(Table 1). These results suggest both that a precursor-to-product relationship doesexist between the pinosome populations and that rapidly labelled or early pinosomesare highly exocytic compartments, as expected from previous studies (e.g. see Adamsetal. 1982).

Morphological appearance of rapidly labelled, early pinosomes

To assess the morphology of the buoyant, early pinosome population after frac-tionation in Percoll gradients, CHO cells were pulsed with HRP for 10 min,fractionated by centrifugation in a 5-6% Percoll gradient and the upper fractionswere pooled according to the distribution of HRP activity. HRP activity in therapidly labelled, early pinosome fraction was localized in round vesicles with thediaminobenzidine reaction product generally found around the periphery of thevesicles and facing inward (Fig. 5, arrows). The peroxidase positive vesicles weretypically devoid of any clathrin coat. Within most early pinosomes, smaller vesiclesrimmed with outward-facing reaction product were also observed. Rapidly labelled,

Fig. 4. Distribution of pinocytized HRP activity in 5-6% Percoll gradients after apulse (lmin) or a 1-3 min chase. Cells were exposed to 2mgml~' HRP for 1 minin 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 andoverlaid with 5-6% Percoll. Following centrifugation, fractions were collected andanalysed for HRP ( • • ) , /3-hexosaminidase (O O), and alkaline phospho-diesterase I (A A) activities. HRP activity in the bottom portion of the gradient wasnon-latent at the end of the pulse and was latent after the chase.

10 h

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Table 1. Effect of chase time on cell-associated HRP activityCell-associated HRP activity

Chase time(min)

0123

Expt 1(%)

100483937

Expt 2(%)

100674445

Cells were incubated with 2mgml ' HRP for 1 min at 37°C, poured onto 0-4vol. of crushedfrozen saline to stop uptake and washed extensively at 4°C. Cells were then chased for various timesat 37°C in complete culture media. The data were derived by correcting the recovery of HRPactivity in postnuclear supernatants for the efficiency of cell homogenization based on the yieldof plasma membrane (alkaline phosphodiesterase I) and lysosomal (j8-hexosaminidase) markerenzyme activities. The HRP uptake period = 1 min.

early pinosomes were often 0-2—0-3//m in diameter and ranged in size up to about1 ;Um in diameter. Also present in the early pinosome fraction were various smoothand rough membranous organelles.

To determine the morphology of rapidly labelled, early pinosomes in situ, CHOcells were pulsed with peroxidase for between 15 s to 1 min, fixed and HRP activity

r\

<~ x:-*QFig. S. Morphological characterization of rapidly labelled, early pinosomes in Percollgradient fractions. Cells were exposed to 2 m g m r ' HRP for lOmin and poured onto0-4 vol. of crushed frozen saline to stop uptake. The total postnuclear supernatant wasmixed with dense sucrose and overlaid with 5-6% Percoll. The early pinosome-enrichedfractions were pooled on the basis of HRP activity and processed for peroxidase cyto-chemistry. Black deposits indicate sites of HRP activity (arrows). X22950.

Fig. 6. Morphological characterization of rapidly labelled, early pinosomes in situ. Cellswere 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 peroxidasecytochemistry. Black deposits indicate sites of HRP activity. In B, the arrowhead pointsto a pinosome-associated microfilament cluster. In C, the arrowhead points to a micro-tubule cluster, and the arrow points to a coated pit. A, X28125;B, x59700;C, X28650;D, X28125.


was localized by the DAB procedure. By electron-microscope cytochemistry of intactcells, HRP activity after a 15 s to 1 min pulse was found in round and elongatevesicles with the DAB reaction product generally found around the periphery of thevesicles (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 DABreaction product was observed in the peroxidase positive vesicles. The labelledvesicles were generally 0-1-0-4 jum in diameter, irrespective of whether the uptakeperiod was 15 s or as long as 1 min. Typically, the diameter of the early pinosomeswas much larger than that of coated pits and the pinosomes were non-coated. For a30 s uptake period, HRP positive vesicles ranged in size from 0-07-1-1 jUm diameterwith a median diameter of 0-30 fim. At higher magnification, a microfilament clusterwas occasionally observed in association with an early pinosome (Fig. 6B). Thefunctional significance of this is unknown.

The morphology of the rapidly labelled, early pinosomes both in situ and afterfractionation resembles that reported previously for endosomes/receptosomes (forreview, see Pastan & Willingham, 1985), suggesting that this rapidly labelledpinosome population is a form of endosome/receptosome.

DISCUSSION

These experiments were done to characterize early vesicles formed during fluid-phase pinocytosis in CHO cells, a cell line of fibroblastic origin. Knowledge of theproperties of early pinosomes should provide information on the initial intracellularsteps in the pinocytic pathway. Our results suggest that rapidly labelled, earlypinosomes are buoyant compartments that can be readily resolved from later pino-cytic compartments by cell fractionation and imply a unique set of molecularproperties for the compartments.

In these experiments, the pinocytic solute tracer was horseradish peroxidase. Forsome 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 con-centrations (mg ml"1) used in the present work, such a receptor, if present, would besaturated and most peroxidase uptake would be by fluid-phase pinocytosis (Sunget al. 1983). For CHO cells we find no evidence of cell surface binding of HRPpreparations. In the present work surface binding was not detected by either bio-chemical or cytochemical assays. In previous work we found HRP uptake by CHOcells to be non-saturable and unaffected by mannose concentrations as high as150 mM (Adams et al. 1982; Storrie et al. 1984). Recently completed work (Sullivan& Storrie, unpublished data) indicates that neither, 19mgml~' yeast mannan norperiodate oxidation of the enzyme to open the ring structure of the sugar side-chainshas any significant effect on HRP uptake by CHO cells.

The identification of rapidly labelled pinosomes as unique pinocytic compart-ments rests chiefly on cell fractionation data combined with a series of control


experiments. The first indication of the compartment was the unexpected outcome ofexperiments using flotation through a 5-6 % Percoll gradient introduced by Sahagian& Neufeld (1983) for the separation of endosomes involved in receptor-mediatedendocytosis from plasma membrane. In their work, also with CHO cells, a singlelow-buoyancy endosome population that overlapped extensively with lysosomes wasobserved after a 2 min period of /3-galactosidase uptake. With this gradient, for CHOcells pulsed with HRP for 10min, we observed two pinosome populations: theexpected low-buoyancy population and an unexpected buoyant population. Thebuoyant population sedimented very similarly to plasma membrane and might havebeen an artifact of HRP trapping during homogenization or cell surface adherence ofHRP. However, in control experiments no trapping or cell-surface-adherent HRPcould be detected. The cell-associated HRP was found to be intracellular by directbiochemical assay, and in the cell fractions it was latent and resistant to removal bytreatment with EDTA.

Because of their similarity in fractionation properties to plasma membrane, themembrane origin of endocytic vesicles, we postulated that buoyant pinosomes wereearly pinocytic compartments. This proved to be the case. In a series of pulse-uptake experiments, the buoyant pinosome population was the major rapidly labelledpinosome population after a 15-60s uptake and in pulse-chase experiments HRPwas rapidly chased from buoyant to low-buoyancy pinosomes. As expected for anearly endocytic compartment, buoyant pinosomes appeared to be a site of reversiblepinocytosis (diacytosis).

The rapidly labelled, early pinosomes were not identified in previous work becauseof the gradient systems and uptake protocols used. Sedimentation through 10 % and20% Percoll gradients, as done earlier in this laboratory (Pool et al. 1983; Storrieet al. 1984), is incapable of resolving very early pinosomes from other pinosomepopulations. The resolution achieved in the 5-6% Percoll gradient is either aconsequence of the very flat density gradient generated or the use of a flotationversus a sedimentation protocol. Recent work indicates that osmotic contraction indense sucrose is not necessary for the separation. Similar resolution occurs withresuspension of organelles in dense Percoll prepared in 0-25M-sucrose (Wirt &Storrie, unpublished observations). The preferential labelling of early pinosomesrequires a very brief uptake protocol. With most uptake protocols, early pinosomeswould be only a small portion of the labelled pinosome population and hence wouldbe easy to miss whatever the resolution of the gradient.

As a fluid-phase marker, HRP should be included in any newly formed endocyticvesicle, 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 andreceptor-mediated endocytosis in fibroblasts is the coated pit. The typically smoothappearance and large size (mean diameter =0'30^m with vesicles as large as 1 [imor more) of HRP positive early pinosomes relative to coated pits after even briefuptake periods (15 s to 1 min) indicates that the kinetics of the precursor-to-productrelationship between these structures must be very rapid. Recent work (Pastan &


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 compart-

ments (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 inter-

pretation 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 withelectron 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 Chinesehamster 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 ofpinocytized 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 inpolymorphonuclear 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).

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{Received 9 September 1985 -Accepted, in revised form, 15 January 1986)

characterization of early compartments in fluid … · cell fractionation washed cells (pooled al....

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