Journal of Cell Science 108, 3611-3621 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
JCS1134
3611
Low temperature reversibly inhibits transport from tubular endosomes to a
perinuclear, acidic compartment in African trypanosomes
Marla Jo Brickman1, J. Michael Cook2 and Andrew E. Balber1,2,*
1Department
of Immunology and 2Comprehensive Cancer Center, Duke University Medical Center, Durham NC 27710, USA
*Author for correspondence (e-mail: [email protected])
SUMMARY
We have used electron microscopy and flow cytofluorimetry to study endocytosis and intracellular transport of
fluid phase bovine serum albumen gold complexes and
membrane bound concanavalin A through endosomal compartments of bloodstream forms of Trypanosoma brucei
rhodesiense. Both markers were rapidly endocytosed from
the flagellar pocket. Within 20 minutes at 37°C the markers
reached a large, vesicular, perinuclear compartment that
stained heavily with the CB1 monoclonal antibody. Neither
marker left the flagellar pocket and entered cells at 4°C.
When cells were incubated at 12°C, both markers entered
the cell and were transported to collecting tubules, a
tubular endosomal compartment that receives endocytosed
material from coated endocytic vesicles. However, no
material was transported from collecting tubules to the
late, perinuclear compartment at 12°C. The morphology of
collecting tubule membranes was specifically altered at
12°C; tubules became shorter and were arrayed near the
flagellar pocket. The morphological alteration and the
block in transport of endocytic markers to the perinuclear
compartment seen at 12°C were reversed 10 minutes after
cells were returned to 37°C. We also used flow cytofluorimetric measurements of pH dependent fluorescence
quenching to measure the pH of the terminal endocytic
compartment. Fluoresceinated lectins accumulated in a
terminal compartment with a pH of 6.0-6.1, a value considerably higher than that of mammalian lysosomes. Fluorescence from fluoresceinated lectins in this terminal
endocytic compartment was dequenched when bloodstream forms were incubated in the presence of chloroquine.
INTRODUCTION
1994a). The unique properties of the FP has raised interest in
targeting anti-trypanosomal agents to components of the FP and
to the internal compartments that communicate with it
(Opperdoes et al., 1986). Consequently, elucidating how BF
control endocytosis and intracellular transport of material and
how these processes differ in trypanosomes and mammalian
cells is of considerable practical, as well as evolutionary,
interest.
The mechanisms that mediate endocytosis and vesicular
transport in BF are beginning to be characterized. Several
macromolecules enter BF by receptor mediated endocytosis; the
receptors bind ligands in the FP (Coppens et al., 1988, 1991,
1992; Webster and Grab, 1988; Grab et al., 1992; Hager et al.,
1994; Sternberg and McGuigan, 1994; Ligtenberg et al., 1994;
Chaudhri et al., 1994). Other molecules, including bovine serum
albumen (BSA) appear to enter BF primarily by fluid phase
endocytosis (Coppens et al., 1987; Webster, 1989). Macromolecules are taken up from the FP in coated vesicles and then move
anteriorly through a series of morphologically distinct compartments towards the nucleus (Langreth and Balber, 1975;
Coppens et al., 1987; Webster, 1989; Webster and Fish, 1989).
The rate of uptake is extremely high, and the entire surface of
the FP may turn over every 2-3 minutes (Coppens et al., 1987;
African trypanosomes offer a unique system in which to study
the structure, function, regulation, and evolution of the
endosomal and lysosomal system. All components of this
system are more active in bloodstream forms (BF) that parasitize humans and other mammals than in procyclic forms that
develop in tsetse flies (Langreth and Balber, 1975; Pamer et al.,
1989; Webster and Fish, 1989; Mbawa et al., 1991a; Brickman
and Balber, 1994b). The developmental activation of endocytic
and hydrolytic activity in BF reflects the dependence of BF on
efficient mechansims that provide macromolecular growth
factors (Coppens et al., 1987, 1988; Gillett and Owen, 1992;
Grab et al., 1992; Sternberg and McGuigan, 1994; Ligtenberg
et al., 1994; Chaudhri et al., 1994). Endocytosis occurs exclusively from the flagellar pocket (FP), a specialized surface
domain at the posterior end of the highly polarized BF; in the
FP the variant surface glycoprotein (VSG) coat that covers the
BF surface is reorganized to permit interaction with growth
factors (reviewed by Balber, 1990; Webster and Russell, 1993).
At least one lysosomal membrane glycoprotein, CB1-gp, is
transported to lysosomes by way of the FP membrane and is
transiently exposed on the surface (Brickman and Balber,
Key words: chloroquine, endosome, endocytosis, flagellar pocket,
intracellular transport, lysosome, pH, Trypanosoma brucei
rhodesiense
3612 M. J. Brickman, J. M. Cook and A. E. Balber
Webster and Griffiths, 1994). The coated vesicles deliver endocytosed material to a compartment composed of smooth, flat
cisternae that have been called collecting tubules, tubular
profiles, or intermediate endosomes (Langreth and Balber,
1975; Webster, 1989; Burleigh et al., 1993). A membrane glycoprotein, gp65, is localized exclusively in this intermediate
compartment of T. vivax (Burleigh et al., 1993). Some evidence
suggests that endocytosed VSG may recycle to the surface from
the intermediate compartment (Webster and Grab, 1988;
Seyfang et al., 1990; Duszenko and Seyfang, 1993). CB1-gp
(Brickman and Balber, 1994a) and LDL receptors may also
recycle from an endosomal compartment (Coppens et al., 1992),
but transferrin receptors probably do not (Grab et al., 1992).
Endocytic markers leave the collecting tubules and pass into
large, heterogeneous vesicular lysosomes just posterior to the
nucleus (Langreth and Balber, 1975; Webster, 1989; Grab et al.,
1992). Serial sections of BF incubated with BSA-gold showed
that tubular membranes with a luminal diameter of roughly 200
nm formed an interconnecting network that in single sections
appeared vesicular or lysosome-like (Webster, 1989). CB1-gp
and Tb292, two lysosomal membrane proteins present in all
compartments of the endosomal and lysosomal pathway, are
abundant in this terminal lysosome (Brickman and Balber,
1993; Lee et al., 1994). Cysteine and serine proteases, acid phosphatase, and other hydrolases that are probably involved in
digesting endocytosed material are present within lysosomes
and endosomes (Langreth and Balber, 1975; Lonsdale-Eccles
and Mpimbaza, 1986; Lonsdale-Eccles and Grab, 1987; Mbawa
et al., 1991b). Leupeptin sensitive proteases in these compartments also process newly sythesized CB1-gp (Brickman and
Balber, 1994a) and activate a component of human high density
lipoprotein preparations called trypanosome lytic factor (TLF),
probably haptoglobin-related protein (Smith et al., 1995), to a
lytic form (Hager et al., 1994). The pH of these endocytic compartments of BF has not been directly measured, but calculations based on chloroquine (Coppens et al., 1993) and radiolabeled weak base (Nolan and Voorheis, 1990) accumulation by
whole cells have yielded pH estimates of 4.4 or 5.6, respectively. Lysosomotropic agents have been reported to inhibit
recycling of LDL receptors and transport and processing of TLF
by BF (Coppens et al., 1993; Hager et al., 1994). The factors
that control transport among these endosomal compartments or
recycling of molecules from them to the BF surface are
unknown.
In this report we compare the kinetics of intracellular transport
of endocytosed markers through the endosomal and lysosomal
compartments of BF of Trypanosoma brucei rhodesiense at 4,
12 and 37°C. We show that when BF were incubated at 12°C,
fluid phase and membrane bound markers accumulated in the
collecting tubules, and transport of material from collecting
tubules to a late, perinuclear compartment that stains heavily
with CB1 was blocked. Inhibition of transport was reversed
when BF were warmed to 37°C. We also show that the late, perinuclear compartment of BF where markers accumulate at 37°C
is maintained at a pH of about 6.1.
MATERIALS AND METHODS
Parasites
The derivation, maintenance and isolation of the DuTat 1.1 clone of
the Wellcome CT strain of Trypanosoma brucei rhodesiense has been
previously described (Frommel and Balber, 1987).
Incubation media
We used several different media to optimize incubation conditions
during endocytosis experiments and now routinely use HMI-9
medium (Carruthers and Cross, 1992) with 1% (v/v) goat serum substituted for fetal bovine serum (abbreviated CM). This medium
preserved parasite integrity and motility better than any other formulation we tried. Modifications for specific experiments are noted as
appropriate.
Temperature control
All operations were done on ice in a cold room using pre-chilled
buffers and glassware to maintain temperature at or below 4°C. For
temperature shift studies, all solutions were equilibrated in water baths
in the cold room at 12°C or 37°C for at least 10 minutes before cells
were added. It was essential to rigorously control temperature at all
steps to observe the uptake kinetics described below.
Labeling endocytic and lysosomal vesicles
BSA was adsorbed to 12 nm colloidal gold as described previously
(Brickman and Balber, 1993). BF were washed into CM and
incubated on ice for 10 minutes in BSA-gold (starting optical density
525 nm = 60.0) diluted 1:1 in CM at 5×108 cells/ml. After 10 minutes,
100 µl of cells were then directly transferred to 1 ml of CM equilibrated at 12°C or 37°C for 2, 5, 20 or 60 minutes. The cells were then
washed twice at 4°C for 30 seconds, pelleted, and put into fixative for
cryoimmuno-electron microscopy. For temperature shift studies, the
cells remained at 12°C for 20-30 minutes, washed once, very quickly
at 12°C, brought up in 100 µl of CM at 12°C, then transferred to 1
ml of CM at 37°C for a total of 10 minutes, with samples taken at
two minute intervals, and fixed as described above.
In some experiments, BF were incubated with 100 µg/ml succinylConA (sConA; all lectins were obtained from Sigma, St Louis, MO)
instead of the BSA-gold. The cells were fixed, sectioned, and blocked
as described above. Thin sections were then incubated with a rabbit
anti-ConA IgG (Cappel, Durham, NC) for 30 minutes at room temperature, washed and incubated with a goat anti-rabbit IgG+IgM conjugated to 10 nm gold (Amersham, UK).
Electron microscopy
BF were incubated on ice in the presence of BSA-gold for 10 minutes
and then transferred to CM at either 4, 12 or 37°C for thirty minutes.
The cells were then pelleted and fixed in 2% glutaraldehyde/0.1 M
sodium cacodylate/0.12 M sucrose/1% (w/v) tannic acid equilibrated
at either 4, 12, or 37°C for 30 minutes. The cells were then processed
as described by Brickman and Balber (1990).
Cryoimmuno-electron microscopy
Trypanosomes were prepared for cryoimmuno-electron microscopy
and labeled with CB1 as described by Brickman and Balber (1993)
with the exception that a goat anti-mouse (IgG + IgM) secondary
antibody conjugated to 5 nm gold (Amersham, UK) was used instead
of 10 nm gold. Sections were also double-labeled with the anti-VSG
monoclonal antibody, 106.1 (Frommel et al., 1987).
Flow cytometry
BF-associated fluorescence was measured using a Becton Dickenson
FACScan or a FACStar Plus flow cytometer as described previously
(Brickman and Balber, 1990). Fluoresence from fluorescein labeled
(Fl-) probes was excited at 488 nm, and emission signals were
measured using a 520-530 nm band pass filter. Tetramethylrhodamine
(Rh-) fluorescence was excited at 514 nm, and fluorescence was
measured behind a 560 nm long pass filter. Mean fluorescence channel
numbers were calculated from 104 gated trypanosomes; channel
numbers are expressed in linear units.
Endosomal transport in trypanosomes 3613
Determining pH of compartments containing Fl-lectins
BF were incubated on ice with 100 µg/ml Fl-wheat germ agglutinin
(WGA), Fl-ConA, or Rh-ConA to label the FP and washed. Portions
of these labeled cells were used to construct a pH standard curve.
These cells were resuspended in ice-cold 100 mM sodium
phosphate-20 mM NaCl buffer containing 1% BSA and either 1 mM
glycerol (ConA experiments) or 16 mM glucose (WGA experiments) at pH values between 5.0 and 8.0. Cell-associated green
fluorescence was determined flow cytometrically. A standard
curve expressing fluorescence intensity as a function of pH was
constructed. The remaining cells that had been labeled in the FP on
ice were incubated at 37°C to permit lectin to enter the cells.
Fluoresence signals were collected from these cells at various
intervals following the temperature shift. The pH of the compartment containing the endocytosed lectin was determined by interpolation from the standard curve as discussed in the text. No correction was made for possible pH dependent changes in
autofluorescence.
RESULTS
Effect of temperature on endocytosis of BSA-gold
Fluid phase endocytosis of BSA-gold has been studied previously by us (Brickman and Balber, 1993) and by Webster
(1989). We reinvestigated the kinetics of uptake at 37°C using
a carefully controlled temperature shift protocol. In general, we
confirmed the results obtained by Webster (1989). No marker
entered cells from the FP when cells were maintained on ice.
At 37°C, the uptake of the BSA-gold and its transport from the
FP through endosomes and collecting tubules to a large, multivesicular region was rapid. After 2 minutes, BSA-gold was
present in the FP, endosomes, and in the collecting tubules
(Fig. 1B and C). A few grains of BSA-gold were present in the
large multivesicular region characterized by dense labeling
with the CB1 monoclonal antibody (Brickman and Balber,
Fig. 1. Cryosections of BF incubated with BSA-gold at
37°C and then immunostained with the CB1
monoclonal antibody. (A,B and C) Incubation with
BSA conjugated to 15 nm gold for 2 minutes; (D) for 5
minutes. CB1 was detected with 5 nm gold-labeled antiimmunoglobulin. After 2 minutes, only a few BSA-gold
grains (large arrowhead) can be detected in the
multivesicular region labeled with CB1-5 nm gold
(small arrowheads). Most of the BSA-gold is present in
the FP (C), and collecting tubules (A,B). By 5 minutes
(D) BSA-gold can be readily detected in the CB1labeled region. FP, flagellar pocket. Bars, 100 nm.
3614 M. J. Brickman, J. M. Cook and A. E. Balber
1993) (Fig. 1A). After 5 minutes, large amounts of BSA-gold
were found in the large, multivesicular region (Fig. 1D). The
late perinuclear region corresponds to the region that has been
called a ‘lysosome-like’ or ‘lysosomal’ region by other
workers (Coppens et al., 1987; Webster and Grab, 1988;
Webster, 1989; Grab et al., 1992; Brickman and Balber, 1993;
Burleigh et al., 1993 among others; see Balber, 1990 for
review).
We next explored the effect of temperature on uptake and
transport of BSA-gold. In these experiments BF were
incubated at 4°C with BSA-gold for 10 minutes. Samples of
these cells were then mixed with 10 volumes of CM equilibrated at either 4, 12 or 37°C and incubated for an additional
30 minutes. The cells were then fixed, and either plastic thin
sections or cryosections were prepared. As expected from
previous work (Webster, 1989), BSA-gold was only detected
in the FP of BF incubated at 4°C (Fig. 2A). At 37°C, the BSAgold accumulated in the large perinuclear compartment. Under
these experimental conditions, cells were effectively pulsed
with the marker; BSA-gold was not seen in the FP or in the
collecting tubules, and all label had moved to the late perinuclear compartment by 30 minutes (Fig. 2C).
Very different results were obtained when BF pulsed with
BSA-gold on ice were transferred to medium at 12°C. After 30
minutes at 12°C, BSA-gold was detected in the FP, in
endocytic vesicles and in collecting tubules that were located
proximal to the FP (Figs 2B and 3B). However, BSA-gold
could not be detected in the multivesicular region densely
stained with CB1 (Fig. 4A and C). Even with incubation times
up to one hour, BSA-gold did not enter the multivesicular
region (Fig. 4C); the BSA-gold remained in the collecting
tubules (Fig. 4D) and appeared to be blocked from further
transport through the endocytic system. Thus, incubation at
12°C caused a specific block in transport from the collecting
tubules to the perinuclear compartment.
Incubation at 12°C also caused a reorganization of the
endosomal system. The collecting tubules of BF maintained for
30 minutes at 37°C appeared as linear, tubular cisternae.
However, when cells were incubated at 12°C, very few linear
tubules were present. Instead rounded, vesicular structures
with increased electron density were abundant around the FP
and proximal to perinuclear, multivesicular regions (Figs 2B
and 4A-D). These changes in morphology in the tubules at
12°C appeared to be specific for the collecting tubules. Other
Fig. 2. Plastic thin sections of BF
pulsed with BSA-gold at 4°C (A);
12°C (B) or 37°C (C). BF were
incubated with BSA conjugated
to 15 nm gold on ice for 10
minutes, then mixed with 10
volumes of medium at the
temperature shown, and
incubated for 30 minutes. BSAgold is only detected within the
flagellar pocket (FP) of BF
incubated at 4°C (A). At 12°C
(B), BSA-gold is present in
vesicles and short, rounded
collecting tubules (arrowheads)
proximal to the FP. At 37°C (C),
BSA-gold is only present in late,
multivesicular, lysosome-like
structures (L) anterior to the FP;
no BSA-gold is present in the FP
or in the collecting tubules (CT)
when this protocol is used. N,
nucleus; G, Golgi. Bars, 100 nm.
Endosomal transport in trypanosomes 3615
structures, such as the FP, the surface membrane, and the Golgi
appeared the same at 12°C and at 37°C. The distribution of the
CB1 (Fig. 4A-D) and anti-VSG monoclonal antibodies (Fig.
3A,B) also were not altered detectably by incubating cells at
12°C.
To determine whether the block in transport to the late, perinuclear compartment and changes in endosomal morphology
at 12°C could be reversed, we incubated BF in the presence
of BSA-gold at 12°C for 30 minutes to allow the BSA-gold
to accumulate in the collecting tubules. The cells were then
washed and rapidly diluted into medium at 37°C. We took
samples of the cells immediately after addition to media at
37°C and then at two minute intervals to determine whether
the BSA-gold could be detected in structures other than the
collecting tubules. Immediately following addition to
medium at 37°C, the BSA-gold was detected in endocytic
vesicles and collecting tubules close to the FP (Fig. 5A).
BSA-gold was not detected in the large vesicles characterized
by dense labeling with CB1 (Fig. 5A and B). After 6 to 8
minutes at 37°C, the BSA-gold was detected in elongated collecting tubules close to the vesicular region labeled with CB1
(Fig. 5C and D). By 10 minutes, BSA-gold was abundant in
the late, multivesicular compartment that stained heavily with
CB1 (Fig. 5E and F). As the length of incubation at 37°C
increased, the early endocytic compartment reassumed the
characterisic morphology of collecting tubules in BF. Stacks
of several collecting tubules, rather than discrete vesicles,
were common, and these tubules again appeared to form an
interconnecting network (Fig. 5C,D, and E). Unlike the
vesicles at 12°C that were arrayed primarily around the FP,
these tubular networks extended anteriorly towards the perinuclear region (Fig. 5E).
Fig. 3. Cryosections of BF incubated with BSA-gold or ConA under various conditions and then immunostained. In immunostaining, CB1 and
anti-VSG 106.1 monoclonal antibodies were detected with 5 nm gold anti-immunoglobulin. (A) BF incubated with ConA (10 nm gold/large
arrowheads) for 5 minutes at 37°C; section immunostained with 106.1 (small arrowheads). Both ConA and VSG are present on the flagellar
pocket membrane and within the collecting tubules. ConA gold is present in forming endocytic vesicles (large arrowheads). (B) BF incubated
with BSA conjugated to 15 nm gold (large arrowhead) for 20 minutes at 12°C ; section immunostained with 106.1 (small arrowheads). Surface
coat is heavily labeled with the anti-VSG antibody. Both BSA-gold and VSG are detected within the same round and distended collecting
tubule. (C) BF incubated with ConA (10 nm gold/large arrowheads) for 20 minutes at 37°C; section immunostained with CB1 (small
arrowheads). ConA is detected within the CB1 labeled region. (D) BF incubated with ConA for 20 minutes at 12°C. ConA is present within the
rounded collecting tubules. F, flagellum; FP, flagellar pocket; S, surface coat. Bar, 100 nm.
3616 M. J. Brickman, J. M. Cook and A. E. Balber
Fig. 4. Cryosections of BF incubated with BSA-gold at 12°C for 20 minutes (A and B) or 60 minutes (C and D). (A,C and D) Immunostaining
with CB1 (5 nm gold, small arrowheads). (A) BSA-gold (15 nm, large arrowheads) clearly accumulates within vesicles and short, round
collecting tubules that surround the flagellar pocket (FP); no BSA-gold enters the more anterior CB1-labeled region (small arrowheads).
(B) Accumulation of round vesicles containing BSA-gold near the FP. (C and D) BSA-gold remains sequestered from CB1-staining regions in
round collecting tubules even after 60 minutes of incubation at 12°C. F, flagellum. Bars, 100 nm.
Effect of temperature on endocytosis and transport
of membrane bound Con A
We previously showed that lectins bind to oligosaccharides
exposed in the FP but do not bind elsewhere on the surface of
living BF (Balber and Frommel, 1988; Brickman and Balber,
1990). We took advantage of this to determine if uptake and
intracellular transport of membrane bound ligands was also
inhibited at 12°C. We used cryoimmunoelectron microscopy
to localize intracellular ConA at various times after endocytosis. BF were incubated with ConA on ice, shifted to 12°C or
to 37°C, and prepared for cryoimmunoelectron microscopy.
After 5 minutes at 37°C, anti-ConA labeled the FP membrane,
and tubular profiles (Fig. 3A). Webster and Grab (1988) have
reported that two other surface-bound probes move from the
FP into the collecting tubules within 5 minutes at 37°C. Fig.
3C shows that after 20 minutes at 37°C, membrane bound
ConA was detected in the collecting tubules and in the multivesicular preinuclear region that stains heavily with the CB1
antibody. Significantly, membrane bound ConA, like BSAgold, remained in the collecting tubules and was not transferred
to the perinuclar region when BF labeled with ConA on ice
were subsequently incubated at 12°C (Fig. 3B,D). Thus, both
BSA and ConA leave the FP and reach the collecting tubules
within 5 minutes, and both enter the mutlivesicular compartment by 20 minutes after transfer to 37°C. Furthermore, neither
marker proceeds into the multivesicular, perinuclear compartment at 12°C.
The endocytic compartments are acidic
The fluorescence from Fl-labeled molecules is quenched in
acidic environments (Ohkuma and Poole, 1978), and flow cytometric measurement of this fluorescence quenching can be
used to estimate the pH of intracellular compartments containing fluoresceinated ligands (Murphy et al., 1984; Wilson
and Murphy, 1989). We have already demonstrated that the
amount of Fl-lectin that binds to the FP of BF maintained on
ice can be detected by flow cytometry (Brickman and Balber,
1990), and we used Fl-lectins to probe the pH of the endocytic
compartments encountered by these lectins at various times
after endocytosis was induced by the temperature shift
protocols described above.
When BF that had been labeled in the FP with Fl-lectin on
ice were washed and then maintained on ice, cell-associated
fluorescence remained constant for at least 3 hours (not
shown). However, when these labeled cells were transferred to
medium at 37°C to induce endocytosis of the lectin, the mean
green fluorescence signal emitted from Fl-ConA and Fl-WGA
decreased progressively and rapidly during the first 15 minutes
following temperature shift and then remained relatively
constant. Fig. 6 shows representative data for Fl-WGA; similar
data were obtained with Fl-ConA.
We did several control experiments to determine if this
apparent quenching of Fl-lectin pH was due to transport of the
probe into an acidic environment: first, we determined that we
could measure pH dependent changes in fluorescence signals
Endosomal transport in trypanosomes 3617
Fig. 5. BSA-gold moves from collecting tubules to the multivesicular, late compartment when BF are transferred from 12 to 37°C. BF were
incubated with BSA-gold (15 nm, large arrowheads) for 30 minutes at 12°C, then rapidly transferred to 37°C. Sections were immunostained
with CB1 (5 nm gold, small arrowheads). BSA-gold is present in the flagellar pocket and within short, round collecting tubules which surround
the flagellar pocket immediately after (A) or 2 minutes after (B) being transferred to 37°C. After 6 (C) and 8 (D) minutes at 37°C, the collecting
tubules appear longer and less distended than they were at 12°C and extend to the heavily CB1-labeled region. After 10 minutes (E and F) at
37°C, BSA-gold is detected within the heavily CB1-labeled region. F, flagellum. Bars 100 nm.
from Fl-lectins bound to the FP by flow cytometry. We did this
by allowing BF to bind Fl-lectin on ice, suspending the cells
in ice-cold buffers at various pH values, and measuring fluorescence flow cytometrically. Fig. 7 shows typical results with
Fl-WGA; similar results were obtained with Fl-ConA. The
dependence of Fl-lectin fluorescence on buffer pH measured
by this method closely ressembles pH titrations published by
several groups using other methods (Ohkuma and Poole, 1978;
Murphy et al., 1984; Wilson and Murphy, 1989).
Next, we did similar experiments using Rh-lectins instead
of Fl-lectins. Fig. 7 shows representative data. In contrast to
the results with Fl-lectins, fluoresence from Rh-lectins did not
decrease when labeled cells were incubated at 37°C.
Rhodamine fluorescence is not generally quenched by low pH
(Murphy et al., 1984; Wilson and Murphy, 1989), and we
confirmed (Fig. 7) that the fluorescence signals from BF
labeled with Rh-ConA on ice and equilibrated with different
buffers was not pH sensitive.
Third, we found that the lysosomotropic agent chloroquine
reversed quenching of fluorescence from intracellular Fl-lectin.
In these experiments we incubated BF with Fl-lectins on ice,
washed out unbound lectin, and then incubated for extended
periods at 37°C to allow the probe to accumulate in the late,
perinuclear compartment. We then rapidly mixed the Fllabeled BF with medium containing chloroquine to obtain final
chloroquine concentrations of 1.0 to 200 µM, incubated an
additional 30 minutes at 37°C, and then measured cell associated fluorescence by flow cytofluorimetry. We set up these
experiments so all tubes were analyzed exactly 30 minutes
after chloroquine addition because BF began to lose normal
morphology and motility (not shown) during extened incubation at 37°C in chloroquine. Fig. 8 shows that BF retained
3618 M. J. Brickman, J. M. Cook and A. E. Balber
350
300
MEAN SCATTER
250
200
150
100
50
0
0
Fig. 6. Quenching of Fl-WGA fluorescence after endocytosis of
lectin from the flagellar pocket. BF were incubated with 100 µg/ml
Fl-WGA on ice for 20 minutes and washed. The labeled cells were
brought up in medium that had been equilibrated at 37°C, and green
Fl-WGA fluorescence signals from the live cells were measured by
flow cytofluorimetry at the times shown on the abscissa.
Fluorescence decreased rapidly for about 10 minutes following
endocytosis. Another experiment was done simultaneously using RhConA; fluorescence remained relatively stable during endocytosis.
Fig. 7. Effect of pH on fluorescence signals from BF labeled in the
flagellar pocket (FP) with Fl-WGA and Rh-ConA. These data were
generated using the same cells as were used for the data in Fig. 6. BF
were incubated with the lectins on ice, washed and resuspended in
buffers at various pH as described in the text. Green fluorescence FlWGA (u) and red Rh-ConA (r) signals were measured and plotted
as a function of pH. Green signals are strongly queched by acidic
pH. By comparing the fluorescence values in Fig. 6 to this standard
curve, we estimate that the apparent pH of the environment of FlWGA drops from a pH of 7.5 in the FP to about 6.1 after 60 minutes
at 37°C. Red fluorescence from BF labeled in the FP with Rh-ConA
was not altered significantly by changes in pH, and Fig. 6 shows that
Rh-ConA fluorescence was not quenched in cells.
normal light scattering properties at concentrations of chloroquine less than 50 µM, but that light scattering changed
markedly during 30 minute incubations at higher concentrations. Fig. 9 shows that cell associated fluorescence increased
when BF containing Fl-lectin in the perinuclear compartment
50
100
150
200
10-6 M CHLOROQUINE
Fig. 8. Light scattering profiles of BF incubated at 37°C in the
presence of chloroquine. BF were labeled with FL-ConA (166
µg/ml) for 15 minutes at 37°C, washed, incubated for 15 minutes at
37°C, and then incubated at 37°C in different concentrations of
chloroquine for 30 minutes as described in the text. Mean side scatter
(r) and mean forward scatter (j) were determined flow
cytometrically and plotted as a function of chloroquine
concentration. At concentrations less than 50 µM chloroquine, BF
appear to maintain normal light scattering profiles. At concentrations
higher than 50 µM chloroquine, light scattering profiles are altered.
were incubated in chloroquine. Fluorescence increased with
increasing chloroquine concentration between 0 and 50 µM
chloroquine but did not increase when chloroquine concentration was increased above 50 µM.
To determine if some direct interaction between chloroquine and fluorescein caused the increased fluorescence in
these experiments, we labeled BF with Fl-lectin on ice, maintained the cells on ice, and then added 100 µM chloroquine.
Under these conditions, fluoroprobe in the FP should have
been accessible to the high concentration of chloroquine.
However, flow cytometric analysis showed that chloroquine
did not change cell associated fluorescence under these conditions (not shown), suggesting that chloroquine did not
directly increase the fluorescence yield from Fl-lectins.
Collectively, these experiments show that lectin conjugates
remain associated with BF when labeled cells are warmed to
37°C; that decrease in Fl-lectin fluorescence reflects changes
in the intracellular environment of the fluoresceinated probe;
and that these environmental changes are likely to involve a
progressive drop in pH.
We were able to estimate the pH of the late perinuclear compartment by comparing the fluorescence signals from BF
incubated with Fl-lectins at 37°C to the pH titration done with
BF labeled in the FP. We did five experiments using this method
to estimate the pH of the late compartment in which the lectin
ultimately accumulated 30-80 minutes after temperature shift
(Fig. 7). The pH values determined in each experiment were
6.1, 6.15, 6.2, 6.2 and 6.35 (mean pH, 6.20; s.d., 0.10).
DISCUSSION
Others have shown (Webster, 1989; Grab et al., 1992), and we
Endosomal transport in trypanosomes 3619
MEAN CHANNEL NUMBER
120
100
80
60
40
20
0
0
20
40
60
80
100
120
140
160
10-6 M CHLOROQUINE
have confirmed here, that endocytosis of fluid phase markers
and membrane bound lectins occurs very rapidly from the FP
in BF incubated at 37°C. Endocytosed material entered the FP,
and was transported via endocytic vesicles to the collecting
tubules within 2 minutes. Within 5 minutes, material entered
the large post nuclear, lysosomal compartment. Material
appeared to remain in this compartment for at least one hour,
confirming other reports (Webster and Grab, 1988; Webster,
1989; Grab et al., 1992).
In this report we show that incubating BF at 12°C had
dramatic effects on intracellular transport of endocytosed
material. Interiorization of immune complexes from the
surface of BF (Russo et al., 1994) and transport of CB1-gp
from the FP to a proteolytic endosome (Brickman and Balber,
1994a) are both slowed at 12°C. Although endocytosis was
much slower at 12°C, readily detectable quantities of both
ConA and BSA-gold were delivered to the collecting tubules
at 12°C. Nevertheless, we never detected either BSA or ConAgold in the multivesicular, perinuclear compartment of BF
incubated at 12°C. Similarly, Hager et al. (1994) have reported
that TLF enters an early endosomal compartment at 17°C but
does not progress to late compartments. Thus, it appears that
transport from the collecting tubules to the late, multivesicular
compartment is blocked more completely than endocytosis and
transfer to collecting tubules. In other cell types, transport from
early to late endocytic compartments has been reported to be
completely inhibited (Dunn et al., 1980; Gruenberg and
Howell, 1989; Lenhard et al., 1992), slowed significantly, or
redirected (Morris and Saelinger, 1986; Roederer et al., 1987;
Sullivan et al., 1987; Harding and Unanue, 1990; Haylett and
Thilo, 1991) by temperatures below 20°C.
We also observed that the morphology of the collecting
tubules was significantly altered at 12°C. The network of long
tubular cisternae characteristic of the collecting tubule region
appeared to break into a series of independent shorter tubules
arrayed near the FP. We did not detect changes in the morphology of any other BF compartment during incubation at
12°C. Furthermore, although we have not done morphometric
analysis, we did not notice any apparent increase in steady state
levels of immunologically detectable VSG or CB1-gp in the
altered collecting tubules where BSA-gold or ConA accumulate at 12°C. Thus, the reorganization of collecting tubules
appears to be a specific change induced by this temperature.
Very similar changes in endosomal structure have been
180
200
Fig. 9. Cell associated fluorescence of BF loaded with
Fl-ConA in the perinuclear compartment and then
exposed to chloroquine for 10 minutes at 37°C. Data
from the experiment shown in Fig. 8. Mean fluorescence
channel number is plotted as a function of chloroquine
concentration. Standard deviations for the fluorescence
distributions ranged between 1.5 (no chloroquine) and
32.9 (50 µM chloroquine) channels with a mean
standard deviation of 22.4 channels. Mean fluorescence
increased with increasing chloroquine concentration
between 0 and 50 µM chloroquine but did not increase
further when chloroquine concentration exceeded
50 µM.
observed when epidermoid carcinoma cells were incubated at
low temperatures (Haylett and Thilo, 1991). Other treatments
can also reversibly convert mammalian lysosomes between
vesicular and tubular structures (reviewed by Heuser, 1989).
Hopkins et al. (1990) have suggested that mammalian tubular
endosomes comprise one continuous reticulum, and serial
sections of BF incubated at 37°C showed that the collecting
tubules comprise an anastomosing network (Webster, 1989).
Webster and Grab (1988) suggested that functionally discrete
subregions exist within these collecting tubules. The maintenance of specific microenvironments within this reticulum may
be necessary for proper intraendosomal transport. Low temperatures may alter these microenvironments, compromise
tubule integrity, and thereby interfere with transport of ligands
within the endosomes into the late, multivesicular compartment.
The endosomal system of BF resembles that of most eukaryotic cells in that early compartments are less acidic than later
compartments. The FP rapidly equilibrated with the pH of the
suspending medium when BF were maintained on ice. When
the BF were diluted into medium at 37°C, Fl-lectins entered
the cells from the FP, and fluorescein fluorescence was rapidly
and progressively quenched. Rhodamine labeled probes were
not quenched, and quenching was reversed by chloroquine.
These results confirm the work of others suggesting that BF
maintain an acidic endocytic system (Nolan and Voorheis,
1990; Coppens et al., 1993; Hager et al., 1994).
We could not unambiguously measure the pH of the early
endocytic compartments. At 37°C transport of probe throughout the endocytic system was so rapid that any measurement
we made represented an average of several compartments; we
can only state that this average pH decreased rapidly as probes
moved anteriorly from the FP. We could, however, measure
the pH of the late, multivesicular, perinuclear compartment that
stains heavily with the CB1 monoclonal antibody by chasing
fluoroprobes into this terminal compartment. Even when fluoroprobes were chased for several hours they behaved as if
they were in an environment with a pH of about 6.2. The earlier
endosomal compartments are even less acidic.
Other workers have reported the pH of unspecified endocytic
compartments in BF is maintained at pH 4.4 (Coppens et al.,
1993) or 5.6 (Nolan and Voorheis, 1990). Our studies differ
from this previous work in several ways. In particular, we have
specifically loaded the perinuclear compartment with the flu-
3620 M. J. Brickman, J. M. Cook and A. E. Balber
oroprobes we used to report compartmental pH. Fl-lectin
remaining in early compartments is unlikely to have raised the
apparent pH of the terminal compartment significantly, for we
could not detect lectin on the surface, in the FP, or in early
compartments by fluorescence or electron microscopy after a
one hour chase. Although lectins can inhibit
endosome/lysosome fusion in some mammalian cells
(Goldman et al., 1976; Keilian and Cohn, 1981; Rabinowitz et
al., 1992), we have found that the lectins and the fluid phase
marker BSA-gold end up in the same terminal compartment.
The perinuclear compartment we have identified as the
terminal compartment appears to be the structure where other
endocytic markers accumulate in BF (Coppens et al., 1987;
Webster and Grab, 1988; Webster, 1989; Grab et al., 1992).
This suggests that the lectin probes are transported normally
through BF and that the measurements we made reflect the
normal pH in the endosomal system.
Another important feature of our studies is that we loaded
BF in the absence of lysosomotrophic agents. In previous
studies (Coppens et al., 1993; Nolan and Voorheis, 1990),
endosomal pH was estimated by measuring the partitioning of
lysoosmotrophic agents between cells and exposing medium.
BF were incubated with high concentrations of these agents
over extended periods, and pH was calculated based on several
assumptions concerning intracellular drug distribution. We
have confirmed the observation of Hager et al. (1994) that concentrations of chloroquine in excess of 50 µM are rapidly toxic
to BF. We detected light scattering changes in cells incubated
in 100 µM chloroquine for only 10 minutes. Indeed, chloroquine has been used as a trypanocide in vivo (Otigbuo and
Woo, 1988). We observed dequenching of the Fl-signals with
chloroquine concentrations as low as 5 µM; about 15 µM
chloroquine dequenched fluorescence by 50%, and dequenching was maximal at 50 µM chloroquine. Dequenching of intracellular fluoroprobe fluorescence and inhibition of TLF activation in endosomes (Hager et al., 1994) show remarkably
similar dependence on chlorquine concentration. Both activities are inhibited 50% by 10-15 µM chloroquine, and >95% by
about 50 µM chloroquine. The activity of relatively low concentrations of chloroquine in our dequenching assay and in the
TLF inhibition assay suggests that BF have an easily neutralized endocytic compartment; the rapid toxicity of chloroquine
suggests that maintenance of a mildly acidic endosomal system
is physiologically important to BF.
Other parasitic protozoa maintain endosomal or lysosomal
compartments that differ in pH from those in many vertebrate
cells. T. cruzi epimastigotes have a specialized pre-lysosomal
compartment called a reservosome that is maintained at pH 6.0,
but this structure is not present in mammalian forms of this
parasite (Soares et al., 1992). Entameoba histologica retains
endocytosed material in a neutral prelysosomal compartment
for several hours before transporting it to acidic lysosomes or
out of the cell (Aley et al., 1984). The existence of such
organelles in other protozoan parasites of mammals and our
finding that BF retain endocytosed ligands in a late compartment maintained at pH 6.0 suggest that the trypanosome
endocytic system, particularly the late, perinuclear, multivesicular region that has been assumed to be lysosome-like, has properties that cannot be predicted from studies on mammalian cells.
However, it is important to point out from the perspective of
drug design that not all mammalian cells maintain terminal
lysosomes in the highly acidic pH 4 range characteristic of
macrophage and other frequently studied cell types. Neurons,
for example, have terminal endocytic compartments that are pH
6.0 (Augenbraun et al., 1993), similar to BF.
The system described in this paper provides a way to study
discrete steps in the intracellular transport of endocytosed
material by BF. During incubation at 12°C, detectable amounts
of material are endocytosed from the FP and delivered to
vesicular endosomes that collect near the FP. When cells that
have accumulated material in these vesicles are transferred to
37°C, tubular endosomes reform, and transport to the late, multivesicular compartment proceeds. This system, coupled with
our finding that late endocytic compartments of BF maintain
higher pH values than those of some mammalian cells, should
be useful in developing therapeutic agents that target the
endocytic pathway of these parasites.
The authors thank Susan Hester for assistance with the electron
microscopy. This work was supported by NIH grant AI28427-01A4
to AEB.
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(Received 8 June 1995 - Accepted 3 August 1995)