1 Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA
2 Max-Planck-Institut für Biochemie, D82152 Martinsried, Germany
3 Washington University School of Medicine, St Louis, Missouri, USA
* Author for correspondence (e-mail: clarkem{at}omrf.ouhsc.edu )
Accepted 7 May 2002
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Summary |
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Key words: Dictyostelium, V-ATPase, Contractile vacuole, Endosome, Phagosome, GFP
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Introduction |
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The V-ATPase consists of two functional subcomplexes, V1 and
V0. V1 is a peripheral complex of at least eight
subunits that is responsible for ATP hydrolysis. V0 is an integral
membrane complex of at least five subunits that carries protons across the
membrane. Mutational analysis of Vph1p, a S. cerevisiae isoform of
the large ( 100 kDa) transmembrane subunit of the V0 complex,
has shown that this protein is important in V-ATPase assembly and proton
translocation (Leng et al.,
1996
; Leng et al.,
1998
). The role of the 100 kDa subunit in proton translocation has
led to the suggestion that it is functionally similar to the 30 kDa `a'
subunit of the evolutionarily related F-ATPase, in spite of the difference in
primary structure (Leng et al.,
1996
). The F-ATPase or ATP synthase of bacteria, mitochondria and
chloroplasts is a rotary motor (Cross and
Duncan, 1996
; Junge et al.,
1996
; Noji et al.,
1997
), with intersubunit rotation leading to energy transduction.
In the V-ATPase, which is thought to operate in a similar manner, Vph1p
provides a link between the V0 and V1 sectors and thus
may serve as a stator for the rotary motor
(Landolt-Marticorena et al.,
1999
; Landolt-Marticorena et
al., 2000
).
The predicted amino acid sequence of Vph1p (and its homologues in many
organisms) contains multiple potential membrane-spanning domains; estimates
range from six or seven (Manolson et al.,
1992; Nelson and Harvey,
1999
) to nine (Leng et al.,
1999
). The hydrophilic N-terminal domain of Vph1p has been shown
to face the cytosolic side of the membrane
(Jackson and Stevens, 1997
;
Landolt-Marticorena, 1999
),
but the orientation of the C-terminal domain is not known with certainty.
Random mutagenesis of Vph1p identified certain amino acids between residues
800 and 814 (...LxWVxxxxxFxxxxG...) that, when mutated, led to a substantial
decrease in the assembly of the V-ATPase complex; mutations of the F and G
residues also appeared to affect targeting of this subunit
(Leng et al., 1998
). All of
these residues lay beyond the last presumptive transmembrane domain of the
protein, near the C-terminus. The finding that the C-terminus of Vph1p is
important for enzyme assembly suggested that the C-terminus extended into the
cytoplasm, where it could associate with the V1 sector
(Leng et al., 1998
). However,
a subsequent study from the same laboratory employing cysteine scanning
mutagenesis and labeling by sulfhydryl reagents of differing membrane
permeabilities led to the opposite conclusion, namely, that the C-terminus of
Vph1 probably resides in the vacuole lumen
(Leng et al., 1999
). In the
latter model, (lumenal) amino acids 800-814 were postulated to regulate
protein conformation in cytoplasmic as well as lumenal domains of the protein
(Leng et al., 1999
).
The eukaryotic microorganism Dictyostelium discoideum appears to
contain a single isoform of the V-ATPase 100 kDa subunit (VatM), as determined
by PCR-based assays (Liu and Clarke,
1996) and indicated by the largely completed sequence of the
Dictyostelium genome. VatM is 40% identical to Vpp1, the rat
clathrin-coated vesicle/synaptic vesicle 116 kDa V-ATPase subunit and is 37%
identical to Vph1p of S. cerevisiae. In Dictyostelium, VatM
has been localized to the membranes of two compartments, the contractile
vacuole complex (Fok et al.,
1993
), an osmoregulatory organelle in which the proton pumps
energize water movement from the cytosol into the vacuole complex
(Heuser et al., 1993
), and the
endolysosomal system (Adessi et al.,
1995
), in which the proton pumps serve to acidify the lumen of
endosomes. These compartments are physically distinct
(Gabriel et al., 1999
),
although mutations have been identified that affect both organelles
(O'Halloran and Anderson,
1992
; Ruscetti et al.,
1994
; Bush et al.,
1996
), suggesting that a membrane trafficking relationship exists
between them. There is a substantial difference in the abundance of proton
pumps in the membranes of these two organelles; the pump density in
contractile vacuole membranes is greater by a factor of about ten
(Rodriguez-Paris et al., 1993
;
Clarke and Heuser, 1997
).
We have created a fusion of VatM to green fluorescent protein (GFP) in order to monitor the activity of the contractile vacuole complex and the trafficking of the V-ATPase in the endocytic pathway of living Dictyostelium cells. The fusion protein, VatM-GFP, is correctly targeted to contractile vacuole and endosomal membranes. This fluorescent V-ATPase subunit has enabled us to visualize the distribution of proton pumps in the contractile vacuole system during the interconversion of vacuolar and tubular elements as this organelle takes up and expels fluid. VatM-GFP has also permitted real-time visualization of the trafficking of proton pumps to phagosomes. This marker has revealed that the principal route of delivery of the V-ATPase to phagosomes is as a component of the membrane of pre-existing acidic endosomal vesicles.
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Materials and Methods |
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Preparation and expression of a VatM-GFP fusion protein
A GFP gene containing the S65T mutation was recovered by PCR from the
plasmid pRSET using DeepVent polymerase (New England Biolabs). The forward
primer was
5'-CCCTGCAG(GGTGCA)5ATGAGTAAAGGAGAAGAACTT-3',
which added a PstI site (underlined) and five tandem (gly ala) codons
to the 5'-end of the GFP gene; the reverse primer was
5'-TCTAGAGTTATTGCTCAGCGGTGG-3' (pRSET Reverse, Invitrogen). The
PCR product was cut with BamHI (which cut at a site in the pRSET
polylinker immediately adjacent to the 3' end of the GFP gene) and
cloned into pBluescript-SK- cut with EcoRV and
BamHI. The resulting plasmid, pSK-/(GA)5-GFP,
was cut with PstI and SalI, and the large fragment
containing the GFP gene plus the vector was isolated. The plasmid
pVM1 (Liu and Clarke, 1996)
was used as a template to generate the VatM coding sequence by PCR, again
using DeepVent polymerase. The primers were
5'-CCGAGCTCATGAGCTTTTTAAGACCATCC-3' (forward) and
5'-CCCTGCAGTTCATC TTCAGAAAGAATAC-3' (reverse), which
added SacI and PstI restriction sites at the 5' and
3' ends of the vatM gene, respectively. The PCR product was
cloned into pBluescript-SK- at its EcoRV site, and the
resulting plasmid was cut with PstI and SalI, releasing the
VatM coding sequence. This was inserted into
pSK-/(GA)5-GFP cut with the same two enzymes (as
described above), yielding an in-frame fusion of the VatM coding sequence with
that for (gly ala)5-GFP, the GFP being positioned at the C-terminus
of the fusion protein. (The C-terminus of VatM normally
ends...SEDDE*; the fusion junction is...SEDEQQ(GA)5....)
The restriction fragment encoding VatM-GFP was excised with SacI and
XbaI and cloned into the Dictyostelium expression vector
pDXA-3H (Manstein et al.,
1995
) cut with the same two enzymes, yielding pDXA/VatM-GFP.
AX3-ORF+ cells (Manstein et
al., 1995) growing in HL5 were harvested at a density of
1x106 cells/ml, pelleted by centrifugation and suspended in
electroporation buffer (8.75 mM NaH2PO4, 1.25 mM
Na2HPO4, 2 mM sucrose) at 1x107
cells/ml. One ml of cells was mixed with 20 µg of the pDXA/VatM-GFP
plasmid. The cells were electroporated using a BioRad Gene Pulser at 1.6
kV/cm. Cells were then transferred to 10 cm tissue culture plates and left
overnight in HL5. The next day G418 was added to a final concentration of 5
µg/ml. The cells were diluted 10-fold and plated in 96-well tissue culture
plates for clonal selection. Transformation of VatMpr cells was carried out as
above except that 20 µg pREP (Manstein
et al., 1995
) was co-transfected with pDXA/VatM-GFP. (pREP
contains the Ddp2 ORF sequence necessary for pDXA replication in strains other
than AX3-ORF+, which carries this sequence on the chromosome.)
Transformants were screened by epifluorescence microscopy to identify clones
with bright GFP labeling of intracellular membranes.
Indirect immunofluorescence methods
D. discoideum cells (VatMpr and VatMpr/VatM-GFP) were grown in
suspension in association with K. aerogenes for two or three days
before being examined. Samples were taken from low-density, exponentially
growing cultures and washed free of bacteria by three cycles of centrifugation
in 17 mM Na/KPO4 buffer (pH 6.4). After agar-overlay, the cells
were fixed in the same buffer containing 2% formaldehyde (5 minutes, room
temperature), followed by 1% formaldehyde in methanol (5 minutes, -15°C).
The cells were stained with N4 hybridoma culture supernatant (diluted 1:20),
which labels the V-ATPase A subunit (Fok
et al., 1993), followed by Cy3-conjugated donkey anti-mouse IgG
(1:500, Jackson ImmunoResearch Laboratories). A detailed description of the
staining methods may be found in Clarke et al.
(Clarke et al., 1987
).
Fluorescence microscopy of living cells
Cells from axenic culture were plated on a glass coverslip (5x5 cm)
within a plastic ring 40 mm in diameter. After the cells had settled and
attached, the axenic medium was replaced with 17 mM Na/KPO4 buffer
(pH 6.0) or with fresh nutrient medium diluted 3- to 10-fold in this buffer,
as indicated. For labeling of the endosomal compartment, cells were incubated
in dilute axenic medium containing 2 mg/ml tetramethylrhodamine isothiocyanate
(TRITC)-dextran (Sigma, Mr 70,000) for 30 to 90 minutes.
In some experiments, cells were observed in this medium; in others, it was
replaced with phosphate buffer prior to observation of the cells. For
monitoring phagocytosis, a suspension of living yeast cells in phosphate
buffer was added to the Dictyostelium cells. Just prior to
observation, the Dictyostelium cells were compressed under a thin
layer of agarose prepared as described by Yumura et al.
(Yumura et al., 1984). In some
experiments, the agarose layer had first been soaked in Neutral Red (2.5 µM
in phosphate buffer) in order to label acidic compartments.
For all endocytosis and phagocytosis experiments, a Zeiss LSM 410 microscope equipped with a 100x 1.3 N.A. Plan Neofluar objective was used to collect simultaneous phase contrast and confocal fluorescence images of the cells. For excitation of S65T-GFP, the 488 band of an argon-ion laser was used together with a 515-565 nm filter for emission. For simultaneous recording of GFP and Neutral Red or TRITC-dextran, the 488 nm laser band was used together with the 543 nm band of a He-Ne laser, and emission was split by the use of a 510-525 nm filter for GFP and a 570 nm high pass filter for the fluorescence of other dyes. Simultaneous fluorescence and interference reflection microscopy (IRM) images showing the dynamics of the contractile vacuole complex in living cells (Fig. 3) were obtained using a BioRad Radiance 2000-AGR3 confocal microscope equipped with a Zeiss 100x, 1.4 N.A. planapochromat objective lens.
|
Purification of TRITC-dextran-loaded endosomes from
Dictyostelium cells
D. discoideum cells expressing VatM-GFP were cultivated in
association with K. aerogenes on SM agar plates and harvested prior
to clearing of the bacterial lawn. The cells were suspended at
5x106 cells/ml in AX2 medium containing 0.2 mg/ml
TRITC-Dextran (Sigma, 70,000 Mr) and incubated on a rotary
shaker at 22°C for 6 hours. The cells were harvested by sedimentation,
washed once in cold phosphate buffer and once in cold homogenization buffer
(50 mM Tris-HCl, pH 7.6, 25 mM KCl, 5 mM MgCl2, 100 mM sucrose),
then suspended in 1.5 ml of the same buffer. The cell suspension was placed in
a syringe and forced twice through a stack of two polycarbonate filters
(Nucleopore, 5 µm pore size). The cell lysate was centrifuged at 500
g for 10 minutes at 4°C to remove unbroken cells. The
supernatant was loaded on a Percoll gradient (2.5 ml each of 70%, 65% and 60%
Percoll in homogenization buffer) and spun for 1 hour at 80,000
g. A faintly visible band was recovered from the middle of the
gradient. Epifluorescence microscopy confirmed that TRITC-dextran-containing
vesicles were present in this fraction.
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Results |
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VatMpr cells were used to assess the biological activity of VatM-GFP. These
cells contain approximately normal levels of VatM (and vacuolar proton pumps)
when they are grown axenically, but when they are cultured on bacteria, the
level of VatM drops to about one-third of normal. This deficiency has several
phenotypic consequences, including slow growth of the cells on bacteria and
redistribution of VatA, the peripheral catalytic subunit of the V-ATPase, from
membranes to the cytoplasm (Liu et al.,
2002). We examined whether VatM-GFP expressed in VatMpr cells
could correct these defects. Bacterially grown VatMpr and VatMpr/VatM-GFP
cells were fixed and stained to visualize the distribution of VatA. Expression
of VatM-GFP proved to be sufficient to correct the mislocalization of VatA
(Fig. 1). In VatMpr/VatM-GFP
cells, antibodies to VatA labeled contractile vacuole membranes
(Fig. 1C), as they do in normal
cells, colocalizing with VatM-GFP (Fig.
1E). This was in contrast to the diffuse cytoplasmic staining by
anti-VatA observed in VatMpr cells not expressing VatM-GFP
(Fig. 1A). These results
demonstrate that VatM-GFP is correctly targeted to contractile vacuole
membranes and that it is competent to direct assembly of the V-ATPase enzyme
complex.
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Nonetheless, VatM-GFP proved to be less effective than wild-type VatM in complementing certain other phenotypic defects of VatMpr cells. VatMpr/VatM-GFP cells continued to grow slowly on bacteria and were unable to complete development, defects characteristic of VatMpr cells (data not shown). Possibly, the presence of the GFP moiety on the C-terminus of VatM interferes to some degree with proton translocation, a key function of this subunit.
Orientation of C-terminal GFP on VatM
To examine the orientation of the C-terminus of VatM with its attached GFP
moiety, we isolated endosomes from cells that had been fed TRITC-dextran, a
fluorescent, membrane-impermeant fluid phase marker (see Materials and Methods
for details). Cells were broken by the gentle technique of forcing them
through 5 µm filters, and the cell lysate was fractionated on a Percoll
gradient. Fractions were examined by epifluorescence microscopy using a
rhodamine filter set, and the peak fraction of vesicles loaded with
TRITC-dextran was identified (Fig.
2A). The membranes surrounding these vesicles were brightly
labeled when viewed with the GFP filter set
(Fig. 2B), confirming the
presence of VatM-GFP in endolysosomal membranes.
|
Proteinase K (final concentration 100 µg/ml) was added to an aliquot of this vesicle fraction. After an incubation period of 7 minutes on ice, the vesicles were viewed by epifluorescence microscopy. The vesicles retained their TRITC-dextran fluorescence, indicating that the endosomal membranes had not been perforated (Fig. 2D). However, the GFP signal in the membranes of these vesicles had completely disappeared (Fig. 2E). The only remaining GFP fluorescence was in the membranes of a few tiny vesicles without red endosomal content, possibly fragments of membrane that had become resealed inside out during isolation. These results suggest that GFP, and thus the C-terminus of VatM, is exposed on the cytoplasmic rather than the lumenal side of the endosomal membrane.
VatM contains the same sequence of amino acids near its C-terminus
(...LxWVxxxxxFxxxxG...) that were demonstrated by random mutagenesis to be
important for association of the V1 and V0 domains in
Vph1p (Leng et al., 1998). If,
owing to the attachment of the GFP moiety, the C-terminus of VatM were
erroneously retained in the cytoplasm rather than being delivered to its
proper location in the vacuole lumen, one would expect the improper location
of these key residues to block the function of the C-terminal domain in enzyme
targeting and assembly. However, VatM-GFP is quite functional in these
respects, as described above. Thus, our results lend support to a model in
which the C-terminus of the 100 kDa V-ATPase subunit resides in the
cytoplasm.
Labeling of the contractile vacuole complex and endosomal membranes
with VatM-GFP
In living Dictyostelium cells, the distribution of the fluorescent
signal from VatM-GFP corresponded to that expected for vacuolar proton pumps
on the basis of other methods of analysis (see Discussion). VatM-GFP strongly
labeled membranes of the contractile vacuole complex (both the vacuolar and
tubular elements) and, less strongly, endosomal membranes.
VatM-GFP provides an excellent marker for visualizing the dynamics of the contractile vacuole system. Fig. 3 and Movie 1 (available at jcs.biologists.org/supplemental) show the formation and discharge of multiple contractile vacuoles in an AX3/VatM-GFP cell. Using a confocal microscope, the cell was viewed simultaneously by fluorescence and interference reflection microscopy (IRM). A time series was recorded for a single focal plane near the substratum, since this is the region of the cell where the contractile vacuole complex is primarily situated. The IRM and fluorescence views are shown separately for a portion of the cell in Fig. 3, so that the conversion of individual vacuoles to a network of tubules after emptying can be seen more clearly. The IRM and fluorescence views are overlaid in Movie 1. The highly dynamic and pleiomorphic nature of the contractile vacuole complex is evident, with continuous interconversion of vacuolar and tubular elements of the system as fluid is accumulated and expelled.
VatM-GFP also labels membranes of endolysosomes. The cell shown in
Fig. 4 was incubated in
nutrient medium containing TRITC-dextran for 70 minutes prior to observation.
TRITC dextran is taken up by macropinocytosis, and, over this time interval,
becomes distributed among vesicles at all stages of the endolysosomal pathway
(Hacker et al., 1997;
Jenne et al., 1998
). A focal
plane near the substratum (Fig.
4A) displayed the contractile vacuole complex, whereas focal
planes higher in the cell (e.g., Fig.
4B) were greatly enriched in endosomes filled with the red
fluorescent fluid phase marker. The periphery of most of the endosomes was
labeled with VatM-GFP, which is best seen after subtraction of the red channel
(Fig. 4C). However, this cell,
and others for which an hour or more had elapsed since first exposure to
TRITC-dextran, also contained some endosomes not labeled with VatM-GFP
(arrowheads). We presume these are late endosomes, from which vacuolar proton
pumps have already been retrieved. Typically, the red fluorescence from such
endosomes appeared more intense, indicating that the TRITC-dextran has become
concentrated, as described previously
(Maniak, 2001
).
|
VatM-GFP in phagosomes
Dictyostelium cells are avid phagocytes that consume not only
bacteria, their natural prey, but also larger particles such as yeast cells,
which are especially well suited for visualization by light microscopy. When
cells expressing VatM-GFP are fed yeast particles, the appearance of GFP
fluorescence in the phagosome membrane provides a marker for the delivery of
vacuolar proton pumps to that membrane. Using this assay, we have monitored
the delivery of proton pumps to phagosomes, their presence during the
digestion of yeast particles and their disappearance from phagosome membranes
prior to expulsion of the indigestible remnants of the particle.
VatM-GFP is delivered to the membrane of newly formed phagosomes in the membranes of small vesicles. These small vesicles begin clustering at the phagosome membrane about 1 to 2 minutes after uptake of the yeast particle. If the Dictyostelium cells are bathed in medium containing Neutral Red, a vital dye that passes through cell membranes and becomes trapped in acidic compartments, these small vesicles have red lumens, indicating that they are acidic (Fig. 5A). If, instead, the Dictyostelium cells are pre-incubated with TRITC-dextran, a membrane-impermeant fluid phase marker that enters cells by endocytosis, the content of the small GFP-rimmed vesicles is also red, demonstrating that they are endosomal vesicles (Fig. 5C). Within three or four minutes of particle uptake, TRITC-dextran from these endosomes can be detected within the phagosome. Both Neutral Red and TRITC-dextran can sometimes be visualized as discrete patches under the phagosome membrane, presumably representing recent sites of vesicle fusion. Fig. 6A shows a cell labeled with Neutral Red, and Fig. 6B shows a cell labeled with TRITC-dextran; the latter cell had been washed free of TRITC-dextran before yeast particles were added. When the red channel is removed from the second image (Fig. 6C), it can be seen that the green VatM-GFP rim is continuous around the outer side of the mound of red marker, confirming that local fusion had occurred between the phagosome membrane and a vesicle filled with TRITC-dextran. Neutral Red labeled presumptive fusion sites along the phagosome membrane in a similar manner (Fig. 6A), indicating that the fusing vesicles were acidic. Thus, vacuolar proton pumps are delivered to phagosomes by pre-existing, acidic, endosomal vesicles, and the content of these vesicles is released into the phagosome.
|
|
Within the phagosome, endosomal fluid-phase markers accumulate
predominantly at the necks of budded yeast cells, because the acute curvature
at a bud neck precludes a snug fit of the phagosome membrane at this point, as
previously described (Schneider et al.,
2000). The yeast strain used here is partially impaired in
cytokinesis, so most yeast particles have one or more buds. Localization of
the marker in the lumen of the phagosome is easier to detect in such cells
than in unbudded yeast, where the marker spreads out in a uniform thin layer
under the phagosome membrane (not shown).
The rapidity with which the fluorescence signal from VatM-GFP in the membrane of a new phagosome reaches an intensity similar to that present in `older' phagosomal membranes seems to depend on the availability of appropriate endosomes. If the endosome pool is large, as in the cell shown in Fig. 5B-D [and the upper cell shown in Movie 2 (available at jcs.biologists.org/supplemental)], then this occurs over a period of 3 or 4 minutes after clustering of the vesicles. If the pool is small, then substantially more time is required. Two cells in the process of phagocytosing yeast particles are shown in Movie 2; the second (lower) cell is also shown in Fig. 7. These cells were preincubated in medium containing TRITC-dextran in order to label their endosomal compartments prior to the addition of yeast. When we began this recording, the upper cell was ingesting its first yeast particle; this cell contained an abundance of VatM-GFP-labeled endosomal vesicles that clustered rapidly at the membrane of the new phagosome (Movie 2). Expression of VatM-GFP, which varies in the cell population, was not high in this cell, so the endosomes were more brightly labeled with TRITC-dextran than with VatM-GFP. TRITC dextran accumulation at the neck of the budded yeast particle was evident at 3 minutes, and several red patches were visible on the phagosome membrane at 3 to 4 minutes after particle uptake. However, VatM-GFP labeling of the phagosome membrane was hazy in this weakly expressing cell.
|
The second cell, just below, was expressing a high level of VatM-GFP. It phagocytosed a yeast particle about two minutes after the first cell had done so (Fig. 7A). This second cell already contained several phagosomes whose membranes were strongly labeled with VatM-GFP, and the budded yeast particles within those phagosomes had bright red collars of TRITC-dextran at the bud necks. In fact, virtually all of the TRITC-dextran and VatM-GFP available in the endosomal compartments of this cell appeared to have been provided to these earlier phagosomes, which had merged to form a multiparticle compartment. Close inspection of this video sequence suggests that fusion between the new phagosome and the pre-existing compartment occurred between 5 and 6 minutes after uptake of the new yeast particle. Starting in that interval, VatM-GFP appeared and grew steadily brighter in the membrane of the new phagosome, reaching an intensity close to that of the pre-existing phagosomes by about 9 minutes after uptake (Fig. 7D). About 6 minutes after uptake, TRITC-dextran could be observed not only at the yeast bud neck but also at the point of contact between the new phagosome and the pre-existing phagosomal compartment, indicating that membrane fusion had taken place (arrow in Movie 2; Fig. 7C,D). We conclude that the delivery of proton pumps to phagosomes occurs predominantly through fusion of pre-existing endosomes and phagosomes with the new phagosome and that this takes place within a few minutes of uptake of a yeast particle.
As is evident in Fig. 6A, Neutral Red does not stain living yeast cells. Thus, a landmark in the phagocytic pathway is the point at which a phagocytosed yeast particle becomes permeabilized, allowing Neutral Red to stain its cytoplasm. Cytoplasmic staining implies that lysosomal enzymes have been at work on the yeast particle. Fig. 8 shows an example of a yeast particle that has reached this stage of digestion. The two images were recorded 30 seconds apart. Note that VatM-GFP was still present in the phagosome membrane at the time when the yeast cytoplasm became accessible to Neutral Red.
|
Late phagosomes and retrieval of VatM-GFP
We found that VatM-GFP disappears from the phagosome membrane before the
cell exocytoses the indigestible remnants of a yeast particle. In cell
populations that had been incubated with yeast particles for more than two
hours, there were many unlabeled phagosomes. If the cells had been
preincubated with TRITC-dextran, the yeast particles within the
VatM-GFP-deficient phagosomes had a collar of TRITC-dextran encircling the bud
neck (Fig. 9, arrows),
indicating that these phagosomes were not newly formed, but had previously
fused with endosomal vesicles and acquired TRITC-dextran and hence also
VatM-GFP. (The cell shown in Fig.
9 also contained a large multi-particle phagosome whose membrane
was brightly labeled with VatM-GFP; this phagosome presumably represents an
earlier stage in the pathway.)
|
It is also possible to capture the actual expulsion event, in which a cell exocytoses the remains of a digested yeast particle. Fig. 10 shows a cell that contained three phagosomes, each with a yeast particle. Two of the yeast particles had buds, allowing TRITC-dextran accumulation at the bud neck to be visualized. One of the budded yeast particles resided in a phagosome whose membrane was devoid of VatM-GFP, whereas the other phagosome containing a budded yeast particle still had a GFP rim. The yeast particle from the unlabeled phagosome was expelled from the cell during the course of observation (Fig. 10C-D and Movie 3; available on-line at www.biologists.org/supplemental ). The rapidity of expulsion (between one frame and the next, 5 seconds apart) is in contrast to uptake, which is more leisurely, extending over about 15 seconds (Movie 2).
|
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Discussion |
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We found that VatM-GFP was correctly targeted to the same
Dictyostelium membranes in which wild-type VatM is found, namely,
membranes of the contractile vacuole complex and the endolysosomal system.
Furthermore, VatM-GFP appeared to be competent to direct assembly of the
enzyme. That is, expression of VatM-GFP in a mutant cell line deficient in
wild-type VatM (Liu et al.,
2002) resulted in redistribution of VatA, the catalytic subunit of
the V1 sector, from a diffuse cytoplasmic distribution in the
mutant cells to a normal location on contractile vacuole membranes in
VatM-GFP-expressing cells. These results indicate that VatM-GFP is targeted to
the proper endomembranes, where it serves as the locus for enzyme
assembly.
The distribution of VatM-GFP within the Dictyostelium contractile
vacuole complex was consistent with that of vacuolar proton pumps observed by
other methods. Both electron microscopy of freeze-dried cells and
immunostaining of fixed cells with anti-V-ATPase antibodies have shown that
vacuolar proton pumps are distributed throughout all membranes of the
contractile vacuole system (Heuser et al.,
1993; Clarke and Heuser,
1997
). Likewise, as we show here, VatM-GFP brightly labeled all
elements of the contractile vacuole system, both vacuolar and tubular,
throughout their cycles of filling and discharge. These data also confirmed
that a reversible interconversion of tubular and cisternal elements of the
system occurs as fluid is accumulated and expelled, as previously inferred
from observations of living cells by interference reflection microscopy and
styryl dye staining (Heuser et al.,
1993
) and by the use of dajumin-GFP as a label for contractile
vacuole membranes (Gabriel et al.,
1999
). In mitotic cells, where the contractile vacuole system
fragments into many small vacuoles (Zhu
and Clarke, 1992
; Gabriel et
al., 1999
), these vacuoles continue to be brightly labeled with
VatM-GFP and remain fully active in fluid accumulation (J.H. and M.C.,
unpublished). Thus, VatM-GFP in the contractile vacuole system of living cells
behaves as expected for a V-ATPase marker.
Population measurements of the trafficking of vacuolar proton pumps
and the evolution of endosomal pH
The presence of vacuolar proton pumps in membranes of endosomes and
phagosomes from Dictyostelium and mammalian cells and their probable
role in acidification of these organelles were first recognized by observing
the effects of inhibitors and antibodies
(Yamashiro et al., 1983;
Padh et al., 1989
;
Sato and Toyama, 1994
).
Immunostaining revealed a rapid enrichment of V-ATPase subunits in phagosome
membranes during the first 10 minutes or so after particle uptake
(Pitt et al., 1992b
;
Temesvari et al., 1994
;
Nolta et al., 1994
; Rebazek et
al., 1997). More sophisticated analytical methods such as microsequencing
(Adessi et al., 1995
) and
peptide mass fingerprinting (Garin et al.,
2001
) verified the presence of V-ATPase subunits in endosomal
membranes. In Dictyostelium, which neutralizes late endosomes and
then exocytoses the indigestible remnants of endocytosed or phagocytosed
material (reviewed by Maniak,
1999
), the level of V-ATPase subunits was found to drop in
membranes of late endosomes (Nolta et al.,
1994
).
The kinetics of pH evolution along the endosomal pathway in
Dictyostelium were defined by Satre and co-workers, who employed both
31P-NMR (Brenot et al.,
1992) and fluorescence spectroscopy
(Aubry et al., 1993
) to analyze
fluid phase endocytosis. Mathematical modeling of the transit of FITC-dextran
in cells incubated in nutrient medium
(Aubry et al., 1997
) indicated
that endosomes are acidified rapidly, reaching a minimal pH value of
5.1
about 15 minutes after uptake. During the next 40 minutes, the pH rises,
stabilizing at
pH 6.3. After a total of about 60 minutes, pseudo-first
order efflux occurs. Taken together, these studies of Dictyostelium
cell populations demonstrated that a temporal correlation exists between high
levels of V-ATPase subunits in endosomal membranes and acidification of
endosomal contents, and between reduced levels of the V-ATPase and subsequent
reneutralization.
Single-cell observations of pH evolution and protein trafficking in
Dictyostelium
The present study and other recent studies discussed below have employed
imaging methods that allow the behavior of individual cells to be tracked.
This approach provides information that the population measurements described
above cannot. Microscopic observations of living cells make apparent the great
heterogeneity of cell behavior, for example, with respect to the elapsed time
between exposure of a cell population to a suspension of particles and the
uptake of a particle by any given cell. For population measurements
(biochemical, enzymatic, or spectroscopic), this heterogeneity has the effect
of broadening the time interval during which a given step in a pathway appears
to occur and of blurring any boundaries between steps. Thus, when dealing with
asynchronous behaviors, observation of individual cells provides a much better
definition of the temporal relationship between events.
Using VatM-GFP as our marker for trafficking of vacuolar proton pumps, we have found that this enzyme is brought to new phagosomes by acidic endosomal vesicles that cluster at the phagosome membrane, beginning 1 to 2 minutes after the uptake of a yeast particle. Judging from the intensity of the GFP signal, the concentration of proton pumps in the membrane of the new phagosome increases over the next few minutes. If a cell contains many VatM-GFP-positive endosomal vesicles, the fluorescence in the membrane of the new phagosome reaches a level similar to that of pre-existing phagosomes by about 4 minutes after particle uptake. Proton pumps can also be acquired through fusion with existing phagosomes.
These data indicate that in Dictyostelium the proton pumps are recycled in the endosomal pathway and that most of the pumps delivered to a new endosome are derived from endosomes that had already progressed to a later stage of the pathway. Of course, the proton pumps must initially be fed into the pathway through Golgi-derived vesicles or tubular extensions of the Golgi apparatus, but the contribution of de novo synthesized pumps seems to be minor relative to recycled ones, and it could not be detected by our methods.
Although the same isoform of VatM populates membranes of endosomes and of the contractile vacuole complex, we saw no evidence for a direct transfer of proton pumps from contractile vacuole membranes to phagosomes. Occasionally yeast-containing phagosomes and elements of the contractile vacuole system abutted each other in a cell, but this seemed to be a random and transient situation. In addition, VatM-GFP labeling of the contractile vacuole complex remained very bright even in cells that had taken up several yeast particles, as is evident in Movie 3 and also in Fig. 7C and D, which shows a contractile vacuole discharge.
A mixture of FITC and TRITC dextrans has been used to monitor pH changes
during endosomal transit in Dictyostelium
(Jenne et al., 1998;
Maniak, 1999
). Since FITC
fluorescence is quenched at low pH, whereas TRITC fluorescence is insensitive
to pH, the differential pH sensitivity of the two probes results in color
changes from yellow-orange (extracellular and newly ingested) to red
(acidified) to yellow-orange (neutralized) as the dextran mixture proceeds
along the endosomal pathway. This technique is suitable not only for
spectroscopic measurements but also for the visualization of pH changes in
individual cells by confocal microscopy. Consistent with our results, Maniak
was able to detect the rapid acidification of a macropinosome 50 to 60 seconds
after uptake (Maniak, 2001
).
He also detected, at an indeterminate time after uptake, the neutralization of
a phagosome over a period of 2 minutes, followed by its exocytosis 16 minutes
later (Maniak, 1999
).
Since most aspects of macropinosome uptake are similar to phagocytosis,
including the changes in membrane composition (reviewed by
Cardelli, 2001) and the
clustering of vesicles at the endosomal membrane during the first few minutes
after uptake (Schneider et al.,
2000
), we expect that the delivery of proton pumps to
macropinosomes and phagosomes proceeds in a similar manner, namely, by fusion
with pre-existing acidic endosomes. However, fusion of a single acidic
endosome, contributing both proton pumps and acidic solutes, may be sufficient
to produce a large impact on the pH of a new macropinosome, whereas fusion of
multiple endosomes may be needed to produce a comparable effect on a large
phagosome. Further studies in individual cells will be needed to determine the
relationship between endosome volume, the time course of acidification, and
the time required for proton pump density to reach maximal levels in an
endosomal membrane.
Fusions of cytoskeletal and signal-transduction proteins with GFP have
allowed several steps in phagosome formation and maturation to be visualized
in living Dictyostelium cells (reviewed by
Maniak, 1999;
Rupper and Cardelli, 2001
).
Particles such as yeast, bacteria and latex beads are taken up in individual
phagosomes through an actin- and G-protein-dependent process (Peracino et al.,
1997). Actin and actin-binding proteins dissociate from the new phagosome
within about one minute of internalization
(Maniak et al., 1995
;
Hacker et al., 1997
;
Rupper et al., 2001a
). Rab7,
which may regulate homotypic fusion events between early endosomes
(Laurent et al., 1998
), labels
the membrane of new phagosomes within 60 seconds of internalization, as
monitored using a GFP-Rab7 fusion protein
(Rupper et al., 2001a
).
Golvesin, a membrane protein, is delivered to the membrane of new endosomes
within 4-5 minutes of uptake. Like VatM-GFP, it is brought by vesicles that
cluster at the endosomal membrane
(Schneider et al., 2000
).
Lysosomal enzymes that contain N-acetylglucosamine-1-phosphate residues (e.g.,
cysteine proteinases) are delivered to phagosomes within 3 minutes of
ingestion, as determined by antibody staining of fixed cells
(Souza et al., 1997
). Hence,
the timing of the delivery of Rab 7, golvesin, and this class of lysosomal
enzymes is similar to what we have observed for VatM-GFP. A clear implication
is that several of these proteins may be delivered as components of the same
vesicle.
In analyzing trafficking behavior, the nature of the phagocytosed particle
must also be considered, as demonstrated previously
(Souza et al., 1997). These
workers found that in cells fed bacteria, two classes of lysosomal enzymes
were delivered to phagosomes with different time courses. The first class
(those modified with N-acetylglucosamine-1-phosphate residues) was found in
endosomes after about 3 minutes, whereas the second (those modified with
mannose-6-phosphate residues; e.g.,
-mannosidase and
ß-glucosidase) was found in endosomes after about 15 minutes.
Importantly, this temporal separation between the delivery of the two classes
of lysosomal enzymes was not observed for phagosomes containing latex beads
(Souza et al., 1997
;
Rupper et al., 2001a
).
Similarly, the V-ATPase was not retrieved from phagosome membranes of cells
fed large polystyrene or latex beads, even after 300 minutes of chase
(Rezabek et al., 1997
). Such
observations suggest that Dictyostelium amoebae may utilize different
trafficking pathways depending on the chemistry of the phagocytosed particle,
as has been reported for macrophages (Oh
and Swanson, 1996
).
Later stages of phagosome processing in Dictyostelium
The transit time of phagocytosed particles is longer than for endocytosed
fluid. We have observed neutralization of yeast-containing phagosomes 75
minutes after uptake (data not shown), and Cardelli's group has reported
neutralization of bacteria-containing phagosomes
60 minutes after uptake
(Rupper et al., 2001b
;
Rupper and Cardelli, 2001
).
The processing of yeast and bacteria is therefore likely to be similar and
delayed relative to the neutralization of macropinosomes, which begins about
45 minutes after fluid uptake (Aubry et
al., 1993
; Maniak et al., 2001). Rupper et al. reported that, late
in the pathway, neutralized phagosomes fuse to form multi-particle
compartments, which they termed `spacious' phagosomes
(Rupper et al., 2001b
). We too
have observed multi-particle phagosomes, but, as shown in Movie 2 and
Fig. 7, these can be formed by
very early fusion events and may even be instrumental in the delivery of
proton pumps to new phagosomes. In our study, multi-particle phagosomes (Figs
7 and
9) were VatM-GFP-positive and
not `spacious' but had a membrane that fit snugly about the yeast particles.
Furthermore, late phagosomes, identified by the presence of TRITC-dextran and
the lack of proton pumps, typically contained single yeast particles (Figs
9 and
10).
We have occasionally observed phagosomes that contained a large volume of fluid around the yeast particle, but these were very rare. In one case, a VatM-GFP-labeled phagosome with a single yeast particle expanded from a snug fit to a roomy, fluid-filled compartment in less than 10 seconds, suggesting that the change occurred through fusion with a large endosome (data not shown). In another case, a roomy multi-particle phagosome containing three yeast particles was converted to a tight fit and then became separated into three vesicles each with a single yeast particle, two of which were consecutively exocytosed (A. Benjak and G.G., unpublished). Thus, in our studies, roomy phagosomes were not confined to a particular stage of the endocytic pathway, and their rarity suggested that they represented an infrequent or highly transient state.
Our data indicate that exocytosis of the indigestible remnants of a yeast
particle occurs after retrieval of proton pumps from the phagosome membrane.
We anticipate that the retrieval of proton pumps will prove to be linked to
the rise in lumenal pH that occurs in late endosomes and phagosomes, although
this has not been explicitly demonstrated. As noted above, Maniak reported
exocytosis of a yeast particle 16 minutes after the phagosome had been
neutralized (Maniak, 1999),
while Rupper et al. reported that spacious phagosomes still persisted more
than an hour after neutralization had been observed
(Rupper et al., 2001b
). Hence,
experimental conditions may have a profound impact on this aspect of cell
behavior. In future studies, it will be important to monitor simultaneously
the retrieval of VatM-GFP and the timing of neutralization and exocytosis.
Relationship to endosome-phagosome interactions in mammalian
cells
There are many common features between the endocytic pathways in
Dictyostelium and in mammalian cells, but in some respects our data
differ from those reported for mammalian cells. A common theme is the
interactions between endosomes and phagosomes. In J-774 macrophages, Mayorga
et al. showed that within 10 minutes of phagocytosis, both endosomes and
lysosomes fused with phagosomes (Mayorga
et al., 1991). This same group reported that radiolabeled protein
originally associated with phagocytosed particles could be found 18 minutes
later in a non-phagosome vesicle population that displayed some endosomal
characteristics (Pitt et al.,
1992a
). Desjardins et al. reported that the fluid content of
latex-bead-containing phagosomes and late endocytic organelles became
completely intermixed within 60-90 minutes after the uptake of the beads
(Desjardins et al., 1994
),
whereas the transfer of a late endosomal membrane protein (lamp-2) required
about 6 hours. Using video microscopy, these workers detected multiple
transient interactions between late endocytic organelles (pre-loaded with gold
particles) and newly formed latex-bead-containing phagosomes. Similarly,
Jahraus et al. demonstrated that there was retrograde traffic of contents
between lysosomes and late endosomes
(Jahraus, 1994
). Together,
these observations suggested that phagosomes acquire markers from endocytic
organelles through successive interactions in a gradual and regulated
process.
On the basis of such data, it has been proposed that the interaction
between endocytic organelles and phagosomes is not necessarily a complete
fusion but rather a partial exchange of fluid and membrane proteins (reviewed
by Tjelle et al., 2000). This
`kiss and run' hypothesis (Desjardins,
1995
) has been supported by the finding that large and small
endocytosed molecules appear in phagosomes with different kinetics
(Wang and Goren, 1987
;
Desjardins et al., 1997
) and
that phagosomes do not acquire endocytic solutes and membrane proteins
simultaneously (Desjardins et al.,
1994
).
The endosome-phagosome interactions in our study were recorded at 5 second intervals and were taken from single focal planes. This temporal and spatial resolution is not adequate to allow us to track individual endosomal vesicles and determine with certainty that a particular endosome-phagosome interaction resulted in complete fusion. However, it is clear that endosomal vesicles clustered at the membrane of a new phagosome during the first few minutes after uptake and that, within the limits of our resolution, VatM-GFP and the TRITC-dextran content of the endosomes were delivered to the new phagosome with the same time course. Thus, the simplest explanation for our results is that endosomal vesicles containing TRITC-dextran in their lumen and VatM-GFP in their membrane, fused completely with the phagosome. We speculate that vesicles with different lumenal content and membrane composition may be capable of fusing with phagosomes at specific stages of their maturation.
The use of multiple probes will be required to determine whether other
proteins are delivered to phagosomes as components of the same vesicle
population as VatM-GFP. The recent demonstration that a fusion of the cysteine
proteinase cathepsin B with GFP is correctly targeted to endosomes
(Lincke et al., 2001) opens
the way for the simultaneous tracking of pairs of proteins labeled with
color-shifted GFP variants. These methods should make it possible to monitor
the delivery and retrieval of vacuolar proton pumps in direct comparison with
the delivery of lysosomal enzymes, thereby providing new insights into the
mechanisms of endo-lysosomal trafficking.
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Acknowledgments |
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References |
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