Department of Microbiology and Immunology and Feist-Weiller Cancer Center, LSU Health Sciences Center, Shreveport, LA 71130, USA
* Author for correspondence (e-mail: jcarde{at}Isuhsc.edu)
Accepted 10 July 2002
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Summary |
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Key words: Phagocytes, Phagosome fusion, Rab14, Dictyostelium
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Introduction |
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Phagosome-phagosome fusion is an important but poorly characterized
maturation process occurring in mammalian cells infected with certain
bacteria. As an example, phagosomes containing virulent strains of
Helicobacter pylori, a human pathogen thought to cause gastritis,
gastric ulcers and gastric adenocarcinomas, fuse to form multiparticle
phagosomes in macrophages. Phagosome fusion requires bacterial protein
synthesis and may facilitate survival of intracellular bacteria. Less virulent
strains (type II) lacking the cag pathogenicity island are killed and
do not stimulate phagosome fusion (Allen et
al., 2000). Phagosomes containing Chlamydia trachomatis
elementary bodies fuse with each other to form an intracellular inclusion
necessary for replication of the bacteria
(Majeed et al., 1999
).
Finally, Coxiella burnetti, the causative agent of Q fever, and
Leishmania amazonensis, the agent of leishmaniasis, can undergo
heterotypic fusion with particle-containing phagosomes as well as undergoing
homotypic fusion (Dermine et al.,
2001
; Howe and Mallavia,
2000
).
Dictyostelium discoideum, a single-celled eukaryotic organism, is
a professional phagocyte and is a useful genetically tractable system in which
to study macropinosome and phagosome formation and maturation
(Cardelli, 2001). For instance,
Legionella replicates and prevents phagosome-lysosome fusion in
Dictyostelium (Hagele et al.,
2000
; Solomon et al.,
2000
), mimicking the situation observed in mammalian cells
(Roy et al., 1998
).
Furthermore, many of the steps involved in particle internalization and
processing appear to be similar to those found in mammalian cells, including
homotypic phagosome fusion (reviewed in Rupper and Cardelli, 2001a).
Phagosomes mature through at least three different stages in
Dictyostelium. First, newly formed phagosomes contain primarily
plasma-membrane-derived proteins and F-actin and associated proteins
(Rezabek et al., 1997
). Next,
many of these early proteins are recycled from phagosomes, which then fuse
sequentially with hydrolase- and glycosidase-rich lysosomes, and the
phagosomal lumens become acidic (Rezabek
et al., 1997
; Rupper et al.,
2001a
). After about 60 minutes, the intra-phagosomal pH rises to
near neutral levels and phagosomes begin to fuse together in a PI 3-kinase-
and protein kinase B (PKB)/Akt-dependent process, creating spacious multiple
particle phagosomes (Rupper et al.,
2001b
). PI 3-kinase and PKB also regulate macropinocytosis and
endosome fusion but not phagocytosis
(Buczynski et al., 1997b
;
Rupper et al., 2001c
). The
increase in pH, regulated by PI 3-kinase, is important in regulating homotypic
fusion, although the mechanisms required for this are still unclear.
Rab GTPases have also been implicated in phagosome formation and maturation
in Dictyostelium. For instance, Rab7 and RabB appear to act in a
positive fashion to regulate particle internalization
(Rupper et al., 2001a) (E.H.,
unpublished). Rab7 is delivered to the phagosome within one minute of
formation and is retained on the phagosome during the entire maturation
process. Cells overexpressing dominant-negative Rab7 are not defective in the
formation of spacious phagosomes, but these phagosomes do not contain normal
levels of glycosidases or the lysosomal membrane protein, LmpA, suggesting
that Rab7 regulates phagosome-lysosome membrane traffic
(Rupper et al., 2001a
). As
observed in mammalian cells, Rab11 is also implicated in the internalization
of particles (Harris et al.,
2001
), although in Dictyostelium this Rab may act in a
negative fashion.
RabD, a Dictyostelium GTPase related to mammalian Rab14, appears
to regulate macropinocytosis and endo-lysosomal fusion
(Bush et al., 1996). RabD is
primarily found on the contractile vacuole network of membranes, an organelle
important in osmotic regulation and, in lesser amounts, in the endo-lysosomal
pathway (Bush et al., 1994
).
Cells expressing dominant-negative RabD (RabDN121I) contained a
morphologically altered contractile vacuole, were osmotically sensitive and
exhibited a reduced rate of endocytosis
(Bush et al., 1996
).
Furthermore, lysosomal fusion to generate post-lysosomes was also delayed
(Bush et al., 1996
). These
studies did not examine the role of RabD in regulating phagocytic processes.
To begin to determine the role of RabD in phagocytosis and phagosomal
maturation, we have biochemically analyzed cells expressing dominant-negative
and constitutively active forms of this GTPase. We demonstrate that RabD (1)
regulates phagocytosis, (2) plays a prominant role in phagosome-phagosome and
endo-lysosome fusion and (3) and acts upstream or in a parallel pathway with
respect to PI 3-kinase to regulate fusion.
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Materials and Methods |
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Phagocytosis assay
To measure the rate of phagocytosis, cells in log phase of growth in T-175
flasks were harvested and resuspended to 3.0x106 cells/ml in
HL-5. After a recovery period of 15 minutes in shaking suspension at 21°C,
cells were incubated with an equal volume of HL-5-containing red fluorescent
beads (Molecular Probes, FluoSpheres carboxylate-modified microspheres 1.0
µm, crimson fluorescent) at a final concentration of 100 beads/cell. At
each time point, 3.0x106 cells were harvested, washed twice
in cold HL-5, washed once in cold sucrose buffer (5 mM Glycine, 100 mM
sucrose, pH 8.5) and lysed in 1 ml of sucrose buffer with 0.5% Triton X-100.
Bead fluorescence was measured with a Hitachi F-4010 spectrofluorimeter at an
excitation of 625 nm and emission of 645 nm. The total amount of particles
internalized was calculated on the basis of fluorescence per microgram of
protein to correct for differences in cell size or titer.
Phagosome and endo-lysosome fusion assay
To measure rates of phagosome fusion, we used FITC-labeled bacteria
(fl-bacteria) or deep blue latex beads (Sigma, L-1398) to visualize individual
phagosomes. Cells were placed in shaking suspension and allowed to phagocytose
fl-bacteria for 10 minutes followed by three washes with fresh HL-5. Next,
cells were allowed to settle down on coverslips in HL-5 to initiate the 30
minute chase period. Coverslips were gently dipped in fresh HL-5 three times
to wash the remaining fl-bacteria off followed by fixation of cells in 1%
formaldehyde in HL-5. Coverslips were dipped again in HL-5 (10 times) to wash
off any remaining fl-bacteria and mounted on slides to be examined with the
fluorescence microscope. Cells with 10 fl-bacteria per cell were
examined, and the number of fl-bacteria in each phagosome was assessed using
both phase-contrast and fluorescence optics. Control experiments indicated
that particles were internalized individually.
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Results |
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Cells expressing constitutively active RabDQ67L accumulate
large endo-lysosome-like vesicles supporting a role for RabD in lysosome
fusion
Cells expressing RabDN121I (a dominant-negative form of this
GTPase) contain a larger number of acidic lysosomes and correspondingly a
smaller number of larger less acidic postlysosomes, as compared with control
cells (Bush et al., 1994;
Bush et al., 1996
), suggesting
a defect in lysosome fusion. To further examine the role of RabD in lysosomal
fusion, a stable mutant strain of Dictyostelium discoideum was
constructed that inducibly expresses HA-tagged RabDQ67L (a
constitutively active mutant that preferentially binds to GTP). Removal of
folate from the medium induces transcription of the discoidin promoter, which
leads to an accumulation of activated RabD. Western blot analysis of a
selected strain indicated that the level of the exogenously expressed mutant
protein was two- to threefold higher than the level of the endogenous form
(Fig. 2) when cells were grown
for two days without folate. Microscopic examination of cells expressing
RabDQ67L (bottom panel; Fig.
2) indicated that they accumulated a greater number of enlarged
vesicular structures as compared with the same clone of cells growing in the
presence of folate (middle panel; Fig.
2), conditions that prevent expression of exogenous RabD. Control
cells growing in the presence or absence of folate contained vesicles
comparable in size to those observed in RabDQ67L-expressing cells
grown in the presence of folate (data not shown for wildtype). Despite the
accumulation of five or more vesicles >1.5 µm in diameter per cell,
RabDQ67L-expressing cells increase in number at the same rate as
control cells in the absence of folate
(Fig. 2, top panel), suggesting
that the presence of these enlarged vesicles did not adversely affect growth
rate.
|
To determine if the large vesicles represented lysosomes, the following
experiments were performed. First, control cells and
RabDQ67L-expressing cells were exposed to FITC-dextran in growth
medium for 5 minutes followed by a chase period in dextran-free growth medium.
A portion of the cells was quickly fixed in formaldehyde immediately following
the pulse, and at 10 and 60 minutes following the initiation of the chase
period. Representative DIC and fluorescent images of these cells are shown in
Fig. 3. The 5 minute pulse
period is sufficient to load primarily macropinosomes with fluid phase
(Seastone et al., 1998),
whereas the 10 minute and 60 minute chase period have been demonstrated to
load lysosomes and post-lysosomes, respectively
(Padh et al., 1993
). Following
a 5 minute pulse with FITC-dextran, control cells
Fig. 3A,B) and
RabDQ67L-expressing cells (Fig.
3G,H) contained one to three fluorescently labeled vesicles
>1.0 µm in diameter, indicating that only a small percentage of the
large vesicles in RabDQ67L expressing cells were newly formed
macropinosomes. Following a chase of 10 minutes, control cells
(Fig. 3C,D) contained primarily
fluorescent vesicles the size of lysosomes as consistent with previous studies
(Bush et al., 1996
). In
addition, most of the large vesicles in RabDQ67L-expressing cells
(Fig. 3I,J) contained
FITC-dextran, suggesting most of these enlarged vesicles might be lysosomes on
the basis of their kinetics of loading with internalized fluid. Finally,
following a 60-minute chase period, control
(Fig. 3E,F) and
RabDQ67L expressing cells (Fig.
3K,L) contained a few fluorescent postlysosomes 1-2 µm in
diameter. Few of these fluorescently labeled post-lysosomes corresponded to
the enlarged vesicles.
|
Next, control and RabDQ67L-expressing cells were pulsed with FITC-dextran for 5 minutes, followed by a chase period of up to 60 minutes in growth medium lacking this fluorescent marker. At the time points indicated in Fig. 4A, cells were collected by centrifugation, and the intra-endosomal pH was measured as a function of FITC fluorescence as described in Materials and Methods. Consistent with the large vesicles being acidic lysosomes, the kinetics of movement of internalized fluid to the most acidic compartments were identical for control and RabDQ67L-expressing cells (Fig. 4A). Internalized fluid reached the most acidic compartments 15 minutes after internalization for both cell lines and accumulated in less acidic compartments over the next 45 minutes with the same kinetics. Together, these data suggest that most of the large endocytic vesicles in RabDQ67L-expressing cells are acidic, and they acquire fluid with the kinetics observed for the loading of smaller lysosomes in control cells.
|
Finally, live cells were incubated with LysoSensor Green DND-189 (Molecular
Probes) for 2 minutes and immediately visualized by fluorescence microscopy.
LysoSensor is only fluorescent under acidic conditions (pH<5.0).
Fig. 4 indicates that most of
the large vesicles in RabDQ67L-expressing cells were acidic (D,E),
suggesting these vesicles had acquired the V-H+-ATPase proton pump.
In the endosomal pathway the proton pump is most enriched in lysosomes,
suggesting that the large vesicles in RabDQ67L-expressing cells may
be lysosomes. As expected, control cells contained a greater number of smaller
acidic vesicles (Fig. 5C). The
large acidic vesicles were also purified using a previously published magnetic
fractionation approach (Temesvari et al.,
1996a) and demonstrated by western blot analysis to be enriched in
lysosomal enzymes and the proton pump (results not shown).
|
GFP-RabDQ67L is enriched in large acidic lysosomes that
arise by fusion
If RabDQ67L is directly involved in the formation of enlarged
acidic lysosomes, one would expect to find this activated Rab associated with
these compartments as observed for phagosomes. To test this hypothesis, stable
cell lines inducibly expressing GFP-RabD and GFP-RabDQ67L were
subjected to the same pulse-chase regiment using FITC-dextran as described in
Fig. 3. Panels B, D and F of
Fig. 5 indicate that the
GFP-RabD wild-type protein distributed in control cells in the reticular
network of the CV, a result consistent with previous published data on the
basis of immunofluorescence microscopy
(Bush et al., 1994). GFP-RabD
only infrequently ringed macropinosomes (compare panel A with B), lysosomes
(compare C with D) or post-lysosomes (compare E with F). By contrast, a small
percentage of GFP-RabDQ67L localized to membranes of macropinosomes
(G,H) and predominantly with membranes of large acidic lysosomes (I,J),
consistent with a direct role for this activated Rab protein in the formation
of these large vesicles. Only rarely was GFP-RabDQ67L found to
associate with post-lysosomes (K,L).
To determine if RabDQ67L increased fusion between endolysosomes, as observed for phagosomes, cells were pulsed with RITC-dextran for 5 minutes, washed with growth medium for 2 minutes and then exposed to FITC-dextran for an additional 5 minutes. Cells were gently fixed after a chase period of 5 minutes, and images were collected using a digital camera attached to a fluorescence microscope. Fig. 6 indicates that a larger percentage of the endocytic vesicles in RabDQ67L-expressing cells (Fig. 6H) contained both RITC and FITC-dextran as compared with control cells (panel G), indicating an increased rate of fusion between different temporal classes of endo-lysosomes. Images, from over 100 cells for each strain were analyzed, and the number of vesicles that contained both fluorescent markers was calculated. The results indicate that at each chase point following the sequential internalization of two different fluid phase markers, the rate of fusion of temporally distinct early endosomes in RabDQ67L-expressing cells was twice that observed for control cells (data not shown). Interestingly, efflux rates of fluid were identical in mutant and wild-type cells, indicating that the accumulation of enlarged lysosomes does not significantly influence endosomal membrane trafficking (data not shown).
|
RabD regulates phagocytosis
The following series of experiments was done to determine the role of RabD
in the phagocytic pathway. To measure the rate of phagocytosis, both
mutant-expressing cell lines and the parent strain, Ax4, were assayed
for their ability to internalize latex beads. A representative experiment
shown in Fig. 7A indicates that
the rate of phagocytosis of latex beads in cells expressing
RabDQ67L was more than twice the rate observed for the Ax4
parent strain. Conversely, cells expressing the dominant-negative mutant
RabDN121I internalized beads at approximately 50% of the rate of
the parent strain. A more complete quantitative analysis of particle uptake
was performed and subjected to statistical analysis.
Fig. 7B reveals that at the 30
minute time point, RabDQ67L cells had internalized more than twice
as many beads as the control (n=5, P<0.0001) and over
five times the number of beads as the dominant-negative cell lines
(n=5, P<0.001). These data suggest that RabD may be
involved in regulating the rate of internalization of beads. Comparable
results were observed using E. coli, suggesting that RabD also
regulates internalization of a physiologically relevant particle (data not
shown).
|
RabD regulates phagosomal fusion
Phagosome-phagosome fusion occurs in Dictyostelium and in
mammalian cells containing a variety of intracellular pathogens, including
species of Helicobacter and Chlamydia. Homotypic phagosome fusion in
Dictyostelium depends on the activities of PI 3-kinase and PKB and is
dependent on increases in pH beginning 45-60 minutes following internalization
of particles (Rupper et al.,
2001b). In preliminary experiments, we observed that bacterially
infected cells expressing RabDQ67L contained more large
multiparticle phagosomes than control cells, suggesting RabD might regulate
homotypic phagosome fusion. To quantify the rate of phagosome fusion, we
pulsed cells in shaking suspension for 10 minutes with FITC-bacteria and
allowed the cells to chase for 60 minutes while they settled on coverslips.
Under these conditions bacteria enter as single particles
(Harris et al., 2002
), and 60
minutes of chase corresponds to the linear portion of the phagosome fusion
curve. A representative fluorescence and phase contrast micrograph is shown in
Fig. 8 and reveals that cells
expressing activated RabD (middle panel) contain a greater number of
multiparticle phagosomes as compared with control cells (left panel). At this
time point, a smaller fraction of wild-type cells have multiparticle
phagosomes containing two to three bacteria, whereas, by contrast, most of the
cells expressing RabDQ67L contained multiple particle phagosomes
each with more than three particles per phagosome. To confirm that phagosomes
were fusing with each other as opposed to particles being internalized as
aggregates, a fusion assay was employed in which cells were sequentially
pulsed with both FITC-labeled bacteria and deep blue latex beads (fluorescing
weakly in the rhodamine channel). Cells were first pulsed with the
FITC-bacteria for 10 minutes, washed for 2 minutes in cold HL-5, pulsed with
latex beads for 10 minutes and then chased in fresh HL-5 for 30 minutes. Most
of the phagosomes in control cells (Fig.
9A,B) contained only one particle, whereas in RabDQ67L
cells (C,D), most phagosomes contained both FITC-bacteria and latex beads,
proving that phagosomes formed at different times and fused together at a
higher rate.
|
|
The average number of particles per cell and particles per phagosome was enumerated, and the rate of fusion was calculated as described in the Materials and Methods. Phagosome-phagosome fusion occurred at five times the rate in the RabDQ67L cells versus control cells (Fig. 10A). We performed separate phagosomal fusion experiments with RabDN121I-expressing cells and found that the rate of fusion in this mutant was less than half the rate found in the control cells (Fig. 10B).
|
PI 3-kinase and PKB play important roles in the regulation of
phagosome-phagosome fusion during the late stages of phagosome maturation
(Rupper et al., 2001a). To
determine if PI 3-kinase was involved in the RabD-mediated fusion pathway, we
treated RabDQ67L-expressing cells with LY294002, a specific
inhibitor for PI 3-kinase, and repeated the phagosome fusion experiments. Figs
8 and
10 indicate that the rate of
phagosome fusion in RabDQ67L cells treated with LY294002 was
significantly reduced compared with the untreated RabDQ67L cells.
Specifically, drug-treated RabDQ67L-expressing cells treated with
PI 3-kinase inhibitors contained phagosomes that were similar in size and
contained the same number of bacteria as the control cell line
(Fig. 8, far right panel).
These results confirm that PI 3-kinase regulates homotypic fusion of
phagosomes and that PI 3-kinase apparently acts downstream of or in parallel
with RabD to regulate fusion.
To determine if multiparticle phagosomes contained RabD, consistent with this GTPase playing a direct role in fusion, we pulsed GFP-RabDQ67L-expressing cells with RITC-labeled bacteria for 10 minutes, followed by a chase in fresh HL-5 for 30 minutes. Live cells were examined using a fluorescence microscope. Fig. 11 indicates that GFP-RabDQ67L ringed multiparticle phagosomes in nearly all of the cells observed, supporting a direct role for RabD in regulating phagosome fusion. In addition, most of the phagosomes in the GFP-RabDQ67L cells contained multiple bacteria when the chase period was extended beyond 30 minutes, consistent with the results presented earlier.
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Discussion |
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This study provides additional evidence supporting a role proposed a few
years ago for RabD in regulating lysosome fusion
(Bush et al., 1996). Cells
expressing RabDQ67L contained enlarged endocytic vacuoles, which
appeared by multiple criteria to be enlarged lysosomes. In support of this
observation, these large vesicles were acidic, they received internalized
fluid with the kinetics of normal lysosomes and they contained the proton pump
and hydrolytic lysosomal enzymes. The data presented here, as well as that
previously reported (Bush et al.,
1996
), are most consistent with a RabD-mediated increase in fusion
of lysosomes. For instance, RabDN121I-expressing cells contained
fewer post-lysosomes (formed by fusion of lysosomes) than control cells,
whereas cells expressing RabDQ67L contained a greater number of
large lysosomes. Furthermore, GFP-RabDQ67L ringed the large
lysosomes consistent with a direct role in regulating fusion. Finally,
sequential pulse-chase periods, using two different fluorescent fluid phase
markers, indicated an increase in the rate of mixing between endo-lysosomal
compartments in cells expressing RabDQ67L.
Lysosome fusion, like phagosome fusion, is also not a process unique to
Dictyostelium. Earlier studies involving cell-fusion assays
demonstrated that lysosomes could exchange membrane proteins and content
proteins in mammalian cells (Ferris et
al., 1987; Deng and Storrie,
1988
). More recently, it has been demonstrated that lysosomes can
undergo heterotypic fusion with late endosomes as well as homotypic fusion
(Ward et al., 2000a
). It has
been proposed that Rab7 may regulate fusion of mammalian lysosomes, although
this remains controversial.
The enlarged lysosomes accumulating in RabDQ67L-expressing cells
are reminiscent of the enlarged lysosomes observed in a variety of cells from
patients with the inherited disorder Chediak-Higashi Syndrome
(Ward et al., 2000a). The
CHS gene encodes a large protein named Beige/Lyst
(Barbosa et al., 1996
) that may
regulate fusion of lysosomes. Disruption of a related gene in
Dictyostelium, lvsB, also led to the accumulation of large acidic
lysosomes (Harris et al.,
2002
) and the formation of multiparticle phagosomes. These
enlarged vesicles are most likely to be generated as a result of an increase
in homotypic lysosome and phagosome fusion, suggesting that LvsB normally acts
to negatively regulate membrane fusion. Thus, homotypic lysosome and phagosome
fusion may be negatively and positively regulated by proteins like LvsB
(Lyst-like) and RabD (Rab14), respectively, and the final size of lysosomes or
number of particles in a phagosome will depend on the relative balance between
the activities of these two opposing groups of protein.
Moderate overexpression of RabDQ67L increased the rate of
phagocytosis, whereas expression of RabDN121I decreased the rate of
phagocytosis, results consistent with RabD acting directly to regulate
internalization of particles. In fact, it has been hypothesized that the
formation of phagosomal membrane cups may require directed exocytosis of
internal membranes, a process most probably regulated by Rab GTPases
(Cox et al., 2000). In support
of this, mammalian and Dictyostelium Rab11 has been demonstrated to
regulate phagocytosis, perhaps by directing recycling endosomal and
contractile vacuole membrane, respectively, to the forming phagocytic cup
(Cox et al., 2000
). RabD, as
well as Rab11, is highly enriched in the CV system of membranes and regulates
the structure and function of this organelle
(Bush et al., 1996
) and
conceivably could traffic membrane to the forming phagocytic cup.
This current study also revealed that RabD regulated the rate of homotypic
phagosome fusion. Homotypic phagosome fusion also has been observed to occur
in cells infected with a variety of intracellular pathogens, including
Helicobacter, Coxiella and Chlamydia
(Allen et al., 2000;
Dermine et al., 2001
;
Howe and Mallavia, 2000
;
Majeed et al., 1999
). It has
been proposed that formation of multiparticle phagosomes may aid the survival
of bacteria in professional phagocytes. Essentially nothing is known
concerning the molecular factors that regulate phagosome fusion in mammalian
cells. Also, no published data is available concerning the function of Rab14,
the human Rab most related to RabD, although a recent report demonstrates that
this Rab is enriched in phagosomes containing latex beads
(Garin et al., 2001
). On the
basis of amino-acid sequence homology, we hypothesize that Rab14 may function
in mammalian cells such as RabD in Dictyostelium to regulate
lysosomal-phagosomal fusion.
Most of what we currently understand about how Rab proteins regulate
membrane fusion comes from studies analyzing the Rab5-mediated homotypic
fusion of early endosomes (Mills et al.,
1999) and ypt7-mediated fusion of yeast vacuoles
(Eitzen et al., 2000
). In the
former case, Rab5 recruits a variety of effector proteins, including EEA1,
rabaptin-5 and rabex-5, during the tethering stage of early endosomal
homotypic fusion (Lippe et al.,
2001
; Stenmark and Gillooly,
2001
). In addition, the action of PI 3-kinase is required to
generate PI-(3)P, a phosphoinositide that facilitates attachment of EEA1 to
early endosomes. Homotypic yeast vacuole fusion has been intensely studied
(Wickner and Haas, 2000
), and
a large complex of proteins has been identified that regulates this process.
Although ypt7 (a Rab7-related GTPase) has been demonstrated to be involved in
the regulation of vacuole fusion in yeast
(Eitzen et al., 2000
), and Rab
GTPases have been implicated in mammalian lysosome fusion
(Ward et al., 2000b
), the
biochemical identity of the mammalian lysosomal fusion-regulating Rab protein
remains unknown.
Interestingly, PI 3-kinases also play a role in regulating lysosome- and
phagosome-homotypic fusion in Dictyostelium
(Buczynski et al., 1997a;
Rupper et al., 2001b
),
although in this latter case the product required appears to be
phosphoinositide (3,4,5)-trisphosphate. PKB/Akt, in addition to the PI
3-kinases PIK1 and PIK2, regulates homotypic phagosome fusion in
Dictyostelium (Rupper et al.,
2001b
). As demonstrated here, phagosome-phagosome fusion is
reduced to control level in cells expressing RabDQ67L exposed to
LY294002, an inhibitor of PI 3-kinases, suggesting that PI 3-kinase acts in
parallel with RabD or as a downstream effector. RabD and PI 3-kinase may
functionally interact in at least two ways. First, RabD may act upstream of PI
3-kinase in a similar manner to Rab5, which binds to two distinct PI
3-kinases, hVPS34 and p85-p110. It is proposed that Rab5 transiently interacts
with these PI 3-kinases and associates with EEA1, Rabaptin-5, Rabenosyn-5 and
Rabex-5 to form a complex that facilitates fusion of early endosomes
(Christoforidis et al., 1999
;
Nielsen et al., 2000
). Second,
RabD may act in a parallel pathway with PI 3-kinase to act upon a common
downstream effector that is primarily responsible for membrane fusion.
A search of the most current Dictyostelium protein database
indicates that proteins homologous to many of the mammaliantethering factors
(described above) exist, and these proteins conceivably participate in
lysosome-lysosome and phagosome-phagosome fusion (J.C., unpublished). Other
proteins already implicated in lysosome fusion in Dictyostelium
include profilin (Temesvari et al.,
2000) and Scar (a WASp-related protein)
(Seastone et al., 2001
), the
ATPase proton pump (Temesvari et al.,
1996b
), LmpA (a lysosomal membrane protein)
(Temesvari et al., 2000
) and
RtoA (Brazill et al., 2000
).
Finally, affinity purification approaches using GST-RabD have identified a
potential number of effector proteins, including a calcium-binding protein and
a dynein subunit that together with the other proteins named above regulates
phagosome fusion. It remains to be determined if any of these proteins act as
RabD effectors and/or activators and if they also regulate phagosome
fusion.
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Acknowledgments |
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References |
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