(Journal of Leukocyte Biology. 2000;68:627-632.)
© 2000
by Society for Leukocyte Biology
Dynamics of rab5 activation in endocytosis and phagocytosis
R. L. Roberts*,
M. A. Barbieri*,
J. Ullrich2 and
P. D. Stahl*
* Department of Cell Biology and Physiology and
Molecular Microbiology, Washington University, School of Medicine, St. Louis, Missouri
Correspondence: Philip D. Stahl, Department of Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110. E-mail: pstahl{at}cellbio.wustl.edu
 |
ABSTRACT
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Fluid-phase endocytosis is stimulated by H-ras-linked growth
factor receptors and this stimulation requires activation of rab5. We
utilized a GFP-rab5a:wt fusion protein to monitor GFP-rab5a:wt
activation in living fibroblasts and in J774 macrophages. Control CHO
cells that expressed GFP-rab5a:wt were cultured in serum-free
conditions and showed GFP-rab5a:wt localized to endosomal vesicles with
a mean diameter of 0.3 ± 0.1 µm. Endosome fusion, membrane
ruffling, and pinosome formation were rarely detected in these cells.
Coexpression of H-ras:G12V, a constitutively active H-ras mutant that
activates rab5a, in cells resulted in marked enlargement of labeled
endosomes (mean diameter 0.7 ± 0.2 µm) and large numbers of
giant GFP-rab5a:wt-positive endosomes were present. Time-lapse
recordings showed abundant fusion among giant labeled endosomes, and
membrane ruffling and pinosome formation were commonly observed.
Alterations in GFP-rab5a:wt endosome structure and activity in cells
expressing H-ras:G12V were linked to rab5a activation because these
changes were identical to those found in cells expressing
GFP-rab5a:Q79L, a constitutively activated rab5a mutant. Furthermore,
cells co-expressing H-ras:G12V and GFP-rab5a:S34N, an inactive rab5a
mutant, exhibited no evidence of H-ras:G12V-induced endosome
enlargement. To observe changes in endosome structure and activity that
directly followed activation of GFP-rab5a:wt, we performed time-lapse
recordings of cells cultured overnight in serum-free media after
addition of EGF. EGF caused a rapid increase in endosome fusion and in
membrane ruffling activity. Membrane ruffling was often associated with
GFP-rab5a:wt-positive vesicle (pinosome) formation at the base of
membrane ruffles. Endosome and pinosome fusion were common in
EGF-stimulated cells. Phagocytosis is also regulated by GFP-rab5a:wt.
J774 macrophages that expressed GFP-rab5a:wt showed transiently
activation and recruitment of GFP-rab5a:wt to newly formed phagosomes
that contained rhodamine-labeled Escherichia coli. These
studies show that GFP-rab5a:wt activation results in dynamic
alterations in the structure and activity of the early endosomal and
early phagosomal elements.
Key Words: Escherichia coli • epidermal growth factor • endosime fusion • membrane ruffling
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INTRODUCTION
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Endocytosis is the process mammalian cells utilize for the uptake
of essential molecules from their external environment. This material
is taken into cells by membranous vesicles derived from the plasma
membrane (endocytic vesicles) and once internalized it is directed to a
system of internal vesicles called endosomes [1
]. In
endosomes a number of important sorting events are carried out and
include the separation and segregation of the material taken into the
cell from the components of the endocytic vesicle. The latter
components are returned (recycled) to the plasma membrane for use in
subsequent rounds of endocytosis, whereas the endocytosed material is
mostly sorted into lysosomes. The endosomal system is composed of a
series of distinct elements, some of which are thought to perform
sorting (rab5-positive endosomes) or recycling (rab11-positive
endosomes) activities [2
]. Late endosomes (rab7-positive
endosomes) appear to participate in routing material destined for
degradation from the rab5-positive sorting compartment to the lysosomal
compartment.
Rab5 is an important regulator of early events in endocytosis and
phagocytosis [3
, 4
]. Over-expression of
wild-type rab5a or a constitutively activated rab5a mutant that is
deficient in GTP hydrolysis (rab5a:Q79L) maximally stimulate
endocytosis in fibroblasts [5
]. This stimulation is in
part a result of increased endosome fusion because rab5 is rate
limiting for in vitro endosome-endosome [6
]
as well as in vitro endosome-phagosome fusion
[7
]. Rab5 activation and endosome fusion are further
linked by the observation that cells expressing rab5a:Q79L develop
giant rab5a-positive endosomal vesicles [5
,
8
]. Time-lapse recordings of cells expressing rab5a:Q79L
have shown that the giant vesicles arise primarily by fusion of smaller
rab5a-positive endosomal vesicles [8
]. These
observations support the idea that the stimulation of endocytosis
caused by rab5 activation is dependent upon an increase in the size of
the endosomal compartment.
It is becoming clear that rab5a activity is linked to the activation of
an H-ras-linked signal transduction pathway. It has long been known
that expression of oncogenic H-ras stimulates endocytosis in cells
[9
] but only recently has this been directly linked to
rab5 activation [11
, 12
]. Previous work in
our laboratory has shown that fluid-phase endocytosis is stimulated
after activation of a signal transduction cascade that includes H-ras,
phosphoinositide-3-kinase and protein kinase B [10
11
12
].
Furthermore, the stimulation of endocytosis by this signal transduction
pathway requires rab5 activation, since a dominant-negative rab5a
mutant deficient in GTP binding (rab5a:S34N) blocks the stimulation of
endocytosis by this signaling pathway. In fibroblasts
expressing H-ras:G12V [12
] in epidermal growth factor
(EGF)-stimulated fibroblasts [13
], the increase in
endocytosis is similar to that observed after overexpression of
rab5a:Q79L. The mechanisms by which H-ras:G12V or EGF stimulation
result in stimulation of endocytosis are currently unknown.
Furthermore, whether H-ras:G12V or EGF activation of rab5a in living
cells is sufficient to result in the formation of giant
GFP-rab5a:wt-positive endosomes is also not known. Further work is
required to determine the identity of the molecules that link receptor
activation and giant endosome formation.
In this report we have explored the dynamic changes in the structure
and activity of the rab5a-positive endosomal and phagosomal
compartments in living cells after activation by either expression of
H-ras:G12V (or exposure to EGF) and rhodamine-Escherichia
coli, respectively. We used green fluorescent protein (GFP) tagged
rab5a:wt and time-lapse confocal microscopy of living cells to examine
the regulation of rab5a activation by H-ras:G12V and EGF. Rab5a
activation by these agents results in the formation of giant
GFP-rab5a:wt-positive vesicles and links stimulated endocytosis with
enlargement of the early endosomal compartment. Furthermore, this
approach has allowed real-time visualization of the changes in membrane
dynamics that have been suggested by previous studies. In addition, the
giant vesicles appeared to form by two distinct processes that include
vesicle fusion and new vesicle (macropinosome) formation. These data
illustrate the importance of rab5a:wt-mediated endosome fusion in
stimulated endocytosis.
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METHODS
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Cell culture and reagents
The light microscopy described utilized either Chinese hamster
ovary (CHO) or a J774 macrophage cell line. Viral infection with
recombinant Sindbis virus was carried out as described previously
[10
] except that serum-free conditions were maintained
throughout the procedure. Sindbis virus-directed expression of
H-ras:G12V CHO cells occurred more slowly than reported in BHK cells
[10
], and in these studies CHO cells were examined 4–8
h post-infection. Sindbis virus infection of J774 macrophages was
inefficient and resulted in expression of GFP-rab5a:wt in less than 1%
of the cells and GFP-rab5a-expressing cells were examined 18–24 h
after addition of virus to the cells. Rhodamine-transferrin and
rhodamine-labeled E. coli were prepared with
carboxytetramethyl-rhodamine succinimidyl ester (Molecular Probes),
following the method suggested by the manufacturer. E. coli
in serum-free media were applied to cells (10 bacteria/cell) on
coverslips and centrifuged for 30 s at 2000 rpm at 4°C. The
coverslips were then rinsed and immediately mounted on glass slides and
imaged.
Construction and expression of eGFP-rab5a and eGFP-rab5a:Q79L
PeGFP-rab5a:wt and peGFP-rab5a:Q79L were constructed by
polymerase chain reaction (PCR) amplification of the human rab5a and
rab5a:Q79L complementary DNA from pH2’JC1. The PCR products were
inserted into a peGFP expression vector [14
] using the
XbaI site. CHO cells were grown to 80% confluency in 10-cm Petri
dishes and transfected with 20–30 µg DNA with the use of Lipofectin
Reagent (GIBCO-BRL). Stably transfected clonal lines were isolated
after incubation in selective growth medium (0.5 µg/mL G418 for 7–10
days and checked for GFP fluorescence on endosomal vesicles.
Time-lapse fluorescence videomicroscopy of GFP-rab5a in living
cells
Cells grown on glass coverslips were inverted on glass slides
made into a narrow flow-cell by two strips of vacuum grease
[8
] and were examined by either differential
interference contrast or confocal microscopy. Time-lapse confocal
microscopy was carried out on a Bio-Rad MRC 1024 confocal microscope
using a x63, 1.4 NA bright field objective and fluorescene and
rhodamine filter sets. Confocal sequences were collected as Bio-Rad pic
files and were converted to bitmaps for use in Photoshop 4.0 for pixel
intensity quantification and endosome measurement.
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RESULTS
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Activation of GFP-rab5a:wt in living fibroblasts by H-ras:G12V
Expression of a H-ras:G12V stimulates endocytosis, and rab5a:wt
activation is required for this effect [11
,
12
]. To characterize changes in the structure and
activity of the early endosomal compartment that occur after
H-ras-dependent activation of rab5a:wt, we performed time-lapse
recordings of CHO cells expressing GFP-rab5a:wt before and after
expression of a constitutively active H-mutant (H-ras:G12V).
GFP-rab5a:wt-expressing CHO cells were cultured overnight in serum-free
medium and then recorded by confocal microscopy. As shown in
Figure 1A
, GFP-rab5a:wt was primarily localized on punctate cytoplasmic
structures uniformly distributed throughout the cytoplasm. These
structures co-localize with internalized rhodamine-transferrin, but
show little co-localization with mannose 6 phosphate receptor and
LAMP-1 immunoreactivity (data not shown), identifying them as early
endosomes. The labeled vesicles were relatively small, uniform in size,
and exhibited a mean diameter of 0.3 ± 0.1 µm. The labeled
vesicles were observed to move laterally over short distances in the
peripheral cytoplasm but docking and fusion events among labeled
vesicles were rarely identified. Membrane ruffling and pinosome
formation were not observed.
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Figure 1. Expression of a constitutively active GFP-rab5a mutant (Q79L) induces
the formation of enlarged endosomes. (A) CHO cell expressing
GFP-rab5a:wt cultured overnight in serum-free medium.
GFP-rab5a:wt-labeled endosomes were small, vesicular, and uniform in
size. Most of the labeled endosomes were less than 1 µm in diameter.
Time-lapse recordings showed that labeled endosomes moved laterally
over short distances in the peripheral cytoplasm but endosome fusion
events were rarely recorded. (B) CHO cell expressing GFP-rab5a:Q79L.
Expression of the activated rab5a mutant (Q79L) caused endosome
enlargement. Time-lapse recordings showed that the endosome enlargement
was secondary to increased endosome fusion. Scale bar = 10 µm.
See supplemental material for QuickTime movie version of Figure 1A
.
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|
We next examined early endosome structure in CHO cells that
expressed a constitutively active, GTPase-deficient, rab5a mutant
(GFP-rab5a:Q79L) in order to document changes that occurred in cells
after activation of GFP-rab5a:wt. As shown in Figure 1B
, cells
expressing GFP-rab5a:Q79L exhibited marked endosome enlargement (mean
diameter 0.8 ± 0.2 µm), and large numbers of giant endosomes
(2–5 µm diameter) were observed in all cells. Endosome fusion was
readily identified but membrane ruffling and pinosome formation were
not commonly seen.
As shown in Figure 2A
, co-expression of H-ras:G12V in the GFP-rab5a:wt-expressing cells
using a Sindbis virus expression system also led to a dramatic
rearrangement of the surface membrane and the early endosomal
compartment. The most notable features included prominent ruffling and
marked endosome enlargement (mean diameter 0.7 ± 0.2 µm). We
confirmed H-ras overexpression in these cells by immunocytochemistry
(data not shown). Giant endosomes with diameters ranging from 2 to 8
µm were present in most cells. To directly test whether activated
rab5a:wt is required for the increased endosome fusion and endosome
enlargement observed in H-ras:G12V-expressing cells, we next
co-expressed GFP-rab5a:S34N, an inactive rab5a mutant, and activated
H-ras (G12V) in fibroblasts. Membrane ruffling was increased in
co-expressing cells, however, there was no evidence of endosome fusion,
and no significant endosome enlargement was present (Fig. 2B)
. These
results confirmed that H-ras:G12V is a strong activator of GFP-rab5a:wt
and resulted in early endosome enlargement of a magnitude similar to
the constitutively active rab5a mutant (rab5a:Q79L). Furthermore,
H-ras:G12V-induced early endosome enlargement required activated
GFP-rab5a:wt because no endosome enlargement was induced in cells
coexpressing activated H-ras and an inactive rab5a mutant
(GFP-rab5aS34N).
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Figure 2. Expression of H-ras:G12V activates GFP-rab5a:wt and induces the
formation of enlarged endosomes. (A) CHO cell co-expressing
GFP-rab5a:wt and H-ras:G12V showed marked endosome enlargement compared
to control cells expressing GFP-rab5a:wt (see Fig. 1A
). Time-lapse
recordings showed that the formation of giant vesicles occurred by
endosome fusion and pinosome formation. Arrowhead, membrane ruffles.
(B) CHO cell co-expressing GFP-rab5a:S34N and H-ras:G12V showed no
evidence of endosome enlargement. Time-lapse recordings showed no
evidence of endosome fusion but membrane ruffling was increased
(arrowheads), consistent with expression of H-ras:G12V. Scale bar = 10 µm. See supplemental material for QuickTime movie versions of
Figure 2A
and 2B
.
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Time-lapse recordings of cells co-expressing GFP-rab5a:wt and
H-ras:G12V showed that the giant endosomal vesicles arose by two
distinct mechanisms that included endosome fusion (Fig. 3
) and macropinosome formation (Fig. 4
). In Figure 3
, note that the diameter of the giant endosome
depicted in the frame labeled 72s is greater than 1.5 times the
diameter of the largest endosome depicted in frame 0s, confirming that
the endosome fusion contributes to the formation of giant endosomes. In
cells co-expressing GFP-rab5a:wt and H-ras:G12V, macropinosomes were
observed to form near regions of membrane ruffling (Fig. 4)
and were
observed to rapidly acquire GFP-rab5a:wt from the cytosol within
seconds after their formation. Newly formed GFP-rab5a:wt labeled
vesicles (macropinosomes) were highly fusogenic, and time-lapse
recordings often demonstrated fusion events between newly formed
pinosomes with other GFP-rab5a:wt-labeled endosomes and pinosomes (see
supplemental material for QuickTime movie version of Figs. 3
and 4
).
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Figure 3. H-ras:G12V activates GFP-rab5a:wt and results in increased endosome
fusion. A montage of individual frames from a time-lapse sequence taken
from the cell depicted in Figure 2A
shows a cluster of endosomes that
undergo a number of endosome fusion events. Note that the diameter of
the giant endosomal vesicle in frame 72s is greater that 1.5 times the
diameter of the largest endosomal vesicle in frame 0s. The endosome
cluster shown was located adjacent to the arrowhead in Figure 2A
. See
supplemental material for Quicktime movie version of Figure 3
.
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Figure 4. H-ras:G12V expression stimulates macropinosome formation. A montage of
individual frames from a time-lapse sequence taken from the cell
depicted in Figure 2A
shows the rapid acquisition of GFP-rab5a:wt on a
newly formed macropinosome. The macropinosome formed in a peripheral
region of the cell adjacent to an area of increased membrane ruffling
activity. Scale bar = 5 µm. See supplemental material for
Quicktime movie version of Figure 4
.
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Activation of GFP-rab5a:wt in living fibroblasts by EGF
We have shown above that H-ras:G12V activates GFP-rab5a:wt and
results in the formation of giant fluorescent endosomes. H-ras has been
shown to be activated by receptors like EGF receptor and we next set
out to directly visualize changes in early endosome structure that
accompany rab5a activation by recording cells immediately after
exposure to EGF. GFP-rab5a:wt-expressing cells cultured overnight in
serum-free medium were exposed to EGF and immediately recorded by
confocal microscopy (Fig. 5
). As shown in Figure 5
, EGF stimulation resulted in increased
membrane ruffling within 1–2 min, and this was accompanied by
increased fusion among labeled vesicles. Also, within a few minutes
after the addition of EGF, GFP-rab5a:wt-labeled vesicles (pinosomes)
formed at the base of membrane ruffles (Fig. 6
). Most often these vesicles were small and ranged between 0.2 and
0.4 µm in diameter. Occasionally, however, the labeled vesicles were
enlarged (arrowhead, Fig. 6
), with diameters that ranged from 2 to 4
µm in diameter. Endosome and pinosome fusion were common in
EGF-stimulated cells (see supplemental material for QuickTime movie
version of Figs. 5
and 6
) and this activity resulted in an increase in
the size of the GFP-rab5a:wt endosomal elements (mean diameter 0.6 ± 0.2 µm).
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Figure 5. EGF stimulation activates GFP-rab5a:wt and induces the formation of
enlarged endosomes. A cluster of GFP-rab5a:wt-expressing cells before
(A) and 18 min after (B) the addition of 10 µg/mL EGF to the medium
is shown. EGF stimulation results in marked endosome enlargement
(arrowheads). A time-lapse recording of this cell cluster shows that
the increase in endosome size resulted from increased endosome fusion
activity that rapidly occurred after the addition of EGF. In addition,
EGF stimulated membrane ruffling and at later times was with
macropinosome formation. See supplemental material for Quicktime movie
version of Figures 5
and 6
.
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Figure 6. EGF stimulation induced membrane ruffling and the accumulation of
GFP-rab5a:wt pinosomes. A montage of individual frames taken from the
middle portion of the EGF-stimulated cell shown in the time-lapse
sequence depicted in Figure 5
. A pinosome rapidly acquires GFP-rab5a:wt
immediately after its formation adjacent to an area of increased
membrane ruffling. Typically, membrane ruffling activity was stimulated
within 90 s after addition of EGF. Times in seconds relative to
first frame are shown. Scale bar = 6 µm. See supplemental
material for Quicktime movie version of Figures 5
and 6
.
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Activation of GFP-rab5a:wt during phagocytosis in J774 macrophages
To determine the dynamic interaction of GFP-rab5a:wt with newly
formed phagosomes we used Sindbis virus to express GFP-rab5a:wt in J774
macrophages. Labeled macrophages were then presented rhodamine-labeled
E. coli, and their interaction with bacteria was recorded by
confocal microscopy. Membrane ruffles were often observed in regions
where bacteria contacted the macrophages (see supplemental material for
QuickTime movie version of Fig. 7
; arrowheads indicate membrane ruffles associated with phagocytosis
events). Phagocytosis was heralded by the rapid accumulation of
GFP-rab5a:wt on phagosomal membranes surrounding internalized bacteria,
most often occurring in association with membrane ruffles. The
fluorescent label appeared to be derived directly from the cytosol
because no labeled donor vesicles were observed. The labeling of early
phagosomes by GFP-rab5a:wt was transient and persisted for only 1–2
min. We next characterized the dynamic interaction of rhodamine-labeled
E. coli with J774 macrophages expressing GFP-rab5a:Q79L.
Phagosomes in macrophages expressing the constitutively active rab 5
mutant showed persistent labeling with the fluorescent protein for up
to 1 h after phagocytosis, and phagosome-phagosome fusion events
were readily observed (data not shown).
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Figure 7. GFP-rab5a:wt is transiently activated in J774 macrophages during
phagocytosis of rhodamine-E. coli. GFP-rab5a:wt was
expressed in J774 macrophages through the use of a Sindbis virus
expression system. Bacteria were added to cells on coverslips, and
phagocytosis events were recorded by time-lapse confocal microscopy.
Two phagocytosis events are depicted and each showed transiently
GFP-rab5a:wt labeling (arrowheads). The phagocytosis events shown
occurred near membrane ruffles that are most apparent in time-lapse
Quicktime movies. See supplemental material for Quicktime movie version
of Figure7
.
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DISCUSSION
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In this study we have used GFP-tagged rab5a:wt and time-lapse
confocal microscopy to follow the cellular localization of rab5a after
its activation by either H-ras:G12V or EGF in living fibroblasts and
during phagocytosis of rhodamine-labeled E. coli in
macrophages. We show that in fibroblasts, co-expression of GFP-rab5a:wt
and activated H-ras:G12V results in the rab5a-dependent stimulation of
endosome fusion and marked endosome enlargement, which fits well with
previous biochemical studies where H-ras activation resulted in
rab5a-dependent stimulation of endocytosis and increased endosome size
[11
, 12
]. We have shown that H-ras:G12V is
a strong activator of GFP-rab5a:wt and resulted in an increase in
endosome size of a magnitude similar to that observed in cells
expressing a constitutively activated rab5a mutant (GFP-rab5a:Q79L).
The requirement for GFP-rab5a:wt activation in the generation of the
H-ras:G12V-induced endosome enlargement was further illustrated by the
experiments that showed that co-expression of an inactive rab5a mutant
(rab5a:S34N) blocked this H-ras:G12V effect. Still, other
H-ras:G12V-dependent changes, including increased membrane ruffling,
were not inhibited in the GFP-rab5a:S34N-expressing cells as previously
reported [11
, 15
]. We also show that EGF
stimulation causes GFP-rab5a:wt activation. However, marked endosome
enlargement was not a consistent feature in EGF-stimulated cells. Thus,
in the experiments reported here it appears that EGF is a more modest
activator of GFP-rab5a:wt than H-ras:G12V. The CHO cells used in these
studies expressed endogenous levels of EGF receptor. It would be
interesting to test whether stronger activation of GFP-rab5a:wt would
result in cells that express increased levels of the EGF receptor.
Another new finding reported here is that pinosomes rapidly acquire
GFP-rab5a:wt after their formation and then undergo fusion with other
GFP-rab5a:wt-labeled pinosomes and endosomes. Macropinosomes are highly
dynamic organelles and are known to be highly fusogenic with other
endosomal vesicular elements [16
, 17
].
Because rab5a regulates early fusion events after endocytosis, rab5a
participation in macropinosome fusion is not unexpected. It has
previously been reported in macrophages that macropinosomes are rab7
positive and rab5 negative at early time points by immunohistochemical
criteria [18
]. One explanation for these differences is
that pinosome formation in macrophages and fibroblasts are regulated
differently. However, it is possible that antibody staining is not
adequately sensitive to detect endogenous levels of rab5a on macrophage
pinosomes or that rab5a release from the macropinosome membrane had
already occurred before the earliest time point examined. We were not
able to distinguish pinosomes from macropinosomes based on the
acquisition of GFP-rab5a:wt because both types of vesicles rapidly
acquire this marker. We were unable to determine whether pinosomes and
macropinosomes formed by similar mechanisms and we are only able to
distinguish them by differences in their respective size. These studies
clearly showed that macropinosomes formed as intact new vesicles,
consistent with the mechanism that has previously been put forth
[16
17
18
], however, it is possible that pinosomes formed
by a completely different mechanism. It cannot be ruled out that
pinosomes formed by fusion among coated vesicles that had lost the
clathrin coat.
GFP-rab5a:wt activation during E. coli phagocytosis is
similar to GFP-rab5a:wt activation during EGF-induced pinosome
formation in that both processes tended to occur near regions of
membrane ruffling. This suggests that rab5a activation and the
activation of membrane ruffling result from highly localized signals
that each occur in the same highly localized regions of the cell. Also,
in both processes, the activated GFP-rab5a that accumulates on newly
formed phagosomes and pinosomes appears to be derived directly from the
cytosol. Infrequent fusion events between GFP-rab5a:wt-negative
macropinosomes and GFP-rab5a:wt-positive endosomes were recorded in
H-ras:G12V-expressing cells. This represents an example of heterotypic
endosome fusion. The GFP-rab5a:wt labeling of early E.
coli-containing phagosomes was always transient, and rapid removal
of rab5a from the phagosome after 1–2 min was always noted. Similarly,
GFP-rab5a:wt labeling of pinosomes in EGF-stimulated cells was also
often transient and of a relatively short duration compared to the
labeling of endosomes in unstimulated cells or cells expressing
GFP-rab5a:Q79L, in which the labeling persisted for hours.
Received January 24, 2000;
revised May 8, 2000;
accepted May 9, 2000.
|
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