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(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

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
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

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
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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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.

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
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 .

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 .

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 .

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 .

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 .

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
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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