GFP Fusion Proteins of Rab4, Rab5, and Rab11 Are Correctly Localized to Their Target Membranes
To demonstrate that endosomal membrane organization visualized via GFP fusion proteins reflects the situation in normal cells, we had to establish that the expressed Rab proteins were correctly localized and did not alter the transferrin cycle. Using human A431 cells in which transferrin recycling has been extensively characterized (Hopkins 1983
), we generated stable cell lines expressing wild-type GFP-Rab4, -Rab5, or -Rab11. Overexpression was moderate, about fivefold over the endogenous levels as estimated by Western blotting (not shown). All three fusion proteins became membrane bound () and could be extracted into the soluble pool by microinjected recombinant Rab GDI (GDP dissociation inhibitor) or by incubation with Rab GDI after permeabilization with streptolysin O (not shown). The GFP-Rab5 cell line has been described elsewhere (Nielsen et al. 1999
). GFP-Rab5 was colocalizing with its effector EEA1, indicating that the GFP tag did not interfere with its correct membrane targeting ( A). GFP-Rab4 localized to endosomes, as defined by the presence of transferrin receptor ( C). Colocalization of GFP-Rab4 with EEA1 was often partial (compare with B). GFP-Rab11 labeling was found in the pericentriolar area as well as on small vesicular structures throughout the cytoplasm. These vesicles were distinct, but often in close proximity to EEA1-containing endosomes ( D). As previously demonstrated by antibody staining (Ullrich et al. 1996
), the pericentriolar membranes colocalized partially with transferrin receptor ( E) and with a marker for the TGN, TGN46 ( F).
Characterization of A431 cell lines expressing GFP-Rab4, Rab5 or Rab11. A431 cells stably expressing GFP-Rab5 (A), GFP-Rab4 (B and C) or GFP-Rab11 (D, E, and F) were labeled with antibodies against EEA1 (A, B and D), transferrin receptor (C and E) or TGN46 (F) followed by secondary antibodies coupled to rhodamine. Images represent individual confocal sections. (G) Wild-type A431 cells and stable cell lines were labeled with biotinylated transferrin for 2 min at 37°C, followed by incubation in the presence of an excess of nonlabeled transferrin for the indicated time periods. Medium and cells were collected, and analyzed for biotinylated transferrin. Recycled transferrin was expressed as a percentage of the total. Bar, 2 μm.
To measure transferrin recycling, biotinylated transferrin was internalized for 2 min at 37°C and chased in the presence of nonlabeled transferrin for up to 30 min ( G). Comparing wild-type A431 cells to all three cell lines, we found no major differences in the amounts of transferrin recycled to the medium. As previously noted (Van der Sluijs et al. 1992
), expression of Rab4-GFP led to a slight increase in the early rates of recycling. After this initial characterization, we concluded that the GFP fusion proteins of Rab4, Rab5, and Rab11 could faithfully represent their endogenous counterparts in A431 cells.
Transferrin Travels through Rab5-, Rab4-, and Rab11-labeled Domains on Endosomes
We then followed the passage of transferrin through endosomes with respect to the machinery represented by Rab4, Rab5, and Rab11. In cells expressing GFP-Rab4, Rab5, or Rab11, rhodamine transferrin was internalized for 2 min at 37°C and chased in the presence of nonlabeled transferrin ( A). After fixation, images were acquired and overlap of fluorescent signal was quantified (see Materials and Methods). Early after internalization, the major population of transferrin-labeled structures (70%) colabeled with Rab5 ( and ). This association was transient and decreased rapidly after the first minutes. The number of transferrin-labeled endosomes containing Rab11 increased slowly from ~20 to 55% during a 30-min chase ( and ), in line with an involvement of Rab11 in late stages of recycling (Ullrich et al. 1996
; Ren et al. 1998
). Rab4 has been implicated in the fast route of recycling (Van der Sluijs et al. 1992
; Sheff et al. 1999
). Consistently, >90% of transferrin-labeled membranes contained Rab4 after a 5-min chase ( and ). Surprisingly, ~70% remained positive for Rab4 at late time points (15 and 30 min after internalization) when >80% of the transferrin had been recycled (compare with G), suggesting that a requirement for Rab4 might not be limited to early stages.
Time course for association of rhodamine transferrin with GFP-Rab4–, -Rab5–, or -Rab11–labeled endosomes in fixed and living cells. (A and B) A431 cells stably expressing GFP-Rab4 (triangles), GFP-Rab5 (diamonds), or GFP-Rab11 (circles) were labeled with rhodamine transferrin for 2 min at 37°C, followed by a chase for the indicated time periods. Images of fixed cells were analyzed for fluorescent overlap (see Materials and Methods). (A) Percentage of transferrin endosomes labeling for GFP-Rab proteins at the indicated time points. n = 4–5 cells. (B) Representative merged images for different time points. (C) Consecutive video images from time-lapse series of A431 cells stably expressing Rab5 (top), Rab4 (middle) or Rab11 (bottom) after internalization of rhodamine transferrin. Bars: (B) 2 μm; (C) 1 μm.
Early endosomes are comprised of globular and tubulovesicular membrane elements, which have been proposed to reflect different functional domains (Mayor et al. 1993
). We hypothesized that if Rab proteins generate distinct domains on the membrane, Rab4, Rab5, and Rab11 might be differentially localized to these membrane elements. Strikingly, visualizing endosomes by time-lapse video microscopy we could distinguish distinct subdomains of GFP-Rab protein on larger transferrin-filled structures ( C and videos 1–3, transferrin in red, Rab proteins in green). Rab5 was mostly restricted to globular domains, and largely excluded from tubules and vesicles. Rapid segregation of transferrin from these Rab5-positive domains could frequently be observed ( C, arrows, and video 1). Both, Rab4 and Rab11 were seen on tubules and small globular domains forming on larger transferrin-labeled structures ( C, arrows, videos 2 and 3). Whereas Rab11 structures localized preferentially towards the cell center, those positive for Rab4 were more widely distributed. Tubules often fragmented into smaller, vesicular profiles ( C and video 3). In summary, these experiments suggested that Rab4, Rab5, and Rab11 are localized to discrete domains on endosomes.
Rab4, Rab5, and Rab11 Label Distinct Domains on the Same Endosomes
We next asked whether Rab4, Rab5, and Rab11 were differentially localized to domains of the same endosome. In other words, were Rab5 negative tubules that emerged from a transferrin-labeled endosome occupied by Rab4 or Rab11? To address this question, combinations of CFP and YFP fusions of the three Rab proteins were coexpressed in A431 cells. Texas red transferrin was internalized for 30 min to reach steady state labeling. After fixation, confocal serial sections were obtained and processed with a deconvolution software before quantification of overlapping fluorescent signals. Single endosomes were defined as structures with continuous transferrin signal (see Materials and Methods).
Interestingly, although residing on the same structure, as defined by the continuous labeling for transferrin, the fluorescent signals for the individual Rab proteins were mostly segregated (, insets). This suggested that Rab4, Rab5, and Rab11 could indeed occupy distinct domains on continuous membranes. Transferrin traverses through Rab5-positive endosomes early after internalization ( A). Consistently, at steady state the cargo was mainly found in Rab4 and Rab11 containing membranes ( and ). Remarkably, the majority (63 ± 5%) of these transferrin structures harbored both Rab4 and Rab11. In contrast, only about a quarter of the transferrin positive endosomes contained Rab4 and Rab5 (23.5 ± 7%, and ). The lowest overlap was seen for Rab5 and Rab11 (19 ± 8%). The time course suggested that after leaving Rab5 endosomes, transferrin rapidly reached Rab4-positive membranes ( A). We therefore speculated that this transit might occur within the same endosomes containing both Rab4 and Rab5. Consistently, when we analyzed cells shortly after internalization, more than twice as many transferrin-filled endosomes contained Rab4 and Rab5, compared with the steady state situation (51 ± 8% vs. 23.5 ± 7%, ). This suggests that the cargo could be sorted from a Rab5 into a Rab4 domain, residing on the same membrane.
Figure 3 Confocal images of Rab4-, Rab5- and Rab11-labeling on transferrin-filled endosomes. A431 cells expressing CFP-Rab5 and YFP-Rab4 (top and bottom row), CFP-Rab5 and YFP-Rab11 (second row), CFP-Rab11 and YFP-Rab4 (third row) after internalization of Texas (more ...)
Quantitation of Rab4, Rab5, and Rab11 on Transferrin-labeled Early Endosomes
So far, we had confirmed that cargo-filled endosomes are structured in domains, represented by different Rab proteins. However, with this approach we could not determine whether in respect to the Rab machinery the composition of endosomes changes as transferrin flows through or whether cargo is transported between endosomes with a stable composition. If Rab proteins were subjected to a similar flow as cargo this should be reflected in a change of overlapping distribution between Rab4, Rab5, and Rab11 at different stages of recycling. To visualize all three Rab proteins together we detected endogenous Rab11 by antibody staining in cells coexpressing CFP-Rab5 and YFP-Rab4 (). All endosomes labeled for a particular Rab protein were counted and the extent of overlap with the other Rab proteins was determined at steady state and early after transferrin internalization. Under both conditions, half of the Rab4-positive endosomes also contained Rab5, suggesting a relatively stable pool of membranes with this composition (). Another pool, similar in size, contained Rab4 and Rab11, and a quarter of the Rab4 endosomes was positive for all three Rab proteins. Interestingly, whereas most of the Rab4 structures colocalized either with Rab5 or Rab11 or both, about half the population of Rab5-positive endosomes did not label for either of the other Rab proteins ().
Quantitation of Rab4, Rab5, and Rab11 Overlap on the Same Endosomes
Taken together, the quantification suggests that transferrin moves through endosomes that are structured in domains labeling for Rab5, Rab4, or Rab11. Via membranes that are mainly Rab5-positive, the cargo enters a pool of endosomes that contain both Rab5 and Rab4. Transfer from this pool is fast, and at steady state transferrin is mostly found in membranes containing Rab4 and Rab11. However, it has to be noted that endosomes containing all three Rab proteins were observed, and likewise membranes containing only Rab4, only Rab11, or Rab5 and Rab11 (Tables I–III). It will probably be necessary to consider all possible combinations of domain arrangements to get a complete view of the pathway.
Electron Microscopy Confirms that Rab Proteins Localize to Distinct Membrane Domains on the Same Endosome
The data presented so far support our model for a domain organization of endosomal membranes. However, the size of the observed structures is at the limit of resolution for light microscopy techniques. We therefore sought to substantiate these findings by electron microscopy. To have the entire surface area available for immunolabeling, we decided to avoid sectioning of the membranes. Cells expressing GFP-Rab4 ( and ) or GFP-Rab5 ( D) were incubated with 5-nm gold-conjugated to BSA for 30 min at 37°C. Membranes from postnuclear supernatants of these cells were adsorbed to copper grids by incubation at room temperature in the presence of an ATP-regenerating system and GTP. The material was labeled with antibodies to GFP and EEA1 or Rab11.
Figure 4 Distinct membrane domains of Rab4, Rab5, and Rab11 visualized by electron microscopy. A431 cells stably expressing GFP-Rab4 (A, B, and C) or GFP-Rab5 (D) were incubated with BSA coupled to 5-nm gold for 30 min at 37°C. Membranes from post nuclear (more ...)
Remarkably, the gold labeling for individual Rab proteins was often localized in discrete patches ( and ). These were only found on the membranes and were not observed when the material was adsorbed at 4°C ( C), excluding artifacts generated by protein A aggregates or overexpression of the GFP fusion proteins. However, we cannot rule out that in some instances these patches appeared particularly tight due to a collapsing of membrane elements during the staining procedure. Nevertheless, labeling for GFP-Rab4 ( A, arrowheads) and the Rab5 effector EEA1 ( A, arrows) were clearly segregated on the same endosomal membrane, as defined by the internalized BSA-gold (asterisks). Likewise, patches of endogenous Rab11, detected by an affinity-purified antibody ( B, arrows), were distinct from GFP-Rab4–labeled areas ( B, arrowheads). In contrast, both Rab5 and its effector EEA1 were localized to the same domains on endosomes from GFP-Rab5–expressing cells ( D, arrows). 54% of the BSA-gold–filled endosomes positive for Rab4 also labeled for Rab11 (). After short internalization for 5 min, 18% of the BSA-gold–filled endosomes labeled for Rab4 and Rab11, whereas 49% contained EEA1 and Rab4 (). These numbers are consistent with the data obtained from the confocal images ( and ). In summary, the ultrastructural analysis confirmed that Rab proteins and their effectors can form discrete domains on continuous membranes.
Quantitation of Rab4, Rab11, and EEA1 on BSA-Gold-filled Endosomes
Arrangements of Distinct Membrane Domains within the Same Endosome Can Be Visualized in Living Cells
In a third approach, we studied the proposed domain organization in living cells, following Rab4, Rab5, and Rab11, coexpressed as YFP and CFP fusion proteins, by time-lapse video microscopy. As expected, the labeled endosomes were highly dynamic. Nevertheless, following individual structures over time, we frequently observed that more than one type of Rab protein occupied continuous membrane elements. More importantly, also in living cells, fluorescent signals were mostly segregated within the same structure ( A and videos 4–6). This was not a consequence of the imaging technique since in cells coexpressing YFP-Rab11 and CFP-Rab11, both signals were clearly overlapping (not shown). As in fixed cells, we mainly found Rab4 and Rab5, or Rab4 and Rab11 residing on the same endosome ( A). Rab5 was restricted to globular domains, which in some instances could be observed to dock or fuse ( A, middle panel, arrows, and video 5). Parts of the Rab4 domain on Rab5-positive endosomes were often seen to segregate into distinct tubular or vesicular structures (arrows, top panel, and video 4). Similar observations were made for endosomes containing Rab4 and Rab11, both proteins entering separate tubules and vesicles (, bottom panel, arrows). Thus, Rab domains are not generated by fixation but can be observed in living cells.
Visualization of Rab proteins on endosomal membrane domains by time-lapse video microscopy. (A) Time-lapse series of A431 cells coexpressing CFP and YFP fusion proteins of Rab4, Rab5, and Rab11. (B) Differential response of Rab4, Rab5, and Rab11 membrane domains to treatment with BFA. Single frames from time-lapse series (compare videos 4–6). (Top) Cells expressing GFP-Rab4 after internalization of rhodamine transferrin for 10 min followed by treatment with 5 μg/ml BFA for 10 min. (Middle and bottom) Cells expressing combinations of Rab4 and Rab11, or Rab4 and Rab5. Bars: (A) 1 μm; (B) 1 μm.
Domains on Rab4 and Rab11 Endosomes Are Sensitive to Brefeldin A
We next asked whether structural alterations on the endosomal membrane would perturb the segregation of Rab4, Rab5, and Rab11. Transferrin and Rab4 have been shown to localize to an extensive tubular network in response to brefeldin A (BFA; Tooze and Hollinshead 1992
; Daro et al. 1996
). Consistently, we found that transferrin rapidly entered long GFP-Rab4–positive tubules after a short treatment with BFA ( B and video 7). In cells expressing CFP-Rab11 and YFP-Rab4 both proteins localized to the same tubular network ( B, arrows, and video 8), but were no longer clearly segregated in distinct domains. Remarkably, a fraction of Rab4-containing endosomes did not form tubules, but rather remained in distinct globular structures. These membranes contained little or no Rab11 ( B, arrowheads). Imaging cells expressing CFP-Rab5 and YFP-Rab4 under these conditions revealed that Rab5-positive endosomes were resistant to BFA-induced tubulation. Likewise, Rab4 remained in the globular part when present on Rab5 endosomes ( B and video 9). The amount of Rab4 on Rab5-positive membranes was slightly decreased compared with control cells (30 ± 8% vs. 43 ± 6%), suggesting that some Rab4 entered the tubular network from Rab5-positive endosomes.
Consistent with this observation, transit of transferrin from Rab4/Rab5-positive endosomes to the Rab4/Rab11 network proceeded when cells were pretreated with BFA to allow tubule formation. Rhodamine transferrin, internalized for 2 min at 37°C, was followed by a chase in the presence of BFA (). The amount of Rab5-positive transferrin structures was slightly higher than in control cells at early time points ( A), however, we found no significant morphological difference to nontreated cells ( B, Rab5 panel, compare with B). Transfer of transferrin into Rab4-labeled endosomes proceeded faster in the presence of BFA ( A, 2 min), but at early stages the cargo remained restricted to globular Rab4-positive endosomes and was largely excluded from the tubular network ( B). After 5 min, transferrin had left Rab5 positive and reached Rab4-labeled membranes to similar levels as seen in control cells ( A). At this point some cargo had entered the BFA-induced tubular network ( B). This was accompanied by a slight increase for Rab11 labeling compared with nontreated cells, consistent with the observation that Rab4 and Rab11 were not clearly segregated in these tubules. At later time points most of the transferrin was found in a membrane network that did not permit quantitation of individual structures (not shown).
Transfer of transferrin from Rab4/Rab5 to Rab4/Rab11-positive endosomes proceeds in BFA-treated cells. (A) A431 cells stably expressing GFP-Rab4, -Rab5, or -Rab11 were labeled with rhodamine transferrin for 2 min followed by a chase for the indicated time points. BFA treatment of the cells was started 5 min before and continued during internalization and chase times. For acquisition and quantitation of the fluorescent images see and Materials and Methods. (B) Representative merged images for BFA-treated cells (compare with B). Bar, 2 μm.
Given that recycling is only marginally effected by BFA (Lippincott-Schwartz et al. 1991
; our unpublished data), the bulk of transferrin might be delivered back to the surface directly from the BFA resistant Rab5/Rab4 endosomes. However, segregation of Rab4 and Rab11 domains might not be strictly required, and recycling could occur after a fast transit into the tubular network.
Wortmannin Treatment Delays Exit of Transferrin from Rab4/Rab5-Positive Endosomes
Cells treated with the phosphatidylinositol-kinase inhibitor wortmannin show a delay in transferrin recycling (Spiro et al. 1996
). The activity of the PI-(3)-kinase hVPS34 is necessary for the membrane recruitment of the Rab5 effector EEA1 (Mills et al. 1998
; Simonsen et al. 1998
; Christoforidis, et al. 1999b), suggesting that a Rab5 domain requires a microenvironment of PI(3)P and active Rab5 with its effectors on the membrane. Therefore, depletion of PI(3)P by wortmannin treatment should perturb the Rab5 domain. To test whether this might interfere with the transfer of transferrin to Rab4-positive domains, we quantitated the codistribution of Rab4, Rab5, and Rab11 with transferrin after 30 min internalization in the presence of 50 nm wortmannin (). Consistent with our hypothesis, we found that compared with control cells twice as many transferrin-labeled endosomes contained Rab4 and Rab5 (47 ± 14% vs. 23 ± 7%, ). Likewise, the number of cargo-filled structures containing Rab4 and Rab11 was reduced to almost half compared with control cells (35 ± 6% vs. 63.5 ± 5%, ), indicating that transferrin was retained in Rab4/Rab5 endosomes upon wortmannin treatment. Moreover, also the domain arrangement was changed as judged by the number of Rab4-positive endosomes containing either Rab5 or Rab11. More Rab4 endosomes were positive for Rab5 (65.5 ± 4% vs. 52 ± 6%) and fewer labeled for Rab11 (34.5 ± 9% vs. 50 ± 1%, ). Sorting of membrane associated cargo has been suggested to occur by segregation from globular into tubular domains of the endosome (Mayor et al. 1993
). The observed delay of transferrin recycling in wortmannin-treated cells might be explained by the perturbation of this domain structure. Consistently, wortmannin treatment frequently resulted in structural changes of Rab5 endosomes (video 10), which were not observed on Rab11 membranes.