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Protein and Vesicle Trafficking, Cytoskeleton
Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, California
Submitted 7 July 2006 ; accepted in final form 17 October 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIAL AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Rab proteins; recycling; transcytosis; guanosine 5'-triphosphatase inhibitor; AG10
2-macroglobulin. This endosomal compartment has been characterized as a tubular network, in contrast with the tubulovesicular sorting endosome, and is concentrated in the perinuclear area of nonpolarized cells, including Chinese hamster ovary cells (CHO), AtT20 cells, and HeLa cells (11, 15, 27, 30, 46, 62). In polarized cells, the cell surface is separated into apical and basolateral plasma membrane domains. They have different lipid and protein composition, and endocytosis can occur from both domains (31, 34). In polarized cells, the basolaterally endocytosed TfR is efficiently sorted from the transcytotic pathway and recycled back to the basolateral membrane. Polarized epithelial cells contain distinct populations of apical early endosomes (AEEs) and basolateral early endosomes (Fig. 1). The majority of Tf (65%) was found to be recycled from basolateral early endosomes within 10 min after internalization from the basolateral membrane in MDCK cells (40). The existence of an endosomal compartment, which is accessible to both apically and basolaterally endocytosed ligands, has been reported in the polarized epithelial cells (17, 22, 52). In addition, transcytosing proteins such as IgA and recycling proteins such as Tf have been found in a "common" endosome (CE), which consists partly of tubules (32). This compartment, which seems to be an ubiquitous feature of the polarized epithelial cells, is termed the common recycling endosome in MDCK cells (41) or the subapical compartment in hepatocytes (19, 36, 44, 50). The CE has been identified as a tubularized endosomal system with a diameter of 60 nm in MDCK cells, and it is from there that the basolaterally endocytosed Tf-TfR complex recycles to the basolateral membrane (Fig. 1, pathway 8) (1, 9). On the other hand, the basolaterally internalized complex formed by polymeric immunoglobulin receptor and dimeric IgA on its route from the basolateral to the apical surface crosses the CE (Fig. 1, pathway 7) and moves to a population of 100- to 150-nm cup-shaped vesicles above the CE, which constitutes the apical recycling endosome (ARE) in MDCK cells (3, 12). It may reach the apical plasma membrane through the Tf recycling pathway (Fig. 1, pathway 4). In hepatocytes, the subapical compartment has been characterized as a subcompartmentalized endosomal compartment that is composed of 60- to 100-nm tubulovesicular structures (analogous to the CE in MDCK) and 150- to 200-nm cup-shaped vesicles in the apical region (analogous to the ARE in MDCK) (36).
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In this report, the effect of AG10 on Tf intracellular trafficking was used to investigate the intracellular processing of endocytosed FITC-Tf conjugates to identify endosomal compartments involved in Tf intracellular pathways in Caco-2 cells. We also study the effects of AG10 on the colocalization of various Rab proteins with endocytosed Tf during its intracellular trafficking and transcytosis in Caco-2 cells. We found that Rab11 is involved in the apical recycling of Tf in Caco-2 cells. Furthermore, AG10 stimulates the trafficking pathway of the apically endocytosed Tf through the Rab11-containing endosomal compartment. On the other hand, the basolaterally endocytosed Tf did not have access to the Rab11-containing endosomal compartment in Caco-2 cells, unless in the presence of AG10. Therefore, results from this report will provide information regarding not only the difference in TfR processing between apical and basolateral membrane, but also the effect of AG10 on the intracellular trafficking of Tf. Elucidating the role of the intracellular controllers of the recycling and transcytosis of Tf, such as Rab proteins, is potentially important for the development of Tf as a vehicle for the increase of intestinal absorption of protein drugs.
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MATERIAL AND METHODS |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Rabbit polyclonal antibodies against Rab5 and Rab11 were purchased from Santa Cruz Biotechnology. The rabbit polyclonal antibody against ZO-1 and Alexa-Fluor-labeled secondary antibodies were purchased from Molecular Probes. AG10 was obtained from Calbiochem.
Preparation of diferric, radiolabeled, and FITC-labeled Tf. To prepare diferric Tf, 100 µl ferric ammonium citrate in H2O with a stock concentration of 10 mg/ml was added to 1 ml of apo-Tf in PBS solution (20 mg/ml) under constant stirring. After incubation of the mixture at 37°C for 2 h, the iron-loaded Tf was dialyzed against 2 L PBS at 4°C for 16 h.
To radiolabel Tf, the diferric Tf was added to Na125I in PBS, followed by the addition of freshly made chloramine T and sodium metabisulfite in PBS solution. The reaction was terminated by addition of potassium iodide to the reaction tube. The radiolabeled Tf was separated by gel filtration through a 10 x 0.5 cm Sephadex G-50 column.
FITC (Sigma) was conjugated to Tf with a molar ratio of 50:1 in 1 M sodium bicarbonate pH 9.0 under constant stirring for 1 h at 25°C. The untreated FITC was removed by dialysis against 2 L PBS at 4°C for 16 h. Protein concentration and the degree of labeling were calculated by using formulas provided by Molecular Probes (Eugene, OR).
Pulse-chase experiments using 125I-labeled Tf. The 2-wk-old confluent Caco-2 cells grown on 24-mm Transwell filters (Costar, Cambridge, MA), which displayed a transepithelial electrical resistance (TEER) of 100 ohms/cm2 as measured by an epithelial voltohmmeter (EVOM; World Precision Instrument, West Haven, CT), were incubated with serum-free DMEM containing 0.1% BSA and 20 mM HEPES for 1 h at 37°C to deplete endogenous Tf. The cell monolayers were further incubated in the presence or absence of AG10 (500 µM) for 2 h at 37°C. 125I-labeled Tf [2–4 µg/ml, 2 x 106 counts per minute (cpm)/ml] was bound to the apical surface of the cells in the presence or absence of AG10 on ice for 15 min, which was followed by incubation at either 16°C or 37°C for 30 min. After removal of the unbound and cell surface-bound 125I-labeled Tf with ice-cold PBS (containing 0.1 mM Ca2+ and 0.05 mM Mg2+) and acid (0.15 M NaCl, 0.5 M acetate, pH 2.2) washes on ice, the cells were chased with or without AG10 and in the presence of 100-fold excess of unlabeled Tf for up to 30 min at 37°C. About 80% of cell-associated Tf was removed by the acid-wash procedure. At indicated time points, the samples from apical and basal media were collected. The collected samples were subjected to 15% trichloroacetic acid precipitation, and the radioactivity level in the protein precipitates and filter membranes recovered in the end of the chase period was measured by using a Packard gamma counter. 125I-labeled Tf recovered from the basal chamber was taken as a measurement of Tf transcytosis, while the radioactive protein recovered from the apical chamber was used as a measurement of Tf recycling. The radioactivity in the filter membrane reflected the internalized and cell membrane-bound 125I-labeled Tf in the end of the chase period. The results were corrected for nonspecific recycled, transcytosed, and cell-associated 125I-labeled Tf, which was determined in the parallel experiments in the presence of 100-fold excess of unlabeled Tf during the pulse period. The results are presented as a percentage of the total internalized 125I-labeled Tf.
125I-labeled Tf uptake assay. Caco-2 cells cultured for 2 wk grown on 24-mm plates were depleted of endogenous Tf and were preincubated in serum-free DMEM containing 0.1% BSA and 20 mM HEPES in the presence or absence of AG10 (500 µM) for 2 h at 37°C. The medium was replaced with a fresh DMEM (0.1% BSA, 20 mM HEPES) containing 125I-labeled Tf (with a final concentration of 2–4 µg/ml, 2 x 106 cpm/ml) in the continued presence or absence of AG10 for 30 min at either 16°C or 37°C. The unbound and surface-bound 125I-labeled Tf was removed as indicated above. The cells were chased in the presence of 100-fold excess of unlabeled Tf and in the continued presence or absence of AG10 for 10 or 30 min at 37°C. At the end of the chase period, the surface-bound 125I-labeled Tf was removed as indicated above and the PBS and acid washes were collected. The cells were lysed in 1 M NaOH, and the radioactivity level in the cells and the collected acid fractions was counted by using a gamma counter. Protein assay was performed by using bicinchoninic acid (Pierce). The data were corrected for nonspecific uptake of 125I-labeled Tf determined in parallel experiments in which an excess of unlabeled Tf was present during the pulse period. The internalized Tf is expressed as ng/mg total protein.
Confocal fluorescence microscopy. Caco-2 cells grown on 12-mm polystyrene filters (Costar) were subjected to pulse-chase assay as described above, except that FITC-Tf was used to replace 125I-labeled Tf. To obtain a better visualization, the cells were used at about >90% confluency. However, the fields with 100% confluency were selected for confocal analysis. After binding of FITC-Tf (45 ng/ml) to the apical or basolateral surface of the cells for 45 min on ice, the cells were incubated at either 16°C or 37°C for 2 or 30 min. The surface-bound FITC-Tf was removed by sequential PBS (containing 0.1 mM Ca2+ and 0.05 mM Mg2+) and acid (0.15 M NaCl, 0.5 M acetate, pH 2.2) washes, and then the cells were chased for 0, 2, or 30 min in the presence of 10-fold Tf at 37°C. At the end of the 30-min chase period, the unbound and surface-bound FITC-Tf was removed by multiple PBS and acid washes, and the cells were fixed by using 3.7% paraformaldehyde (Sigma) in PBS, which was followed by quenching and permeabilization with 0.2% saponin for 5 min. The cells were then blocked with 10% FBS in PBS and incubated with appropriate primary antibodies diluted in 1.5% FBS in PBS. Alexa-Fluor 568 goat anti-rabbit antibody was used for the secondary detection of the proteins. The nucleus was labeled by incubation with 4',6-diamidino-2-phenylindole, dilactate (DAPI, dilactate, Invitrogen) dye for 15 min on a platform shaker. The efficiency of "acid washes" in removing the surface-bound FITC-Tf was demonstrated in control experiments, in which the cells were incubated with FITC-Tf for 30 min at 37°C and were not subjected to the acid washes at the end of the pulse period.
Image processing.
The images were collected from different fields of the prepared slides by using a confocal laser-scanning microscope (LSM 510 Meta NLO imaging system; Zeiss) equipped with argon, red HeNe, and green HeNe lasers for standard confocal fluorescence microscopy and a Ti-Sapphire tunable Coherent Chameleon laser (720–950 nm) for multiphoton excitation of DAPI. The ability of this system to acquire fluorescence emission signals resolved within narrow ranges in multitrack mode and the use of singly labeled control samples ensured the validity of colocalization studies. The acquisition parameters for the collection of images were kept constant between samples to allow comparison of the images taken from different fields and slides. The analysis of the confocal microscopy image by using computer software has already been described by Nunez et al. (33). Caco-2 cell monolayers were optically sectioned in the z-axis from the apical to the basolateral side of the cell. The first z-section from the apical surface of the cells was captured when FITC-Tf (green) was observed. The step size in the z-axis was 0.4 µm. Analysis of the extent of colocalization between markers of interest in confocal microscopy images was done by using the Enhanced Colocalization tool available with the Zeiss LSM 510 software. To quantify the colocalization between FITC-Tf and Rab proteins, the image fields were thresholded. The whole image field was defined as a region of interest, and the system calculated mean intensities + 2 x SD to threshold the images. Colocalization coefficients representing the relative number of colocalizing pixels in either channel compared with the total number of pixels above the threshold irrespective of their intensity were measured through the image sequence in the z-series. To analyze the extent of the colocalization between FITC-Tf and the Rab proteins, we used the colocalization coefficient of FITC determined in each z-section of a z-series that shows the relative number of colocalizing FITC pixels compared with the total number of FITC pixels above the threshold in the z-sections. The average colocalization coefficient of FITC-Tf at any specific height in the cells was obtained by using the FITC-Tf colocalization coefficients of the z-sections, which were collected from different fields of the prepared slide and localized at the same height above either the nucleus or the tight junction. For statistical analysis, the SE was calculated. Student's t-test was used to analyze significance (P 
0.05). In the images labeled with the same fluorophore to detect ZO-1 and Rab protein staining, the single cells were thresholded twice, with and without ZO-1 staining. To obtain the colocalization coefficient, ZO-1 staining was omitted and the inner part of the cells was defined as the region of interest. To look at the distribution of the tight junction, the mean intensity for Alexa-Fluor 568 pixels in the cells thresholded with ZO-1 was considered. Three-dimensional views of the z-series were obtained by using the projection function, which calculated a series of projections after the rotation of the data package around the y-axis. The separate profile for each channel along a three-dimensional view was obtained by using the Profile function. Two-dimensional deconvolution views of the yz- and xz-plane were generated by using the ortho function. All images were compiled by using Adobe Photoshop software (version 7.0; Adobe Systems, Mountain View, CA).
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RESULTS |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Consistent with the previous observations showing the effect of the temperature on the accumulation of Tf in different endosomal compartments, FITC-Tf showed a high level of colocalization with Rab5 in the cells pulsed at 16°C. Galleries of the optical sections from the apical to the basolateral plasma membrane of the cells are presented in Fig. 2, A and B, and in Fig. 3, A and B. The three-dimensional views of these galleries were obtained by using the projection function (Fig. 2, C and D, and Fig. 3, C and D). Comparing the galleries of the optical sections of the cells pulsed at 16°C and 37°C, we found FITC-Tf colocalized with Rab5 to a high degree in the first three z-sections of the gallery of the cells pulsed at 16°C (Fig. 2, A and B), which is more noticeable in one of the cells presented in this gallery. These z-sections are localized about 4.6 µm to 6.7 µm above the intracellular level at which the nucleus begins to appear. An apically peripheral distribution of FITC-Tf, which was absent in the cells pulsed at 37°C, is shown in the three-dimensional view of the gallery of the Caco-2 cells pulsed at 16°C and immunolabeled with the antibody against Rab5 (Fig. 2C). On the other hand, a high degree of colocalization between FITC-Tf and Rab11 was observed in the cells pulsed with FITC-Tf at 37°C. As shown in Fig. 3B, FITC-Tf was colocalized with Rab11 in the z-sections localized only 2 µm to 3 µm above the nucleus of the cells pulsed at 37°C. The three-dimensional views of the galleries confirm also the FITC-Tf presence above the nucleus in the cells pulsed at 37°C (Figs. 2D and 3D). The colocalization between FITC-Tf and Rab11 in Caco-2 cells pulsed at 37°C is shown in Fig. 3D.
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70% of the fields analyzed in the z-sections localized from 6.4 µm to 7.2 µm above the z-section containing the first sign of nucleus labeling in the cells pulsed at 16°C (Fig. 2E). In these z-sections, 55% of FITC-Tf was colocalized with Rab5 at 16°C. The highest level of the colocalization between FITC-Tf and Rab11 was found in the z-sections localized from 2.0 µm to 3.2 µm in the cells pulsed at 37°C (Fig. 3E). In the z-sections localized about 2.4 µm and 2.8 µm above the nucleus, about 35–40% of total FITC-Tf was colocalized with Rab11 at 37°C. FITC-Tf was colocalized with Rab5 and Rab11 in two different intracellular locations above the tight junction. To determine the intracellular location of the z-sections containing the highest level of the colocalization between FITC-Tf and the Rab proteins, the tight junction was labeled by using antibody against ZO-1. Staining of ZO-1 allowed us to follow the FITC-Tf distribution in single cells, and it also eliminated the effect of the uneven filter membrane and different cell height.
Galleries of the optical sections from the apical to the basolateral plasma membrane of the cells pulsed at 16°C and 37°C are shown in Figs. 4A and 5A. Consistent with the results obtained from the cells without any tight junctional labeling, the intracellular location of the FITC-Tf colocalized with Rab5 and Rab11 is different in the cells pulsed at 16°C and 37°C (Figs. 4A and 5A). In the cells pulsed at 16°C, a high level of colocalization between FITC-Tf and Rab5 was observed in the z-sections localized apically and distal from the tight junction. On the other hand, FITC-Tf was colocalized with Rab11 in the z-sections localized above the tight junction in the cells pulsed at 37°C. The intracellular location of the FITC-Tf colocalized with the Rab proteins relative to the tight junction is also shown in the two-dimensional deconvolution views of the yz- and xz-plane (Figs. 4B and 5B). These results were also confirmed by analyzing the distribution of FITC-Tf, the Rab proteins, and the ZO-1 along the three-dimensional views of the galleries (Figs. 4C and 5C). The distribution profile for each channel was obtained by using the Profile function. At 16°C, the green and red peaks that represent FITC-Tf and Rab5 overlapped far from the peaks representing the ZO-1 and the nucleus (Fig. 4C). On the other hand, an overlap between the peaks representing FITC-Tf and Rab11 was observed adjacent to the ZO-1 in the cell pulsed at 37°C (Fig. 5C). The animated three-dimensional projections are also available in the online version of this article (supplemental movies 1 and 2).
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The effect of AG10 on transcytosis, recycling, and cell association of Tf was observed only in the cells pulsed at 37°C. The recycling, transcytosis, and cell association of Tf was measured in the presence and absence of AG10. As shown in Caco-2 cells, which were pulsed with 125I-labeled Tf at 16°C, the recycling and transcytosis of Tf were not affected by the treatment with AG10, but the cells pulsed at 37°C showed a statistically significant increase in the recycling and transcytosis of Tf in the presence of AG10 (Fig. 6, A and B). The cell association of Tf did not change in the presence of AG10 in the cells pulsed at 16°C for 30 min (Fig. 6C). On the other hand, a statistically significant decrease in the amount of cell-associated Tf was observed in the cells that were treated with AG10 and pulsed at 37°C for 30 min (Fig. 6C). In control cells, the transcytosis, recycling, and cell association of Tf reached the plateau after the 10-min chase, whereas AG10 showed a continuous effect through the chase period (Fig. 6, A–C).
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1.0 µm above the intracellular level at which ZO-1 begins to appear (Fig. 11C). On the other hand, we observed that the FITC-Tf was colocalized with Rab11 in the z-section located 1.6 µm above the tight junction (Fig. 11D, arrows). As shown in Fig. 11E, the average colocalization coefficient of the FITC-Tf increased significantly in the z-sections localized apically and above the tight junction. FITC-Tf was detected in the z-sections localized about 2.0 µm to 2.4 µm above the tight junction, only in the cells that have been treated with AG10 (Fig. 11F).
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DISCUSSION |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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On the other hand, FITC-Tf showed a higher level of colocalization with Rab11 in the cells which were pulsed at 37°C (Fig. 3, A–E). This observation indicated the transfer of FITC-Tf from Rab5-containing endosomal compartments to a Rab11-containing endosomal compartment in the cells pulsed at 37°C (Fig. 1, pathway 3). The existence of a population of cup-shaped vesicles containing Rab11 and Rab25, which constitute the ARE, has been reported in the apical region of MDCK cells (5, 12). We found Rab11 in the vesicles in the apical region of Caco-2 cells and above the z-sections containing ZO-1 staining, which could be analogous to the ARE in MDCK cells (Fig. 4, A–D). Studies in MDCK cells and rat hepatocytes suggested the subdivision of a single endosomal compartment (subapical compartment) into CE and ARE (9, 51). The involvement of Rab11 in the recycling of internalized Tf has been shown in nonpolarized cells (Table 1). Ren et al. (37) reported the inhibition of Tf recycling from perinuclear recycling endosomes in TRVb cells expressing the dominant-positive (Q70L) or dominant-negative (S25N) mutant forms of Rab11.
Tyrphostins are a group of low-molecular-weight organic compounds that have been used to specifically inhibit the GTPase activity of protein tyrosine (10, 23, 24, 61). The effect of AG10 (tyrphostin A8) on the transport of insulin-Tf conjugate has already been shown. The TfR-mediated transcytosis of insulin-Tf increased by 20-fold in AG10-treated Caco-2 cells (59). An enhancement in the hypoglycemic effect of orally administered insulin-Tf conjugate was also reported in diabetic rats (59).
To investigate the effect of AG10 on intracellular processing of Tf among the endosomal compartments, we made use of the observation that endocytosed Tf accumulates in the endosomal compartments containing Rab5 and Rab11 at 16°C and 37°C, respectively (Table 1 and Figs. 2–5). The cells were pulsed with 125I-labeled Tf at two different temperatures, and, consistent with the results obtained from confocal microscopy, the passage of the endocytosed Tf from endosomal compartments containing Rab5 to those containing Rab11 was blocked at 16°C and the majority of Tf (90%) was chased out of the cells within the first 10 min (Fig. 1, pathway 2, and Fig. 6A). As shown in Fig. 6A, in the cells pulsed with 125I-labeled Tf at 37°C, the recycling of Tf followed the criteria of the slow-phase recycling, i.e., an average of only 20% of Tf was recycled in the first 10 min (Fig. 1, pathway 4). The effect of AG10 was pronounced at 37°C, and the results obtained from pulse-chase and uptake assays suggested that AG10 affects the late process of Tf transport, after the exit of Tf from endosomal compartments containing Rab5. The significant increase in the apical-to-basolateral transcytosis of Tf (Fig. 6B) in the presence of AG10 may explain the effect of AG10 on the transport of insulin-Tf conjugate in Caco-2 cells, as previously reported (58). The increase in the mean intensity of the apically endocytosed FITC-Tf, which was observed in the more basolateral regions of the Caco-2 cells treated with AG10 (Fig. 9D), may also be explained by the effect of AG10 on TfR-mediated transcytosis. The amount of cell-associated Tf decreased by an average of 40% after a 30-min chase in the presence of AG10 (Fig. 6C). As shown in Fig. 7A, AG10 decreased the uptake of Tf by 15% after a 10-min chase, which can be explained by the increase in the amount of recycled and transcytosed Tf in Caco-2 cells. From the uptake assay in the presence and absence of AG10, there was no significant difference in the amount of surface-bound Tf, as shown in the radioactivity recovered in the acid-wash fractions (Fig. 7B). Since a high level of the cellular uptake of Tf at 37°C has been shown in many cell types and the existence of an internal TfR pool has been suggested (21, 26), a potential effect of AG10 on the TfR internal pool can be considered. In the presence of AG10, the internalized Tf-TfR complexes may be depleted from Caco-2 cells resulting in an increase in both recycling and transcytosis of Tf. The effect of AG10 on the late processes of Tf trafficking was confirmed by the demonstration of a higher degree of colocalization between FITC-Tf and Rab11 in the presence of AG10 (Fig. 9), suggesting the involvement of Rab11 in the recycling of apically endocytosed Tf at 37°C and possibly explaining the effect of AG10 on apical recycling of Tf (Fig. 6A). In the presence of AG10, Tf was transported to a higher degree in the endosomal compartments containing Rab11 before the apical delivery. AG10 may increase the transport of Tf-TfR complexes from intracellular compartments containing the internalized Tf at 37°C, which are localized before the Rab11-containing endosomal compartment, to the endosomal compartments from which transcytosis and recycling of Tf occur. This may explain as well the observed high colocalization between the apically endocytosed FITC-Tf and Rab11 in the presence of AG10 (Fig. 9C). An overloading of these endosomal compartments may potentially result in an increase in both transcytosis and recycling of Tf.
To further investigate the effect of the duration of the pulse period on the colocalization between the apically endocytosed FITC-Tf and Rab proteins, Caco-2 cells were pulsed with FITC-Tf in the absence or presence of AG10 (500 µM) for 2 min at 37°C and were then chased in the continued absence or presence of AG10 for 2 min. As shown in Fig. 10, FITC-Tf was colocalized with Rab5 in a higher extent than with Rab11. However, the highest level of the colocalization between FITC-Tf and Rab5 was observed in Caco-2 cells pulsed at 37°C for 2 min without any chase (data not shown). The presence of the Rab5 in the z-sections localized above the tight junction indicates that Rab5 leaves AEE in the cells pulsed at 37°C. This may be due to the involvement of Rab5 in the vesicular transport between AEE and the endosomal compartments that are involved in the later steps of Tf trafficking. At 37°C, pathway 3 is not blocked, and Rab5 may be involved in the transport of vesicles containing FITC-Tf out of AEE and toward CE and ARE. It may also be explained by the existence of a subcompartmental structure for the endosomes. A 19% overlap between Rab5 and Rab11 has been reported in A431 cells (43). A small population of Rab5 may be localized in the ARE but not at the same domain as Rab11. Most important, AG10 did not show any effect on the colocalization between FITC-Tf and Rab5 (Fig. 10, C–E). Consistent with previous results (Fig. 9), AG10 increased the colocalization between FITC-Tf and Rab11 by 20% (Fig. 10, A, B, and E). This confirms the effect of AG10 on the trafficking of the apically endocytosed Tf in which Rab11 is involved. AG10 did not show any effect on the distribution pattern of Rab11 in Caco-2 cells. This was confirmed by quantitative analysis of the mean intensity of Rab11 through the optical sections from the apical to the basolateral side of the cells (Fig. 10E). A condensed Rab11-containing endosomal compartment, which was observed in the AG10-treated cells, was also found in the control cells. However, the effect of AG10 on Rab11 protein needs further investigation.
As shown in the present study, the apically endocytosed FITC-Tf was colocalized with Rab11 at 37°C in Caco-2 cells (Table 1; Fig. 3, B, D, and E; and Fig. 5, A–D). However, others have reported that there was no colocalization between the basolaterally endocytosed Tf and Rab11 in MDCK cells (3, 53). Therefore, the Tf trafficking endocytosed from the basolateral membrane in Caco-2 cells was further investigated in the absence or presence of AG10. Consistent with the reported observations by Brown et al. (3) in MDCK cells, the basolaterally endocytosed FITC-Tf did not reach the Rab11-containing endosomal compartment localized above the tight junction of Caco-2 cells (Fig. 11). This finding indicates the existence of different intracellular pathways for the apically and basolaterally endocytosed Tf in Caco-2 cells. The majority of the basolaterally endocytosed Tf recycles back to the basolateral cell membrane after the exit from CE (Fig. 1, pathway 8) and does not have access to the Rab11-containing endosomal compartment above the tight junction. On the other hand, the apically endocytosed Tf, after the exit from CE, moves toward the Rab11-containing endosomal compartment and reaches the apical membrane through the pathway 4 (Fig. 1). Interestingly, in AG10-treated Caco-2 cells, a fraction of the basolaterally endocytosed FITC-Tf was transported to the apically localized z-sections above the tight junction (Fig. 11B). In the presence of AG10, the basolaterally endocytosed FITC-Tf was colocalized with Rab11, which, as expected, was localized in the z-sections above the tight junction (Fig. 11, D and E). The pulse-chase experiment also showed that the effect of AG10 on 125I-labeled Tf trafficking is not limited to the apically endocytosed Tf. AG10 increases the recycling and also the basolateral-to-apical transcytosis of the basolaterally endocytosed Tf in Caco-2 cells (F. Norouziyan, unpublished observations). The observed colocalization between FITC-Tf and Rab11 may be due to the effect of AG10 on the basolateral-to-apical transcytosis of Tf. Interestingly, the basolaterally endocytosed Tf may reach the apical membrane in the presence of AG10 through pathway 4, which normally is accessible only to the apically endocytosed Tf.
In summary, in the present study, we took advantage of the effect of temperature on the colocalization between the apically endocytosed Tf and Rab proteins, Rab5 and Rab11, to study the effect of AG10 on the intracellular trafficking of Tf. The effect of AG10 on Tf intracellular trafficking was observed only at 37°C, but not 16°C, which indicates that the effect of AG10 on Tf trafficking occurred after the exit of Tf from the AEE. We found that, in Caco-2 cells, the apically endocytosed Tf crosses the Rab11-containing endosomal compartment after the exit from the CE on its way toward the apical membrane. The transport of the apically endocytosed Tf to the Rab11-containing endosomal compartment was stimulated in AG10-treated Caco-2 cells, which may be due to the involvement of Rab11 in the apical recycling of Tf. By shortening the pulse period to 2 min at 37°C, and also a chase of 2 min, a colocalization between Rab5 and the apically endocytosed FITC-Tf was observed above the tight junction. This may be due to the involvement of Rab5 in the transport of the vesicles containing Tf or the existence of a subcompartmental structure for the Rab5 and Rab11-containing endosomes. The level of the colocalization between Rab5 and FITC-Tf was not affected in the presence of AG10. The existence of different intracellular pathways for the apically and basolaterally endocytosed Tf was shown in Caco-2 cells. The basolaterally endocytosed Tf did not have access to the Rab11-endosomal compartment. Interestingly, in the presence of AG10, FITC-Tf was found in the apically located z-sections and was further colocalized with Rab11. This may be explained by the effect of AG10 on the transcytosis of the basolaterally endocytosed Tf, which may reach the apical membrane also through pathway 4.
Tyrphostins specifically inhibit the GTPase activity of protein tyrosine kinases (10, 23, 24, 61), and AG10 was also reported to inhibit the GTPase activity of transducin and calcineurin (25, 56). The ability of tyrphostins to inhibit the GTP-utilizing enzymes raises the possibility of their inhibitory effect on GTPase proteins such as Rab proteins. The involvement of Rab proteins in the effect of AG10 on Tf intracellular trafficking needs further investigation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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