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First published online 31 October 2006
doi: 10.1242/jcs.03238

Journal of Cell Science 119, 4758-4769 (2006)
Published by The Company of Biologists 2006
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Macropinocytosis: regulated coordination of endocytic and exocytic membrane traffic events

Sestina Falcone1,2,*, Emanuele Cocucci3,*, Paola Podini2, Tomas Kirchhausen4, Emilio Clementi1,2 and Jacopo Meldolesi2,3,5,{ddagger}

1 University of Milan, Department of Preclinical Sciences, via G.B. Grassi 74, 20157 Milan, Italy, and E. Medea Scientific Institute, 23842 Bosisio Parini, Italy
2 Scientific Institute San Raffaele, Stem Cell Research Institute
3 Vita-Salute San Raffaele University, Department of Neuroscience, Center of Excellence on Cell Development, via Olgettina 58, 20132 Milan, Italy
4 Harvard Medical School, The CBR Institute for Biomedical Research, Longwood Avenue, Boston, MA 02115, USA
5 IIT Research Unit of Molecular Neuroscience, via Olgettina 58, 20132 Milan, Italy


Figure 1
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  		Fig. 1. Tracer and subcellular marker distribution in DC macropinosomes. Panel A shows the accumulation of FITC latex Bds into DCs, revealed by FACS histograms of cell preparations incubated with the tracer, from left to right, for 0, 5, 10, 20 and 60 minutes. The conversion in time-dependent curves of the FACS data, obtained in three experiments with either Bds or Dex, is shown in panel B. Panels C and D show merged confocal images of DCs exposed for 10 minutes to the styryl dye FM4-64 (red), alone (C) or together with FITC-conjugated Bds (green, D). The almost complete negativity of C shows that without the Bds endocytosis is weak, whereas with the Bds the labelled puncta are numerous, mostly positive for both the dye and the tracer (yellow). The merged images of panels E and F show the two tracers (green), Bds (E) and Dex (F), administered for 10 minutes, localized mostly in a fraction of the puncta positive for the early endosome marker EEA1 (red). Panel G illustrates the co-localization of Bds and Dex administered together for 10 minutes to DCs. Bar, 10 µm (C), also valid for D. Bar, 10 µm (E), also valid for F and G. Panels H and I show the ultrastructure of two cytoplasmic areas in DCs loaded with Bds for 10 minutes. Vacuoles of variable size and shape packed with the tracer (macropinosomes) are indicated by arrows. Bar, 0.5 µm (H).

 

Figure 2
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  		Fig. 2. Kinetics of transferrin and latex bead uptake in DCs immunolabelled for macropinosome-endosome markers: rabankyrin-5 and EEA1. This figure shows quantitative morphometric results (mean ± s.d. from at least ten fields each), obtained as described in Materials and Methods. Corresponding representative images and additional quantitative data with Dex and Bds are shown in supplementary material Fig. S2. Panel A compares the kinetics of uptake by DCs of two tracers, Tf and Bds, taken up by clathrin-dependent endocytosis and macropinocytosis, respectively. Notice that the first appears monophasic, whereas the second shows a considerable increase in rate after a few minutes of delay. Panel B illustrates the time course of Bd accumulation in puncta positive also for Rbk, EEA1 or both; panel C shows the accumulation of Tf in puncta positive for EEA1, and of Bds in puncta positive for Tf, EEA1 or both. Notice that the macropinocytic tracer becomes co-localized with the markers with a few minutes' delay with respect to Tf; and that its co-localization with the two markers is sequential: first Rbk-5 (newly generated macropinosomes and an early endosome subfraction), and then EEA1 (early endosomes).

 

Figure 3
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  		Fig. 3. Macropinosome generation: dependence on PI3K and Ca2+, and effects of tracers on [Ca2+]i. Panels A-B show DCs loaded with Bds without pretreatments (A), after pretreatment with amiloride (B), after preloading with BAPTA (C), and after pretreatment with the PI3K-blocker drugs Wort (D) and LY-294002 (E) or the inactive analogue LY-303511 (F). Bar, 10 µm (B), also valid for A-D. Panels G and H show representative fura-2 ratiometric [Ca2+]i traces of DC suspensions exposed at the arrows to non-fluorescent Bds (G) or Dex (H). Panel I summarizes the average increases (results of four experiments ± s.d.) induced by the 5 minute addition of either tracer to DCs non-pretreated (NPT) or exposed for the same time to Bds after pretreatment with Wort, Vac1 or BAPTA-AM.

 

Figure 4
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  		Fig. 4. Effects of ionomycin on DCs: [Ca2+]i increase and surface area expansion. Panel A shows the increase of the fura-2 ratiometric [Ca2+]i signal in a DC suspension exposed to IONO (arrow). The average increases at 1 minute (results of four experiments ± s.d.) in DCs, non-pretreated (NPT) and pretreated with Wort, Vac1 or BAPTA-AM, are shown in B. Panel C shows the prompt IONO-induced (arrow) increase of FM1-43 fluorescence recorded in a DC suspension analyzed in the presence of the dye, documenting cell surface expansion; panel D shows the average 1 minute FM1-43 fluorescence increases in DC suspensions pretreated as in panel B (results of four experiments ± s.d.).

 

Figure 5
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  		Fig. 5. Ionomycin-induced discharge of macropinosomes. Panel A shows the confocal merged images of DCs loaded for 10 minutes with Bds (green) before fixation, followed by EEA1 immunolabelling (red). The large co-localization of Bds and EEA1 (yellow) is similar to that in Fig. 1E. Panel B shows DCs as in A, but exposed for 1 minute to IONO before fixation and EEA1 immunolabelling. Notice the complete disappearance of the Bds-positive puncta with apparent no change in the puncta positive for EEA1 alone. Bar, 10 µm (A), also valid for B. Panel C shows mean ± s.d. of results on Bd-EEA1-positive puncta such as those in A and B, quantified morphometrically in at least ten fields. Notice that the Bd-EEA1-positive puncta, which before IONO were almost 45% of the whole EEA1-positive population, dropped to <3% in 1 minute of application of the ionophore. Panels D and E illustrate results such as those in A and B, respectively, but at the electron microscope level. The macropinosome vacuoles packed with Bds (arrows in D), similar to those of Fig. 1H,I, are no longer visible after 1 minute of IONO administration (E). Bar, 0.5 µm (D), also valid for E.

 

Figure 6
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  		Fig. 6. Blockade by vacuolin1 of the ionomycin-induced discharge of macropinosomes. Panel A shows merged images of DCs pretreated with Vac1 and loaded for 10 minutes with Bds (green) and immunolabelled for EEA1 (red), showing extensive co-localization of the two signals (yellow) in fluorescent puncta distinctly larger than those of non-pretreated cells (Fig. 1E and Fig. 5A). Panel B shows DCs as in A but stimulated with IONO for 1 minute. The puncta positive for both Bds and EEA1 appear unaffected by the ionophore. Morphometric quantification of the data in the VAC1-pretreated cells before and after IONO (D) reveals an almost doubling of the percentage of dually positive puncta, with respect to DCs non-pretreated with the drug (Fig. 5C), and no significant decrease induced by the ionophore. At the ultrastructural level (panels E,F), the VAC1-pretreated cells show swollen macropinosomes. Their Bd density is similar in IONO-treated and untreated cells and distinctly lower than that of macropinosomes of non-pretreated DCs (Fig. 1H,I and Fig. 5D). Panel C shows VAC1-pretreated DCs loaded with Bds as in A, immunolabelled for Lamp1 (red). Lamp-1-positive puncta are larger than in non-pretreated cells, however their lack of co-localization with the tracer is as in untreated cells (supplementary material Fig. S1B,D). Bar, 10 µm (A), also valid for B and C. Bar, 0.5 µm (F), also valid for E.

 

Figure 7
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  		Fig. 7. Macropinocytosis-macropinosome discharge coupling. The two left histograms of panel A summarize FACS results with DCs loaded for 20 minutes with FITC-conjugated Bds and then split in two aliquots, one fixed immediately (left), the other after an additional 10 minutes of incubation with TRITC-conjugated Bds (centre). Notice the decrease of the FITC signal (over 30%) during the second incubation compensated by an analogous accumulation of the TRITC signal, indicating replacement of the discharged green Bds with red Bds. The histogram to the right of A shows that the 20 minutes of accumulation of FITC-conjugated Bds in DCs pretreated with Vac1 is almost 30% larger than that of non-pretreated cells, as expected for a drug-induced blockade of macropinosome discharge. The DCs of panels B and C, first pulsed for 20 minutes with FITC-conjugated Bds, were chased for 10 or 20 minutes with non-fluorescent Bds and then immunolabelled for EEA1 and TfR. Morphometric analyses of confocal images (at least ten fields) revealed considerable decreases of Bd fluorescence during chase (B), largely because of the discharge of Bd-rich puncta positive for EEA1 (C). By contrast, Bd-rich puncta positive for TfR alone increased proportionally (from ~10% to ~40% of the puncta present at 0 and 20 minutes of chase, respectively, panel C).

 

Figure 8
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  		Fig. 8. Enlargeosome exocytosis in DCs exposed to the tracers or ionomycin. Panels A-D show cell images obtained from DCs permeabilized after fixation. The enlargeosome marker, d/A (red), which appears in small cytoplasmic puncta of resting DCs (A), is redistributed to the cell surface after exposure to Bds, as shown in the same cells: (B) redistribution of d/A (red); (C) the Bds taken up in 10 minutes incubation (green); (D) the merged image, which reveals the negativity for d/A of the intracellular Bd-rich puncta and the lack of coincidence of the Bds and the d/A also at the cell surface. Panels E-F and H-M show merged, red-green images of cells that were not permeabilized before immunolabelling. In these preparations, d/A (red), an enlargeosome lumenal membrane protein, is labelled only after exocytosis, at the external surface of the plasma membrane. No surface d/A signal is appreciable in resting DCs (E), whereas after Bd application (green, 10 minutes) a strong surface d/A signal becomes appreciable (F). Panel G shows the time-course of the d/A surface labelling in DCs fixed from 1 to 20 minutes after application of Bds or Dex, revealed by FACS analysis and expressed as arbitrary units. Panel H shows that the surface redistribution of d/A induced by 10 minutes of exposure to Bds was not decreased by DC pretreatment with Vac1 (quantification in ten fields), whereas it was blocked by pretreatment with Wort (panel I) and preloading with BAPTA (panel J), Panels K-M show that the d/A surface redistribution induced by IONO (K) was unaffected by pretreatment of the cells with Wort (L) or Vac1 (M). Bar, 10 µm (D), also valid for A-C. Bar, 10 µm (F) and H-M.

 

Figure 9
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  		Fig. 9. Bead uptake and d/A surface redistribution in DCs exposed to PIP3. Panels A-D are merged images of DCs exposed to phosphoinositides for 20 minutes, loaded or not with the Bds for 10 minutes, fixed and surface immunolabelled for d/A. With PIP3, Bd uptake and d/A surface appearance took place even after pretreatment with Wort (A) or preloading with BAPTA (B). Notice the large size of the PIP3-induced Bd-rich puncta mostly located near the plasma membrane (tridimensional reconstruction in supplementary material Fig. S5); (compare with the cells exposed to the beads only: Fig. 1D,E; Fig. 5A, Fig. 8D,F,H). In PIP3-exposed DCs pretreated with Wort (not shown) or preloaded with BAPTA (C) the d/A surface redistribution occurred even without Bd loading. By contrast, the PIP3 precursor, PIP2, had no effect in DCs, not-pretreated and pretreated with Wort (not shown) or with BAPTA (D). Panels E-G show merged images of DCs pretreated with PIP3 as in A-C and then loaded for 10 minutes with Bds (green) together with FM4-64 (FM, red). The large Bd-rich puncta of E were sealed, discrete vacuoles because after washing they retained their FM staining. Some loss of the FM signal was visible in contrast to DCs pretreated with Wort (F) and, even more, in those preloaded with BAPTA (G). The Bd-rich puncta induced by PIP3 were negative for EEA1 (red in H). Bar, 10 µm (C), also valid for A,B,D,H. Bar, 10 µm (E), also valid for F and G.

 

Figure 10
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  		Fig. 10. Macropinocytic membrane traffic in tracer-exposed DCs. The model illustrates three sequential membrane traffic phases (specified at the top and dealt with in the Discussion), occurring in DCs during the first 30 minutes of tracer (orange dots) exposure. Phase 1 starts with the application of the tracers (Bds or Dex) and ends 4 minutes later, with the change of the kinetics of tracer uptake (see Fig. 2A). During this time [Ca2+]i begins to rise in DCs (see Fig. 3D,E). In addition to the low-rate macropinocytosis, the main traffic event of Phase 1 is the early exocytosis of enlargeosomes (yellow membrane), initiated within 1 minute and continued thereafter (see Fig. 8G). Some specialization of the plasma membrane by sorting of specific components, anticipating the generation of macropinosomes (purple), is assumed to take place at the bending ruffles sticking out from the cell surface. The increased rate of tracer-positive macropinosome (purple membrane) generation, which seems to depend on [Ca2+]i (blocked by cell preloading with BAPTA) and PI3K activity (blocked by Wort) (see Fig. 2 and supplementary material Fig. S2), is illustrated in Phase 2. Macropinosomes, progressively enlarged (probably by fusion) and packed with the tracer, acquire in sequence various markers: first Rbk-5, then EEA1 (early endosomes, blue membrane) and finally TfR (recycling endosomes, green membrane) (Fig. 2B,C; supplementary material Fig. S2). During this phase tracer uptake predominates, however macropinosomes can undergo regulated exocytosis (regurgitation, blocked by Vac1, Fig. 7A), with discharge of their segregated tracer. This process depends on [Ca2+]i. In the case of µM [Ca2+]i increases, such as those induced by IONO, exocytosis becomes prompt and complete (see Fig. 5). The alternative possibility, more frequent for macropinosomes positive for TfR and no longer for EEA1 (Fig. 7C), is entering in a `deep pathway', presumably leading to processing and finally to presentation of the antigens. Phase 3, in which uptake and discharge of tracer approach equilibrium, begins after ~20 minutes of application (Fig. 1B and Fig. 7) and probably continues as long as the tracer is applied. During this phase, even if macropinocytosis continues at similar rate, the number of macropinosomes per DC remains approximately stable (Fig. 1A,B and Fig. 7A) so that cell overloading is prevented.

 

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