QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

doi: 10.1242/10.1242/jcs.00259

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Duclos, S. Articles by Desjardins, M. Search for Related Content PubMed PubMed Citation Articles by Duclos, S. Articles by Desjardins, M. Social Bookmarking                         What's this?

Remodeling of endosomes during lysosome biogenesis involves `kiss and run' fusion events regulated by rab5

Département de pathologie et biologie cellulaire, Université de Montréal, CP 6128, Succ. Centre ville, Montréal, QC, H3C 3J7, Canada

View larger version (97K): [in a new window]   Fig. 1. Rab5(Q79L) induces the formation of giant endosomes in RAW264.7 macrophages. The effect of the rab5(Q79L) mutation on the morphology of endosomes (e) in RAW264.7 macrophages was examined at the electron microscope. Internalization of HRP (10 mg/ml) for 30 minutes in control (A) or rab5(Q79L)-expressing cells (B) led to the accumulation of this tracer in small vesicles in control cells (A), while it was found in very large endosomes (e) in mutant cells (B). Bar, 0.5 µm.

View larger version (145K): [in a new window]   Fig. 2. Giant endosomes kinetically correspond to both early and late endosomes. To determine the nature of the large endosomes, control or rab5(Q79L)-expressing cells were allowed to internalize the fluid phase endocytic tracer BSA-rhodamine (1.5 mg/ml) for 2 minutes, followed by the indicated times of chase. While BSA-rhodamine was observed in small vesicles (small arrows) at each time point studied in control cells, in mutant cells this tracer was present in giant endosomes (large arrows) for at least 30 minutes after its internalization. However, chase of the fluorescent tracer for 60 minutes resulted in its transfer to smaller vesicles similar to those observed in control cells. The inset represents large vacuoles (arrowheads) not displaying the fluorescent tracer that could be observed at this time point in rab5(Q79L)-expressing cells. n, nucleus. Bar, 10 µm.

View larger version (121K): [in a new window]   Fig. 3. Giant endosomes do not correspond to lysosomes. To further demonstrate the nature of the giant endosomes in rab5(Q79L) expressing macrophages, cells were incubated with 16 nm BSA-gold for 30 minutes, followed or not by a 16 hours chase period. After an internalization of 30 minutes (A), BSA-gold particles were found in large endocytic organelles (e). As in the kinetic experiment with the fluid phase tracer, a longer chase period (16 hours) resulted in the transfer of the BSA-gold particles to smaller electron dense vesicles (l) (B), while large vacuoles now devoid of BSA-gold particles could be observed. Bars, 100 nm.

View larger version (166K): [in a new window]   Fig. 4. Bafilomycin A1 induces fragmentation of early endosomes in control cells. To determine the effect of the proton pump inhibitor bafilomycin A1 (Baf) on endocytic organelles, macrophages were incubated in the presence of 16 nm BSA-gold particles for the indicated times in order to form endosomes of various ages. Cells were then treated for 30 minutes with 500 nM of bafilomycin A1 (right panels) or DMSO as a control (left panels), and the resulting morphology of the endocytic structures was observed at the electron microscope. As shown in the top right panel, only early endosomes have been disrupted by the bafilomycin A1 treatment. Bar, 550 nm (bottom-right panel) or 500 nm (all other panels).

View larger version (165K): [in a new window]   Fig. 5. Bafilomycin A1 does not induce fragmentation of giant late endosomes in rab5(Q79L)-expressing cells. To investigate the effect of bafilomycin A1 (Baf) on the giant endosomes in rab5(Q79L)-expressing cells, late endosomes (le) were formed by a 1 hour/4 hour pulse-chase of 35 nm BSA-gold particles, and the same cells were then allowed to internalize 5 nm BSA-gold particles for 30 minutes in order to load mostly early endosomes (ee) (A). The two sizes of particles are found in different endosome populations, both representing quite large endosomes. Panel B corresponds to an inset of panel A, allowing a better visualization of early endosomes containing only 5 nm BSA-gold particles. The same experiment was repeated, with cells incubated in the presence of 500 nM bafilomycin A1 for 30 minutes after the pulse-chase of gold particles. In C, we show a large late endosome filled with 35 nm BSA-gold particles, while the 5 nm BSA-gold particles are now found in very small vesiculo-tubular structures, better seen at a higher magnification (D). Bars, 250 nm (A,C); 50 nm (B,D).

View larger version (43K): [in a new window]   Fig. 6. Giant endosomes display early and late endosome/lysosome markers in RAW264.7 macrophages. To better confirm the nature of the large endocytic structures, rab5(Q79L)-expressing macrophages were grown on coverslips, fixed and probed with antibodies against different endosomal markers. Cells were then observed at the epifluorescence microscope. The early endosomal markers EEA1 and rabaptin-5 decorated large vesicles in a punctate pattern (arrows). The same phenomenon was observed with rab7, as well as with the recently described late endosome marker flotillin1. The large vacuoles were also positive for the late endosomal/lysosomal markers LAMP1 and LAMP2. However, these markers defined a continuous pattern around the large endosomes. Co-localization of rab5(Q79L) (designated as EGFP-rab5) with LAMP1 was observed using confocal fluorescence microscopy in cells expressing EGFP-rab5(Q79L). Arrowhead in the merge panel points at the section magnified in the inset, where arrows point at the yellow dots representing endosomal membrane domains on which EGFP-rab5(Q79L) and LAMP1 co-localize. n, nucleus; e, endosome. Bar, 10 µm.

View larger version (88K): [in a new window]   Fig. 7. Rab5 regulates the `kiss and run' fusion between early/late endosomes. To determine whether rab5 modulates `kiss and run' interactions between endosomes, cells were incubated with mixtures of dextrans (FDx10 and TRDx70 or FDx70 and TRDx70) for 30 minutes, then washed and further incubated for the indicated times. The distribution of dextrans was observed by confocal fluorescence microscopy. Immediately after internalization, the different-sized dextrans (FDx10, green signal and TRDx70, red signal) were co-localizing in the same organelles, labeled in yellow, in control and rab5(Q79L)-expressing cells. After a 30 minute chase, distinctly labeled endosomes were observed in control cells allowed to internalize the mixture of FDx10 and TRDx70. At the same time point in mutant cells, the different-sized dextrans were still co-localizing in the endocytic compartments. After a 120 minute chase, the segregation of different-sized dextrans was almost complete in control cells, in contrast with the mutants, where yellow endosomes showing the co-localization of the FDx10 and TRDx70 could still be observed. After a longer chase period (240 minutes), the size-dependent segregation was still observed in control cells, while the separation of the different-sized dextrans started to become apparent in mutant cells. The inset represents a control cell after a pulse-chase of 30/240 minutes of FDx70 and TRDx70, which clearly demonstrates that the segregation of dextrans depends on the size of the molecules, and not on the properties of the different fluorophores, since similar-sized dextrans co-localize in the same vesicles. Bar, 10 µm.

View larger version (102K): [in a new window]   Fig. 8. `Kiss and run' fusion between early/late endosomes. To further characterize and quantitate the transient `kiss and run' interactions between endosomes, cells were pulse-chased with a mixture of 100 nm BSA-coated latex beads and 16 nm BSA-gold particles for the indicated times. Endosomes containing either a mixture of 100 nm (arrows) and 16 nm (arrowheads) particles, or only one of the tracers were quantified at the electron microscope. The results obtained indicate that, at the earliest time point observed (15 minutes of internalization), the majority of endosomes contained the mixture of particles in control and rab5(Q79L)-expressing cells. At 150 minutes after endocytosis, the two tracers were segregated in different endosomes in contol cells, while a large proportion of endosomes still contained both of them in rab5(Q79L)-expressing cells. Quantitative analysis of these results is presented in the bottom panel. Bar, 100 nm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter