Plasticity of early endosomes

Abstract

We observed that the structural organization of early endosomes was significantly modified after cell surface biotinylation followed by incubation in the presence of low concentrations of avidin. Under these conditions early endosomes increased in size to form structures which extended over several micrometers and which had an intra-luminal content with a characteristic electron-dense appearance. The modified early endosomes were not formed when either avidin or biotinylation was omitted, suggesting that they resulted from the cross-linking of internalized biotinylated proteins by avidin. Accumulation of a fluid-phase tracer was increased after the avidin-biotin treatment (145% after 45 min). Both recycling and transport to the late endosomes still occurred, albeit to a somewhat lower extent than in control cells. Quantitative electron microscopy showed that the volume of the endosomal compartment was increased approximately 1.5-fold but that the surface area of the compartment decreased relative to its volume after avidin-biotin treatment. Finally, overexpression of a rab5 mutant, which is known to inhibit early endosome fusion in vitro, prevented the formation of these structures in vivo and caused early endosome fragmentation. Altogether, our data suggest that early endosomes exhibit a high plasticity in vivo. Cross-linking appears to interfere with this dynamic process but does not arrest membrane traffic to/from early endosomes.

Photoconversion of FM1-43 Reveals Differences in Synaptic Vesicle Recycling and Sensitivity to Pharmacological Disruption of Actin Dynamics in Individual Synapses

“…How these two drugs affected the capability of FM-dye to drive DAB polymerization and to form an osmiophilic precipitate when photoactivated was then analyzed in stimulated cells (Figure A). , In terms of ultrastructure, presynaptic terminals contained both photoconverted (PC + ) vesicles with an electrodense lumen and nonphotoconverted vesicles (PC – : Figure B). Indeed, presynaptic boutons contained many SVs of ∼40 nm diameter (both PC + and PC – ) juxtaposed to postsynaptic densities, as well as other endosomal structures with a distinct morphology. − We quantified the photoconverted vesicles relative to the total pool in sections of cells subjected to the different treatments (Figure C) and consistent with the aforementioned results, JSK and LA significantly reduced the proportion of PC + SVs (control 68.27 ± 11.65%; JSK 60.13 ± 14.28%; LA 48.92 ± 13.62%: p < 0.001, one-way ANOVA followed by Holm–Sidak method). The distance of the photoconverted (PC + ) and nonphotoconverted (PC – ) vesicles from the AZ was measured, and although JSK or LA did not alter the distribution of nonphotoconverted (PC – ) vesicles (data not shown), there was a significant decrease in the proportion of photoconverted (PC + ) vesicles within 50 nm of the synapse in LA treated cells (control 8.79 ± 1.37%; JSK 9.55 ± 1.27%; LA 5.18 ± 1.15%: p = 0.004, one-way ANOVA followed by Dunn’s method; Figure D).…”

“…To obtain a readout of the terminaĺs ultrastructure when the drugs were applied after stimulation, cells were dye-labeled, washed for 5 min, incubated with either JSK or LA, and then fixed (Figure A). When photoconverted and analyzed by TEM (Figure B), many ∼40 nm diameter SVs (PC + and PC – ) and pleomorphic endosomal structures were evident in presynaptic terminals . We quantified the photoconverted SVs relative to the total pool in the cells and while JSK did not modify the proportion of PC + SVs, LA did clearly reduce this number (control 57.53 ± 14.29%; JSK 62.70 ± 13.14%; LA 47.99 ± 14.49%: p < 0.001, Kruskal–Wallis test followed by Dunn’s method; Figure C).…”

“…When photoconverted and analyzed by TEM (Figure 8B), many ∼40 nm diameter SVs (PC + and PC − ) and pleomorphic endosomal structures were evident in presynaptic terminals. 66 We quantified the photoconverted SVs relative to the total pool in the cells and while JSK did not modify the proportion of PC + SVs, LA did clearly reduce this number (control 57.53 ± 14.29%; JSK 62.70 ± 13.14%; LA 47.99 ± 14.49%: p < 0.001, Kruskal−Wallis test followed by Dunn's method; Figure 8C). There was no difference in the distance of the photoconverted (PC + ) and nonphotoconverted (PC − ) vesicles from the AZ in the control synapses or the synapses of cells exposed to JSK, although there were significantly fewer recycled (PC + ) vesicles within 150 nm of the synapses of LA treated cells (0−50 nm bin, control 10.99 ± 1.41%; JSK 11.08 ± 2.04%; LA 6.89 ± 1.49%: p = 0.003 Kruskal−Wallis test followed by Dunn's method; 50−100 nm bin, control 12.00 ± 1.32%; JSK 12.35 ± 1.50%; LA 7.16 ± 1.22%: p = 0.01 Kruskal−Wallis test followed by Dunn's To determine the rate of dye release, the fluorescence remaining in each bouton at the end of the stimulation was subtracted from all the values and then normalized to the initial fluorescence.…”

Mapping lesion-specific response and progression dynamics and inter-organ variability in metastatic colorectal cancer

“…These organs are known to have discontinuous or fenestrated endothelial membranes, which may lead to higher drug exposure, potentially conferring high treatment responses 43,44 . In contrast, the organs bearing poorly-responding lesions are usually those with continuous endothelial membranes and thus more limited drug distribution, such as the muscle and brain/CNS [45][46][47][48] . Some organs that bear poorly responding metastatic lesions, such as kidney and muscle, have relatively dense tissue matrices.…”

Mapping Intrapatient Response Heterogeneity and Lesion-specific Relapse Dynamics in Metastatic Colorectal Cancer

“…These organs have discontinuous or fenestrated endothelial membranes, which may lead to higher drug exposure, potentially conferring high treatment responses 47,48 . In contrast, the organs bearing poorly-responding lesions are usually those with continuous endothelial membranes and thus more limited drug distribution, such as the muscle and brain/CNS [49][50][51][52] . Some organs that bear poorly-responding metastatic lesions, such as kidney and muscle, have relatively dense tissue matrices.…”