Fluorescent labeling endocytic vesicles

The development of amphipathic fluorescent dyes that label endocytic vesicles has permitted the study of endo-cytosis in nerve terminals in real time [25,26], The probe FM1-43 equilibrates between the aqueous phase and the membrane but is not membrane-permeating. The plasmalemma becomes fluorescent (Fig. 10-8). Upon endocytosis, the labeled membrane is internalized. When removed from the extracellular medium, the dye is retained by the endocytic vesicles but lost from the plasmalemma. Endocytic vesicles are transformed into synaptic vesicles containing FM1-43. Importantly, recycled synaptic vesicles lose the probe upon exocytosis. 

Fig. 2. Intracellular localization of TAT-liposomes. OVCAR-3 cells are incubated with 150 nmol of double fluorescently labelled TAT-liposomes for 1 h and subsequently incubated for 1 h (a) or 23 h (b) in liposomes-free medium. Thirty minutes before visualization the endocytic pathway is labelled with Lysotracker Red. Live cell imaging is performed with confocal laser scanning microscopy. Double labelled liposomes are used to study the integrity of the liposome during the uptake process co-localization of both liposomal labels would indicate that the liposomes are intact. 1 h both liposomal labels are localized at the plasma membrane, which represent intact cell-bound TAT-liposomes. The electronically merged image clearly shows lack of co-localization with the endocytic pathway marker, Lysotracker Red. This opposite to the 24 h incubation, both liposomal labels can be seen intracellularly in a punctuate pattern. In the electronically merged image, co-localization with Lysotracker Red is clearly visible. This indicates that the TAT-peptide modified liposomes bind to the plasma membrane and after internalization are present in endocytic vesicles. Therefore, we conclude that the liposomes are internalized by endocytosis. (Reproduced from (12) with permission from Elsevier Science)...

Figure 27 Block copolymers In which one block serves as an AID can be internalized by cells. Fluorescence microscopic images of live HeLa cells incubated with the rhodamine-labeled polymer for varying time points at 37 °C are shown. The block copolymer localizes in both endocytic vesicles (punctate staining) and the cytoplasm (diffuse fluorescence). Scale = 25 pm.

Certain viruses and bacterial toxins are endocytosed by a specialized pathway that involves invaginations of the plasma membrane known as caveolae. Recent studies have traced out this caveolar endocytic pathway by jointly tagging the cellular machinery with GFP and labeling viruses or bacterial toxins with fluorescent dyes, which enables their dual visualization in real time (23, 24). Such work has revealed several unanticipated properties of this pathway, such as the ability of viruses to induce formation of actin comets that propel virus-containing vesicles (23) and the stable, immobile nature of the caveolar coat that encases these vesicles (24). 

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