Fig 1.
TEM micrographs of UROtsa cells demonstrate the lack of any internalization of colloidal gold particles in absence of liposomes by absence of any dark grains inside the cells.
Compared to faint to dark grey color of uranyl acetate staining acquired by other cell organelles and electron dense gold particles appear as dark grains. UROtsa cells incubated at either 37°C (Panel A&C) or at 4°C (Panel B&D) showed similar absence of any dark grains inside the endosome marked by E in all the panels to indicate uptake was independent of temperature. Inscribed area of panel A and B in white rectangle is magnified 3 times further in panel C and D, respectively. UROtsa cells have prominent large nucleus shown by N and cells are marked by finger like projections called lateral interdigitation (shown by LI) for connecting with adjacent cells. Plain gold is marked by red colored G, Magnification is shown by the scale bar in each panel.
Fig 2.
TEM micrographs showing endocytosis mediated uptake of liposome encapsulated gold marker at 37°C (Panel A&C).
Higher magnification image of inscribed area from panel A showed that vesicle like structures in endosomal compartment (marked by E) contained cluster of electron dense dark particles inside a single cell (Panel C). In contrast, only extracellular binding of liposomes containing dark grains was observed at 4°C (Panel B) and corresponding higher magnification image (Panel D) showed that vesicle like structures in a single cell were devoid of dark gold particles, which indicates absence of internalization due to temperature dependent inhibition of endocytosis. Gold encapsulated in liposomes is marked by red colored G inside a red circle, nucleus is marked by N, endocytic vesicles are marked by E and finger like projections called as lateral interdigitation are marked by LI. Inscribed area of panel A and B in white rectangle is magnified further in panel C and D, respectively and magnification is shown by scale bar in each panel.
Fig 3.
TEM micrographs showing endocytosis mediated uptake of liposome encapsulated gold in rat bladder (A) as revealed by electron dense dark grains consistent with uptake of gold across the urothelium.
(B) Dense black grains binding to the cell surface were noted in rat group instilled with colloidal gold in absence of liposomes. Gold encapsulated in liposomes are marked by red colored G inside a red circle and plain gold is marked by yellow G in the images. (C) Untreated rat bladder is marked by (absence of dark black gold grains. Magnification of 8900x was used in all the images and is shown by the scale bar.
Fig 4.
Effect of different endocytic inhibitors on cellular uptake of fluorescent liposomes.
Flow cytometry analysis suggest clathrin mediated endocytosis as the dominant pathway for the internalization of fluorescently labeled liposomes by UROtsa cells at 37°C. Chlorpromazine (inhibitor of clathrin) dose dependently inhibited the internalization as cell fluorescence from externally bound non-internalized liposomes was quenched by sodium dithionite. Fluorescence intensity of liposomes in cells untreated with inhibitors was taken as 100%.
Fig 5.
Illustration depict temperature dependent endocytoic uptake of liposome encapsulated gold (Lipo-gold).
The cellular process of clathrin and caveolin mediated endocytosis are energy dependent and therefore only occur at 37°C and are inhibited at 4°C. Compared to mhCD, chlorpomazine was more efficient in blocking endocytosis of liposomes, which indicates a predominance of clathrin mediated endocytosis as a mechanism of endocytosis.