Mapping cholesteryl ester analogue uptake and intracellular flow in Paramecium by confocal fluorescence microscopy

Abstract

SummaryIn Paramecium primaurelia the uptake and intracellular flow of cholesteryl ester was studied by fluorescence confocal laser scanning optical microscopy and by the fluorescent analogue cholesteryl-BODIPY ® FL C 12 (BODIPY-CE). The BODIPY FL fluorophore has the characteristic of emitting green fluorescence, which is red-shifted as the probe concentrates. In cells incubated with 25 µ m BODIPY-CE for 30 s, fluorescence is found in vesicles located around the cytopharynx in the posterior half of the cell. Successively, the lipid is internalized by food vacuoles, the fluorescent vesicles are distributed throughout the cell and the intracellular membranes are labelled. The food vacuole number is maximum after 10-15 min of continuous labelling, then it decreases until no food vacuoles are found in 30-min fed cells. BODIPY-CE accumulates in red-labelled cytoplasmic droplets located in the anterior half of the cell. When food vacuole formation is inhibited by trifluoperazine, fluorescence is found on cellular membranes and in small green-labelled vesicles at the apical pole. The inhibition of clathrin-mediated endocytosis does not interfere in P. primaurelia with BODIPY-CE intracellular flow: intracellular membranes and storage droplets in the cell anterior part are dyed. Conversely, the use of sterol-binding drugs prevents the lipid accumulation in droplets, stopping the lipid within the cytoplasmic membranes. Furthermore, the cells treated with monensin and cytochalasin B show a labelling of the cellular membranes and lipid droplets, whereas NH 4 Cl reduces the lipid storage. Low temperature (4 ° C) does not prevent the internalization of BODIPY-CE that, however, is localized at the cytoplasmic membrane level and does not accumulate in storage droplets. In addition, BODIPY-CE inhibits phagocytosis, as evidenced by comparing the kinetics of food vacuole formation of control cells, only fed with latex particles, with that of cells fed with latex particles and BODIPY-CE. In conclusion, this study points out that in P. primaurelia the cholesteryl ester enters the cell via food vacuoles and through the plasma membrane and, inside the cell, it alters cell functions.

Transport Mechanisms of Squalenoyl-Adenosine Nanoparticles Across the Blood–Brain Barrier

“…When NAs were incubated on the transwells at 4 °C, no detectable passage of the BODIPY dye could be observed (Figure B), suggesting an energy-dependent transcytosis mechanism across the cell monolayer. Finally, incubating the cells with the BODIPY-FL probe alone resulted in a reduced passage of the dye, comparatively to BODIPY-embedded NAs (Supporting Information, Figure 20), which was consistent with the storage of the free fluorescent probe into lipidic droplets inside the cell cytoplasm . More importantly, this observation demonstrated that the NAs triggered the transcytosis of the BODIPY dye.…”

Introducing the concept of biocatalysis in the classroom: The conversion of cholesterol to provitamin D3

“…Figure 5A shows co-localization between fluorescent E. coli and bodipy-cholesterol, suggesting that cholesterol is taken up via phagocytosis. These results are in accordance with previous experiments carried out in the ciliate Paramecium [27].…”

“…The appearance of red-stained particles in the ciliate (a well-known bacteriovorus organism) would confirm the use of this pathway. These results are in accordance with previous experiments carried out in the ciliate Paramecium [27]. These results are in accordance with previous experiments carried out in the ciliate Paramecium [27].…”

Identification, Localization, and Functional Implications of the Microdomain-Forming Stomatin Family in the Ciliated Protozoan Paramecium tetraurelia

“…Endocytosis via coated pits was tested as indicated by Ramoino et al (46). Briefly, phagocytosis was inhibited by trifluoperazine (2.5 g/ml; Sigma-Aldrich, St. Louis, MO), and internalization of endocytic vesicles from the cell surface was followed with wheat germ agglutinin labeled with AlexaFluo-488 (Sigma-Aldrich, St. Louis, MO) for fluorescence analysis in a Zeiss Axiovert 100TV.…”