Filamentous actin in Paramecium cells: mapping by phalloidin affinity labeling in vivo and in vitro.
In living Paramecium cells, microinjected rhodaminyl (R)-phalloidin rapidly labels a thin cortical layer. This can be more clearly resolved with microinjected and fixed cells (allowing for better resolution) as well as with isolated pellicles (surface membrane complexes with trichocysts, microfilaments, and mitochondria attached). Labeling of a longitudinal and perpendicular pattern, reflecting the relief of the cell surface, and labeling of ciliary basal bodies then becomes clearly visible. Other structures labeled by R-phalloidin are the surfaces of food vacuoles of different sizes and, although inconsistently, the borders of the buccal cavity. Small acidic compartments (as identified by acridine orange fluorescence vital staining), probably representing acidosomes and small lysosomes, were not labeled. F-actin on food vacuole surfaces may somehow be involved in intracellular transport or fusion processes. No labeling was observed in association with the osmoregulatory system (contractile vacuoles and their ampullae and radial canals). The specificity of in vivo labeling obtained was supported by the abolition of R-phalloidin labeling when isolated pellicles were pretreated with unlabeled phalloidin or with DNAse I. It was also possible to discriminate among different layers of R-phalloidin binding in the cortex by detaching different layers of the surface complex from each other. Since localization of F-actin in ciliates has raised a considerable amount of dispute in the past, we also repeated all these experiments with RITC-labeled HMM, but we obtained essentially the same labeling pattern as with R-phalloidin. Ciliary basal bodies therefore clearly contain some F-actin. Our data shed some light on aspects of surface structuring and motility in these cells.
H
H
Kersken
J
J
Vilmart-Seuwen
M
M
Momayezi
H
H
Plattner
D008841:Actin Cytoskeleton / Q000648:ultrastructure*; D000199:Actins / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D003599:Cytoskeleton / Q000648:ultrastructure*; D008845:Microinjections; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D009842:Oligopeptides / Q000378:metabolism*; D010247:Paramecium / Q000166:cytology*; D010590:Phalloidine / Q000378:metabolism*; D011485:Protein Binding; D013194:Staining and Labeling
D000199:Actins; D009842:Oligopeptides; D010590:Phalloidine