Immunolabeling analysis of biosynthetic and degradative pathways of cell surface components (glycocalyx) in Paramecium cells

Abstract

Biosynthetic and degradative pathways of glycocalyx components are largely unknown in Paramecium and in some related parasitic protozoa. We isolated cell surface (glyco-)proteins, i.e., surface antigens (SAg) and used them in the native (nSAg) or denatured (dSAg) state to produce antibodies (AB) for immunolocalization by confocal imaging and by quantitative immunogold EM-labeling of ultrathin sections or of freeze-fracture replicas. Antibodies against nSAg or dSAg, respectively, yield different labeling densities over individual structures, thus indicating biosynthetic or degradative pathways, respectively. We derive the following biosynthetic way: ER --> Golgi apparatus --> non-regulated/non-dense core vesicle transport --> diffusional spread over non-ciliary (somatic) and ciliary cell membrane. For degradation we show the following pathways: Concentration of nSAg in the cytostome --> nascent digestive vacuole --> mature vacuoles --> release of dSAg at cytoproct, with partial retrieval by "discoidal vesicles". A second internalization pathway proceeds via coated pits ("parasomal sacs") --> early endosomes ("terminal cisternae") --> digestive vacuoles. Dense packing of SAg in the glycocalyx may drive them into the endo-/phagocytic pathway. Still more intriguing is the site of nSAg integration into the cell membrane by unstimulated exocytosis. We consider unconspicuous clear vesicles relevant for nSAg export, probably via sites which most of the time are occupied by coated pits. This could compensate for membrane retrieval by coated pits, while scarcity of smooth profiles at these sites may be explained by the much longer time period required for coated pit formation as compared to exocytosis.