Microdomain Ca2+ Activation during Exocytosis in Paramecium Cells. Superposition of Local Subplasmalemmal Calcium Store Activation by Local Ca2+ Influx

In Paramecium tetraurelia, polyamine-triggered exocytosis is accompanied by the activation of Ca2+-activated currents across the cell membrane (Erxleben, C., and H. Plattner. 1994. J. Cell Biol. 127:935– 945). We now show by voltage clamp and extracellular recordings that the product of current × time (As) closely parallels the number of exocytotic events. We suggest that Ca2+ mobilization from subplasmalemmal storage compartments, covering almost the entire cell surface, is a key event. In fact, after local stimulation, Ca2+ imaging with high time resolution reveals rapid, transient, local signals even when extracellular Ca2+ is quenched to or below resting intracellular Ca2+ concentration ([Ca2+]e ⩽ [Ca2+]i). Under these conditions, quenched-flow/freeze-fracture analysis shows that membrane fusion is only partially inhibited. Increasing [Ca2+]e alone, i.e., without secretagogue, causes rapid, strong cortical increase of [Ca2+]i but no exocytosis. In various cells, the ratio of maximal vs. minimal currents registered during maximal stimulation or single exocytotic events, respectively, correlate nicely with the number of Ca stores available. Since no quantal current steps could be observed, this is again compatible with the combined occurrence of Ca2+ mobilization from stores (providing close to threshold Ca2+ levels) and Ca2+ influx from the medium (which per se does not cause exocytosis). This implies that only the combination of Ca2+ flushes, primarily from internal and secondarily from external sources, can produce a signal triggering rapid, local exocytotic responses, as requested for Paramecium defense

Hydrophobic and hydrophilic radio-iodination, crosslinking, and differential extraction of cell surface proteins in Paramecium tetraurelia cells

We combined widely different biochemical methods to analyze proteins of the cell surface of P. tetraurelia since so far one can isolate only a subfraction of cell membrane vesicles enriched in the GPI-anchored surface antigens ( immoblization or i-AGs ). We also found that i-AGs may undergo partial degradation by endogenous proteases. Genuine intrinsic membrane proteins were recognized particularly with lipophilic 5-[125I]-iodonaphthalene-1-azide (INA) labeling which reportedly sees integral proteins and cytoplasmic cell membrane-associated proteins. With INA (+DTT), bands of ≤ 55 kDa were similar as with hydrophilic iodogen (+DTT), but instead of large size bands including i-AGs, a group of 122, 104 and 94 kDa appeared. Several bands of the non i-AG type are compatible with integral (possibly oligomeric) or associated proteins of the cell membrane of established molecular identity, as we discuss. In summary, we can discriminate between i-AGs and some functionally important minor cell membrane components. Our methodical approach might be relevant also for an analysis of some related protozoan parasites

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

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