The contractile properties of the myonemes of Stentor are very similar to caltractin (centrin)-containing fibers of other organisms. We investigated whether the calcium-binding protein caltractin was present in Stentor by using three different antibodies to caltractin or caltractin-related proteins, in conjunction with immunofluorescence microscopy and protein blotting. Immunofluorescence demonstrated that a protein immunologically similar to caltractin is present in the myonemes and in the bases of the membranelles of Stentor. The localization to the myonemes is observed in intact cells, osmotically lysed cells, and isolated cortices. Double-label immunofluorescence with anti-alpha-tubulin and anti-caltractin antibodies showed that the fluorescence in the myonemes was not in the overlying Km fibers. The myonemes in the posterior one-third of the cell appear as thick fibers with no cross-bridging. They become thinner as they approach the anterior end of the cell and show extensive cross-bridging here. Staining in the bases of the membranelles shows a distinct comma-like immunofluorescence pattern similar to that seen with protargol-stained cells and SEM views of the membranellar band reported by others. Western blots demonstrated that the caltractin-like protein in Stentor has an apparent molecular weight of 23 kDa compared with the 20-kDa protein from Chlamydomonas and is a calcium-binding protein.

Regenerating Stentor, exposed to the plant lectin phytohemagglutinin, are significantly delayed in completing oral regeneration. All of the delays are restricted to the earliest stages of regeneration prior to stage 3. The effects of phytohemagglutinin are reversible once the drug is removed. The addition of the presumed sugar receptor for phytohemagglutinin, N-acetylgalactosamine, at the start of oral regeneration could not reverse the effects of phytohemagglutinin, but the addition of excess (10 mM) extracellular calcium could. When the addition of the excess calcium was delayed for various times after phytohemagglutinin exposure, the effectiveness of the calcium in reversing the phytohemagglutinin-induced delays was reduced. Based on the mechanism of action of PHA in other cells, these results suggest that membrane proteins may be involved in controlling oral regeneration, possibly through mechanisms involving Cat+.PON the loss of its oral feeding apparatus, the ciliate Sten-

We have found cyclic AMP in the large, heterotrichous ciliate Stentor coeruleus in amounts per milligram protein similar to those found in another ciliate, Tetrahymena pyriformis. The possible function of cyclic AMP in Stentor was first examined by determining its effects on oral regeneration, the process by which Stentor can replace a missing oral apparatus in eight to ten hours. Once begun (by brief exposure to a 15% sucrose solution, causing shedding of the oral apparatus) regeneration follows eight specific morphological stages visible with the dissecting microscope. Continuous exposure of regenerating cells to either N6, Z'-O-dibutyryl adenosine cyclic 3':5'-monophosphate (DBC) or theophylline begun a t the onset of oral regeneration (stage 0) caused delays in the completion of regeneration. The delays induced by DBC occurred in the early stages prior to stage 5. Regenerating cells exposed to DBC or theophylline at various stages of development were delayed, even at stages 5 and 6. Both DBC and theophylline reversibly bleached the cortical pigment of the cells. Guanosine 3':5'-cyclic monophosphate (cyclic GMP), AMP, GMP, and sodium butyrate neither delayed oral regeneration nor bleached the cortical pigment. Excess extracellular calcium ions alone had no effect on oral regeneration, but 10 mM calcium and DBC caused more delay than DBC alone. Thus, the delay of oral regeneration in Stentor caused by cyclic AMP may involve calcium ions.To determine if cyclic AMP can retard in situ ciliary regeneration by Stentor, as it does in Tetrahymena, a new technique, more accurate than past methods, was developed to monitor ciliary regrowth. Using this procedure we found that both DBC and theophylline significantly delayed the in situ ciliary regeneration by Stentor.

Concanavalin A (Con A) has been shown to induce delays in oral regeneration in the ciliate Stentor coeruleus. Associated with the delayed oral regeneration is a shedding of the cell's extracellular pellicle with the loss of some pigment granules. It is shown that the delays in oral regeneration are not the result of the pigment shedding. The delays are localized in the earliest stages of oral regeneration prior to stage 4. The delays caused by Con A are completely reversible by the addition of 2 mg/ml alpha-D-methyl mannoside either at the time of Con A exposure or 5 min later. Con A clearly binds to the cell surface as shown by the binding of FITC-Con A and its reversal by alpha-methyl mannoside. Crosslinking of Con A receptor molecules may be responsible for the effects of Con A since succinyl Con A, which does not crosslink these receptors, has no effect on oral regeneration even at double the Con A concentration. Calcium ions are also implicated in the action of Con A because an excess of extracellular calcium (10 mM) completely eliminates the Con A delays when added simultaneously with Con A. Examination of the minimum extracellular calcium concentration required for this effect showed that 2 mM calcium can reverse most of the delays but that 5 mM is necessary to completely reverse the delays caused by Con A. If the addition of calcium is delayed for various times after Con A addition, the extracellular calcium is progressively less effective in reversing the Con A delays.

This study suggests that membrane perturbations can affect oral morphogenesis in Stentor, possibly by a mechanism involving calcium ions. Exposure of regenerating Stentor to micromolar concentrations of the membrane active local anesthetics dibucaine, tetracaine, or procaine greatly delayed the progress of oral regeneration. In the case of tetracaine and dibucaine the greatest delays were observed in the early stages of regeneration prior to stage 4, when the majority of essential synthetic activity is occurring. The effects of dibucaine were generally readily reversible upon removal of the cells from the drug, with some residual effects occurring at higher dibucaine concentrations. Regenerating cells in the presence of dibucaine and excess extracellular calcium were not delayed, suggesting that the effects of dibucaine were reversible by calcium ions. The effects of tetracaine were not reversible by calcium ions, however. Exposure of regenerating cells to medium either lacking in, or containing an excess of, extracellular calcium had no effect on the time required to complete oral regeneration. The plant lectin, phytohemagglutinin, can also delay oral regeneration. The possible implications of these findings on the control of oral regeneration are discussed.