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Centrioles, cilia, and flagella are ancestral conserved organelles of eukaryotic cells. Among the proteins identified in the proteomics of ciliary proteins in Paramecium, we focus here on a protein, Bug22p, previously detected by cilia and basal-body high-throughput studies but never analyzed per se. Remarkably, this protein is also present in plants, which lack centrioles and cilia. Bug22p sequence alignments revealed consensus positions that distinguish species with centrioles/cilia from plants. In Paramecium, antibody and green fluorescent protein (GFP) fusion labeling localized Bug22p in basal bodies and cilia, and electron microscopy immunolabeling refined the localization to the terminal plate of the basal bodies, the transition zone, and spots along the axoneme, preferentially between the membrane and the microtubules. RNA interference (RNAi) depletion of Bug22p provoked a strong decrease in swimming speed, followed by cell death after a few days. High-speed video microscopy and morphological analysis of Bug22p-depleted cells showed that the protein plays an important role in the efficiency of ciliary movement by participating in the stroke shape and rigidity of cilia. The defects in cell swimming and growth provoked by RNAi can be complemented by expression of human Bug22p. This is the first reported case of complementation by a human gene in a ciliate.

In the ciliate Paramecium tetraurelia, 3',5'-cyclic GMP (cGMP) is one of the second messengers involved in several signal transduction pathways. The enzymes for its production and degradation are well established for these cells, whereas less is known about the potential effector proteins. On the basis of a current Paramecium genome project, we have identified a multigene family with at least 35 members, all of which encode cGMP-dependent protein kinases (PKGs). They can be classified into 16 subfamilies with several members each. Two of the genes, PKG1-1 and PKG2-1, were analyzed in more detail after molecular cloning. They encode monomeric enzymes of 770 and 819 amino acids, respectively, whose overall domain organization resembles that in higher eukaryotes. The enzymes contain a regulatory domain of two tandem cyclic nucleotide-binding sites flanked by an amino-terminal region for intracellular localization and a catalytic domain with highly conserved regions for ATP binding and catalysis. However, some Paramecium PKGs show a different structure. In Western blots, PKGs are detected both as cytosolic and as structure-bound forms. Immunofluorescence labeling shows enrichment in the cell cortex, notably around the dense-core secretory vesicles (trichocysts), as well as in cilia. Immunogold electron microscopy analysis reveals consistent labeling of ciliary membranes, of the membrane complex composed of cell membrane and cortical Ca2+ stores, and of regions adjacent to ciliary basal bodies, trichocysts, and trafficking vesicles. Since PKGs (re)phosphorylate the exocytosis-sensitive phosphoprotein pp63/pf upon stimulation, the role of PKGs during stimulated exocytosis is discussed, in addition to a role in ciliary beat regulation.

The kinetics of loss from the cytoplasm and changes in ultrastructure of symbiont lambda particles after treatment of axenically cultivated lambda-bearing Paramecium aurelia with penicillin G was investigated. Low concentrations (1 to 2 unit/ml) of the antibiotic caused many particles within the cell to become filamentous; high concentrations (2,000 unit/ml) caused lysis of the particles without noticeably affecting the protozoan. The ED(50) value (2 to 3 unit/ml) was within the range of values found to cause lysis of many gram-negative bacteria. Rapidly dividing lambda were more vulnerable to the action of the antibiotic than slowly dividing particles. Nondividing particles were not affected by exposure to the antibiotic. Ultrastructural changes observed in lambda during lysis by penicillin G were consistent with the view that penicillin interferes with the synthesis of a vital component of the cell envelope of the particle, possibly a peptidoglycan similar to that found in the cell walls of bacteria. The deoxyribonucleic acid of lambda was dispersed throughout the particle as electron dense fibers enclosed within electron transparent areas. The cell envelope appeared to consist of at least two morphologically distinguishable layers, an inner layer homologous to the plasma membrane of bacteria and an outer layer homologous to the bacterial cell wall. Lambda may be regarded as a randomly distributed population of bacteria growing and dividing synchronously within the collective cytoplasm of its protozoan host.