Detection of Ca2+-dependent cyclic GMP binding protein in frog rod outer segments

For the identification of the cGMP-sensitive ion channel protein of frog rod outer segments (ROS), we analyzed cGMP binding proteins in the ROS by photoaffinity labeling with [3H]cGMP. We found three cGMP binding polypeptides (66 kDa, 92 kDa and 100 kDa) in the membrane protein fraction of ROS. cGMP binding to the 66 kDa polypeptide required the addition of 2 mM CaCl2. We propose that this polypeptide corresponds to the cGMP-activated channel protein reported by Cook et al. [(1987) Proc. Natl. Acad. Sci. USA 84, 585-589]. The 100 kDa and 92 kDa polypeptides are subunits of the cGMP phosphodiesterase.     

Culture of Stentor coeruleus on Colpidium campylum*

SYNOPSIS. A method is described for culturing Stentor coeruleus in agar-lined petri dishes using the smaller ciliate Colpidium campylum as food organism.     

The Effects of Colchicine and Colcemid on Oral Differentiation in Stentor coeruleus*†

SYNOPSIS. Oral regeneration, oral reorganization and cell division in the ciliate Stentor coeruleus can be temporarily blocked at any stage of development by exposure to colchicine. The implications of this observation, presented as an assumption that colchicine exerts its effects by specifically preventing the formation of microtubular elements in the cells, suggest that microtubules are essentially involved in many aspects of these complicated processes of cellular differentiation.     

Ultraviolet-Microbeam Irradiation of Regenerating Stentor*

SYNOPSIS. Regeneration of missing feeding organelles in the ciliate Stentor coeruleus can be delayed by ultraviolet (UV) irradiation. In an attempt to identify the radiation-sensitive cellular component(s), cells in various stages of regeneration were immobilized, flattened and irradiated with a microbeam of 265 nm UV light. Targets included regions of either the macronucleus or the cytoplasm at varying distances from the ramifying zone of the cortex, which is the region where the new oral organelles are formed. During the early stages of regeneration the macronucleus is more sensitive to UV-induced regeneration delay than the cytoplasm, but it is not possible to distinguish between direct and indirect effects. Damage to cortical primordia in middle or late stages leading to structural abnormalities appears to result only from direct effects.     

Interactions between divalent cations and the gating machinery of cyclic GMP-activated channels in salamander retinal rods

The effects of divalent cations on the gating of the cGMP-activated channel, and the effects of gating on the movement of divalent cations in and out of the channel's pore were studied by recording macroscopic currents in excised membrane patches from salamander retinal rods. The fractional block of cGMP-activated Na+ currents by internal and external Mg2+ as well as internal Ca2+ was nearly independent of cGMP concentration. This indicates that Mg2+ and Ca2+ bind with similar affinity to open and closed states of the channel. In contrast, the efficiency of block by internal Cd2+ or Zn2+ increased in proportion to the fraction of open channels, indicating that these ions preferentially occupy open channels. The kinetics of block by internal Ni2+, which competes with Mg2+ but blocks more slowly, were found to be unaffected by the fraction of channels open. External Ni2+, however, blocked and unblocked much more rapidly when channels were mostly open. This suggests that within the pore a gate is located between the binding site(s) for ions and the extracellular mouth of the channel. Micromolar concentrations of the transition metal divalent cations Ni2+, Cd2+, Zn2+, and Mn2+ applied to the cytoplasmic surface of a patch potentiated the response to subsaturating concentrations of cGMP without affecting the maximum current induced by saturating cGMP. The concentration of cGMP that opened half the channels was often lowered by a factor of three or more. Potentiation persisted after the experimental chamber was washed with divalent-free solution and fresh cGMP was applied, indicating that it does not result from an interaction between divalent cations and cGMP in solution; 1 mM EDTA or isotonic MgCl2 reversed potentiation. Voltage-jump experiments suggest that potentiation results from an increase in the rate of cGMP binding. Lowering the ionic strength of the bathing solution enhanced potentiation, suggesting that it involves electrostatic interactions. The strong electrostatic effect on cGMP binding and absence of effect on ion permeation through open channels implies that the cGMP binding sites on the channel are well separated from the permeation pathway.     

The Kinase Regulator Mob1 Acts as a Patterning Protein for Stentor Morphogenesis

Morphogenesis and pattern formation are vital processes in any organism, whether unicellular or multicellular. But in contrast to the developmental biology of plants and animals, the principles of morphogenesis and pattern formation in single cells remain largely unknown. Although all cells develop patterns, they are most obvious in ciliates; hence, we have turned to a classical unicellular model system, the giant ciliate Stentor coeruleus. Here we show that the RNA interference (RNAi) machinery is conserved in Stentor. Using RNAi, we identify the kinase coactivator Mob1—with conserved functions in cell division and morphogenesis from plants to humans—as an asymmetrically localized patterning protein required for global patterning during development and regeneration in Stentor. Our studies reopen the door for Stentor as a model regeneration system.     

Mechanosensitive channel of Thermoplasma, the cell wall-less archaea

By using a functional approach of reconstituting detergent-solubilized membrane proteins into liposomes and following their function in patch-clamp experiments, we identified a novel mechanosensitive (MS) channel in the thermophilic cell wall-less archaeon Thermoplasma volcanium. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the enriched protein fractions revealed a band of approx 15 kDa comparable to MscL, the bacterial MS channel of large conductance. 20 N-terminal residues determined by protein microsequencing, matched the sequence to an unknown open reading frame in the genome of a related species Thermoplasma acidophilum. The protein encoded by the T. acidophilum gene was cloned and expressed in Escherichia coli and reconstituted into liposomes. When examined for function, the reconstituted protein exhibited properties typical of an MS ion channel: 1) activation by negative pressure applied to the patch-clamp pipet, 2) blockage by gadolinium, and 3) activation by the anionic amphipath trinitrophenol. In analogy to the nomenclature used for bacterial MS channels, the MS channel of T. acidophilum was termed MscTA. Secondary structural analysis indicated that similar to MscL, the T. acidophilum MS protein may have two transmembrane domains, suggesting that MS channels of thermophilic Archaea belong to a family of structurally related MscL-like ion channels with two membrane-spanning regions. When the mscTA gene was expressed in the mscL ⁻ knockout strain and the MscTA protein reconstituted into liposomes, the gating of MscTA was charaterized by very brief openings of variable conductance. In contrast, when the mscTA gene was expressed in the wild-type mscL ⁺ strain of E. coli, the gating properties of the channel resembled MscL. However, the channel had reduced conductance and differed from MscL in its kinetics and in the free energy of activation, suggesting that MscTA and MscL can form functional complexes and/or modulate each other activity. Similar to MscL, MscTA exhibited an increase in activity in liposomes made of phospholipids having shorter acyl chain, suggesting a role of hydrophobic mismatch in the function of prokaryotic MS channels.     

Perinuclear organization in the heterotrich ciliateStentor coeruleus

Summary Different fixation techniques were employed to obtain satisfactory fixation of the endoplasm ofStentor coeruleus for ultrastructural investigations. The nuclei ofS. coeruleus are surrounded by a flattened fenestrated cisterna. The space between the nuclear envelope and the cisterna (= perinuclear space) is continuous with the cytoplasm via channels. The envelopes of both, micronucleus and macronucleus, are connected to the fenestrated cisterna by filamentous material. This organization accounts for the close association between micronucleus and macronucleus inStentor coeruleus. The fenestrated cisterna is compared to similar structures occurring in other organisms, and its possible function is discussed.Abbreviations fC fenestrated cisterna - FV food vacuole - km km fibers - MaNu macronucleus - MiNu micronucleus - My myonome - NE nuclear envelope - PC perinuclear cisterna - PfC pore of fenestrated cisterna - PS perinuclear cytoplasmic space - EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N-tetraacetic acid) - GA glutaraldehyde - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PEG polyethylene glycol - PIPES piperazine-N,N-bis[2-ethanesulfonic acid] - PTA phosphotungstic acid     

Four cases of direct ion channel gating by cyclic nucleotides

Four different nucleotide-gated ion channels are discussed in terms of their biophysical properties and their importance in cell physiology. Channels activated directly by cGMP are present in vertebrate and invertebrate photoreceptors. In both cases cGMP increases the fraction of time the channel remains in the open state. At least three cGMP molecules are involved in channel opening in vertebrate photoreceptors and the concentration of the cyclic nucleotide to obtain the half maximal effect is about 15 µM. The light-dependent channel of both vertebrates and invertebrates is poorly cation selective. The vertebrate channel allows divalent cations to pass through 10–15-fold more easily than monovalent ions. In agreement with their preference for divalent cations, this channel is blocked byl-cis Dialtazem, a molecule that blocks certain types of calcium channels. In olfactory neurons a channel activated by both cAMP and cGMP is found and, as in the light-dependent channel, several molecules of the nucleotide are needed to open the channel with a half maximal effect obtained in the range of 1–40 µM. The channel is poorly cationic selective. A K⁺ channel directly and specifically activated by cAMP is found inDrosophila larval muscle. At least three cAMP molecules are involved in the opening reaction. Half-maximal effect is obtained at about 50 µM. This channel is blocked by micromolar amount of tetraethylammonium applied internally. Interestingly, this channel has a probability of opening 10–20-fold larger in the mutantdunce, a mutant that possesses abnormally elevated intracellular cAMP level, than in the wild type.     

Cortical Control over the Direction of Macronuclear Elongation in the Heterotrich Ciliate Stentor coeruleus1

Earlier experimental work involving macronuclear implants in Stentor coeruleus has shown that the cytoplasmic cortex of the nuclear site 1) attracts the macronucleus and 2) holds it in place during interphase. Now experiments indicate macronuclei transferred with overlying cortex elongate in the direction of the transferred cortical pigment stripes, whether or not the transferred stripes realign in the direction of the host stentor's stripes. Therefore the third function of the cortex is to determine the direction of elongation and thus assure that both daughter cells at division receive part of the macronucleus.     

hERG ion channel pharmacology: cell membrane liposomes in porous-supported lipid bilayers compared with whole-cell patch-clamping

The purpose of this study was to obtain functional hERG ion channel protein for use in a non-cell-based ion channel assay. hERG was expressed in Sf9 insect cells. Attempts to solubilise the hERG protein from Sf9 insect cell membranes using non-ionic detergents (NP40 and DDM) were not successful. We therefore generated liposomes from the unpurified membrane fraction and incorporated these into porous Teflon-supported bilayer lipid membranes. Macroscopic potassium currents (1 nA) were recorded that approximated those in whole-cell patch-clamping, but the channels were bidirectional in the bilayer lipid membrane (BLM). Currents were partially inhibited by the hERG blockers E4031, verapamil, and clofilium, indicating that the protein of interest is present at high levels in the BLM relative to endogenous channels. Cell liposomes produced from Sf9 insect cell membranes expressing voltage-gated sodium channels also gave current responses that were activated by veratridine and inhibited by saxitoxin. These results demonstrate that purification of the ion channel of interest is not always necessary for liposomes used in macro-current BLM systems.     

Survival of ciliate protozoa under starvation conditions and at low bacterial levels

Under starvation conditions, 50% survivorship times displayed no significant relationship with cell size in 2 ciliate species in this study and 5 protozoan species from the literature. Differences in survival ability were attributed to differences in weight-specific respiratory rate and relative motility among these 7 species. At low bacterial levels, 4 ciliate species in this study displayed significant differences in survivorship. High survivorship ofEuplotes patella relative to that ofParamecium caudatum andParaurostyla sp. at low ciliate densities was attributed to the lower individual energy requirements of this smaller species. High survivorship ofStentor coeruleus was interpreted as an effect of its large quantity of reserves and low respiratory rate. The survivorship ofE. patella was reduced at a higher population density. Four ciliate species survived longer at 15C than at 22C. Q₁₀ values based on 50% survivorship times at these 2 temperatures were much lower than Q₁₀ values based on respiratory rates and growth rates of well-fed ciliates over a similar temperature range.     

Deoxyribonucleic Acid Base Composition of Stentor coeruleus*

SYNOPSIS. The deoxyribonucleic acid of Stentor coeruleus has of 33 moles percent guanine plus cytosine. a buoyant density in CsCl of 1.692 gcm^?3 and a base composition of 33 moles percent guanine plus cytosine.     

The endosymbiotic unit ofStentor polymorphus andChlorella sp. morphological and physiological studies

Summary The greenStentor polymorphus harbours unicellular coccoid chlorophycean algae. They are located in food vacuoles, where they show various states of digestion, as well as in individual so-called perialgal vacuoles. According to their characteristic morphological properties the algae belong to the genusChlorella. They can be isolated from the ciliate and cultivated in mass cultures in a sterile defined inorganic medium supplemented with vitamins B₁ and B₁₂. The algae have no secondary carotenoids and excrete maltose by a pH-dependent mechanism. They thus show a conspicuous physiological similarity to the symbiotic chlorellae ofParamecium bursaria andHydra viridis, which also excrete maltose.A comparison of the properties of the chlorellae isolated fromStentor polymorphus and of the intactStentor polymorphus-Chlorella unit with the characteristic features of symbiotic chlorellae and with endosymbiotic systems containingChlorella sp. in general, lead to the conclusion that the greenStentor polymorphus is also a true endosymbiotic system.     

Voltage-sensitive ion channel ofEscherichia coli

Summary A voltage-sensitive, cation-selective ion channel ofEscherichia coli has been reconstituted into liposomes and studied with the patch-clamp method. The single channel conductance was 91 pS in symmetric solutions of 150mm KCl. Many channels were open most of the time, with frequent brief transitions to closed levels. Multiple conducting units could close and reopen simultaneously, and this apparent cooperativity in gating was increases with depolarizing voltages. Above a voltage threshold, the channels closed irreversibly, often in groups.     

Microtubules and Filaments Beneath the Fission Furrow of Stentor coeruleus1

ABSTRACT. The ultrastructure of the cortex beneath the fission furrow of dividing Stentor coeruleus was examined using scanning and transmission electron microscopy. During division, basal bodies, axonemes, and km fibers beneath the furrow were absorbed near the moving primordial oral apparatus, and a circumferential band of microtubules and filaments was formed at the base of the furrow. The location and orientation of this fibrous band suggest that it may be an important component of the cytokinetic machinery. Treatment with vinblastine sulfate (4 × 10^-5 M) disrupted the circumferential microtubules and blocked division, which is consistent with this hypothesis.     

Differentiation of X chromosomes in early female mouse embryos

The stalk segment of the heterotrich ciliate, Stentor coeruleus, appears nearly isotropic in the contracted state and develops a characteristic birefringence during extension. The birefringence occurs in stripes and is associated with the myonemes, one of the two longitudinally running subpellicular fiber systems. Electron microscopical investigations reveal changes in the ultrastructure of the myonemes from the extended to the contracted state. The relaxed myonemes consist mainly of 3 nm filaments running in the longitudinal direction, while the contracted myonemes show 10 nm tubular-like filaments, more randomly oriented. It is suggested that during elongation the randomly oriented tubular filaments undergo a conformational change to more regularly arranged thin filaments, thus causing development of birefringence.     

Cytochemical Studies on the Distribution of DNA in the Macronucleus of Stentor coeruleus*

SYNOPSIS. The DNA content of the different macronucleus nodes of Stentor coeruleus has been studied by microspectrophotometry on Feulgen-stained isolated macronuclei. No relation between the position of the nodes in the macronucleus row and their DNA content could be found. Sister animals showed differences in distribution of the DNA in their macronuclei.     

A rhodopsin immunoanalog in the related photosensitive protozoans Blepharisma japonicum and Stentor coeruleus

Immunoblotting of isolated cell membrane fractions from ciliates Blepharisma japonicum and Stentor coeruleus with a polyclonal antibody raised against rhodopsin revealed one strong protein band of about 36 kDa, thought to correspond to protozoan rhodopsin. Inspection of both ciliates labeled with the same antibody using a confocal microscope confirmed the immunoblotting result and demonstrated the presence of these rhodopsin-like molecules localized within the cell membrane area. Immunoblot analysis of the ciliate membrane fractions resolved by two-dimensional gel electrophoresis identified two distinct 36 kDa spots at pIs of 4.5 and 7.0 for Blepharisma, and three spots at pIs of 4.4, 5.0 and 6.0 for Stentor, indicating a possible mixture of phosphorylated rhodopsin species in these cells. The obtained results suggest that both Blepharisma and the related ciliate Stentor contain within the cell membrane the rhodopsin-like proteins, which may be involved as receptor molecules in the sensory transduction pathway mediating motile photoresponses in these protists as in other species of lower eukaryota.     

Inactivation of cGMP-dependent conductance of rod outer segment plasma membrane induced by cGMP.

Integral cGMP-dependent currents as well as activity of single cGMP-activated channels in plasma membrane of rod outer segment (ROS) were recorded using the patch-clamp method. The dependence of integral currents on cGMP concentration is shown to be bell-shaped. Decrease in cGMP-dependent conductance at high cGMP concentration results from a decrease in channel opening probability. Thus, cGMP in high concentrations inactivates cGMP-dependent conductance.