Paramecium Cilia Research Articles

CAMP-stimulated phosphorylation of an axonemal polypeptide that copurifies with the 22S dynein arm regulates microtubule translocation velocity and swimming speed in Paramecium.

In Paramecium tetraurelia, cyclic nucleotides are important physiological second messengers that could regulate dynein mechanochemistry by phosphorylation. A 29-kDa polypeptide that is phosphorylated in a cAMP- and Ca(2+)-sensitive manner in permeabilized cells and isolated axonemes is the only significant phosphorylated moiety that consistently copurifies with 22S dynein from paramecium cilia. It is not a component of 14S dynein. This polypeptide can be thiophosphorylated in a cAMP-sensitive manner, and this form of 22S dynein is stable when stored at -70 degrees C. cAMP-mediated thiophosphorylation of the 29-kDa polypeptide significantly increases the velocity with which 22S dynein causes microtubules to glide in vitro. The increase is abolished, together with the thiophosphorylation of the 29-kDa polypeptide, by preincubation with high Ca2+. Pretreatment with high Ca2+ does not alter the thiophosphorylation pattern of, or the velocity of microtubule translocation by, 14S dynein. The same preincubation conditions that permit or abolish the increase in velocity of microtubule translocation by 22S dynein permit or fail to permit swimming speed of permeabilized cells to increase on reactivation even after cAMP is removed. The effect of cAMP on swimming speed can therefore be accounted for by changes in the mechanically coupled 22S dynein activity via phosphorylation or thiophosphorylation of the 29-kDa polypeptide, which could act as a regulatory dynein light chain.

Alkaline phosphatase from Paramecium cilia and cell bodies: purification and characterization

A soluble alkaline phosphatase was purified 10 000-fold in an overall yield of 8% from both of the cilia and cell bodies of the protozoan Paramecium tetraurelia. The concentration in cilia (1.7 μM) was 6-fold higher than in cell bodies, although the latter contained most of the activity due to their much greater volume. The purified protein showed a single (36 kDa) protein staining band on SDS-PAGE. This value, in conjunction with the apparent molecular mass of 66 kDa for the native enzyme (gel filtration) suggests a dimeric structure. The specific activity of the purified phosphatase ranged from 10 to 70 μmol · min−1 · mg−1 at the pH-optimum of 8.0 and the Km for p-nitrophenyl phosphate was 81 μM. Basal enzyme activity was inhibited by metal chelators and stimulated up to 12-fold by addition of divalent cations. Mg2+ acted as a non-essential mixed-type activator with a half-maximal effect at 7 μM. Ca2+ was inhibitory, the extent of inhibition was dependent on the concentration of Mg2+ in the assay. Furthermore, the kinetics of inhibition by Ca2+ varied with the Mg2+ concentration. Phosphate, pyrophosphate, and SH-group blocking agents also strongly inhibited. The enzyme did not dephosphorylate Tyr- or Ser-/Thr-phosphoproteins. The Paramecium enzyme is not of lysosomal origin and its properties are quite different from all known phosphatases. It is a novel type of phosphatase since it (i) shows F−-inhibition like Ser/Thr-phosphatases but (ii) is inhibited by vanadate and molybdate like Tyr-phosphatases, and (iii) inhibition by Ca2+ has not been reported for any other phosphatase.

Cyclic AMP-dependent protein kinases of Paramecium. II. Catalytic and regulatory properties of type II kinase from cilia

The type II cAMP-dependent protein kinase (cAMP-PK-II) from cilia of Paramecium, purified free of type I cAMP-PK (cAMP-PK-I) and of cGMP-dependent protein kinase (cGMP-PK), phosphorylated several basic proteins and a heptapeptide containing serine (Kemptide). The enzyme was partially inhibited by the protein kinase inhibitor (Walsh inhibitor), but only at relatively high inhibitor concentrations. Half-maximal activation of cAMP-PK-II occurred at 15-25 nM cAMP. Several cAMP analogs were tested for ability to bind and activate the enzyme. 8-bromo-cGMP, a potent activator of Paramecium cGMP-PK, was a poor activator of Paramecium cAMP-PK-II. Activation of cAMP-PK-II was influenced by the phosphorylation assay buffer. Phosphate buffers provided increased activation by cAMP but decreased total activity relative to that measured in Mops-Tris buffer. The kinase was cAMP-independent when the pH of the assay buffer was high. Preincubation of cAMP-PK-II with histones also activated the enzyme in the absence of cAMP. The cAMP-PK-II bound cAMP with a Kd of 23 nM, and bound cAMP was released with a biphasic time course, suggesting two non-identical binding sites. The properties of the cAMP-PK of this ciliated protozoan appear to be closely similar to those of vertebrates.

A novel cGMP-dependent protein kinase from Paramecium.

An unusual monomeric cGMP-dependent protein kinase, enriched in cilia, was isolated from Paramecium cilia and whole cells. Cilia and whole cell extracts had relatively high ratios of cGMP-dependent to cAMP-dependent protein kinase activity (1:2). The calculated molecular weight of the native enzyme was 88,000. The enzyme was identified on sodium dodecyl sulfate-polyacrylamide gels as a 77,000 molecular weight band based on copurification of this protein with enzyme activity, 8-N3-[32P]cAMP labeling, and autophosphorylation. Based on the size of the native enzyme, it was concluded that the kinase is a monomer with cGMP-binding and catalytic activities on the same polypeptide. Dimer-sized cGMP-dependent protein kinase, like that of the well characterized mammalian enzyme, was never seen, despite stringent efforts to control proteolysis. The structure of the Paramecium cGMP-dependent protein kinase supports a model in which the dimeric vertebrate form of the enzyme evolved from an early monomeric form. The catalytic properties of the Paramecium enzyme differed in several respects from those of the mammalian enzyme: it could use GTP or ATP as the phosphoryl donor, it did not phosphorylate Kemptide effectively, and it had poor histone kinase activity with high Mg2+ concentrations. Quercertin, 5'-guanylyl imidodiphosphate, indomethacin, and the isoquinolinesulfonamide drug H7 inhibited Paramecium cGMP-dependent protein kinase activity. The enzyme had fast and slow binding sites (with kd values of 5-10 x 10(-3)s-1 and 0.44 x 10(-3)s-1) and showed an order of preference for cyclic nucleotides and cyclic nucleotide analogs similar to that of the mammalian enzyme.

Purification and properties of dyneins from Paramecium cilia

Dynein ATPases purified from Paramecium cilia by salt extraction followed by sucrose density gradient centrifugation and anion exchange chromatography. The two major dyneins sedimented in sucrose gradients as species of 22 S and 12 S. After purification by anion exchange chromatography, their specific activities were about 0.4 and 0.5 μmol/min per mg, respectively. The dyneins could be distinguished by subunit composition and immunological crossactivity. Sucrose density gradient centrifugation revealed additional ATPase activity in the region between the 22 S and 12 S dyneins, including a 19 S activity. Mg 2+-ATPase activities of the dyneins and the 19 S activity were inhibited by vanadate and Zn 2+, and were activated by Triton X-100. Antibodies against the 22 S dynein from Paramecium reacted on immunoblots with most of the polypeptides of 22 S dynein, and showed that the heavy chains of 22 S dynein are not indentical to those that sediment at 19 S and 12 S. Several minor ATPase activities were revealed by anion exchange chromatography of fractions from the 22 S, 19 S and 12 S regions of sucrose gradients. These minor activities were stimulated by Mg 2+, inhibited by vanadate, and could be distinguished from each other by their elution positions and polypeptide compositions.

Regulation of axonemal Mg 2+-ATPase from Paramecium cilia: effects of Ca 2+ and cyclic nucleotides

Ciliary activity is regulated by Ca 2+ and cyclic nucleotides, but the molecular mechanisms of the regulation are unknown. We have tested the ability of Ca 2+ and cyclic nucleotides to alter ciliary Mg 2+-ATPase or to stimulate phosphorylation of axonemal dynein. Mg 2+-ATPase activity in cilia and axonemes from Paramecium was stimulated 2-fold by micromolar Ca +, but this Ca 2+ sensitivity was lost upon solubilization of the dyneins from the axoneme. The Ca 2+-sensitive component of ciliary Mg 2+-ATPase activity was inhibited by the dynein inhibitors vanadate and Zn 2+, but was insensitive to the calmodulin antagonists calmidazolium and melittin. Dynein activity in the high-salt extract from axonemes was also insenstive to calmidazolium. Calmodulin did not sediment with 22 S or 12 S dyneins on sucrose gradients containing Ca 2+, but it did sediment in the region from 19 S to 14 S. Mg 2+-ATPase activity in ciliary fractions was unaltered in the presence of cAMP or cGMP. However, polypeptides associated with the 22 S and 12 S dyneins, as well as proteins of 19 S, 15 S, and 8 S, were substrates for endogenous ciliary kinases. High molecular weight polypeptides that sedimented at 22 S and 19 S were phosphorylated in a cyclic nucleo-stimulated manner.

Voltage-Dependent Calcium Channels from Paramecium Cilia Incorporated into Planar Lipid Bilayers

Two different divalent cation-selective channels from Paramecium cilia were incorporated into planar lipid bilayers. Both channels were much more permeable to divalent than univalent cations, and one of them discriminated significantly among the divalent cations. The selectivity and voltage dependence of the latter channel are comparable to those of voltage-dependent calcium channels found in a variety of cells. A combined biochemical, biophysical, and genetic study of calcium channels is now possible.

Calcium Channels in Excitable Cell Membranes

The existence of calcium permeability in excitable cells has been appreciated for 30 years (3 I)-almost as long as we have known about voltage-depend­ ent permeabilities to sodium and potassium (46, 47). Yet, to this day, our understanding of Ca channels has lagged far behind our knowledge of Na or K channels. This situation is ironic because Ca channels are widely distributed and rival their better-characterized counterparts in diversity of biological function (42). Ca channels play a crucial role in coupling mem­ brane excitation to cellular responses such as secretion or contraction. They are primarily responsible for some specialized but very interesting forms of excitability-e.g. in the nodal regions of the heart (14, 76), the cilia of Paramecium (11, 12), or the dendrites (64) or growth cones (6) of certain neurons. Unlike Na channels, Ca channels are often modulated by hor­ mones and neurotransmitters. For many years, biophysical or biochemical analysis of Ca channels has been seriously hampered by experimental problems (e.g. 42, 81). No prepa­ ration has been as suitable and no drug as specific for Ca channels as the squid axon and tetrodotoxin are for Na channels. But the state of the art has improved recently with the development of powerful methods for recording Ca channel activity and of blocking drugs that may interact with Ca channels in a potent and specific manner. We now know that Ca channels are pores, capable of transferring millions of permeant ions per second, whose voltage-dependent properties clearly distinguish them from pumps or exchange mechanisms described elsewhere in this volume. There is growing appreciation that Ca channels can be

Electron probe microanalysis of pyroantimonate precipitates in the cilia ofParamecium

Electron-dense deposits were observed at the bases of the cilia ofParamecium fixed in 1% OsO4 solution containing 2% potassium pyroantimonate. The deposits were shown to contain Ca and Sb by X-ray microanalyzer.

The pair of central tubules rotates during ciliary beat in Paramecium

UNLIKE the one-dimensional movement of striated muscle, the beat of a cilium is typically three dimensional1. Thus, the dynein arms, which are situated on the peripheral tubules around the circumference as well as longitudinally along the cilium, must be temporally coordinated in their actions. The mechanism for coordination is not known. The present study was undertaken to see whether the pair of central microtubules exhibits any systematic movement during the ciliary beat. We conclude that the central pair of tubules rotates anticlockwise 360° per beat cycle and that this rotation may regulate the dynein arms. Paramecium cilia were used because: markers distinguish the two central tubules so that their orientation can be unambiguously determined; and the metachronal waves of cilia can be ‘instantaneously fixed’ for the analysis of sequential phases of the beat.

Control of ciliary activity in paramecium—IV. Ca 2+ modification of Mg 2+ dependent dynein ATPase activity

1. 1. Dynein proteins were solubilized from demembranated cilia of Paramecium by extraction at high ionic strength. 2. 2. Mg 2+-dependent ATPase (EC 3.6.1.3) activity of crude dynein extracts was inhibited by micromolar concentrations of Ca 2+ ions. 3. 3. Sepharose 4B chromatography of the crude extracts yields three dynein fractions. The major fraction contains a single protein and is insensitive to Ca 2+ ions. Two other fractions, both heterogeneous in composition, show opposing Ca 2+ ion sensitivity expressed as a Ca 2+ dependent alteration in MgATP 2− dependent ATPase activity. The Ca 2+ ion sensitive forms show altered electrophoretic mobility on native polyacrylamide gels in the presence and absence of Ca 2+ ions. 4. 4. The data provides evidence for a Ca 2+ ion dependent concomitent alteration in both molecular form and hydrolytic activity of the dyneins. The results are discussed in terms of a possible molecular mechanism for Ca ion regulation of ciliary activity in terms of the sliding microtubule model.

Localization of calcium channels in Paramecium caudatum.

1. Electrical recordings from Paramecium caudatum were made after removal of the cilia with chloral hydrate and during ciliary regrowth to study the electrical properties of that portion of the surface membrane enclosing the ciliary axoneme. 2. Removal of the somatic cilia (a 50% reduction in membrane surface area) results in an almost complete elimination of the regenerative Ca response, all-or-none Ba2+ spike, and delayed rectification. 3. A twofold increase in input resistance resulted from the 50% reduction in membrane surface area. 4. The electrical properties remained unchanged, despite prolonged exposure to the chloral hydrate, until the cilia were mechanically removed. 5. Restoration of the Ca response accompanied ciliary regrowth, so that complete excitability returns when the cilia regain their original lengths. 6. It is concluded that the voltage-sensitive Ca channels are localized to that portion of surface membrane surrounding the cilia. 7. Measurements of membrane constants before and after deciliation and estimations of the cable constants of a single cilium suggest that the cilia of Paramecium may be fully isopotential along their length and with the major cell compartment.

Electron microscopy and electron probe analysis of the Ca-binding sites in the cilia of Paramecium caudatum.

Electron-dense deposits were observed at the base of cilia ofParamecium fixed in glutaraldehyde solution containing 5 mM CaCl2. The deposits were probed by X-ray microanalyzer and it was clearly demonstrated that the deposits consisted of calcium and phosphorus.

Contractile events in the cilia of Paramecium, Opalina, Mytilus, and Phragmatopoma

The motion of the abnormal cilia of Opalina and Mytilus can be described by the recently developed model for ciliary motion, provided the activation of the contractility during the effective stroke is reduced by three- to fivefold compared with that in the recovery stroke. The stiffness of the Mytilus cilium during the effective stroke is found several hundred times larger than that predicted by the model, however. The stiffness of the cilia of Paramecium, Opalina, Phragmatopoma, and of Mytilus in the recovery phase, is predicted approximately correctly by the model. The activation of contractility in Mytilus and Phragmatopoma cilia increases with the viscosity of the medium, as the velocity of the ciliary motion slows down. This leads to the equivalent of a force-velocity relation. The velocity of propagation of the bend in the cilia during the recovery stroke is shown to be dependent only on the elastic properties of the ciliary shaft, and to be independent of the contractile activiey.

Control of the orientation of cilia by adenosinetriphosphate, calcium, and zinc in glycerol-extracted Paramecium caudatum.

The predominant orientation of cilia in glycerol-extracted Paramecium is toward the posterior of the specimen in a KCl solution. The cilia became reoriented toward the anterior shortly after transfer of the extracted cell to a mixture of ATP, calcium, and zinc. The degree of response was graded as a function of the concentration of each of the three essential factors. Minimum concentrations for the maximum response were 0.2 mM in ATP, 0.8 mM in calcium, and 0.0002 mM in zinc. The observations support the hypothesis that cation-induced ciliary reversal in live specimens is initiated by calcium ions which become displaced from an inferred cellular cation exchanger system.

Reversal response elicited in nonbeating cilia of paramecium by membrane depolarizatin.

Ciliary reversal occurs in response to electrical and chemical stimuli in specimens of Paramecium caudatum in which ciliary beat has been completely inhibited by external application of nickel ions. The mechanism underlying ciliary reversal appears, therefore, to differ from that of ciliary beat. The cessation of ciliary beat has no effect on the intracellular potential of Paramecium. However, depolarizing action potentials are associated with ciliary reversals in paramecia, treated with nickel, without ciliary beat. Thus, membrane depolarization in this species seems specifically concerned with the ciliary reversal, and not with ciliary beat.

ELECTRON MICROSCOPE OBSERVATIONS OF THE TRICHOCYSTS AND CILIA OF PARAMECIUM

Electron micrographs of shadow-cast trichocysts of Paramecium show that the dried trichocyst shaft is flattened on the supporting film, while the pointed tip is apparently more resistant to collapse on dehydration. Accentuation, by the metal, of the cross striation previously observed in the shaft indicates that the periodicity is accompanied by corrugation of the dried surface. A cross striation in the tip is also visible in some micrographs of shadow-cast specimens. In the few cases where the periodicity could be measured, the average spacing was about 300 A, as compared to about 550 A for the well-defined shaft striation.In electron micrographs of shadow-cast specimens of Paramecium cilia, the component fibrils are seen with greatly increased clarity.

Response to electricity in Volvox and the nature of galvanic stimulation

1. Volvox orients fairly precisely in a constant galvanic current. 2. It is positive to the cathode whenever it is photopositive and positive to the anode whenever it is photonegative. Anything which causes reversal in the direction of orientation in light causes reversal in the direction of orientation in a galvanic current, e. g. light, temperature, chemicals, hydrogen-ion concentration. 3. Galvanic orientation is due to cessation or diminution in the activity of the flagella on the side of the colony toward which it turns. Photic orientation is brought about largely, if not entirely, by change in the direction of the stroke of the flagella due to change in the amount of light received by the photosensitive tissue in the eyes, owing to rotation on the longitudinal axis. 4. When the circuit is closed the activity of the flagella in colonies which are not rotating, diminishes or ceases on the surface facing the pole toward which the colonies are positive, but the decreased activity continues only a few seconds after which the activity increases to that which obtained before the circuit was closed. If the circuit is now opened a similar response is obtained on the opposite side. 5. If the colonies are positive to the cathode the flagella on the cathode side respond after the circuit is closed and those on the anode side after it is opened. If they are positive to the anode the reverse obtains. 6. The galvanic response in the individual zooids in Volvox is only momentary. But in colonies which are swimming freely and rotating on the longitudinal axis the galvanic response on the side toward which they turn is continuous, owing to the continuous transfer of zooids to this side, from the opposite side, i. e. from the side where they are not affected by the current to the side where they are affected. 7. Reversal in the direction of galvanic orientation is probably accompanied by a change in the electric charge of the colonies. Colonies which are positive to the cathode usually drift cataphoretically toward the anode, indicating a negative charge; and those which are positive to the anode usually drift toward the cathode, indicating a positive charge. This has, however, not been unequivocally established. 8. A galvanic current induces in the lower organisms various chemical and physical changes at the anode and at the cathode surfaces but the changes induced at these two surfaces differ greatly. In some the changes at the surface toward the cathode are followed by certain conspicuous phenomena, e. g. reversal in the direction of the stroke of the cilia in Paramecium; in others those at the surface toward the anode are followed by equally conspicuous but different phenomena, e. g. contraction in Amoeba and bioluminescence in ctenophores; and in still others those on one side are followed by the same conspicuous phenomena if the organisms are in a given state, as those on the other side, if the organisms are in different state, e. g. decrease in the effectiveness of the stroke of the flagella in Volvox. 9. The reversal in galvanic response observed in Volvox is not in accord with Pfluger's law. Whether or not the responses observed on the anode side in amoeba and ctenophores also violate this law, depends upon its interpretation. 10. Galvanic stimulation in Volvox is probably associated with decrease in surface polarization and decrease in water-content.