Podophyllotoxin,Colcemid and Cold Temperature Interfere with Cilia Regeneration in Stentor*

SYNOPSIS Stentor coeruleus, induced to shed their ciliary membranellar bands, regenerate these and associated oral structures within a few hours after treatment. In cells placed in media containing optimal concentrations of mitotic spindle inhibitors, regeneration of the ingestive organelles is reversibly inhibited. Inhibitory effects of Colcemid, podophyllotoxin, and cold temperature reported here are compared with previous results using colchicine, griseofulvin and isopropyl-n-phenyl carbamate on regenerating oral membranellar cilia and cell growth.     

Oral Regeneration in the Ciliate Stentor coeruleus: A Scanning and Transmission Electron Optical Study

SYNOPSIS. Elaboration of ciliated feeding organelles in the protozoon Stentor coeruleus was reinvestigated for the first time by scanning electron microscopy which gives the most realistic 3-dimensional images. Parallel transmission EM studies of synchronized regenerating stentors gave further ultrastructural details of stomatogenesis, while also confirming the expectation that in the structure of its kineties this now classical experimental object does not differ from other species of Stentor previously studied. Within 2 hr after the stimulus to regeneration, several generations of new kinetosomes for the oral primordium are produced, first in association with kinetosomes of kineties at the restricted primordium site. These kinetosomes rapidly sprout membranellar cilia as well as subpellicular microtubules but are still randomly oriented (anarchic field). The forming membranellar band increases from its center-line to both sides while it grows in length. Young cilia are blunt-ended. Recession of the early anlage occurs without rupture of the pellicle; soon apparent is the clear border stripe of unknown function along the right side of the membranellar band. Instantaneous fixation of beating cilia in early primordia revealed random beating, with coordination and presumably membranellar organization not yet attained. In late anlagen there are 2 types of metachronal rhythm: transversely from cilium to cilium across any given membranelle, as well as the easily observable serial beating of membranelles along the entire band. A single file of cilia leads the subsequent cytostomal invagination. The posterior end of the membranellar band then follows to line the cytopharynx.     

The Effect of Colchicine on Regenerating Membranellar Cilia in Stentor coeruleus

SYNOPSIS. Stentors treated with toxic substances can be induced to shed their oral bands (19, Fig. 1), complex structures composed of many cilia organized into membranelles. Regenerating membranellar bands were observed in control stentors removed from toxic (urea-containing) medium at about 3.5 hours. At 8 hours regenerated control organisms were indistinguishable from normal unshed stentors. Experimental animals replaced into colchicine medium were inhibited from regeneration at low, nontoxic concentrations of this mitotic spindle inhibitor. Upon removal of the colchicine and replacement of the shed animals into normal medium or normal medium to which GTP had been added, complete and normal regeneration of the membranellar band ensued. Our observations are consistent with many suggesting that colchicine acts by reversibly binding with a protein during processes involving microtubule formation. Colchicine inhibition of membranellar band formation further indicates that oral membranelles are specialized evolutionary homologs to other centriole (= basal body, = kinetosome) derivatives such as mitotic spindle fibers, cilia and flagella, axopods, etc. (structures containing the ubiquitous microtubules of eukaryotic cells).     

Con A Binds to the Membranellar and Somatic Cilia of Stentor and to the Developing Oral Primordium During Oral Regeneration1

Binding sites for Concanavalin A have been located in the ciliate Stentor coeruleus by utilizing FITC-Con A and fluorescence microscopy. When both nonregenerating and regenerating Stentor are fixed prior to FITC-Con A exposure, FITC-Con A binds intensely to the cilia of the membranellar band and to the somatic cilia that cover much of the cell surface. No binding is observed between the ciliary rows. The FITC-Con A also binds to the developing oral primordia of regenerating cells. Binding of FITC-Con A in the early stages of regeneration (prior to stage 4) appears to be less intense than that in the later stages. Additional FITC-Con A binding appeared as a granular fluorescence in the area of the developing buccal cavity beginning at about stage 4 and disappearing around stages 6–7. The presence of α-D-methyl mannoside prevented the binding of FITC-Con A to either regenerating or nonregenerating cells. If nonregenerating Stentor are exposed to FITC-Con A prior to fixation, the binding pattern is entirely different with the fluorescence primarily in the form of random, granular patches spread over much of the cell but with no binding to either type of cilia. These results demonstrate that membrane glycoproteins capable of binding Con A are located primarily in the membranellar and somatic cilia and in the developing oral primordia during oral regeneration in Stentor. Concanavalin A binding to these sites may be involved in the Con A-induced inhibition of oral regeneration observed in earlier studies.     

Mitotic arrest by melatonin

We are testing the hypothesis that migration of the newly formed mouth (i.e., oral membranellar band) in stentor is homologous to mitotic chromosomal movement and that both types of movement within single cells depend directly on microtubule elongation. The following compounds synchronously delay the migration of the oral membranellar band as an exponential function of concentration: Colcemid, podophyllotoxin, β-peltatin and vinblastine. Delay for these compounds can be described by the equation, y = kxⁿ, where y is delay in hours and x is concentration of mitotic spindle inhibitor in moles/l. We discovered that the animal pineal gland hormone, melatonin (5-methoxy n-acetyl tryptamine), also specifically and reproducibly delays oral band regeneration according to an equation of this form. Thus we predicted that melatonin would arrest mitosis. We report here a colchicine-type disruption of the mitotic apparatus in onion root tips by melatonin. Two closely related tryptamine derivatives, n-acetyl serotonin and serotonin were inactive in both the stentor and onion assays: they neither delayed band migration in stentor as an exponential function nor induced mitotic arrest in onion.     

CILIA REGENERATION IN TETRAHYMENA AND ITS INHIBITION BY COLCHICINE

The cilia of Tetrahymena were amputated by the use of a procedure in which the cells remained viable and regenerated cilia. Deciliated cells were nonmotile, and cilia regeneration was assessed by scoring the percentage of motile cells at intervals following deciliation. After a 30-min lag, the deciliated cells rapidly recovered motility until more than 90% of the cells were motile at 70 min after amputation. Cycloheximide inhibited both protein synthesis and cilia regeneration. This indicated that cilia formation in Tetrahymena was dependent on protein synthesis after amputation. Conversely, colchicine was found to inhibit cilia regeneration without affecting either RNA or protein synthesis. This observation suggested the action of colchicine to be an interference with the assembly of ciliary subunit proteins. The finding that colchicine binds to microtubule protein subunits isolated from cilia and flagella (13) supports this possibility. The potential of the colchicine-blocked cilia-regenerating system in Tetrahymena for studying the assembly of microtubule protein subunits during cilia formation and for isolating ciliary precursor proteins is discussed.     

Localization of mechanoreceptors in the protozoan,Stentor coeruleus

Summary Mechanoreceptor channels were localized by using the ligands, tubocurarine (TC), decamethonium (Deca), and gallamine (Gall), which have been shown to bind specifically to these channels. The binding of radioactively labeled TC (TC^*) was found to be directly proportional to the cell surface area suggesting that the channels are uniformly distributed over the cell surface. Intracellular TC and Gall injections did not depress mechanical stimulus sensitivity though these drugs did depress sensitivity when applied extracellularly at the same concentrations; therefore, the ligand binding sites are on or near the external surface of the cell. Autoradiographs revealed that radioactively labeled Deca (Deca^*) bound to the pigmented stripes but not to the ciliary stripes or membranellar band. Further,Stentor induced to shed their membranellar band through exposure to 8% urea were more sensitive to mechanical stimuli than were controls; therefore, the membranellar cilia do not appear to contain mechanoreceptor channels. Collectively, these data indicate that the mechanoreceptor channels are located in the somatic surface covering the pigmented stripes. The density of mechanoreceptor channels in the plasma membrane covering the somatic surface is tentatively estimated to be between 5500 and 14500 m^–2 based on the density of TC^* binding, the apparent number of TC molecules binding per mechanoreceptor channel, and data suggesting that only one fifth to one fourth of the bound TC^* is bound to structures in the plasma membrane.Abbreviations TC Tubocurarine - TC ^{* 14} C-tubocurarine - Deca Decamethonium - Deca ^{* 3}H-decamethonium - Gall Gallamine - DAPA Bis(3-aminopyridinium)-1,10-decane diiodide - DAPA ^{* 3}H-bis(3-ammopyridinium)-1,10-decane diiodide     

Ultrastructure and membrane topography of special ciliary organelles in the ciliate Eufolliculina uhligi (Protozoa)

Summary In Eufolliculina uhligi and other folliculinid ciliates, a territory has been identified that differs ultrastructurally from other areas of the cell, and that is especially sensitive to mechanical stimuli. This territory is located around the anterior oral apparatus of the loricate trophont and posterior to the membranellar spiral of the swarmer. Each cilium in this territory is closely apposed to a small membrane-covered pin that is supported by transverse microtubules of the cilium. In front of the pin, the base of the cilium bulges out; the ciliary membrane is interconnected with the axoneme by filamentous material. Freeze-fractured cilia show a large rectangular particle array at the site of the basal swelling. Only scattered particles have been observed in the pin membrane. It is suggested that the cilium and the pin act as a unit, which has therefore been named the ciliumpin-complex. Comparison with ciliary organelles of unicellular and multicellular organisms indicates that, because of their polar organization, the complexes are involved in the transduction of oriented, presumably mechanical, stimuli.     

Melatonin: Inhibition of Microtubule-Based Oral Morphogenesis in Stentor coeruleus

SYNOPSIS. The temporal separation of macromolecular syntheses from protein assembly into microtubular structures has permitted the use of the Stentor ciliated oral membranellar band regenerating system as an assay for mitotic spindle inhibitors (Banerjee & Margulis, 1971). Melatonin, the pineal gland hormone, is additive to Colcemid in this assay; like Colcemid it specifically inhibits band migration as a sensitive function of developmental stage and concentration. Altho the entire process of band formation and cilia regeneration (stages 0–8) is inhibited by melatonin, stage 3 is especially sensitive.
Delay in morphogenesis at stage 3 can be measured as a precise function of inhibitors (Colcemid, podophyllotoxin, melatonin) of the form y=kxⁿ, where y is delay in hours and k is the concentration of inhibitor in moles. Riboflavin (0.2 μ M ) and nicotinamide (0.2 μM) in combination reversed Colcemid-induced delay in band regeneration, but (unlike melatonin) the vitamins alone were totally without effect on the regenerating system. Therefore melatonin probably interacts with microtubule protein whereas the B vitamins interact in some way with Colcemid to nullify its activity.     

Gamma Irradiation of Stentor polymorphus

SYNOPSIS. Stentor polymorphus was irradiated with ⁶⁰Co gamma rays at 525 rads/minute to examine the effect on survival, cell division, oral membranellar frequency and oxygen uptake. Both survival and cell division were studied on single cells. The LD₅₀ is 285 kilorads but cell division is inhibited below this dose; 110 kilorads doubles the 1st post-irradiation division interval, and a delay of 240 hours occurs at the maximum tolerated dose of 310 kilorads. Conjugating cells are more sensitive, with an LD₅₀ of approximately 40 kilorads.
The frequency of cilia in the membranellar band, measured stroboscopically, is reduced by 30% after 14.5 kilorads, and stopped by higher doses. Recovery has a similar time scale to recovery of fission. A Clarke electrode was used to measure changes in oxygen uptake after irradiation. Depressions of up to 50% were found. Recovery followed a similar pattern to that of ciliary activity and cell division.     

The Kinetid Structures of the Choreotrichous Ciliate Strobilidium velox and an Assessment of Its Evolutionary Lineage1

The ciliary (kinetid) structures of the ciliate Strobilidium velox have been examined with scanning and transmission electron microscopes. Somatic kineties consist of a linear row of kinetosomes (monokinetids) and short cilia lying partially beneath a thin fold of cytoplasm. The only fibrillar kinetid structure extending from the kinetosomes is a transverse ribbon of microtubules. The paroral membrane is a single-file polykinetid possessing a possible transverse ribbon of microtubules and a nematodesma. The oral polykinetids or membranelles are complex, with microtubules extending from both anterior and posterior rows of cilia. While the kinetid structures do not satisfy the criteria for the order Choreotrichida, they are similar to the tintinnids in several other relevant ways. Strobilidium velox is proposed to be an unusual ciliate that is an exception to the concept that somatic kinetids are conservative and reliable phylogenetic indicator structures.     

Use of a Novel Cell Adhesion Method and Digital Measurement to Show Stimulus‐dependent Variation in Somatic and Oral Ciliary Beat Frequency in Paramecium

When Paramecium encounters positive stimuli, the membrane hyperpolarizes and ciliary beat frequency increases. We adapted an established immobilization protocol using a biological adhesive and a novel digital analysis system to quantify beat frequency in immobilized Paramecium. Cells showed low mortality and demonstrated beat frequencies consistent with previous studies. Chemoattractant molecules, reduction in external potassium, and posterior stimulation all increased somatic beat frequency. In all cases, the oral groove cilia maintained a higher beat frequency than mid‐body cilia, but only oral cilia from cells stimulated with chemoattactants showed an increase from basal levels.     

Cilia regeneration in Tetrahymena : A simple reproducible method for producing large numbers of regenerating cells

We have developed an improved medium in which Tetrahymena can be deciliated by gentle shearing. The cells remain viable and regenerate a new complement of cilia. Unlike previous methods for viable deciliation of Tetrahymena, this method is easily adaptable to large numbers of cells, to cells in different stages of the life cycle (growing, starved, conjugating), and to both commonly studied species, T. thermophila and T. pyriformis. Starved T. thermophila deciliated by this method regained motility by 1 h, regenerated oral apparatus by 4.0 h and restored tubulin in cilia at a linear rate of about 3 pg h⁻¹ cell⁻¹.     

Stomatogenesis during sexual and asexual reproduction in an amicronucleate strain of Paramecium caudatum.

Amicronucleate cells of Paramecium caudatum, whose micronuclei have been artifically removed by micropipetting, are characterized by the appearance of a deciliated area at the posterior part of the buccal opening. These cells form food vacuoles at a slightly lower rate than micronucleate cells. Their mean interfission time is longer than that in micronucleates. The exconjugants of amicronucleate cells can not form food vacuoles and eventually die witout fission, though conjugation proceeds normally in them as well as in their micronucleate mate. The oral apparatus of amicronucleate exconjugants seems to be shallower than that of micronucleates. The membranellar cilia, therefore, can be seen through the buccal overture by scanning electron microscope. The results obtained from the cross of micronucleate and amicronucleate strains and from the induction of autogamy in amicronucleate strains suggest that the micronucleus has a primary role in developing the normal oral apparatus after nuclear reorganization.     

Palp morphology in two species of Prionospio (Polychaeta: Spionidae)

The palp morphology of Prionospio fallax Söderström, 1920 from Sweden and Prionospio cf. saldanha Day, 1961 from Thailand was examined with a scanning electron microscope. Prionospio fallax was also studied in vivo using light-microscopy. Both species have grooved feeding palps, adorned with up to five ciliary characters: frontal cilia, transverse ciliary bands (or bandlets), latero-frontal cirri, lateral cilia and randomly distributed non-motile cirri. All, except the frontal cilia and non-motile cirri, are asymmetrically arranged relative to the long axis of the palps. Prionospio fallax possesses transverse bandlets and the other four groups, while P. cf. saldanha has transverse bands (consisting of several contiguous bandlets), frontal cilia and some randomly scattered cirri. Asymmetrical palp ciliation was previously only known in Marenzelleria viridis (Verril, 1873) and the genus Scolelepis Blainville, 1828. The newly recognised transverse ciliary bands and bandlets are considered to be homologous with the transverse ciliary rows found basally on the palps of Paraprionospio pinnata (Ehlers, 1901). This multistate character (named transverse cilia) may prove useful in elucidating the phylogeny of the Prionospio-complex of genera.     

Hydropathic analysis and biological evaluation of stilbene derivatives as colchicine site microtubule inhibitors with anti-leukemic activity

The crucial role of the microtubule in cell division has identified tubulin as a target for the development of therapeutics for cancer; in particular, tubulin is a target for antineoplastic agents that act by interfering with the dynamic stability of microtubules. A molecular modeling study was carried out to accurately represent the complex structure and the binding mode of a new class of stilbene-based tubulin inhibitors that bind at the αβ-tubulin colchicine site. Computational docking along with HINT (Hydropathic INTeractions) score analysis fitted these inhibitors into the colchicine site and revealed detailed structure–activity information useful for inhibitor design. Quantitative analysis of the results was in good agreement with the in vitro antiproliferative activity of these derivatives (ranging from 3?nM to 100?μM) such that calculated and measured free energies of binding correlate with an r² of 0.89 (standard error ± 0.85?kcal mol^?1). This correlation suggests that the activity of unknown compounds may be predicted.     

EFFECT OF VINBLASTINE AND COLCHICINE ON UPTAKE AND RELEASE OF PUTATIVE TRANSMITTERS BY SYNAPTOSOMES AND ON BRAIN ACTOMYOSIN-LIKE PROTEIN

Abstract— The effects of several inhibitors, including vinblastine and colchicine, on the accumulation of a number of putative transmitters by a rat brain synaptosomal preparation and their subsequent release by excess K⁺ was examined. In addition, the effect of the alkaloids on the ATPase activity of the actomyosin-like protein, neurostenin, isolated from the synaptosomal preparation, was studied. The uptakes of radioactive glutamate, GABA, dopamine and norepinephrine were energy-dependent, as evidenced by their susceptibility to 0.01 mM carbonyl cyanide m-chlorophenylhydrazone (Cl-CCP), 01 mM ouabain and temperature. The active accumulations of GABA, dopamine and norepinephrine were also greatly inhibited by 1 mM6-hydroxydopamine (6-OHDA), 01 mM mersalyl, 0.05–0.25mM vinblastine and 0.1–1.0 mM colchicine. Vinblastine was approximately 10-fold more potent (K₁, ?0.1 mM) than colchicine as an inhibitor. The release of actively accumulated dopamine or norepinephrine by excess K⁺ (increasing the [K⁺] from 5 to 30 mM) was inhibited somewhat when vinblastine was present during the entire incubation period. If the synaptosomes were preloaded with the radioactive compounds prior to addition of vinblastine, there was no discernible effect on the relative amount of material released by excess K⁺. However, the addition of inhibitor under the latter conditions caused a leakage of radioactivity into the medium even without excess K⁺ being present. Glutamate accumulation was somewhat different from that of GABA, dopamine or norepinephrine. Although it required energy for uptake, 6-OHDA, mersalyl, vinblastine or colchicine were not inhibitory. Studies of the oxidative metabolism of glutamate and GABA by this synaptosomal preparation indicated that the mechanisms of inhibition by vinblastine was not attributable to a metabolic effect. Both vinblastine and colchicine inhibited the Mg²⁺-stimulated, but not the Ca²⁺-activated ATPase of neurostenin. This effect was probably attributable to an interaction of the vinblastine with the neurin moiety of this actomyosin-like protein. We suggest that the inhibitory phenomena exhibited by vinblastine and colchicine in this synaptosomal preparation arose from the effect of these alkaloids on the neurin associated with the synaptic membrane.     

Caenorhabditis elegans ciliary protein NPHP-8, the homologue of human RPGRIP1L, is required for ciliogenesis and chemosensation

Nephronophthisis (NPHP) is the most frequent genetic cause of end-stage renal failure in children and young adults. NPHP8/RPGRIP1L is a novel ciliary gene that, when mutated, in addition to causing NPHP, also causes Joubert syndrome (JBTS) and Meckel syndrome (MKS). The exact function of NPHP8 and how defects in NPHP8 lead to human diseases are poorly understood. Here, we studied the Caenorhabditis elegans homolog nphp-8 (C09G5.8) and explored the possible function of NPHP-8 in ciliated sensory neurons. We determined the gene structure of nphp-8 through rapid amplification of cDNA ends (RACE) analysis and discovered an X-box motif that had been previously overlooked. Moreover, NPHP-8 co-localized with NPHP-4 at the transition zone at the base of cilia. Mutation of nphp-8 led to abnormal dye filling (Dyf) and shorter cilia lengths in a subset of ciliary neurons. In addition, chemotaxis to several volatile attractants was significantly impaired in nphp-8 mutants. Our data suggest that NPHP-8/RPGRIP1L plays an important role in cilia formation and cilia-mediated chemosensation in a cell type-specific manner.     

The oral apparatus of Tetrahymena pyriformis, strain WH-6. IV. Observations on the organization of microtubules and filaments in the isolated oral apparatus and the differential effect of potassium chloride on the stability of oral apparatus microtubules.

This report is an ultrastructural analysis of the organization of the isolated oral apparatus of Tetrahymena pyriformis, strain WH-6, syngen 1. Attention has been focused on the organization of microtubules and filaments in oral apparatus membranelles. Oral apparatus membranellar basal bodies were characterized with respect to structural differentiations at the distal and proximal ends. The distal region of membranellar basal bodies contains the basal plate, accessory microtubules and filaments. The proximal end contains a dense material from which emanate accessory microtubules and filaments. There are at least two possibly three different arrangements of accessory structures at the proximal end of membranellar basal bodies. All membranellar basal bodies appear to have a dense material at the proximal end from which filaments emanate. Some of these basal bodies have accessory microtubules and filaments emanating from this dense material. A possible third arrangement is represented by basal bodies which have lateral projections, from the proximal end, of accessory microtubules and filaments which constitute cross or peripheral connectives. There are at least three examples of direct associations between oral apparatus microtubules and filaments: (1) filaments which form links between basal body triplet microtubules, (2) filaments which link the material of the basal plate to internal basal body microtubules, (3) filaments which link together microtubule bundles from membranellar connectives. KCl extraction of the isolated oral apparatus resulted in the selective solubilization of oral apparatus basal bodies, remnants of ciliary axonemes and fused basal plates. Based on their response to KCl extraction two distinct sets of morphologically similar micro tubules can be identified: (a) microtubules which constitute the internal structure of basal bodies and ciliary axonemes, (b) microtubules which constitute the fiber connectives between basal bodies.