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Biomedical subjects

W L Dentler

Publications and source records attributed to W L Dentler.

At least 37 records · Page 2Linked to original sources
PubMed1985

Development and ciliation of the palate in two frogs, Bombina and Xenopus; a comparative study.

The morphological changes that occur during metamorphosis in the palates of two types of anuran larvae (a discoglossid, Bombina orientalis, and a pipid, Xenopus laevis) are compared. In B. orientalis the structural changes are accompanied by the ciliation of the palate epithelium. Ciliation begins in the anterior region of the palate and continues in a posterior direction throughout metamorphosis. By contrast, the palate of X. laevis never becomes ciliated during its development. Instead, two ciliated grooves develop between the choanae (nasal openings) and the esophageal opening. The grooves transport mucus and trapped objects out of the internal nares and toward the esophagus. These grooves are compared to similar structures on the palate of adult B. orientalis. The timing and pattern of ciliogenesis during metamorphosis in each of these anurans is also described relative to well-established staging series for external frog development. We show that the onset and location of ciliogenesis are consistent and predictable in these anurans and, therefore, make the frog palate an excellent system for the study of ciliogenesis.

Animals↗
PubMed1985

Multiple forms of tubulin in the cilia and cytoplasm of Tetrahymena thermophila.

Most higher eukaryotic tubulins are separated into alpha- and beta-tubulin when electrophoresed in NaDodSO4- denaturing gels, while many lower eukaryotic tubulins are poorly resolved under these conditions, which include a stacking gel (pH 6.80) and a separating gel (pH 8.80). By lowering the pH of the separating gel to 8.25, we have found that tubulin isolated from the protozoan Tetrahymena thermophila is resolved by one-dimensional polyacrylamide gel electrophoresis into two alpha-tubulins and one beta-tubulin. Moreover, at least five alpha- and two beta-tubulin isotypes are identified in Tetrahymena by isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis. Three of these alpha isotypes and one beta isotype are found specifically in ciliary microtubules, while the other two isotypes are found only in the cytoplasmic tubulin pool that was isolated and induced to self-assemble into microtubules in vitro. Peptide mapping by limited proteolytic digestion indicates that the tubulins are closely related. Possible mechanisms for the generation and selection of these tubulin isotypes are discussed.

Animals↗
PubMed1984

Asymmetrical microtubule capping structures in frog palate cilia.

Capping structures at the distal tips of frog palate cilia are attached to the A- and central pair microtubules by electron-dense plug structures similar to those found in protozoan cilia and flagella and in epithelial cilia from invertebrates and vertebrates (W.L. Dentler, 1980, J. Cell Sci. 42, 207-220; W.L. Dentler and E.L. LeCluyse, 1982, Cell Motil. 2, 549-573). The caps in frog palate cilia are composed of a proximal shelf, to which doublets Nos. 1-3, 8, 9 and the central microtubules are attached and a larger distal cap to which doublets Nos. 4-7 are bound. The smaller proximal shelf is positioned to one side of the cilium and gives the cap an asymmetrical appearance. Striated ciliary rootlets attached to the basal bodies are also described. The smaller cap is placed on the same side of all cilia on the palate relative to both the direction of the effective stroke and the position of the rootlets. These results confirm that capping structures are common to most, if not all, cilia and provide direct evidence that they are precisely positioned on specific microtubules.

Aging↗
PubMed1984

Attachment of the cap to the central microtubules of Tetrahymena cilia.

The central microtubule cap is bound to the ends of the two central microtubules in Tetrahymena thermophila cilia by plug-shaped structures similar in appearance to the distal filament plugs attached to the ends of the A-microtubules. The caps have been separated from the microtubules and are composed of a bead, two plates, and two peg-like plugs to which the microtubules are attached. The structure of the cap is discussed in relation to the directionality of microtubule assembly in vivo.

Animals↗
PubMed1982

Microtubule capping structures at the tips of tracheal cilia: evidence for their firm attachment during ciliary bend formation and the restriction of microtubule sliding.

The distal tips of the central pair and A-microtubules are capped in mammalian and avian tracheal cilia. The capping structures are similar to those found in protozoan cilia and flagella [Dentler, 1981], and consist of a central microtubule cap that links the central microtubules to the membrane or to the ciliary crown and A-microtubule plugs that insert into the lumen of each of the A-microtubules. In contrast to the protozoan capping structures, each of the A-microtubule plugs is bound to the central microtubule cap by distal filaments. The ends of the central and outer doublet microtubules are tightly bound to the cap in both intact and in demembranated and reactivated tracheal cilia. Analysis of the displacement of the microtubule tips in cilia fixed at various bend angles revealed that the displacements of A-microtubules are only partially in agreement with those predicted by the sliding filament model [Satir, 1968]. These results are discussed with respect to the regulation of microtubule sliding in capped cilia and the role of the microtubule capping structures in microtubule assembly.

Animals↗
PubMed1982

Association between endocrine pancreatic secretory granules and in-vitro-assembled microtubules is dependent upon microtubule-associated proteins.

By use of dark-field light microscopy, secretory granules isolated from the anglerfish endocrine pancreas were observed to attach to and release from microtubules assembled in vitro from brain homogenates. Secretory granules only bound to microtubules assembled in the presence of microtubule-associated proteins (MAPs) and not to microtubules assembled from purified tubulin. The addition of a MAP fraction to purified tubulin restored secretory granule binding. The secretory granules were released from MAP-containing microtubules by the addition of Mg-ATP but not by other nucleotides. The number of secretory granules bound to MAP-containing microtubules was increased in the presence of cyclic AMP. In addition to the associations of secretory granules with microtubules, MAP-containing microtubules also associated with each other. These laterally associated microtubules were dispersed by the addition of Mg-ATP. Electron micrographs confirmed that the associations between MAP-containing microtubules and secretory granules as well as the associations of microtubules with one another were mediated by the high molecular weight MAPs known to project from the surface of in-vitro-assembled microtubules.

Animals↗
PubMed1981

Microtubule-membrane interactions in ctenophore swimming plate cilia.

The cilia in ctenophore swimming plates are organized into long rows and the cilia within each of the rows are connected to one another by interciliary bridges. The interciliary bridges form a type of intracellular junction and are periodically spaced at 15 nm intervals along the long axis of a cilium. The bridges bind adjacent cilia together even after dissolution of the ciliary membrane by non-ionic detergent. Interciliary bridges are attached to the compartmenting lamellae, which are paracrystalline structures composed of spherical particles which are periodically attached to the outer doublet microtubules at the sites to which the microtubule-membrane bridges are bound. It is proposed that the compartmenting lamellae are modifications of the ciliary microtubule-membrane bridge found in other eukaryotic cilia and that it is associated with a junctional complex that binds adjacent cilia together in swimming plates.

Animals↗
PubMed1981

Microtubule-associated proteins (MAPs) and the organization of actin filaments in vitro.

When purified muscle actin was mixed with microtubule-associated proteins (MAPs) prepared from brain microtubules assembled in vitro, actin filaments were organized into discrete bundles, 26 nm in diameter. MAP-2 was the principal protein necessary for the formation of the bundles. Analysis of MAP-actin bundle formation by sedimentation and electrophoresis revealed the bundles to be composed of approximately 20% MAP-2 and 80% actin by weight. Transverse striations were observed to occur at 28-nm intervals along negatively stained MAP-actin bundles, and short projections, approximately 12 nm long and spaced at 28-nm intervals, were resolved by high-resolution metal shadowing. The formation of MAP-actin bundles was inhibited by millimolar concentrations of ATP, AMP-PCP (beta, gamma-methylene-adenosine triphosphate), and pyrophosphate but not by AMP, ADP, or GTP. The addition of ATP to a solution containing MAP-actin bundles resulted in the dissociation of the bundles into individual actin filaments; discrete particles, presumably MAP-2, were periodically attached along the splayed filaments. These results demonstrate that MAPs can bind to actin filaments and can induce the reversible formation of actin filament bundles in vitro.

Actins↗
PubMed1980

Microtubule-membrane interactions in cilia. I. Isolation and characterization of ciliary membranes from Tetrahymena pyriformis.

Tetrahymena ciliary membranes were prepared by four different techniques, and their protein composition was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electron microscopy, and two-dimensional thin-layer peptide mapping. Extraction of the isolated cilia by nonionic detergent solubilized the ciliary membranes but left the axonemal microtubules and dyneine arms intact, as determined by quantitative electron microscopy. The proteins solubilized by detergent included a major 55,000-dalton protein, 1-3 high molecular weight proteins that comigrated, on SDS-PAGE, with the axonemal dynein, as well as several other proteins of 45,000-50,000 daltons. Each of the major proteins contained a small amount of carbohydrate, as determined by PAS-staining; no PAS-positive material was detected in the detergent-extracted axonemes. The major 55,000-dalton protein has proteins quite similar to those of tubulin, based on SDS-PAGE using three different buffer systems as well as two-dimensional maps of tryptic peptides from the isolated 55,000-dalton protein. To determine whether this tubulin-like protein was associated with the membrane or whether it was an axonemal or matrix protein released by detergent treatment, three different methods to isolate ciliary membrane vesicles were developed. The protein composition of each of these differetn vesicle preparations was the same as that of the detergent-solubilized material. These results suggest that a major ciliary membrane protein has properties similar to those of tubulin.

Animals↗
PubMed1980

Microtubule-membrane interactions in cilia. II. Photochemical cross-linking of bridge structures and the identification of a membrane-associated dynein-like ATPase.

Photochemical cross-linking of both Tetrahymena and Aequipecten ciliary membrane proteins with the lipophilic reagent 4,4'-dithiobisphenylazide links together a high molecular weight dynein-like ATPase, membrane tubulin, and at least two other proteins. Electron microscopy of detergent-extracted cilia reveals that the cross-linked complex remains attached to the outer-doublet microtubules by a microtubule-membrane bridge. Cleavage of the reagent's disulfide bond releases the bridge-membrane complex and the dynein-like membrane-associated ATPase. Electron microscopy was used to ensure that the dynein-like protein did not result from the solubilization of the dynein arms attached to the outer-doublet microtubules. The dynein-like protein has been isolated using sucrose gradients and is similar to axonemal dynein with respect to its sedimentation characteristics nucleotide specificity, and divalent cation requirements. Photochemical cross-linking of ciliary membrane porteins in vivo results initially in the modification of ciliary beat and, eventually, in the cessation of ciliary movement. These results suggest that a dynein-like ATPase comprises the bridge which links the ciliary membrane to the outer-doublet microtubules and that this bridge is involved in the modulation of normal ciliary movement.

Adenosine Triphosphatases↗
PubMed1980

Structures linking the tips of ciliary and flagellar microtubules to the membrane.

The distal tips of both central pair and outer doublet microtubules are linked to flagellar membranes of Chlamydomonas and to ciliary membranes of both Tetrahymena and Aequipecten by the central microtubule cap and by distal filaments, respectively. The distal filaments are linked to ciliary membranes at their distal ends and, at their proximal ends, form a carrot-shaped plug which inserts into the lumen of the A-tubule of each outer doublet. Both types of tip structures are present in regenerating cilia and flagella. At least 2 different types of distal filaments are present in Tetrahymena. Certain cilia in Tetrahymena oral apparatuses and in certain molluscan gills contain distal filaments which join with the central microtubule cap and with the ciliary membrane to form a cap-filament-membrane complex.

Animals↗
PubMed1977

Structure and organization of radial spokes in cilia of Tetrahymena pyriformis.

The structure and organization of radial spokes, the principal components between each of the peripheral doublet microtubules and the central sheath which surrounds the central pair of microtubules have been described in Tetrahymena pyriformis cilia. The radial spokes are grouped in triplets and are attached to the A-microtubule of each peripheral doublet at intervals of 200/280/360 A, the 200 A spacing being most distal to the base of the cilium. The radial spoke triplets are organized in the axoneme in a double helix with a pitch of 4,680 A. A method for determining the helical disposition by correcting for doublet sliding is presented.

Animals↗
PubMed1977

Fine structural localization of phosphatases in cilia and basal bodies of Tetrahymena pyriformis.

Cytochemical localization of ATPase activities in cilia and basal bodies of Tetrahymena pyriformis revealed a number of possible sites of ATPases. In basal bodies, reaction product was localized on the periphery of basal body microtubules, in the core of the B-microtubules, on the dense basal body core, and on the basal plate; some reaction product was associated with the postciliary and basal microtubules. In the cilium, reaction product was associated with the ciliary membrane, the basal granule, the periphery of the outer doublet microtubules, in the core of the B-microtubules, and on the arms and either the central microtubules or the radial spoke heads. Reaction product deposition required ATP and either Ca2+ or Mg2+ or ADP and Mg2+. When incubated in the presence of ATP and Na+, reaction product was only found at the base of the cilium in the region of the ciliary necklace. Implications of the various sites of activity are discussed with respect to possible mechanisms of ciliary motility.

Adenosine Triphosphatases↗
PubMed1977

Flagellar elongation and shortening in Chlamydomonas. III. structures attached to the tips of flagellar microtubules and their relationship to the directionality of flagellar microtubule assembly.

Two structures on the distal ends of Chlamydomonas flagellar microtubules are described. One of these, the central microbutule cap, attaches the distal ends of the central pair microtubules to the tip of the flagellar membrane. In addition, filaments, called distal filaments, are observed attached to the ends of the A-tubules of the outer doublet microtubules. Inasmuch as earlier studies suggested that flagellar elongation in vivo occurs principally by the distal addition of sublnits and because it has been shown that brain tubulin assembles in vitro primarily onto the distal ends of both central and outer doublet microtubules, the presence of the cap and distal filaments was quantitated during flagellar resorption and elongation. The results showed that the cap remains attached to the central microtubules throughout flagellar resorption and elongation. The cap was also found to block the in vitro assembly of neurotubules onto the distal ends of the central microtubules. Conversely, the distal filaments apparently do not block the assembly of neurotubules onto the ends of the outer doublets. During flagellar elongation, the distal ends of the outer doublets are often found to form sheets of protofilaments similar to those observed on the elongating ends of neurotubules being assembled in vitro. These results suggest that the outer doublet microtubules elongate by the distal addition of subunits, whereas the two central microtubules assemble by the addition of subunits to the proximal ends.

Chlamydomonas↗
PubMed1976

Microtubule-associated proteins and the stimulation of tubulin assembly in vitro.

Microtubules, purified by cycles of assembly and disassembly in vitro, are composed of tubulin and several microtubule-associated proteins (MAPs). When the MAPs were separated from the tubulin by phosphocellulose chromatography, the tubulin by phosphocellulose chromatography, the tubulin no longer assembled at 37 degrees C as measured by turbidity. If the MAPs and tubulin were recombined and warmed to 37 degrees C, microtubules assembled. MAPs stimulated tubulin assembly by affecting both the initiation and elongation processes. The effect on initiation was indicated by results showing an increase in initial rate and a decrease in average microtubule length as the MAP:tubulin ratio was increased. The initiation and elongation activities of the MAPs at 4 degrees C during which time the initiating activity decreased while the ability to affect the total amount of assembly remained constant. The decrease in initiating ability was correlated with the loss of the two major components of the MAP fraction, MAPs 1 and 2.

Animals↗