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P Satir

Publications and source records attributed to P Satir.

At least 19 recordsLinked to original sources
PubMed1995

Ciliary beat frequency is controlled by a dynein light chain phosphorylation.

cAMP-dependent phosphorylation of a 29-kDa axonemal polypeptide (p29) increases the swimming speed of permeabilized Paramecium and in vitro translocation velocity of bovine brain microtubules over 22S dynein extracted from Paramecium axonemes. A quantitative relationship between microtubule translocation velocity and beat frequency is developed. We conclude that p29 acts as a regulatory light chain of outer arm dynein in the control of ciliary beat frequency.

Animals↗
PubMed1992

A role for microtubules in sorting endocytic vesicles in rat hepatocytes.

The vectorial nature of hepatocyte receptor-mediated endocytosis (RME) and its susceptibility to cytoskeletal disruptors has suggested that a polarized network of microtubules plays a vital role in directed movement during sorting. Using as markers a well-known ligand, asialoorosomucoid, and its receptor, we have isolated endocytic vesicles that bind directly to and interact with stabilized endogenous hepatocyte microtubules at specific times during a synchronous, experimentally initiated, single wave of RME. Both ligand- and receptor-containing vesicles copelleted with microtubules in the absence of ATP but did not pellet under similar conditions when microtubules were not polymerized. When 5 mM ATP was added to preparations of microtubule-bound vesicles, ligand-containing vesicles were released into the supernatant, while receptor-containing vesicles remained immobilized on the microtubules. Release of ligand-containing vesicles from microtubules was prevented by monensin treatment during the endocytic wave. Several proteins, including the microtubule motor protein cytoplasmic dynein, were present in these preparations and were released from microtubule pellets by ATP addition concomitantly with ligand. These results suggest that receptor domains within the endosome can be immobilized by attachment to microtubules so that, following monensin-sensitive dissociation of ligand from receptor, ligand-containing vesicles can be pulled along microtubules away from the receptor domains by a motor molecule, such as cytoplasmic dynein, thereby delineating sorting.

Adenosine Triphosphate↗
PubMed1992

Mechanisms of ciliary movement: contributions from electron microscopy.

A brief review of important contributions of electron microscopy to the study of ciliary motility is presented. The electron microscope was used to show the universality of axonemal structure of cilia, and to develop the sliding microtubule model of ciliary motility and later the switch point hypothesis to explain the conversion of sliding into bending. Unexpectedly, insights into the importance of cilia in human health have stemmed from these studies.

Cell Movement↗
PubMed1991

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.

Animals↗
PubMed1991

Computer modelling of Tetrahymena axonemes at macromolecular resolution. Interpretation of electron micrographs.

A computer-generated model of the structural arrangement of the complete 9+2 ciliary axoneme of Tetrahymena at macromolecular resolution (4 nm) is presented. The model reconciles detailed information about subcomponents from negative-stained, thin-section and freeze-fracture electron micrographs, integrating the images into a consistent three-dimensional picture. This illuminates problems such as the requirement for compaction of dynein to form the arm, difficulties in visualization of the circumferential links, construction of the central sheath, and the comparative periodicities of the inner and outer arms. The model is pragmatic in that it is flexible and easily changed, as new information becomes available. It is also useful in the development of dynamic concepts, such as a spatial description of the dynein cross-bridge cycle, which is illustrated, or relationships between adjacent doublets during sliding and bending.

Animals↗
PubMed1991

Analysis of Ni(2+)-induced arrest of Paramecium axonemes.

This study examines the molecular basis for paralysis of ciliary motility by Ni2+. At concentrations above 0.1 mM, Ni2+ slowed and subsequently stopped swimming of living, axenically grown Paramecium tetraurelia. However, some cilia still beat in the presence of 0.1 mM Ni2+. When permeabilized and reactivated with 4 mM ATP at pCa greater than 7, cells resumed ciliary beat and swam forward at approximately 170 +/- 28 microns s-1; swimming speed increased in the presence of 10 microM cyclic AMP. Addition of Ni2+ (pNi less than 5) caused rapid arrest of all ciliary beat in a single position. This was fully reversible when EGTA was added to raise the pNi. Axonemes were then isolated and sliding was observed in the presence of trypsin and ATP. When pNi was lowered to about 5, sliding was reduced dramatically. This too was reversible with EGTA. Dynein was then extracted from the axonemes and used for in vitro translocation assays. At concentrations of Ni2+ where microtubule-sliding and axonemal beat were greatly inhibited or absent, microtubule translocation in vitro by 22 S dynein was only slightly affected. However, translocation by 14 S dynein was stopped completely. When pNi was raised by repeated washing with solutions containing EGTA, microtubule translocation by 14 S dynein resumed. We conclude that Ni2+ induces a reversible paralysis by a direct effect on 14 S dynein while 22 S dynein is not a primary target.

Adenosine Triphosphatases↗
PubMed1990

A physical model of microtubule sliding in ciliary axonemes.

Ciliary movement is caused by coordinated sliding interactions between the peripheral doublet microtubules of the axoneme. In demembranated organelles treated with trypsin and ATP, this sliding can be visualized during progressive disintegration. In this paper, microtubule sliding behavior resulting from various patterns of dynein arm activity and elastic link breakage is determined using a simplified model of the axoneme. The model consists of a cylindrical array of microtubules joined, initially, by elastic links, with the possibility of dynein arm interaction between microtubules. If no elastic links are broken, sliding can produce stable distortion of the model, which finds application to straight sections of a motile cilium. If some elastic links break, the model predicts a variety of sliding patterns, some of which match, qualitatively, the observed disintegration behavior of real axonemes. Splitting of the axoneme is most likely to occur between two doublets N and N + 1 when either the arms on doublet N + 1 are active and arms on doublet N are inactive or arms on doublet N - 1 are active while arms on doublet N are inactive. The analysis suggests further experimental studies which, in conjunction with the model, will lead to a more detailed understanding of the sliding mechanism, and will allow the mechanical properties of some axonemal components to be evaluated.

Animals↗
PubMed1989

In vitro phosphorylation of Paramecium axonemes and permeabilized cells.

This study seeks to identify phosphoproteins in axonemes from Paramecium tetraurelia whose phosphorylation responses to adenosine 3', 5'-cyclic monophosphate (cAMP) and Ca2+ parallel responses induced by these agents in ciliary behavior in this cell. In purified axonemes, over 15 bands ranging from Mr greater than 300 kDa to 19 kDa on SDS-PAGE incorporate 32P from adenosine 5'-gamma-[32P]triphosphate (gamma-32P-ATP) at pCa 7 in the absence of cAMP. A major band whose label turns over rapidly was identified at Mr 43 kDa. In the presence of 5 microM cAMP, more than eight bands, but not the Mr 43 kDa band, were labeled additionally or enhanced their labeling. These phosphoproteins and their kinases are structural components of the axoneme. Overall, some of the same major bands are labeled in the presence of cAMP in Triton X-100-permeabilized paramecia that retain their behavioral responses and in axonemes mechanically isolated from these cells. In particular, two major bands have been identified whose phosphorylation is greatly enhanced by cAMP at low concentrations: 1) a 29 kDa polypeptide whose cAMP-dependent phosphorylation is diminished at pCa 4 compared with pCa 7 and 2) a 65 kDa polypeptide whose phosphorylation is pCa insensitive. These polypeptides meet minimal criteria for signal-sensitive regulators of motility parameters in the Paramecium axoneme.

Animals↗
PubMed1989

Splitting the ciliary axoneme: implications for a "switch-point" model of dynein arm activity in ciliary motion.

In the presence of specific inhibitors of beat. 20 microM VO4(3-) or pCa 4, mussel gill lateral (L) cilia can be arrested in two positions--"hands down" or "hands up"--at opposite ends of the stroke cycle. Cilia move to these positions by doublet microtubule sliding. Axonemes of arrested cilia, still tethered to the cell, are intact after demembranation and protease treatment. When reactivated by 4 mM ATP with inhibitors present, about 40% split apart. Splits are not random but occur preferentially between different specific doublets in the two opposite arrest positions. Several different related patterns of splitting are observed; for every pattern in "hands down" axonemes, there is a corresponding complementary split pattern in "hands up" axonemes. In some split patterns two doublets remain firmly attached to the central pair; these also differ depending on axonemal position. Although some of the patterns seen may be artifactual or difficult to explain, the complementary splitting patterns are predictable with simple assumptions by a "switch point" hypothesis of ciliary activity where, during each recovery stroke, doublets 6-8 have active dynein arms, while during each effective stroke, arms on doublets 1-4 become active, and arms 6-8 are turned off. Because of a difference between the patterns seen and the predictions, the status of the arms on doublet 9 is unresolved. The patterns also suggest that a spoke-central sheath attachment cycle may correlate with switching of arm activity during the generation of an asymmetric beat.

Adenosine Triphosphatases↗
PubMed1989

The role of axonemal components in ciliary motility.

1. The axoneme is the detergent-insoluble cytoskeleton of the cilium. 2. All axonemes generate movement by the same fundamental mechanism: microtubule sliding utilizing ATP hydrolysis during a mechanochemical cycling of dynein arms on the axonemal doublets. 3. Structure, fundamental biochemistry and physiology of the axoneme are conserved evolutionarily, but the phenotypes of beating movements and the responses to specific cytoplasmic signals differ greatly from organism to organism. 4. A model of asynchronous dynein arm activity--the switch point hypothesis--has been proposed to account for cyclic beating in the face of unidirectional sliding. The model suggests that the diversity of beat phenotype may be explicable by changes in the timing of switching between active and inactive states of doublet arm activity. Evidence of axonemal splitting in arrested axonemes provides new support for the hypothesis.

Animals↗
PubMed1988

Ultrastructure and motion analysis of permeabilized Paramecium capable of motility and regulation of motility.

Structural and behavioral features of intact and permeabilized Paramecium tetraurelia have been defined as a basis for study of Ca2+ control of ciliary reversal. Motion analysis of living paramecia shows that all the cells in a population swim forward with gently curving spirals at speeds averaging 369 +/- 19 microns/second. Ciliary reversal occurs in 10% of the cell population per second. Living paramecia, quick-fixed for scanning electron microscopy (SEM), show metachronal waves and an effective stroke obliquely toward the posterior end of the cell. Upon treatment with Triton X-100, swimming ceases and both scanning and transmission electron microscopy reveal cilia that uniformly project perpendicularly from the cell surface. Thin sections of these cells indicate that the ciliary, cell, and outer alveolar membranes are greatly disrupted or entirely missing and that the cytoplasm is also disrupted. These permeabilized paramecia can be reactivated and are capable of motility and regulation of motility. Motion analysis of cells reactivated with Mg2+ and ATP in low Ca2+ buffer (pCa greater than 7) shows that 71% swim forward in straight or curved paths at speeds averaging 221 +/- 20 microns/second. When these cells are quick-fixed for SEM the metachronal wave patterns of living, forward swimming cells reappear. Motion analysis of permeabilized cells reactivated in high Ca2+ buffers (pCa 5.5) shows that 94% swim backward in tight spirals at a velocity averaging 156 +/- 7 microns/second. SEM reveals a metachronal wave pattern with an effective stroke toward the anterior region. Although the permeabilized cells do not reverse spontaneously, the pCa response is preserved and the Ca2+ switch remains intact. The ciliary axonemes are largely exposed to the external environment. Therefore, the behavioral responses of these permeabilized cells depend on interaction of Ca2+ with molecules that remain bound to the axonemes throughout the extraction and reactivation procedures.

Animals↗
PubMed1988

Dynein as a microtubule translocator in ciliary motility: current studies of arm structure and activity pattern.

The dynein arms of ciliary doublet microtubules cause adjacent axonemal doublets to slide apart with fixed polarity. This suggests that there is a unique mechanochemistry to the dynein arm with unidirectional force generation in all active arms and also that not all arms are active at once during a ciliary beat. Negative stain and thin-section images of arms in axonemes treated with beta, gamma methylene adenosine triphosphate (AMP-PCP) show a consistent subunit construction where the globular head of the arm interacts with subfiber B of doublet N+1. This interpretation differs from that provided by freeze etch and STEM interpretations of in situ arm construction and has implications for the mechanochemical cycle of the arm. A computer model of the arms in relation to other axonemal structures has been constructed to test these interpretations. Attachment of the head of the arm subfiber B is directly demonstrable in splayed axonemes in AMP-PCP. About half of the doublets in an axoneme show such attachments, while half do not. This might imply that about half the doublets in an axoneme are active at any given instant and can be identified as such. This information may be useful in probing questions of how active arms differ biochemically from inactive arms and of how microtubule translocators in general become active.

Adenosine Triphosphatases↗
PubMed1988

Immunogold localization of the regulatory subunit of a type II cAMP-dependent protein kinase tightly associated with mammalian sperm flagella.

We have shown previously that the regulatory subunit (RII) of a type II cAMP-dependent protein kinase is an integral component of the mammalian sperm flagellum (Horowitz, J.A., H. Toeg, and G.A. Orr. 1984. J. Biol. Chem. 259:832-838; Horowitz, J.A., W. Wasco, M. Leiser, and G.A. Orr. 1988. J. Biol. Chem. 263:2098-2104). The subcellular localization of this flagellum-associated RII in bovine caudal epididymal sperm was analyzed at electron microscope resolution with gold-conjugated secondary antibody labeling techniques using anti-RII monoclonal antibodies. By immunoblot analysis, the flagellum-associated RII was shown to interact with mAb 622 which cross reacts with both neural and nonneural isoforms of RII. In contrast, a neural specific monoclonal antibody (mAb 526) failed to interact with flagellar RII. In the midpiece of the demembranated sperm tail, gold label after mAb 622 incubation was primarily associated with the outer mitochondrial membrane. Although almost all specific labeling in the midpiece can be assigned to the mitochondria, in the principal piece, there is some labeling of the fibrous sheath. Labeling of the outer dense fibers and the axoneme was sparse. Specific labeling was virtually absent in the sperm head. Sections of sperm tails incubated in the absence of primary antisera or with mAb 526 showed little labeling. A beta-tubulin monoclonal antibody localized only to the 9 + 2 axoneme. These results raise the possibility that a type II cAMP-dependent protein kinase located at the outer mitochondrial membrane plays a role in the direct cAMP stimulation of mitochondrial respiration during sperm activation.

Adenosine Triphosphatases↗
PubMed1987

Dynein arm attachment probed with a non-hydrolyzable ATP analog. Structural evidence for patterns of activity.

The dynein arms that power ciliary motility are normally permanently attached by one end exclusively to subfiber A of each axonemal doublet (N) while the other (head) end transiently attaches to the subfiber B of the adjacent doublet (N + 1) to produce sliding of the doublets. In Tetrahymena axonemes, sliding of contiguous groups of doublets is induced by ATP suggesting that, in the absence of exogenous protease, there may be sets of potentially active and potentially inactive or refractory arms in a single axoneme. In the presence of a non-hydrolyzable analog of ATP, beta,gamma-methylene adenosine 5'-triphosphate (AMP-PCP), about half the doublets in an axonemal preparation retain all arms bound to subfiber A, but half the doublets show long regions where some arms are pulled away from subfiber A of doublet N and attached to subfiber B of doublet N + 1 by their head ends. In AMP-PCP-induced splaying, positional information regarding arm state is retained. Analysis reveals that throughout regions where B subfiber attachment is found, small groups of about four subfiber B attached arms alternate with groups of about four arms that remain attached to subfiber A. This unique pattern of attachment suggests that arms function co-operatively in groups of four. Further, the repetition of the pattern is reminiscent of metachronal activity seen at higher levels of biological organization. This suggests that in these regions we have instantaneously preserved groups of arms capable of attaching to and detaching from doublet N + 1 in rapid succession. This appearance could be used to delineate the potentially active sets of arm, primed for mechanochemical activity, within an axoneme.

Adenosine Triphosphatases↗
PubMed1986

Spreading ciliary arrest in a mussel gill epithelium: characterization by quick fixation.

Spreading ciliary arrest, induced by local laser microinjury, in freshwater mussel (e.g., Elliptio) gill lateral (L) cell cilia, has been characterized by quick fixation with osmium tetroxide, which permits the correlation of known features of the response with structural features of the gill epithelium. Quick fixation reliably preserves the state of the epithelium including the activity state of the L cilia at the moment of fixation. From a disrupted region, the stimulus that triggers arrest spreads outward along an undamaged filament preferentially from L cell to L cell for more than 300 microns to either side of the lesion. In physiological salt solutions transverse spread across the filament via heterologous cells is insufficient to elicit L ciliary arrest on the opposite side of the filament. The spread of arrest is dependent upon the structural integrity of the L epithelium, normally terminates at a boundary between adjacent L cells, and does not spread past a focal break. Arrest occurs asynchronously because cilia in different stroke positions respond to the stimulus with different time courses. The cilia stop in a uniform "hands up" position, i.e., pointing frontally. The arrest response is inhibited by reducing the concentration of extracellular Ca2+ (less than 10(-7) M) or by adding extracellular La3+ (1 mM) or K+ (15 mM). Recovery begins at the margin of a segment of arrested L cilia and spreads back toward the lesion at a constant initial velocity of ca. 60 microns/sec. About 300 microns from the lesion the recovery velocity rapidly falls to ca. 5 microns/sec. Recovery of ciliary beat precedes the recovery of metachronal coordination. Neither spread of the stimulus nor recovery require ciliary beat. The data support the hypothesis that the microinjury-induced arrest is initiated by an injury potential that triggers a graded regenerative depolarization that is propagated electrotonically along the epithelium from L cell to L cell, triggering Ca2+ influx into the axoneme and consequent Ca2+-induced L ciliary arrest as it spreads. A temporary non-linear gradient of intracellular Ca2+ concentration is established along the injured L epithelial tract. As individual cells recover, they lower their intracellular Ca2+ concentration from pCa 5 to pCa 7 in about 10 seconds.

Animals↗
PubMed1986

Structures attached to doublet microtubules of cilia: computer modeling of thin-section and negative-stain stereo images.

With a single set of positional coordinates for longitudinal and transverse attachment of the inner and outer rows of dynein arms with respect to the doublet microtubules of Tetrahymena ciliary axonemes, a computer model has been constructed at 4-nm resolution that reconciles negative-stain en face stereo images of arm and spoke positions to traditional images of tannic acid/glutaraldehyde-fixed sections. In this model, inner and outer arms correspond in substructure; both repeat with a 24-nm periodicity without stagger between rows, and a pair of arms is in exact alignment with the first spoke (S1) in each doublet spoke group. The model and the supporting micrographs suggest that each arm cycles in three dimensions and that, during cycling, the inner and outer arms move in opposite directions with respect to the center of subfiber A of the doublet (N). Attachment is off-center with respect to subfiber B of the adjacent doublet (N + 1), causing the sliding doublets to skew with respect to one another.

Adenosine Triphosphatases↗