Summary Flagellar scales from the green flagellateTetraselmis striata (Prasinophyceae) were isola... more Summary Flagellar scales from the green flagellateTetraselmis striata (Prasinophyceae) were isolated, purified by isopycnic cesium chloride-gradient and zonal sucrose gradient centrifugation and their structure and biochemical composition investigated. Three types of flagellar scales were purified to more than 90% purity, a fourth type up to 75% purity. In addition to the previously known types of flagellar scales (pentagonal scales, rod-shaped
This study confirms and extends previous work on the lateral cilia of the fresh-water mussel, Ell... more This study confirms and extends previous work on the lateral cilia of the fresh-water mussel, Elliptio complanatus, in support of a "sliding filament" mechanism of ciliary motility wherein peripheral filaments (microtubules) do not change length during beat (see ). Short sequences of serial sections of tips are examined in control (nonbeating) and activated (metachronal wave) preparations. Several different tip types, functional rather than morphogenetic variants, are demonstrated, but similarly bent cilia have similar tips. The peripheral filaments are composed of two subfibers: a and b. The bent regions of cilia are in the form of circular arcs, and apparent differences in subfiber-b length at the tip are those predicted solely by geometry of the stroke without the necessity of assuming filament contraction. Various subfibers b apparently move with respect to one another during beat, since small systematic variations in relative position can be detected from cilium to cilium. While subfiber-b lengths are uniform throughout, subfiber-a lengths are morphologically different for each filament: 8 and 3 are about 0.8 A longer than 1, 4 and 5, but each unique length is independent of stroke position or tip type. Subfiber-a does not contract, nor does it move, e.g. slide, with respect to subfiber-b of the same doublet. The central pair of filaments extends to the tip of the cilium where its members fuse. Subunit assembly in ciliary microtubules is evidently precise. This may be of importance in establishing the relationships needed for mechanochemical interactions that produce sliding and beat.
A brief review of important contributions of electron microscopy to the study of ciliary motility... more 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.
In earlier studies, Hamasaki et al. (Proc. Natl. Acad. Sci. USA. 88:7918-7922, 1991) and Barkalow... more In earlier studies, Hamasaki et al. (Proc. Natl. Acad. Sci. USA. 88:7918-7922, 1991) and Barkalow et al. (J. Cell Biol. 126:727-735, 1994) found that cAMP- and Ca2+-sensitive phosphorylation of a 29 kDa dynein light chain (p29) extracted from 22S axonemal dynein of Paramecium, regulates the velocity of in vitro microtubule translocation and ciliate swimming speed. In this study we report evidence of recombination of p29 to cytoplasmic dyneins from both rat liver and Paramecium, as well as to a 22S dynein precursor molecule, based on immunoprecipitation and force filtration data. Immunoprecipitation also provides additional evidence for the binding of p29 to 22S axonemal dynein. The results suggest that p29 might regulate cytoplasmic dynein, as well as axonemal dynein function in Paramecium, and that a homologue of p29 may exist in rat liver and other mammalian cells.
Analysis by confocal microscopy has revealed features of the microtubule network of rat hepatocyt... more Analysis by confocal microscopy has revealed features of the microtubule network of rat hepatocytes in culture, establishing the three-dimensional disposition of the microtubule-based cytoskeleton, its relation to the actin-based cytoskeleton and to ligand-containing endosomes during receptor-mediated endocytosis and the alterations in its structure and disposition by the microtubule pertubant, Taxol. By co-localization studies, we have been able to demonstrate that the microtubules have a significant role in receptor-mediated endocytosis of asialoglycoproteins in this cell. Asialoorosomucoid-containing endosomes attach to widely spaced arrays of microtubules running under the baso-lateral surface of the hepatocytes 5-15 minutes after the initiation of endocytosis and then travel along microtubule paths to become concentrated with microtubules near the centrosome and at bile canaliculi after 30-60 minutes of receptor-mediated endocytosis. Receptor-mediated endocytosis is affected, b...
... New York: Appleton Century Crofts (Meredith Corp.) pp. 194242. Eakin RM, Westfall JA. 1962. ... more ... New York: Appleton Century Crofts (Meredith Corp.) pp. 194242. Eakin RM, Westfall JA. 1962. Fine structure of photoreceptors in amphioxus. ... Get PDF (272K). More content like this. Find more content: like this article. Find more content written by: Peter Satir. ...
cAMP-dependent phosphorylation of a 29-kDa axonemal polypeptide (p29) increases the swimming spee... more 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.
Using potassium pyroantimonate as a Ca2+ chelating agent, we have been able to open the belt desm... more Using potassium pyroantimonate as a Ca2+ chelating agent, we have been able to open the belt desmosome that mechanically couples adjacent ciliated lateral (L) gill epithelial cells of freshwater mussels. Subsequent transfer of the mechanically uncoupled epithelia to solutions containing millimolar Ca2+ results in dramatic alterations in cell shape and partial disruption and apical reorientation of L cell septate junction. The calls remain viable throughout the procedure as determined by the persistence of ciliary activity, although disruption is accompanied by a separation of neighboring groups of cilia and a switch from metachronal to synchronous L cell ciliary beat co-ordination.
Rapid initiation of ion transport occurs in the lower Malpighian tubule of the insect Rhodnius pr... more Rapid initiation of ion transport occurs in the lower Malpighian tubule of the insect Rhodnius prolixus following feeding in vivo or stimulation with 5-hydroxytryptamine (5-HT) in vitro. Using the electron microscope, we have conducted a morphometric analysis of cells in the lowest one-third of the lower tubule, demonstrating that 5-HT also induces mitochondrial movement and microvillar growth simultaneously with, but independent of, the onset of ion transport. Mitochondria move from a position below the cell cortex to one inside the microvilli within 10 min of stimulation with 5-HT, resulting in an 8- to 10-fold increase in the volume of mitochondria within the microvilli. Previous findings indicated that mitochondrial movement is dependent on actin-containing microfilaments, but not microtubules. As the mitochondria enter the microvillus, the core microfilaments are reorganized into a sheath of microfilaments, which extends closely parallel to the outer mitochondrial membrane down...
This study considers the relationship between two structural forms of the 22S dynein arm of Tetra... more This study considers the relationship between two structural forms of the 22S dynein arm of Tetrahymena thermophila: the bouquet and the compact arm. The compact arm differs from the bouquet and from other proposed forms (e.g., the "toadstool") in that the globular domains are situated transversely across the interdoublet gap with one globular subunit, the head, proximal to the adjacent doublet microtubule. The other models place all three globular domains proximal to the neighboring doublet microtubule. When sliding of an isolated axoneme is induced, at least 57% of total attached arms on exposed doublets are in the compact form within dimensions of 24 x 24 x 12 nm, and only about 2% of the arms are bouquets. Toadstools are incompatible with the images seen. Bouquets are not found in regions of the doublet protected by a neighboring doublet. When axonemes with exposed doublets are treated with 0.5 M KCl for 30 min, the compact arms and the dynein heavy (H)-chains disappear, while isolated bouquets and dynein H-chains appear in the medium, suggesting that the compact arms give rise to the bouquets as they are solubilized. The bouquet is the predominant form of isolated 22S dynein molecules, which are found in two apparently enantiomorphic forms, within dimensions 45 x 39 x 13 nm; bouquets attached to doublets have dimensions similar to those of isolated bouquets. Computer modeling indicates that in an intact standard-diameter axoneme, these dimensions are incompatible with the interdoublet volume available for an arm; the bouquet therefore represents an unfolded compact arm. A plausible sequence of changes can be modeled to illustrate the conversion of an attached compact arm to an attached and then free bouquet. The toadstool is probably an artifact that arises after unfolding. Consistent with the conformational difference, H-chains of attached compact arms differ from those of isolated bouquets in their susceptibility to limited proteolysis. These results suggest that the compact arm, rather than the unfolded bouquet or the toadstool, is the functional form of the outer arm in the intact axoneme.
The dominance of viscous forces in the generation of propulsive thrust by cilia is emphasised. Fo... more The dominance of viscous forces in the generation of propulsive thrust by cilia is emphasised. Fourier analysis indicates that ciliary bends consist of circular arcs joined by linear segments; this arc-line shape appears to be a property associated with the molecular mechanism responsible for bending the cilium and is unchanged by variations in the external viscous loading on the organelle. The flexibility of a computer-generated model of axonemal structure is demonstrated by the incorporation of recent data concerning the surface lattice of the microtubules. Computer simulations using the model show that predictions based on stochastic, rather than co-ordinated, dynein arm activity provide a qualitative match to experimental observations of microtubules gliding over fields of dynein molecules.
The construction and assessment of a three-dimensional computer-generated model of inner dynein a... more The construction and assessment of a three-dimensional computer-generated model of inner dynein arms on a 96-nm repeat unit of an axonemal doublet is described. The model is based on published electron micrographs of axonemes from Tetrahymena cilia and eel sperm, which were prepared using several different techniques: negative stain, freeze etch, and thin section. The inner arm structure is represented as three inner dynein arm complexes containing four inner dynein arms (IDAs), three dyads, and one single-headed arm, each capable of bridging the interdoublet gap. The IDA structures in the model have been correlated with the domains containing dynein heavy-chain isoforms mapped by several authors using genetic analyses of Chlamydomonas mutants. The model is consistent with micrographic evidence from axonemes of cilia and flagella from other organisms that led previously to conflicting structural interpretations. In this reconciling interpretation, the different alignments of the IDAs relative to the corresponding outer dynein arms observed in micrographs of differently prepared samples, result from the IDAs being arrested at different stages of their cycles of activity in each preparation. By interpolating between these positions of arrest, cycles of activity are proposed for each of the IDAs during which the arms attach to the neighbouring doublet microtubule and drive it tipwards.
A physical model developed to explain microtubule sliding patterns in the trypsin-treated ciliary... more A physical model developed to explain microtubule sliding patterns in the trypsin-treated ciliary axoneme has been extended to investigate the generation of bending moments by microtubules sliding in an axoneme in which the doublets are anchored at one end. With sliding restricted, a bending moment is developed by the polarized shearing interaction between neighbouring doublets, effected by the activity of dynein arms on doublet N pushing N + 1 in a tipward (+) direction. In arrested axonemes in which arms on several contiguous doublets are active, the bending moment causes splitting of the 9 + 2 microtubule array into two or more sets of doublets. In the absence of special constraints, splitting depends only on breaking the circumferential interdoublet links most distorted by the bending moment. The analysis, which permits assignment of arm activity to specific microtubules in each of the observed patterns of splitting, indicates that the axoneme will split between doublet N and N + 1 if arms on doublet N are inactive and arms on either N + 1 or N-1 are active. To produce the observed major splits, dynein arms on the microtubules of roughly one-half of the axoneme are predicted to be active, in a manner consistent with the switch-point hypothesis of ciliary motion. Electron microscopic examination indicates that virtually every set of doublets in the split axonemes retains its cylindrical form. Maintenance of cylindrical symmetry can be ascribed to the mechanical properties of the unbroken links, which may resist both tensile and compressive stress, and to active dynein arms.
A dynein arm attachment cycle produces sliding between adjacent doublet microtubules (N and N + l... more A dynein arm attachment cycle produces sliding between adjacent doublet microtubules (N and N + l ) of cilia. In intact axonemes, in the absence of ATP, almost all arms appear attached at both ends (rigor). When ATP is added, most arms detach from doublet N + 1. In ATP and vanadate, the arms do not return to rigor, suggesting that ATP hydrolysis is required for re-extension and reattachment of the dynein arm, but not for detachment. Using solutions containing dynein to decorate dynein-less axonemal doublets, we confirm this interpretation. In the absence of ATP, both sides of each doublet decorate with arms. Addition of ATP, ATP and vanadate or AMP-PNP causes immediate arm detachment, but only in the first instance, where extensive ATP hydrolysis can occur, does decoration eventually reappear. Dynein decorates heterologous axonemal doublets and brain microtubules, as well as homologous doublets, suggesting that this mechanochemical cycle may have general applicability in microtubule-based cell motility.
Early studies of migrating fibroblasts showed that primary cilia orient in front of the nucleus a... more Early studies of migrating fibroblasts showed that primary cilia orient in front of the nucleus and point toward the leading edge. Recent work has shown that primary cilia coordinate a series of signaling pathways critical to fibroblast cell migration during development and in wound healing. In particular, platelet-derived growth factor receptor alpha (PDGFRa) is compartmentalized to the primary cilium to activate signaling pathways that regulate reorganization of the cytoskeleton required for lamellipodium formation and directional migration in the presence of a specific ligand gradient. We summarize selected methods in analyzing ciliary function in directional cell migration, including immunofluorescence microscopy, scratch assay, and chemotaxis assay by Methods in Enzymology, Volume 525 # 2013 Elsevier Inc.
Cell cycle control and migration are critical processes during development and maintenance of tis... more Cell cycle control and migration are critical processes during development and maintenance of tissue functions. Recently, primary cilia were shown to take part in coordination of the signaling pathways that control these cellular processes in human health and disease. In this review, we present an overview of the function of primary cilia and the centrosome in the signaling pathways that regulate cell cycle control and migration with focus on ciliary signaling via platelet-derived growth factor receptor alpha (PDGFRalpha). We also consider how the primary cilium and the centrosome interact with the extracellular matrix, coordinate Wnt signaling, and modulate cytoskeletal changes that impinge on both cell cycle control and cell migration.
Summary Flagellar scales from the green flagellateTetraselmis striata (Prasinophyceae) were isola... more Summary Flagellar scales from the green flagellateTetraselmis striata (Prasinophyceae) were isolated, purified by isopycnic cesium chloride-gradient and zonal sucrose gradient centrifugation and their structure and biochemical composition investigated. Three types of flagellar scales were purified to more than 90% purity, a fourth type up to 75% purity. In addition to the previously known types of flagellar scales (pentagonal scales, rod-shaped
This study confirms and extends previous work on the lateral cilia of the fresh-water mussel, Ell... more This study confirms and extends previous work on the lateral cilia of the fresh-water mussel, Elliptio complanatus, in support of a "sliding filament" mechanism of ciliary motility wherein peripheral filaments (microtubules) do not change length during beat (see ). Short sequences of serial sections of tips are examined in control (nonbeating) and activated (metachronal wave) preparations. Several different tip types, functional rather than morphogenetic variants, are demonstrated, but similarly bent cilia have similar tips. The peripheral filaments are composed of two subfibers: a and b. The bent regions of cilia are in the form of circular arcs, and apparent differences in subfiber-b length at the tip are those predicted solely by geometry of the stroke without the necessity of assuming filament contraction. Various subfibers b apparently move with respect to one another during beat, since small systematic variations in relative position can be detected from cilium to cilium. While subfiber-b lengths are uniform throughout, subfiber-a lengths are morphologically different for each filament: 8 and 3 are about 0.8 A longer than 1, 4 and 5, but each unique length is independent of stroke position or tip type. Subfiber-a does not contract, nor does it move, e.g. slide, with respect to subfiber-b of the same doublet. The central pair of filaments extends to the tip of the cilium where its members fuse. Subunit assembly in ciliary microtubules is evidently precise. This may be of importance in establishing the relationships needed for mechanochemical interactions that produce sliding and beat.
A brief review of important contributions of electron microscopy to the study of ciliary motility... more 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.
In earlier studies, Hamasaki et al. (Proc. Natl. Acad. Sci. USA. 88:7918-7922, 1991) and Barkalow... more In earlier studies, Hamasaki et al. (Proc. Natl. Acad. Sci. USA. 88:7918-7922, 1991) and Barkalow et al. (J. Cell Biol. 126:727-735, 1994) found that cAMP- and Ca2+-sensitive phosphorylation of a 29 kDa dynein light chain (p29) extracted from 22S axonemal dynein of Paramecium, regulates the velocity of in vitro microtubule translocation and ciliate swimming speed. In this study we report evidence of recombination of p29 to cytoplasmic dyneins from both rat liver and Paramecium, as well as to a 22S dynein precursor molecule, based on immunoprecipitation and force filtration data. Immunoprecipitation also provides additional evidence for the binding of p29 to 22S axonemal dynein. The results suggest that p29 might regulate cytoplasmic dynein, as well as axonemal dynein function in Paramecium, and that a homologue of p29 may exist in rat liver and other mammalian cells.
Analysis by confocal microscopy has revealed features of the microtubule network of rat hepatocyt... more Analysis by confocal microscopy has revealed features of the microtubule network of rat hepatocytes in culture, establishing the three-dimensional disposition of the microtubule-based cytoskeleton, its relation to the actin-based cytoskeleton and to ligand-containing endosomes during receptor-mediated endocytosis and the alterations in its structure and disposition by the microtubule pertubant, Taxol. By co-localization studies, we have been able to demonstrate that the microtubules have a significant role in receptor-mediated endocytosis of asialoglycoproteins in this cell. Asialoorosomucoid-containing endosomes attach to widely spaced arrays of microtubules running under the baso-lateral surface of the hepatocytes 5-15 minutes after the initiation of endocytosis and then travel along microtubule paths to become concentrated with microtubules near the centrosome and at bile canaliculi after 30-60 minutes of receptor-mediated endocytosis. Receptor-mediated endocytosis is affected, b...
... New York: Appleton Century Crofts (Meredith Corp.) pp. 194242. Eakin RM, Westfall JA. 1962. ... more ... New York: Appleton Century Crofts (Meredith Corp.) pp. 194242. Eakin RM, Westfall JA. 1962. Fine structure of photoreceptors in amphioxus. ... Get PDF (272K). More content like this. Find more content: like this article. Find more content written by: Peter Satir. ...
cAMP-dependent phosphorylation of a 29-kDa axonemal polypeptide (p29) increases the swimming spee... more 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.
Using potassium pyroantimonate as a Ca2+ chelating agent, we have been able to open the belt desm... more Using potassium pyroantimonate as a Ca2+ chelating agent, we have been able to open the belt desmosome that mechanically couples adjacent ciliated lateral (L) gill epithelial cells of freshwater mussels. Subsequent transfer of the mechanically uncoupled epithelia to solutions containing millimolar Ca2+ results in dramatic alterations in cell shape and partial disruption and apical reorientation of L cell septate junction. The calls remain viable throughout the procedure as determined by the persistence of ciliary activity, although disruption is accompanied by a separation of neighboring groups of cilia and a switch from metachronal to synchronous L cell ciliary beat co-ordination.
Rapid initiation of ion transport occurs in the lower Malpighian tubule of the insect Rhodnius pr... more Rapid initiation of ion transport occurs in the lower Malpighian tubule of the insect Rhodnius prolixus following feeding in vivo or stimulation with 5-hydroxytryptamine (5-HT) in vitro. Using the electron microscope, we have conducted a morphometric analysis of cells in the lowest one-third of the lower tubule, demonstrating that 5-HT also induces mitochondrial movement and microvillar growth simultaneously with, but independent of, the onset of ion transport. Mitochondria move from a position below the cell cortex to one inside the microvilli within 10 min of stimulation with 5-HT, resulting in an 8- to 10-fold increase in the volume of mitochondria within the microvilli. Previous findings indicated that mitochondrial movement is dependent on actin-containing microfilaments, but not microtubules. As the mitochondria enter the microvillus, the core microfilaments are reorganized into a sheath of microfilaments, which extends closely parallel to the outer mitochondrial membrane down...
This study considers the relationship between two structural forms of the 22S dynein arm of Tetra... more This study considers the relationship between two structural forms of the 22S dynein arm of Tetrahymena thermophila: the bouquet and the compact arm. The compact arm differs from the bouquet and from other proposed forms (e.g., the "toadstool") in that the globular domains are situated transversely across the interdoublet gap with one globular subunit, the head, proximal to the adjacent doublet microtubule. The other models place all three globular domains proximal to the neighboring doublet microtubule. When sliding of an isolated axoneme is induced, at least 57% of total attached arms on exposed doublets are in the compact form within dimensions of 24 x 24 x 12 nm, and only about 2% of the arms are bouquets. Toadstools are incompatible with the images seen. Bouquets are not found in regions of the doublet protected by a neighboring doublet. When axonemes with exposed doublets are treated with 0.5 M KCl for 30 min, the compact arms and the dynein heavy (H)-chains disappear, while isolated bouquets and dynein H-chains appear in the medium, suggesting that the compact arms give rise to the bouquets as they are solubilized. The bouquet is the predominant form of isolated 22S dynein molecules, which are found in two apparently enantiomorphic forms, within dimensions 45 x 39 x 13 nm; bouquets attached to doublets have dimensions similar to those of isolated bouquets. Computer modeling indicates that in an intact standard-diameter axoneme, these dimensions are incompatible with the interdoublet volume available for an arm; the bouquet therefore represents an unfolded compact arm. A plausible sequence of changes can be modeled to illustrate the conversion of an attached compact arm to an attached and then free bouquet. The toadstool is probably an artifact that arises after unfolding. Consistent with the conformational difference, H-chains of attached compact arms differ from those of isolated bouquets in their susceptibility to limited proteolysis. These results suggest that the compact arm, rather than the unfolded bouquet or the toadstool, is the functional form of the outer arm in the intact axoneme.
The dominance of viscous forces in the generation of propulsive thrust by cilia is emphasised. Fo... more The dominance of viscous forces in the generation of propulsive thrust by cilia is emphasised. Fourier analysis indicates that ciliary bends consist of circular arcs joined by linear segments; this arc-line shape appears to be a property associated with the molecular mechanism responsible for bending the cilium and is unchanged by variations in the external viscous loading on the organelle. The flexibility of a computer-generated model of axonemal structure is demonstrated by the incorporation of recent data concerning the surface lattice of the microtubules. Computer simulations using the model show that predictions based on stochastic, rather than co-ordinated, dynein arm activity provide a qualitative match to experimental observations of microtubules gliding over fields of dynein molecules.
The construction and assessment of a three-dimensional computer-generated model of inner dynein a... more The construction and assessment of a three-dimensional computer-generated model of inner dynein arms on a 96-nm repeat unit of an axonemal doublet is described. The model is based on published electron micrographs of axonemes from Tetrahymena cilia and eel sperm, which were prepared using several different techniques: negative stain, freeze etch, and thin section. The inner arm structure is represented as three inner dynein arm complexes containing four inner dynein arms (IDAs), three dyads, and one single-headed arm, each capable of bridging the interdoublet gap. The IDA structures in the model have been correlated with the domains containing dynein heavy-chain isoforms mapped by several authors using genetic analyses of Chlamydomonas mutants. The model is consistent with micrographic evidence from axonemes of cilia and flagella from other organisms that led previously to conflicting structural interpretations. In this reconciling interpretation, the different alignments of the IDAs relative to the corresponding outer dynein arms observed in micrographs of differently prepared samples, result from the IDAs being arrested at different stages of their cycles of activity in each preparation. By interpolating between these positions of arrest, cycles of activity are proposed for each of the IDAs during which the arms attach to the neighbouring doublet microtubule and drive it tipwards.
A physical model developed to explain microtubule sliding patterns in the trypsin-treated ciliary... more A physical model developed to explain microtubule sliding patterns in the trypsin-treated ciliary axoneme has been extended to investigate the generation of bending moments by microtubules sliding in an axoneme in which the doublets are anchored at one end. With sliding restricted, a bending moment is developed by the polarized shearing interaction between neighbouring doublets, effected by the activity of dynein arms on doublet N pushing N + 1 in a tipward (+) direction. In arrested axonemes in which arms on several contiguous doublets are active, the bending moment causes splitting of the 9 + 2 microtubule array into two or more sets of doublets. In the absence of special constraints, splitting depends only on breaking the circumferential interdoublet links most distorted by the bending moment. The analysis, which permits assignment of arm activity to specific microtubules in each of the observed patterns of splitting, indicates that the axoneme will split between doublet N and N + 1 if arms on doublet N are inactive and arms on either N + 1 or N-1 are active. To produce the observed major splits, dynein arms on the microtubules of roughly one-half of the axoneme are predicted to be active, in a manner consistent with the switch-point hypothesis of ciliary motion. Electron microscopic examination indicates that virtually every set of doublets in the split axonemes retains its cylindrical form. Maintenance of cylindrical symmetry can be ascribed to the mechanical properties of the unbroken links, which may resist both tensile and compressive stress, and to active dynein arms.
A dynein arm attachment cycle produces sliding between adjacent doublet microtubules (N and N + l... more A dynein arm attachment cycle produces sliding between adjacent doublet microtubules (N and N + l ) of cilia. In intact axonemes, in the absence of ATP, almost all arms appear attached at both ends (rigor). When ATP is added, most arms detach from doublet N + 1. In ATP and vanadate, the arms do not return to rigor, suggesting that ATP hydrolysis is required for re-extension and reattachment of the dynein arm, but not for detachment. Using solutions containing dynein to decorate dynein-less axonemal doublets, we confirm this interpretation. In the absence of ATP, both sides of each doublet decorate with arms. Addition of ATP, ATP and vanadate or AMP-PNP causes immediate arm detachment, but only in the first instance, where extensive ATP hydrolysis can occur, does decoration eventually reappear. Dynein decorates heterologous axonemal doublets and brain microtubules, as well as homologous doublets, suggesting that this mechanochemical cycle may have general applicability in microtubule-based cell motility.
Early studies of migrating fibroblasts showed that primary cilia orient in front of the nucleus a... more Early studies of migrating fibroblasts showed that primary cilia orient in front of the nucleus and point toward the leading edge. Recent work has shown that primary cilia coordinate a series of signaling pathways critical to fibroblast cell migration during development and in wound healing. In particular, platelet-derived growth factor receptor alpha (PDGFRa) is compartmentalized to the primary cilium to activate signaling pathways that regulate reorganization of the cytoskeleton required for lamellipodium formation and directional migration in the presence of a specific ligand gradient. We summarize selected methods in analyzing ciliary function in directional cell migration, including immunofluorescence microscopy, scratch assay, and chemotaxis assay by Methods in Enzymology, Volume 525 # 2013 Elsevier Inc.
Cell cycle control and migration are critical processes during development and maintenance of tis... more Cell cycle control and migration are critical processes during development and maintenance of tissue functions. Recently, primary cilia were shown to take part in coordination of the signaling pathways that control these cellular processes in human health and disease. In this review, we present an overview of the function of primary cilia and the centrosome in the signaling pathways that regulate cell cycle control and migration with focus on ciliary signaling via platelet-derived growth factor receptor alpha (PDGFRalpha). We also consider how the primary cilium and the centrosome interact with the extracellular matrix, coordinate Wnt signaling, and modulate cytoskeletal changes that impinge on both cell cycle control and cell migration.
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