Axonemal Components Research Articles

Calcium control of waveform in isolated flagellar axonemes of Chlamydomonas.

The effect of Ca(++) on the waveform of reactivated, isolated axonemes of chlamydomonas flagella was investigated. Flagella were detached and isolated by the dibucaine procedure and demembranated by treatment with the detergent Nonidet; the resulting axomenes lack the flagellar membrane and basal bodies. In Ca(++)-buffered reactivation solutions containing 10(-6) M or less free Ca(++), the axonemes beat with a highly asymmetrical, predominantly planar waveform that closely resembled that of in situ flagella of forward swimming cells. In solutions containing 10(-4) M Ca(++), the axonemes beat with a symmetrical waveform that was very similar to that of in situ flagella during backward swimming. In 10(-5) M Ca(++), the axonemes were predominantly quiescent, a state that appears to be closely associated with changes in axomenal waveform or direction of beat in many organisms. Experiments in which the concentrations of free Ca(++), not CaATP(--) complex were independently varied suggested that free Ca(++), not CaATP(--), was responsible for the observed changes. Analysis of the flagellar ATPases associated with the isolated axonemes and the nonidet- soluble membrane-matrix fraction obtained during preparation of the axonemes showed that the axonemes lacked the 3.0S Ca(++)-activated ATPase, almost all of which was recovered in the membrane-matrix fraction. These results indicate that free Ca(++) binds directly to an axonemal component to alter flagellar waveform, and that neither the 3.0S CaATPase nor the basal bodies are directly involved in this change.

High-voltage electron microscopy reveals new components in the axonemes and centroplast of the centrohelidian Raphidiophrys amibigua.

A high-voltage electron microscope was used to study the structure of the centroplast and axonemes in the centrohelidian Heliozoan Raphidiophrys ambigua. In Raphidiophrys the microtubules (Mts) of each axoneme are arranged so that they describe the apices of an array of hexagonally shaped prisms and all axonemes originate from a centroplast located near the centre of the cell. The centroplast was found to consist of 3 components: (1) a tripartite central disk, (2) a sphere of electron-opaque material, approximately 0.75 micron in diameter, which immediately surrounds the central disk, and (3) a slightly less electron-opaque centroplast shell, approximately 2-3 micron in diameter which consists primarily of hitherto undescribed components associated with the proximal 1-2 micron of each axonemal base. The new axonemal components described in this study include: (1) a 20-nm rod positioned in the centre of each hexagonal prism of Mts comprising the axonemal pattern, and (2) an electron-opaque linkage material which connects each Mt of the axonemal pattern to its 4 nearest neighbours. Both of these structures are associated with the base of each axoneme within the centroplast shell, both form crossbridges with axonemal Mts, and both terminate at the periphery of the centroplast shell once the complete axonemal Mt pattern is established. It is suggested that these structures are involved in forming and maintaining the Mt pattern seen in the axopodia of Raphidiophrys.

Observations of the structural components of flagellar axonemes and central pair microtubules from rat sperm

The structure and spatial disposition of the matrix elements of the rat spermatozoan flagellar axoneme were investigated by thin sectioning, negative staining, and image analysis techniques. In order to visualize axonemal components by negative staining, spermatozoa were demembranated with Triton X-100 and the flagellum was either frayed by mechanical agitation or actively disintegrated by adding 1 m M ATP to the pH 9.0 extraction solution. In the dissociated flagella each axonemal doublet microtubule remained adjoined to its associated outer dense fiber, and the central pair remained as an intact complex. Flagella extracted in the presence of ATP displayed a specific pattern of axonemal disintegration: doublet tubules—dense fibers numbers 4, 5, 6, and 7 were extruded in a proximal direction from the fibrous sheath, while numbers 8, 9, 1, 2, and 3 remained associated as a half cylinder together with the central pair in the fibrous sheath. Of the extruded fibers, doublet tubules numbers 5 and 6 always remained adjoined to each other suggesting the possibility of a structural linkage similar to that found in typical 9 + 2 flagella. The radial spokes are arranged along the doublet tubule in sets of three; the spoke sets show a center to center spacing of 96 nm. Within a set, two spokes (nos. 1 and 2) are spaced 32 nm center to center, while spokes 2 and 3 are spaced 24 nm center to center. The polarity of the spoke triplets is such that the 32-nm spoke space is directed toward the anterior of the flagellum. An additional axonemal structure associated with the doublet tubule is located between spokes 1 and 2; this globular component lies close to the tubule surface and successive globules are spaced 96 nm center to center. Fine filamentous linkages extending between adjacent outer dense fiber—doublet tubule complexes were found to approach and join the doublet tubule at the level of the globule or spoke number 1; successive filamentous linkages are spaced at 96-nm intervals. The relationship of these components to the circumferential nexins is discussed. In negatively stained preparations, central pair microtubule complexes were frequently found which appeared to retain periodic “sheath” components. Optical transforms of negatively stained central pairs show a regular set of layer lines indexing on a fundamental 32-nm repeat. By masking selected areas of the specimen it is shown that the 32-nm reflection arises from only one member of the central pair and that in negatively stained images and optically filtered reconstructions the 32-nm periodicity arises from a set of “barb-shaped” structures which originate from the lateral edge of one central pair microtubule. The “barb-shaped” projections are pointed toward the proximal end of the flagellum and are attached along the tubule nearly in register with alternating “bridges” and “projections,” both of which are spaced axially at 16 nm along the central pair complex. Filtering of images where the central pair retains its structural integrity with a doublet microtubule provides evidence of structural interactions between the 32-nm periodic sheath components and the radial spokes. The implications of these structural findings to flagellar motility are discussed.

Two-dimensional analysis of flagellar proteins from wild-type and paralyzed mutants of Chlamydomonas reinhardtii.

Flagellar polypeptides of Chlamydomonas reinhardtii were analyzed in two-dimensions by isoelectric focusing and electrophoresis in the presence of sodium dodecyl sulfate. In addition to flagellar tubulin, over 130 polypeptides were resolved and 100 of these were identified as axonemal components in wild-type organisms. Flagella of two nonconditional paralyzed mutants, pf 14 and pf 1, were also analyzed and, at the same time, electron microscopic studies were carried out. pf 14 flagella, which completely lack radial spokes and associated spokeheads, are missing 12 polypeptides. Six of these polypeptides are also missing from pf 1 flagella in which spokes are clearly present but spoke heads appear to be absent.

A functional flagella with a 6 + 0 pattern

The male gamete of the Gregarine Lecudina tuzetae has been studied with transmission electron microscopy and microcinematography. It is characterized by a flagellar axoneme of 6 + 0 pattern, a reduction of the chondriome, and the abundance of storage polysaccharide or lipid bodies. The movements of the flagella are of the undulating type and they are performed in the three dimensions of space. They are very slow, with a cycle time of about 2s. The structure of the axoneme components are similar to those of flagella with a 9 + 2 pattern. Each doublet has overall dimensions of 350 x 220 A; the space between the adjacent doublets is about 160 A. The A subfiber bears arms like dynein arms. The diameter of the axoneme is about 1,000 A. The basal body consists of a cylinder of dense material 2,500 A long and 1,300-1,400 A in diameter; a microtubule 200 A in diameter is present in the axis. This study shows that a 6 + 0 pattern can generate a flagellar movement. The mechanism of the flagellar movement of the male gamete of L. tuzetae does not require the presence of central microtubules and it would include molecular interactions of the dynein-tubulin type between the adjacent peripheric doublets. The slowness of the movements is discussed in terms of the axoneme's structure and its energy supply. Finally, the phylogenetic significance of this flagella is examined on the basis of the morphopoietic potentialities of the centriolar structures.

Further observations on the ultrastructure of cilia from Tetrahymen a pyriformis

Optical transforms of negatively stained central and outer-doublet microtubules from the cilia of Tetrahymena pyriformis show characteristic differences which may be attributed to the axial periodicities of the subsidiary axonemal components. A fundamental periodicity of 32 nm is observed in the central pair and this is not present in the outer doublets which show a fundamental repeat of 48 nm. The basic axial spacing of both the arms and radial spokes is 24 nm. The spokes are attached to each doublet microtubule in longitudinal groups of three, the distance between groups being 48 nm giving a total repeat of 96 nm; each spoke is T-shaped. A model for the arrangement of arm attachment sites is given in which one row is staggered longitudinally, with respect to the other, by 12 nm, and a possible geometrical linkage (generating a 32 nm periodicity at the centre) between the outer and inner parts of the axoneme is also described.

Chlamydomonas flagella. I. Isolation and electrophoretic analysis of microtubules, matrix, membranes, and mastigonemes.

Methods were developed for the isolation of Chlamydomonas flagella and for their fractionation into membrane, mastigoneme, "matrix," and axoneme components. Each component was studied by electron microscopy and acrylamide gel electrophoresis. Purified membranes retained their tripartite ultrastructure and were shown to contain one high molecular weight protein band on electrophoresis in sodium dodecyl sulfate (SDS)-urea gels. Isolated mastigonemes (hairlike structures which extend laterally from the flagellar membrane in situ) were of uniform size and were constructed of ellipsoidal subunits joined end to end. Electrophoretic analysis of mastigonemes indicated that they contained a single glycoprotein of approximately 170,000 daltons The matrix fraction contained a number of proteins (particularly those of the amorphous material surrounding the microtubules), which became solubilized during membrane removal. Isolated axonemes retained the intact "9 + 2" microtubular structure and could be subfractionated by treatment with heat or detergent. Increasing concentrations of detergent solubilized axonemal microtubules in the following order: one of the two central tubules; the remaining central tubule and the outer wall of the B tubule; the remaining portions of the B tubule; the outer wall of the A tubule; the remainder of the A tubule with the exception of a ribbon of three protofilaments. These three protofilaments appeared to be the "partition" between the lumen of the A and B tubule. Electrophoretic analysis of isolated outer doublets of 9 + 2 flagella of wild-type cells and of "9 + 0" flagella of paralyzed mutants indicated that the outer doublets and central tubules were composed of two microtubule proteins (tubulins 1 and 2) Tubulins 1 and 2 were shown to have apparent molecular weights of 56,000 and 53,000 respectively

AN EFFECT OF ADENOSINE TRIPHOSPHATE ON THE LIGHT SCATTERED BY SUSPENSIONS OF CILIA

Cilia were isolated from Tetrahymena pyriformis by an ethanol-calcium method. Addition of adenosine triphosphate to a suspension of intact or digitonin-extracted cilia caused a decrease of about 20 per cent in turbidity. Study of fractionated cilia showed that the presence of two distinct axonemal components, the outer fibers and the 30S dynein (the axonemal ATPase protein), was necessary for this effect on turbidity to occur. The decrease in turbidity is interpreted as a result of a specific interaction of ATP with these protein components causing an effective increase in hydration. The high nucleotide specificity suggests that the change in hydration is closely related to the processes responsible for motility. The outer fibers themselves swell when suspended in media of very low ionic strength. The concentration of salt needed to prevent this swelling (2 mM MgSO(4) or 30 mM KCl) is about the same as that needed to keep dynein bound to the fibers. The recombination of purified 30S dynein with the outer fibers can be followed by the rise in turbidity resulting from increased dry mass of the particles.