Axonemes

FIGURE 17.5 The structure of an axoneme. Note the manner in which two microtubules are joined in the nine outer pairs. The smaller-diameter tubule of each pair, which is a true cylinder, is called the A-tubule and isjoined to the center sheath of the axoneme by a spoke structure. Each outer pair of tubules isjoined to adjacent pairs by a nexin bridge. The A-tubule of each outer pair possesses an outer dynein arm and an inner dynein arm. The larger-diameter tubule is known as the B-tubule. 

The cytosolic dyneins bear many similarities to axonemal dynein. The protein isolated from C. elegans includes a heavy chain with a molecular mass of approximately 400 kD, as well as smaller peptides with molecular mass ranging from 53 kD to 74 kD. The protein possesses a microtubule-activated ATPase... 

FIGURE 17.7 A mechanism for ciliary motion. The sliding motion of dyneins along one microtnbnle while attached to an adjacent microtnbnle resnlts in a bending motion of the axoneme. 

Some specialized eukaryotic cells have cilia that show a whiplike motion. Sperm cells move with one flagella, which is much longer than a cilium but has a nearly identical internal structure called axoneme. It is composed of nine doublet MTs that form a ring around a pair of single MTs. Numerous proteins bind to the MTs. Ciliary dynein motors generate the force by which MTs slide along each other to cause the bending of the axoneme necessary for motion. 

Axoneme Movement Is Produced by a Sliding Microtubule Mechanism... 

Parallel arrays of microtubules are found in the axoneme of cilia and flagella of eukaryotic cells, and these are of constant pattern throughout the phylogenetic scale. 

The axoneme consists of a cylinder of nine outer doublets of fused microtubules and a pair of discrete central microtubules (commonly referred to as the 9 + 2 arrangement of microtubules). The outer doublets each consist of a complete A-microtubule and an incomplete B-microtubule, the deficiency in the wall of the latter being made up by a sharing of wall material with the former. The tip of the axoneme contains the plus ends of all of the constituent microtubules. Two curved sidearms, composed of the MAP protein dynein, are attached at regular intervals to the A-microtubules of each fused outer doublet (Figures 1 and 2). 

Figure 2. Electron micrograph of cross section of flagellum of mouse sperm, taken near the tip. The axoneme contains nine outer pairs of doublet microtubules and two central singlet microtubules. Several dynein arms and the fibrous sheath of the sperm are also shown.

Even though dynein, kinesin, and myosin serve similar ATPase-dependent chemomechanical functions and have structural similarities, they do not appear to be related to each other in molecular terms. Their similarity lies in the overall shape of the molecule, which is composed of a pair of globular heads that bind microtubules and a fan-shaped tail piece (not present in myosin) that is suspected to carry the attachment site for membranous vesicles and other cytoplasmic components transported by MT. The cytoplasmic and axonemal dyneins are similar in structure (Hirokawa et al., 1989 Holzbaur and Vallee, 1994). Current studies on mutant phenotypes are likely to lead to a better understanding of the cellular roles of molecular motor proteins and their mechanisms of action (Endow and Titus, 1992). 

Asai, D.J. Brokaw, C.J. (1993). Dynein heavy chain isoforms and axonemal motility. Trends Cell Biol. 3. 398-402. 

The most important molecules so far identified from this screen include a likely transduction channel, an extracellular molecule that could gate channels, and several molecules known to be important for axonemal structure and function. Although the set of molecules is less complete than that identified for C. elegans touch receptors, the diversity of mechanotransduction in Drosophila and the apparent similarity of these receptors to those in vertebrates, including hair cells (see below), demonstrates the significance of this model system. 

Hair cells are neuroepithelial cells their large basolat-eral surface includes synaptic contacts with afferent and efferent nerve fibers, while the mechanically sensitive hair bundle is located on their apical surface. The hair bundle is an ensemble of 30-300 actin-filled stereocilia and a single axonemal kinocilium (Fig. 51-2). The kinocilium,... 

Fig. 4. Lattice structure of an A-type" singlet microtubule from ciliary axonemes. [Redrawn from Amos (1979). In Microtubules (J. Hyams, ed.), pp. 2-64. Academic Press, New York.]...

The above theory allows one to estimate the off-rate constant for biopolymer disassembly, but it does not take into account the possibility of having distinctly different rate constants at each end of the polymer. Thus, one obtains the sum of the off-rate constants rather than the exact constants for each end. Another limitation is that one does not know how many growing points there are at each microtubule end, and from the discussion of the multiplicity of helical starts (Amos et al., 1976), the observed off-constant should represent the product of n (the number of growing points) and the actual microscopic rate constant for each of these growing points. As will be seen below, axoneme-promoted assembly of microtubules (Bergen and Borisy, 1980) may help to obviate the former limitation, but the latter requires further characterization of the true growing points. 

The basic approach with the axoneme-based analysis is to combine tubulin and axonema fractions and then to quench the reaction with glutaraldehyde. Samples of sufficient axoneme concentration are then added directly onto Formvar-coated sample grids for staining and electron microscopy. In some cases where the axoneme count is too low, samples may be sedimented onto grids by the method of Gould and Borisy (1977). With the methodology perfected by Borisy and Bergen (1982) samples can be taken as frequently as every 20 seconds, and the... 

Summary of the Kinetic and Equilibrium Constants Characterizing Microtubule Elongation from Chlamydomonas Flagellar Axonemes"... 

Bergen and Borisy (1980) used the axoneme-hased approach to explore the commitment to treadmilling, and they also found that the efficiency was quite low. An 5 value of 0.07 0.04 was obtained, corresponding to the net addition of 1.6 0.8 tubulin protomers/second. Interestingly, their estimates of the dissociation constants for the two ends were 2.2 0.6 and 3.2 0.6 iiM for the assembly and disassembly ends, respectively. We calculate that this corresponds to only about 0.2 kcal/mol. In a more recent investigation from the same laboratory (Cote and Borisy, 1981), porcine tubulin was found to exhibit an s value of about 0.26, and the rate of the tubulin flux was about 28 protomers/ second. The authors of the latter study suggested that the discrepancy might be accounted for in terms of the need to use MAP-depleted tubulin with the axoneme system to prevent self-nucleation, or in terms of the temperature differences in the two studies. 

The threshold concentration of monomer that must be exceeded for any observable polymer formation in a self-assembling system. In the context of Oosawa s condensation-equilibrium model for protein polymerization, the cooperativity of nucleation and the intrinsic thermodynamic instability of nuclei contribute to the sudden onset of polymer formation as the monomer concentration reaches and exceeds the critical concentration. Condensation-equilibrium processes that exhibit critical concentration behavior in vitro include F-actin formation from G-actin, microtubule self-assembly from tubulin, and fibril formation from amyloid P protein. Critical concentration behavior will also occur in indefinite isodesmic polymerization reactions that involve a stable template. One example is the elongation of microtubules from centrosomes, basal bodies, or axonemes. 

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