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Chapter 11. Tubulin and microtubule-mediated processes11.1. Discovery of microtubules in cilia and flagella and the mechanism of movementImagine the excitement Antony van Leeuwenhoek (1677,1678) felt when he first looked at a drop of water through the microscope he made with his own hands, and saw a whole new world of little playful swimming creatures. Leeuwenhoek (1677) saw that “when these animalcula or living atoms did move, they put forth two little horns, continually moving themselves” and he noticed that others were “furnished with diverse incredibly thin feet, which moved very nimbly.” Two hundred years later, with the advantage of better microscopes, cytologists could see that the flagella and cilia that powered the little protozoa were composed of fibers (Figure 11-1; Ballowitz, 1888), and Prénant (1913) suggested that these little fibers were contractile. The fibrous nature of the filaments were confirmed with electron microscopy (Figure 11-2;Jakus and Hall, 1946; Grigg and Hodge, 1949). With the resolution attainable at the time, the filaments seemed similar to those found in muscle (Hall et al., 1946; Draper and Hodge, 1949). However, the introduction of the ultramicrotome allowed Fawcett and Porter (1954) to section cilia transversely, and thin enough to reveal a structure different from that of muscle. A structure that has come to be known as the 9 + 2 arrangement of tubules with which we are familiar today (Figure 11-3). I will use the terms cilia and flagella interchangeably to describe the whip-like structures of eukaryotic cells. At one time, Shmagina suggested that the appendages be called undulipodia, but that term never caught on (Margulis, 1980; Corliss, 1980). Others have suggested that flagella be used to describe the whip-like appendages of prokaryotes, and cilia be used to describe those of eukaryotes. This suggestion, which also did not catch on, was based on the facts that prokaryotic and eukaryotic appendages are composed of different proteins, and have different structures. Thus in eukaryotes, we are stuck with two terms for organelles with identical internal structure and composition. Many people consider flagella to be longer than cilia, more sparsely arranged on a cell, and to have a symmetrical beating motion, compared with the asymmetrical beat of cilia. However, as I will discuss below, the flagellar and ciliary beat can occur at different times in the same structure. This, combined 375with the fact that there are intermediates in all the characteristics (Figure 11- 4), and that there is no universally accepted distinction, I will use the terms interchangeably.While ciliary motion is widespead in unicellular plants, fungi and animals, it also occurs in multicellular organisms, although it is restricted to specializedcells (Gray, 1928; Sleigh, 1962,1974). In the plant kingdom, the sperm of many embryophytic plants, including Ginkgo, the cycads, ferns, fern allies and mosses are ciliated (Manton, 1950,1959; Hepler, 1976; Wolniak and Cande, 1980; Paolillo, 1981; Li et al., 1989). In animals, sperm are powered by flagella, and cilia line the respiratory tract where they sweep mucus, dead cells and dust up toward the mouth; and the oviduct, where they move the eggcells along to the uterus. The structure of cilia and flagella in all the above examples are extremely similar, which led Peter Satir (1961) to state, “cellularstructure, down to its minute details, remains constant as long as function is constant.” The exceptions often prove the rule and some cilia, including the rods and cones in our retina, as well as our olfactory and auditory cells have highly modified structures, and consequently have lost their motile abilities (Porter, 1957). Cilia, like muscle, require ATP for movement, as was shown by Hartmut Hoffmann-Berling (1955) using glycerinated cell models. Isolated cilia containeverything they need to beat except Mg2+-ATP. When isolated cilia are given Mg2+-ATP they beat by themselves exactly as if they were intact and still attached to a cell. In order to understand how cilia transduce the chemical energy of ATP into the mechanical energy that causes the cilia to beat, Tibbs (1957) and Child (1959) took a biochemical approach and discovered that a protein isolated from the cilia of algae and protozoa has ATPase activity.Given the success of the structural approach in elucidating the mechanism of muscle contraction, Gibbons and Grimstone (1960) and Satir (1961) used electron microscopy to understand ciliary motion. Cilia are structurally complex membrane-enclosed organelles approximately 0.2 m in diameter and 10 m long. Cilia can be as short as 5 m and as long as 150 m. The internal structure is known as the axoneme, and is mainly composed of nine doublets of tubules that surround a central pair. The central pair is composed of two complete tubules, while each doublet is composed of one complete and one partial tubule called the A tubule and the B tubule, respectively (Pease, 1963; Andre and Thiery, 1963). Each tubule in the axoneme is 376approximately 24 nm in diameter and as long as the cilium. The cilia are asymmetric in every way, and thus the individuality of each doublet can be unambiguously recognized. With the introduction of better fixation procedures, new structures appeared in the electron micrographs that hinted at how the cilia and flagellar may produce force in order to generate movement. For example Afzelius (1959) discovered radial spokes that extended from the A tubule toward a central sheath. He also found arms along the length of the A tubule that form cross bridges with the adjacent B tubule. Afzelius suggested that these arms, like the heads of myosin, may generate force by inducing sliding between adjacent doublets. Gibbons and Grimstone (1960) suggested that the current microscopic data could not distinguish between the possibilities that the bending movement is due to a localized shortening of longitudinal contractile elements, or to sliding of the tubules in a manner similar to that described by the sliding filament model of muscle contraction. Peter Satir (1965) observed that there was no change in the tubule length during ciliary motion, as would be predicted if the tubules were contractile proteins. He also noticed that some tubules, which were now universally called “microtubules”, extended further than others at the tip of the cilia. The time of their extension correlated


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CORNELL BIOPL 4440 - Study Notes

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