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Evidence for Four Classes of Microtubules in Individual Cells

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Evidence for Four Classes of Microtubules in Individual Cells J. Cell Sci. 2, 169-192 (1967) 169 Printed in Great Britain EVIDENCE FOR FOUR CLASSES OF MICROTUBULES IN INDIVIDUAL CELLS O.BEHNKE Department of Anatomy, Royal Dental College, Universitetsparken 4, Copenhagen 0, Denmark AND A. FORER# Carlsberg Foundation, Biological Institute, Tagensvej 16, Copenhagen N, Denmark SUMMARY Experiments were performed on crane-fly spermatids (Nephrotoma suturalis Loew), rat- sperm, and rat tracheal cilia to test whether all microtubules respond in the same way to different treatments. Crane-fly spermatids contain cytoplasmic microtubules, accessory tubules, and the 9 + 2 complex of tubules; rat sperm and rat tracheal cilia contain only the 9 + 2 tubules. Crane-fly spermatid tubules responded to the experimental treatments as follows. After colchicine treatment, or storage at o°C, the cytoplasmic microtubules disappeared, while the 9 + 2 tubules were normal. After storage at 50 CC the cytoplasmic microtubules disappeared, and then the 9 + 2 tubules were affected: first the central tubules and B-tubules were affected, and later the A-tubules. After brief pepsin treatment, the 9 doublet tubules disappeared, while the other tubules appeared normal; after prolonged pepsin treatment the accessory, central, and cytoplasmic tubules disappeared. After negative staining at pH 7, the cytoplasmic microtubules were never seen, the central tubules were only sometimes seen, the B-tubules were sometimes fragmented, and the A-tubules were intact. On the basis of these responses, it was concluded that there are 4 classes of tubules in crane-fly spermatids, namely cytoplasmic microtubules; accessory tubules and central tubules (of the 9 + 2 complex); B-tubules (of the 9 + 2 complex); and A-tubules (of the 9 + 2 complex). At least some of the different responses appeared to be due to intrinsic physical and/or chemical differences between the tubules themselves. Pepsin digestion and negative staining of rat sperm tails gave results similar to those with crane-fly spermatids. In addition, the 9 + 2 tubules responded differently to pepsin digestion at different points along their length. This gradient of sensitivity was attributed to synthesis of new tubules occurring at one end of the sperm tail. Pepsin digestion and negative staining of rat tracheal cilia gave results similar to those with crane-fly spermatids and rat sperm tails. All the tubules had a similar substructure, as revealed by negative-staining techniques. It was concluded that microtubules are proteinaceous, at least in part, and that microtubules are different in composition from membranes. It is suggested that the walls of the B-tubules are composed of two materials—(1) the por- tions adjacent to the A-tubules, and (2) the remaining portion. INTRODUCTION The term ' microtubule' is currently used to designate cylindrical cellular structures with electron-dense walls and less dense cores, and with an outer diameter ranging from about 180 to 300 A (Slautterback, 1963; Ledbetter & Porter, 1963). Micro- tubules have been found in the cytoplasm of almost all cell types studied (see Slautter- • Present address: Department of Zoology, Downing Street, Cambridge 170 O. Behnke and A. Forer back, 1963; Pitelka, 1963; Byers & Porter, 1964; Silveira & Porter, 1964; Behnke & Forer, 1966a). They are universally associated with spermatid tail9, 9perm tails, cilia, flagella, and mitotic spindles. Microtubules are generally straight and can often be followed for distances of microns, though sometimes they are evenly curved (Fawcett & Witebsky, 1964; Behnke, 19656; Haydon & Taylor, 1965; Hoffman, 1966) or wavy (Vivier & Andre", 1961; Barnicot & Huxley, 1965; Carasso & Favard, 1965; Newcomb & Bonnett, 1965; Vivier, 1965; Behnke & Forer, 19666). They can sometimes be identified by the patterns in which they are arranged, or the organelles with which they are associated. For example, microtubules arranged in a consistent '9 + 2' pattern are regularly found in cilia and flagella, and in axial filaments of spermatids and sperm (Manton & Clarke, 1952; Fawcett & Porter, 1954; Afzelius, 1959; Gibbons & Grimstone, i960; Fawcett, 1961). Other consistent patterns of microtubules are found in other cells (see, for example, Tilney & Porter, 1965; Batisse, 1965; Lumsden, 1965; Grimstone & Gibbons, 1966; Nilsson & Williams, 1966). Some microtubules are not arranged in such a precise way, but can nonetheless be identified because they are located in specific regions of the cell, such as near nuclei (Burgos & Fawcett, 1955), in axostyles (Grimstone & Cleveland, 1965), in spindles (Harris, 1962; Kane, 1962; Roth & Daniels, 1962; Dales, 1963; Ledbetter & Porter, 1963), etc. For lack of other distinguishing characteristics, microtubules which are not arranged in any specific pattern and are not located in any specific region are designated ' cytoplasmic micro- tubules'. The function of microtubules is not known. From studies of sectioned material it has been suggested that microtubules are involved in cell motility, or are involved in cytoplasmic streaming, or provide skeletal support, or transport water and small ions. There is, however, no conclusive evidence to support these suggestions, nor is it known if all microtubules are equivalent, or have the same function. Some workers (for example, Slautterback, 1963; H. Moor, unpublished observations) have tried to classify microtubules by size, but because of technical and biological variations this idea has not been generally accepted. Some have suggested a classification by fixation properties (Behnke & Zelander, 1966; Sheffield, 1966), to account for the observation that some microtubules are well preserved with osmium tetroxide fixation while others require special fixation, such as glutaraldehyde, or low pH of the osmium fixative together with addition of cations (Harris, 1962; Roth & Daniels, 1962). In general, however, it is thought that all microtubules are the same. Because of their morphological similarities, various authors have suggested that tubules in cilia and flagella may be the same as cytoplasmic microtubules (Ledbetter & Porter, 1963; Bawa, 1964; Byers & Porter, 1964; Porter, Ledbetter & Badenhausen, 1964; Silveira & Porter, 1964; Anderson, Weissmann & Ellis, 1966; Robison, 1966) or the same as spindle microtubules (Ledbetter & Porter, 1963; Pease, 1963; Harris & Bajer, 1965; Krishan & Buck, 1965; Kiefer, Sakai, Solari & Mazia, 1966), and also that spindle microtubules are the same as cytoplasmic microtubules (Ledbetter & Porter, 1963; de-The", 1964; Anderson et al. 1966). We report here experiments designed to show whether or not all microtubules are Classes of microtubules 171 the same. We conclude that they are not, because various experimental treatments produce different reactions with different microtubules. The results show: there are at least 4 classes of microtubules; within at least 3 of the classes the microtubules differ along their length; microtubules have a similar substructure, as revealed by negative staining techniques; and microtubules are proteinaceous, at least in large part, and their walls are different in composition from cell membranes. Further data suggest that different microtubules contain different material in their less-dense cores, and that the walls of some individual tubules have two components. MATERIALS AND METHODS Crane-fly testes Crane flies (Nephrotoma suturalis Loew) were reared in the laboratory. Last-instar larvae were dissected at a time when their testes contained meiotic cells and young spermatids, or contained spermatids 1-4 days after the second meiotic division (see Behnke & Forer (19666) or Forer (1964, 1965) for details of the animals and spermatocytes). Larvae were dissected at room temperature under Kel-F 10 oil (Minnesota Mining and Mfg Co.), which prevents dehydration of the testes during dissection. Individual testes (and a surrounding film of oil) were either placed directly into fixative, at room temperature, and processed for electron microscopy, or transferred to an experimental solution. In both cases, the testes sank under the surface of the solution as the surrounding film of oil floated on top. Fixation. Except for experiments in which different fixation techniques were studied, all testes were fixed in glutaraldehyde and processed as follows. Testes were fixed by immersion in 2-4 % glutaraldehyde solution ino-iM cacodylate buffer, at pH between pH 6-5 and 7-4. They were post-fixed for 1 h in 1 % osmium tetroxide solution (in o-i M cacodylate or veronal-acetate buffer, at pH 7), and then dehydrated through a series of alcohols and embedded in Epon (Luft, 1961). Thin sections were double stained, with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963), and examined in a Siemens Elmiskop I. Some testes were fixed at room temperature in 1 % osmium tetroxide solution, which was prepared either in o-i M cacodylate buffer at pH 7, with CaCl, added to a final concentration of Ca1+ of 0-002 M, or in o-i M cacodylate buffer at pH 6-5 without Ca1+ added, or in veronal- acetate buffer, at pH 7^4, without Ca1+. Experimental treatments of testes. Some testes were exposed to different temperatures. These were placed in insect Ringer solution (Ephrussi & Beadle, 1936) which was previously equili- brated at the experimental temperature, 0°, 40°, or 50° C. At various times (10-30 min) an equal volume of 4 % glutaraldehyde solution at room temperature was added, and the fixation continued for 2 h at room temperature. The testes were then processed as described above,
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