Directionality and Rate of Assembly of Chick Brain Tubulin Onto Pieces of Neurotubules, Flagellar Axonemes, and Basal Bodies

Abstract

INTRODUCTIONFlagellar elongation in the flagellated protozoans Chlamydomonas and Ochromonas occurs primarily by the addition of material to the distal ends of the flagella (tip growth).', The original studies of this process were carried out by pulsing cells with radioactive amino acids during flagellar regeneration, and by using light microscope autoradiography to determine the localization of the radioactivity along the length of the flagella.l, 2 Since the autoradiography was carried out on intact flagella, it was not possible with this technique to distinguish the radioactivity in the flagellar microtubules from the radioactivity in the flagellar membranes. Witman, however, using electron microscope autoradiography in combination with techniques of flagellar fractionation, has now shown that the microtubules of the flagella are being assembled primarily by distal addition during flagellar regeneration in Chlavnydom~nas.~ Recently, with the development of methods for the assembly of microtubules in vitro,4 it has become possible to investigate the directionality of microtubule assembly in vitro.5 In this report studies are described on the directionality and rates of assembly of chick brain tubulin onto pieces of brain microtubules (neurotubules), flagellar axonemes, and basal bodies. The results show that the assembly in vitro of microtubules onto flagellar axonemes, basal bodies, or pieces of neurotubules, occurs primarily by unidirectional addition of tubulin. Furthermore, in the cases of flagellar axonemes and basal bodies, the end onto which brain tubulin most readily assembles is, as with flagellar formation in vivo, the distal end.6-9 MATERIALS AND METHODS Chick Brain Tubulin Subunit IsolationIn all of the studies reported here, tubulin from the brains of 1 to 3-day-old chicks was used. Three different types of tubulin preparations were used. The first was crude, low-speed supernatant tubulin (LSS). Chick brains were homogenized in a glass homogenizer with a motor-driven Teflon@ pestle at 0" C in a volume of PM (1 mM MgSO,, 2 mM EGTA, 1 mM GTP, and 100 mM PIPES, at pH 6.9) equal to 1.2-1.5 times the weight of the brains and centrifuged at 35,000 x g (17,000 rpm in a Sorvall RC-2 centrifuge, rotor 147 148 Annals New York Academy of Sciences SS-34) for 1 hour at 4" C. The supernatant was removed and used as a crude, low-speed preparation of tubulin subunits (LSS) , The second tubulin preparation was crude,.high-speed supernatant (HSS) : HSS was prepared by centrifuging the chick brain homogenate at 105,000 X g (40,000 rpm in a Spinco L2-65B, rotor Ti-50) for 1 hour at 4" C. The third was purified tubulin (Cl, C,, C,) : To obtain more purified tubulin preparations, the LSS was incubated at 37" C for 30 min to assemble microtubules. The microtubules were harvested by centrifugation at 27,000 X g (15,000 rpm in a Sorvall SS-1 centrifuge) for 15 min at 25" C, resuspended in PM (%-% the original volume of US), and incubated at 0" C for 30 rnin to depolymerize the microtubules. This suspension was centrifuged a...

Evidence for tektins in centrioles and axonemal microtubules.

“…Several antibodies have been reported to cross-react with centriole components (4-7), but the nature and function of these components in relation to the microtubules have not been determined. Perhaps the most basic function of the centriole, in the capacity of a basal body, is to act as a template for the assembly of ciliary and flagellar axonemes (1,2,(8)(9)(10). In particular, the A-and B-subfibers of centriolar triplet microtubules are templates for the assembly of the A-and Bsubfibers of ciliary and flagellar doublet microtubules.…”

Colchicine reversibly inhibits electrical activity in arthropod mechanoreceptors

“…Each protofilament is composed of dimers, and each dimer composed of one alpha and one beta tubulin monomer. The molecular weight of the dimer is approximately 110,000 daltons, with each of the tubulins contributing 55,000 daltons (Rosenbaum, 1975;Erickson, 1975).…”

“…Microtubules also function in intracellular transport, including the movement of organelles, metabolites, and biosynthetic precursors to various areas of the cell (Burnside, 1975). An important neuronal function is the transfer of neurotransmitter precursors to synapses (De Robertis et al, 1971;Olmsted and Borisy, 1973;Rosenbaum et al, 1975).…”

“…Sensitivity to various disruptive agents is due to the constant turnover of subunits within the tubules (Behnke and Forer, 1967;Wittman, 1975;Rosenbaum et al, 1975). A "dynamic equili brium" (Inoue' and Sato, 1967) is maintained between formed MT and free tubulin pools.…”

“…The tubule lengthens when the dynamic equilibrium shifts toward the associated state, in which alpha and beta tubulin attach to the preformed micro tubule. The second method involves alternating attachment of the alpha and beta tubulins, 55,000 daltons each (Burnside, 1975;Rosenbaum et al, 1975;Erickson, 1975), forming a coiled filament, a protofilament. The protofila ments attach to one another side by side, as they uncoil forming a sheet of filaments.…”