Kevin W. Farrell

Comparison of proteolytic cleavage patterns of α-tubulins and β-tubulins from taxonomically distant species

Abstract The α and β-chains of tubulins from several taxonomically distant species were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and isolated by a procedure that avoided stains and acidic conditions. All the β-chains had the same electrophoretic mobility, but the α-chains of some of the lower eukaryotes migrated faster than the rest. Limited proteolytic digestion of the subunits with Staphylococcus aureus protease, followed by electrophoresis on 15% acrylamide gels, resulted in very similar peptide patterns for all the β-chains, but a variety of patterns for the α-chains. The peptide patterns of α-tubulins from sea urchin egg and sperm flagellar outer doublets were markedly different, whereas those of bovine brain and kidney were identical. Bovine and dogfish brain α-tubulin peptide patterns were also identical, in contrast to the very different one of squid brain. Strong similarities were found between the α-chain peptide patterns of sperm flagellar tubulin from the echiurid worm Urechis caupo and the sea urchins Lytechinus pictus and Strongylocentrotus purpuratus , indicating that functionally similar tubulins from very different species can be more closely related than functionally different tubulins from the same organism. Evidence for the evolution of plant sperm flagella from protistan flagella was provided by the distinctive and very similar α-chain peptide patterns of tubulin from sperm flagella of the bracken fern Pteridium aquilinum and the flagella of the unicellular biflagellate alga Chlamydomonas reinhardtii .

Microtubule reassembly in vitro of Strongylocentrotus purpuratus sperm tail outer doublet tubulin.

Abstract Strongylocentrotus purpuratus outer doublet microtubules were prepared by extraction of sperm tail axonemes with 0.6 m -KCl. Sonication of the outer doublet microtubules in 5 m m -2-(N-morpholino)ethanesulphonic acid, 1 m m -ethyleneglycol-bis-(β-aminoethyl ether) N,N′-tetraacetic acid, 1 in m -MgSO4 (pH 6.7) solubilized up to 35% of the outer doublet protein, depending on the power input, in a manner which was non-selective for either subfiber. Tubulin comprised 75 to 85% of the total solubilized protein in a 200,000 g supernatant obtained from the sonicated suspension. Colchicine-binding assays demonstrated that the tubulin was largely in a native form (KA = 106, liters mole−; 0.74 mole of colchicine bound per mole of tubulin at infinite concentration of colchicine). Microtubule self-assembly from the 200,000 g supernatants in the absence of added seeds or glycerol was quantitated by light-scattering at 350 nm. The critical protein concentration for assembly was 0.55 mg ml−1 at 37 °C and the reaction occurred optimally in the presence of 2 m m -GTP and 150 m m -KCl. The solubilized outer doublet tubulin formed singlet microtubules upon reassembly under our in vitro conditions. The authenticity of the microtubules was verified by both negative stain and thin-section electron microscopy. Polymerization was prevented by colchicine and podophyllotoxin, and depolymerization occurred rapidly on cooling the microtubules to 0 °C. The susceptibility of the reassembled microtubules to low temperature suggested that they could be “recycled” by the warm assembly-cold disassembly procedure developed for vertebrate brain (Borisy et al., 1974). Twice recycled outer doublet tubulin was devoid of high molecular weight microtubule-associated proteins, as judged by gel electrophoresis in the presence of sodium dodecyl sulfate. However, trace amounts (less than 5%) of intermediate molecular weight material was visible on heavily overloaded gels. The function of this material is uncertain, but it is not chemically equivalent to the tau factor of vertebrate brain (Weingarten et al., 1975), since it cannot be separated from the tubulin by phosphocellulose adsorption. In addition, phosphocellulose-treated tubulin reassembled to the same extent as untreated tubulin, suggesting that the reassembly of outer doublet tubulin does not require the protein equivalents of brain microtubule-associated proteins or tau factor. If accessory proteins are required for the reassembly of outer doublet tubulin, they are not removed by phosphocellulose under the conditions employed, and they must comprise less than 5% of the total protein.

Differential radiolabeling of opposite microtubule ends: Methodology, equilibrium exchange-flux analysis, and drug poisoning

We describe a method which allows opposite microtubule ends to be distinguished by differentially labeling the microtubules with [3H]- and [14C]guanine nucleotides. Assembly-disassembly reactions at opposite microtubule ends can therefore be measured simultaneously and without modification of the tubulin dimers or microtubules. The method is predicated on experimental observations which demonstrate that net dimer addition to steady-state microtubules must be predominantly unidirectional. This does not preclude, however, some bidirectional dimer addition to steady-state microtubules by an equilibrium-exchange mechanism. We therefore calculated the relative contribution to dimer incorporation of bidirectional equilibrium exchange in a unidirectional microtubule system (s = 0.06). Under our conditions bidirectional dimer incorporation is negligible; net dimer addition to steady-state microtubules is overwhelmingly unidirectional. We used this method to study the effects of colchicine and podophyllotoxin on assembly-disassembly at opposite microtubule ends. Both drugs inhibit substoichiometrically net dimer addition to one microtubule end and, to a lesser extent, net dimer loss from the opposite end.

Chapter 5 Purification and Reassembly of Tubulin from Outer Doublet Microtubules

Publisher Summary The chapter discusses the purification and reassembly of tubulin from outer doublet microtubules. The most commonly used microtubule system is that of vertebrate brain. This system has the advantage of yielding large quantities of native tubulin; however, the tubulin derives from the total brain microtubule population, which shows considerable heterogeneity. The optimal in vitro assembly requirements of outer doublet and vertebrate brain tubulins are very similar, as are the properties of the assembled microtubules. This appears to be generally true for all tubulins so far examined, irrespective of the properties, functions, cellular location, or phylogeny of the microtubules from which the tubulin derives. The reconstituted microtubules are “labile” singlet microtubules, archetypical of which are reconstituted vertebrate brain microtubules. Structurally bonafide singlet microtubules can be formed in vitro from outer doublet and dogfish brain tubulins in the absence of associated proteins. Direct evidence that tubulin conformational changes alter the microtubule lattice is lacking. Studies on the mechanism of colchicine poisoning of microtubule assembly are consistent with this hypothesis. Colchicine poisons microtubule assembly by first complexing with tubulin dimers, which then copolymerize with uncomplexed tubulin into the microtubules and decrease the apparent rate of subsequent drug-free dimer addition.