Pig Brain Tubulin Research Articles

Immunofluorescence microscopy of tubulin and microtubule arrays in plant cells. I. Preprophase band development and concomitant appearance of nuclear envelope-associated tubulin.

The development of the preprophase band (PPB) of microtubules (MT) in meristematic plant cells was studied by using antibodies to pig brain tubulin and indirect immunofluorescence microscopy. The PPB is first visible as a wide band of MT that are arranged only slightly more densely than flanking MT of the cortical interphase array. MT progressively become more tightly packed together, and other cortical MT are no longer seen as the PPB matures. The surface of the nuclear envelope (NE) displays no tubulin fluorescence during interphase but begins to fluoresce in the early stages of PPB development, and its intensity progressively increases thereafter. The pattern at the NE is usually diffuse at first, suggesting the presence of nonpolymerized tubulin, but fibers along the NE can be resolved at later stages. MT, arranged either radially or as a meshwork, can occur between the nucleus and cell cortex, and sometimes appear to connect the PPB and NE directly. Isolated preprophase nuclei from cells ruptured during processing often retain the PPB in its normal orientation, indicating stable linkages between the nucleus and PPB. Fluorescent cross-bars perpendicular to the axis of the MT were resolved in some PPB, suggesting lateral linkages. This suggestion is reinforced by the presence of PPB that hold together as a ribbon of MT in certain preparations, allowing PPB to be isolated from the rest of the cytoplasm and the nucleus.

An immunocytochemical method for the visualization of tubulin-containing structures in the egg of Xenopus laevis.

Tubulin can be isolated and purified from Xenopus laevis eggs through modification of Olmstedt's (1970) tubulin isolation method, viz. by repeating the vinblastin precipitation step after resuspension of the sediment in a detergent-containing stabilizing medium. By this we overcome the deleterious influence of the yolk granules in the isolation procedure. From 11 of Xenopus laevis eggs 25 mg VB-paracrystals can be obtained. The apparent molecular weight of the purified tubulin is 52,800. Antiserum against the purified Xenopus VB-paracrystals, raised in 2 Chinchilla rabbits, cross-reacts in immunodiffusion tests in agar gels with rat brain tubulin and with tubulin isolated from Xenopus laevis eggs by the described procedure. Specific indirect fluorescence staining and appropriate control reactions reveal that cilia of Tetrahymena pyriformis, cytoplasmic networks in cultured mouse Leydig cells, as well as mitotic spindles and nuclear regions in paraffin sections of Xenopus laevis blastulae, react with the antibodies against Xenopus laevis egg tubulin as well as with monoclonal antibodies against pig brain tubulin. These results provide additional evidence for the view that tubulin antibodies are neither species nor tissue specific and show that under appropriate conditions tubulin containing structures can be visualized in paraffin sections.

Biochemical and structural analyses of microtubules in the pellicular membrane of Leishmania tropica.

The structure of the major protein of the pellicular membrane of Leishmania tropica was investigated. This protein is composed of two polypeptides, of ca. 50,000 d molecular weight, that were found to cross-react immunologically with the alpha and beta subunits of pig brain tubulin. The polypeptides and pig brain tubulin subunits were partially digested with S. aureus V8 protease, and the peptides obtained analysis by SDS-polyacrylamide gel electrophoresis. A comparison of the patterns showed that the beta subunits of Leishmania and pig tubulin have very similar primary structures, while the alpha subunits have evolved divergently. These experiments demonstrate that the major polypeptides found in the pellicular membrane of L. tropica are alpha and beta subunits of tubulin. Immunoelectron microscopy indicates that the tubulin is located in the microtubules associated with the pellicular membrane of Leishmania. Arrays of microtubules were prepared by nonionic detergent treatment of the cells and observed by electron microscopy after negative staining. Optical diffraction reveals a 5 nm spacing between protofilaments in the microtubule and a 4 nm axial periodicity corresponding to the tubulin subunits. The pitch of the shallow left-hand three-start helix is 12 degrees. A distance of 47 nm separates each microtubule from the next. These data show that the dimensions and supramolecular organization of the tubulin subunits in the microtubules are identical in the pellicular membrane of L. tropica and in mammalian brain.

Tubulin carbamoylation. Functional amino groups in microtubule assembly.

The characteristics of the carbamoylation of pig brain tubulin were examined by using the modification conditions with cyanate described previously [Mellado, Slebe + Maccioni (1980) Biochem. Int. I, 584--590]. The carbamoylation reaction resulted in an inhibition of microtubule assembly, which was dependent on the concentration of the modifying agent. This tubulin modification appears to inhibit the growth of microtubules. The presence of GTP did not protect tubulin against this inhibition. Electron microscopy showed a marked decrease in the number of tubules after carbamoylation, but no alterations were observed in the microtubule morphology. The incorporation of KN14CO into alpha- and beta-subunits with similar kinetics was also shown, and the carbamoylated residues were identified as epsilon-N-carbamoyl-lysine residues.

Arginyl residues involvement in the microtubule assembly

Modification of pig brain tubulin with 2,3-butanedione, an arginine-specific reagent, resulted in a decrease of its microtubule formation capacity, with apparent first-order kinetics. However, microtubules already assembled were not affected by the reagent. The relation between the polymerization inhibition rate constant and the butanedione concentration followed a saturation curve whereas the colchicine binding activity remained unchanged over that concentration range. GTP partially prevented the decrease of tubulin polymerization induced by the butanedione treatment. This protective effect of GTP was increased by glycerol. The butanedione inhibition of tubulin polymerization appears to be related to the modification of no more than three arginyl residues. These data suggest that at least one of the arginyl residues plays an essential role in tubulin polymerization, probably through its interaction with the negatively charged phosphate moiety of the nucleotide.

Tubulin sequence conservation

Various aspects of the primary structure of tubulin are discussed and tubulin sequence data are compared. A hypothesis concerning the evolution of tubulin is also presented. The main points raised are: (1) Identical glycyl rich regions in α- and β-tubulin, which are similar in both sequence and predicted secondary structure to a region in several nucleotide binding enzymes, may be involved in binding GTP. (2) Small regions of homology are present to actin, myosin and troponin T. These homologous regions may have the same function, resulting in a convergence of their sequences, or they may have arisen by a pathway of protein evolution which is still only very poorly understood. (3) The mutation rate between pig and chick brain tubulin is 0.22 PAMs (accepted point mutations/100 residues) per hundred million years, which is comparable to that of the histones. At an early time in its history, however, the tubulin heterodimer appears to have had a relatively high rate of mutation. This may have been during the evolution of the first eukaryotes.

Identification of a second β chain in pig brain tubulin

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Kinetic analysis of cooperativity in tubulin polymerization in the presence of guanosine di- or triphosphate nucleotides.

In vitro polymerization of pig brain tubulin, highly purified and deprived of microtubule-associated proteins, was followed by turbidimetry. Treatment of the data yielded the relation existing between the observed turbidity and the amount of polymer formed. This allowed a kinetic analysis, according to Oosawa's theories, of the polymerization process, which consisted of a slow spontaneous nucleation followed by the growth process. The apparent elongation rate constant was closely related to the nucleation process and exhibited a highly cooperative variation with tubulin concentration. The cooperativity was indicative of the size of the nucleus which appears to remain the same whether sheets or microtubules are formed. Magnesium ions appear to play a role in the polymorphism of tubulin polymers, the proportion of microtubules to sheets increasing with magnesium ion concentration. From kinetic experiments evidence was provided for GDP binding in competition with GTP, with a sixfold lower affinity. The tubulin-GDP complex could participate in microtubules elongation, but was not able to form nuclei. The critical concentration of tubulin in the presence of GDP was roughly twice as high as in the presence of GTP.