Heterogeneity of microtubules recognized by monoclonal antibodies to alpha-tubulin

A set of four monoclonal antibodies against tubulin (TU-01, TU-02, TU-03, and TU-04) were produced using pig brain microtubule protein as antigen. Their characterization shows that all recognize antigenic determinants located on the tubulin alpha-subunit. However, peptide mapping of isolated alpha-tubulin, followed by immunoblotting with the monoclonal antibodies, shows that the antigenic determinants are located on different peptide fragments in at least three cases. The immunoreactivity with tubulins from different cells and tissues, ranging from eukaryotic microorganisms to man, was studied by immunoblotting and immunofluorescence. The antigenic determinants recognized by the antibodies are not uniformly distributed but, in some instances, are absent from tubulins of lower eukaryotic cells. These antibodies also make it possible to distinguish between different sets of microtubules within individual cells. Antigenically different microtubules are particularly evident in mouse spermatozoa and in some protozoa (T. vaginalis, H. muscarum, L. tropica, N. gruberi) possessing different sets of microtubules with different functions. These monoclonal antibodies can clearly identify the heterogeneity of tubulin or microtubules both from different organisms and within the same cell.     

Trypanosoma cruzi: cell type dependent distribution of a tubulin domain in mammalian stages

The distribution of tubulin domains in the mammalian stages of Trypanosoma cruzi was investigated by using monoclonal antibodies elicited against bovine brain tubulin. Western blotting performed on T. brucei trypomastigotes and T. cruzi epimastigotes showed that the monoclonal antibodies 16D3 and 24E3 reacted only with tubulin in these cell types. Indirect immunofluorescence revealed that, whereas 16D3 stained all microtubules, including subpellicular microtubules, the epitope defined by 24E3 was found in only a part of the tubulin pool of amastigotes and intermediate stages infecting murine fibroblasts and of broad trypomastigotes; the staining was limited to the basal bodies and the distal region of the flagellar adhesion zone in these developmental forms. By contrast, slender trypomastigotes did not exhibit any reaction with 24E3. These results are consistent with a transformation of broad trypomastigotes into slender trypomastigotes during which the tubulin domain recognized by 24E3 would undergo modifications leading to its complete masking in slender forms. The morphogenesis of the mammalian stages of T. cruzi would involve modifications of the tubulin molecule.     

Change in the heterogeneous distribution of tubulin isotypes in mitotic microtubules of the sea urchin egg by treatment with microtubule depolymerizing or stabilizing drugs.

In the mitotic sea urchin egg, the spindle microtubules were composed of different tubulin isotypes from those of astral microtubules using monoclonal antibodies [Oka et al. (1990) Cell Motil. Cytoskeleton, 16, 239-250]. Three of the antibodies, D2D6, DM1B, and YL1/2, were specific for spindle microtubules, astral microtubules and reactive with both microtubules, respectively. The mitotic sea urchin egg was treated with microtubule depolymerizing (colcemid and nocodazole) and stabilizing (hexylene glycol) drugs and change in the heterogeneous distribution of the tubulin isotypes was investigated by the immunofluorescence procedure using these three monoclonal anti-tubulin antibodies. We observed that: (1) the microtubule depolymerizing drugs caused quick depolymerization of most mitotic microtubules, and a small number of spindle microtubules remaining were stained with all three antibodies; (2) hexylene glycol induced many microtubules in the mitotic apparatus, which was stained with D2D6 but was not stained with DM1B; (3) hexylene glycol also induced a great number of miniasters in the cytoplasm, and they were stained with three antibodies. These results suggest that these drugs altered the distribution of tubulin isotypes in the mitotic microtubules during depolymerization or polymerization within a short time.     

MAP3: characterization of a novel microtubule-associated protein

Using monoclonal antibodies we have characterized a brain protein that copurifies with microtubules. We identify it as a microtubule-associated protein (MAP) by the following criteria: it copolymerizes with tubulin through repeated cycles of microtubule assembly in vitro; it is not associated with any brain subcellular fraction other than microtubules; in double-label immunofluorescence experiments antibodies against this protein stain the same fibrous elements in cultured cells as are stained by antitubulin; and this fibrous staining pattern is dispersed when cytoplasmic microtubules are disrupted by colchicine. Because it is distinct from previously described MAPs we designate this novel species MAP3. The MAP3 protein consists of a closely spaced pair of polypeptides on SDS gels, Mr 180,000, which are present in both glial (glioma C6) and neuronal (neuroblastoma B104) cell lines. In brain the MAP3 antigen is present in both neurons and glia. In nerve cells its distribution is strikingly restricted: anti-MAP3 staining is detectable only in neurofilament-rich axons. It is not, however, a component of isolated brain intermediate filaments.     

Heterogeneity among microtubules of the cytoplasmic microtubule complex detected by a monoclonal antibody to alpha tubulin

Three monoclonal antibodies specific for tubulin were tested by indirect immunofluorescence for their ability to stain cytoplasmic microtubules of mouse and human fibroblastic cells. We used double label immunofluorescence to compare the staining patterns of these antibodies with the total microtubule complex in the same cells that were stained with a polyclonal rabbit antitubulin reagent. Two of the monoclonal antitubulin antibodies bound to all of the cytoplasmic microtubules but Ab 1-6. 1 bound only a subset of cytoplasmic microtubules within individual fixed cells. Differential staining patterns were observed under various fixation conditions and staining protocols, in detergent-extracted cytoskeletons as well as in whole fixed cells. At least one physiologically defined subset of cytoplasmic microtubules, those remaining in cells pretreated for 1 h with 5 microM colcemid, appeared to consist entirely of Ab 1-6. 1 positive microtubules. The same was not true of the microtubules that remained in either cold-treated cells or in cells that had been exposed to hypotonic medium. The demonstration of antigenic differences among microtubules within single fixed cells and the apparent correlation of this antigenic difference with at least one "physiologically" defined subset suggests that mechanisms exist for the differential assembly or postassembly modification of individual microtubules in vivo, which may endow them with different physical or functional properties.     

Quantitative biochemical analysis of microtubule content in normal and transformed 3T3 cells

Microtubules in normal and transformed BALB 3T3 cells were preserved in a stabilizing medium and measured by a [3H]colchicine-binding tubulin assay, and compared to total cellular tubulin measured under nonstabilizing conditions. Essentially no change in tubulin or microtubule content was seen with changes in cell density or with changes in cellular morphology at various stages of growth of normal or transformed cells or induced by dibutyryl cAMP treatment of transformed cells. Of five cell lines transformed by a variety of agents, four had a significantly higher total tubulin content than untransformed 3T3 cells and all of them had an increased microtubule content. None of the transformed lines had a lower fraction of tubulin recoverable as sedimentable microtubules compared to untransformed cells, and in three of them this fraction was significantly higher. These results establish that microtubules are present in transformed cells to at least the extent (if not greater) than in normal cells but that there are variations in the total amount of tubulin and microtubules as well as the fraction of the total tubulin present as microtubules which are not strictly correlated with transformation or cell morphology.     

Immunofluorescence colocalization of the 90-kDa heat-shock protein and microtubules in interphase and mitotic mammalian cells

A mouse monoclonal antibody (AC88) that was raised against the 88-kDa heat-shock protein of the water mold, Achlya ambisexualis, and that cross-reacts with the 90-kDa mammalian heat-shock protein (hsp90), and an antibody against tubulin were used to localize hsp90 and microtubules, respectively, in the same cultured rat endothelial and PtK1 epithelial cells by indirect immunofluorescence. AC88 and tubulin antibodies labeled the same structures in cells at all stages of the cell cycle, regardless of whether cells were permeabilized before or after fixation. Labeling of cell structures by both AC88 and anti-tubulin antibodies was identically affected by treating cells with colcemid. Double labeling with AC88 and anti-tubulin antibodies in interphase and mitotic cells is consistent with the conclusion that all microtubules are labeled and that no subclass of microtubules is preferentially labeled. Fluorescent labeling by AC88 was prevented by preabsorption of the antibody with purified rat hsp90 but was unaffected by preabsorption with purified 6S tubulin dimer. In contrast to AC88, fluorescent labeling by an anti-tubulin antibody was prevented by preabsorption with tubulin dimer but was unaffected by preabsorption with rat hsp90. Western-blot analysis demonstrated no cross-reactivity of AC88 for tubulin and no cross-reactivity of the anti-tubulin antibody for hsp90. A polyclonal antiserum fraction from a rabbit immunized with the 89-kDa heat-shock protein from chicken also labeled the mitotic apparatus in dividing cells and, somewhat less distinctly, fibrous structures in interphase cells. Labeling by hsp89 anti-serum was prevented by absorption with hsp90. AC88 also labeled microtubules in cultured mouse (L929 and 3T3), rat (endothelium and TRST), hamster (CHO) and primate (BSC, COS-1 and HeLa) cell lines. The demonstration of colocalization of hsp90 with microtubules should provide a valuable clue to eventual understanding of the cellular function of this ubiquitous, conserved and abundant stress-response protein.     

The display of microtubules in transformed cells.

Monospecific tubulin antibodies have been used in indirect immunofluorescence microscopy on a variety of well characterized, transformed cell lines grown in tissue culture. Networks of colcemid-sensitive fibers are seen in SV40-transformed 3T3 cells, SV40-transformed rat embryo cells, HeLa cells and other transformed cell lines. In each case, greater than 90% of the cells contain visible microtubular networks, and where individual microtubules can be distinguished, they run for long distances. Documentation of these metworks is more difficult in transformed than in normal cells, because transformed cells are in general more rounded and have less well spread cytoplasm. In addition, the microtubular networks can be readily visualized in "cytoskeletons" of both normal and transformed cells, obtained by treatment of cells with nonionic detergents in a buffer which stabilizes microtubules in vitro. Addition of calcium to this buffer results in in situ fragmentation and destruction of the microtubular network. In view of these results, we conclude that transformed cells contain significant numbers of microtubules, and that in transformed cells, as in normal cells, microtubules are arranged in networks.     

Radioimmunoassay for tubulin: a quantitative comparison of the tubulin content of different established tissue culture cells and tissues

A quantitative estimate of the cellular tubulin concentration can be obtained by the use of a radioimmunoassay based upon the competition between tubulin in cell extracts and a known amount of radioactively labeled homogeneous tubulin during binding to a limited amount of antitubulin antibodies. This assay shows that a variety of widely used tissue culture cells (mouse L cells, mouse 3T3 cells, chick embryo fibroblasts) have a tubulin content which corresponds to approximately 2.5-3.3% of their total protein. Transformation of mouse 3T3 cells by the DNA virus SV40, and of chick embryo cells by the RNA Rous sarcoma virus, does not change the intracellular tubulin concentration. Transformed cells of brain origin, such as some glia tumor cell lines and some neuroblastoma cell lines, have a much lower tubulin content than does normal brain tissue.The intracellular concentration of tubulin in mouse 3T3 cells is discussed in relation to the number of microtubules detected during interphase by immunofluorescence microscopy. These results are also discussed in view of a mechanism of microtubule elongation in vivo driven by self-assembly.     

Where and when is microtubule diversity generated inParamecium? Immunological properties of microtubular networks in the interphase and dividing cells

Summary Ciliates are highly differentiated cells which display extensive deployment of microtubular systems. Because genetic diversity of tubulin is extremely reduced in these cells, microtubule diversity is mostly generated at the post-translational level either through direct modification of tubulin or through the binding of associated proteins to microtubules. We have undertaken a systematic exploration of microtubule diversity in ciliates by way of production of monoclonal antibodies. Previously we reported the biochemical characterization of these antibodies. In addition to antibodies directed against primary sequences, we obtained antibodies directed against post-translational modifications. In this paper, we report a detailed analysis of the distribution of the various epitopes on the microtubular networks ofParamecium, both in interphase cells and during division morphogenesis. Each of these antibodies decorates a subset of microtubules. Acetylation, recognized by antibodies TEU 318 and TEU 348, is detected on stable microtubules early after microtubule assembly. Epitopes recognized by two other antibodies (TAP 952 and AXO 58) are found on a subset of stable microtubules; in addition, the TAP 952 antibody is also found on labile microtubules; both epitopes are detected as soon as microtubule assembly occurs. In contrast, the epitope of the antibody, AXO 49, is associated with only a restricted subset of stable microtubules in the interphase cell, and is detected a lag-time after microtubule assembly during division morphogenesis. These data show that microtubule diversity is generated through a time-dependent sequence and according to a definite spatial pattern.     

Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line

Hybrid myeloma cell lines secreting monoclonal antibodies to tubulin have been prepared using rat myelomas and spleen cells from rats immunized with yeast tubulin. A comparison between the results obtained with the rat myeloma Y3-Ag 1.2.3., which secretes a light chain, and a new line, YB2/O, which does not, shows that they are both excellent parental lines and that the second produces hybrids with no myeloma chain components. The antitubulin antibodies in the serum of rats bearing two of the hybrid myeloma tumors gave titers of up to 1:10(6) from which large amounts of monoclonal antibodies could be easily purified. They recognized tubulin from yeast as well as from birds and mammals. The two antibodies gave clear immunofluorescent staining of yeast mitotic spindles as well as the interphase microtubule network of tissue culture cells. Some difference in the pattern of immunofluorescence staining of yeast cells and nuclei was observed between the two antibodies. The purified antibodies could be conjugated to colloidal gold particles and used for direct labeling of yeast microtubules for electron microscopy.     

Post-translational tubulin modifications in spermatogeneous cells of the pteridophyteCeratopteris richardii

Summary Acetylation and tyrosinization are post-translational modifications of tubulin generally associated, respectively, with highly stable or dynamic microtubule arrays in animals and protists. Little is known of these modifications in land plants, however. We examined the presence and distribution of post-translational tubulin modifications in developing spermatogenous cells of the pteridophyteCeratopteris richardii by immunofluorescence and immunogold, utilizing antibodies specific for acetylated and tyrosinated tubulin. Acetylated tubulin is found in mid to late stage spermatogenous cells in stable microtubule configurations: the spline, flagella, and basal bodies. Tyrosinated tubulin, a modification associated with dynamic microtubule arrays, is also present in these structures as well as all other microtubules in the cell. The lamellar strip of the multilayered structure, a body previously described as tubulin-containing, was not labelled by any of the tubulin antibodies or antiserum. Treatment of cultures with the microtubule stabilizer taxol results in the appearance of new arrays of microtubules, including bundles in the cytoplasm. Only those new taxol-induced microtubule arrays present in mid to late stage cells (i.e., those with other normally acetylated tubulin arrays) have acetylated domains. Younger spermatogenous cells had similar microtubule bundles but no acetylated tubulin. Tyrosinated tubulin was found in all these taxol-stabilized arrays. These data indicate that, although these pteridophyte cells have the ability to acetylate tubulin, that this ability is limited to stages after the final spermatogenous cell mitosis and is limited to the highly stable spline and flagella microtubules.Abbreviations LS lamellar strip of multilayered structure - MTOC microtubule organizing center     

Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms

Seven monoclonal antibodies raised against tubulin from the axonemes of sea urchin sperm flagella recognize an acetylated form of alpha-tubulin present in the axoneme of a variety of organisms. The antigen was not detected among soluble, cytoplasmic alpha-tubulin isoforms from a variety of cells. The specificity of the antibodies was determined by in vitro acetylation of sea urchin and Chlamydomonas cytoplasmic tubulins in crude extracts. Of all the acetylated polypeptides in the extracts, only alpha-tubulin became antigenic. Among Chlamydomonas tubulin isoforms, the antibodies recognize only the axonemal alpha-tubulin isoform acetylated in vivo on the epsilon-amino group of lysine(s) (L'Hernault, S.W., and J.L. Rosenbaum, 1985, Biochemistry, 24:473-478). The antibodies do not recognize unmodified axonemal alpha-tubulin, unassembled alpha-tubulin present in a flagellar matrix-plus-membrane fraction, or soluble, cytoplasmic alpha-tubulin from Chlamydomonas cell bodies. The antigen was found in protein fractions that contained axonemal microtubules from a variety of sources, including cilia from sea urchin blastulae and Tetrahymena, sperm and testis from Drosophila, and human sperm. In contrast, the antigen was not detected in preparations of soluble, cytoplasmic tubulin, which would not have contained tubulin from stable microtubule arrays such as centrioles, from unfertilized sea urchin eggs, Drosophila embryos, and HeLa cells. Although the acetylated alpha-tubulin recognized by the antibodies is present in axonemes from a variety of sources and may be necessary for axoneme formation, it is not found exclusively in any one subset of morphologically distinct axonemal microtubules. The antigen was found in similar proportions in fractions from sea urchin sperm axonemes enriched for central pair or outer doublet B or outer doublet A microtubules. Therefore the acetylation of alpha-tubulin does not provide the mechanism that specifies the structure of any one class of axonemal microtubules. Preliminary evidence indicates that acetylated alpha-tubulin is not restricted to the axoneme. The antibodies described in this report may allow us to deduce the role of tubulin acetylation in the structure and function of microtubules in vivo.     

Organization of tubulin in normal and transformed rat kidney cells

We have carried out a quantitative biochemical and ultrastructural study of tubulin and microtubules in a normal rat kidney (NRK) cell line and its viral transformant (442) in culture. Under equivalent culture conditions, both cell lines contain the same amount of tubulin according to a colchicine-binding assay. The normal and transformed cells differ significantly, however, with respect to the state of organization of their tubulin. Counts of microtubules in sectioned cells indicate that NRK cells have almost twice as many microtubules per unit area of cytoplasm as the 442 cells. Centrifugation studies, on the other hand, show that 442 cells have almost twice as much pelletable tubulin as the NRK cells. We propose, therefore, that the transformed cells contain a large amount of tubulin which is in some alternative aggregate form that is not morphologically detectable as microtubles in the cytoplasm     

A radiolabeled monoclonal antibody binding assay for cytoskeletal tubulin in cultured cells

To detect changes in the extent of tubulin polymerization in cultured cells, we have developed a radioactive antibody binding assay that can be used to quantitate total cytoskeletal tubulin or specific antigenic subsets of polymerized tubulin. Fibroblastic cells, grown to confluence in multiwell plates, were permeabilized and extracted with 0.5% Triton X-100 in a microtubule-stabilizing buffer. These extracted cytoskeletons were then fixed and incubated with translationally radiolabeled monoclonal antitubulin antibody (Ab 1-1.1), an IgM antibody specific for the beta subunit of tubulin. Specific binding of Ab 1-1.1 to the cytoskeletons was saturable and of a single apparent affinity. All specific binding was blocked by preincubation of the radiolabeled antibody with excess purified brain tubulin. Specific Ab 1-1.1 binding appeared to represent binding to cytoskeletal tubulin inasmuch as: pretreatment of cells with colchicine decreased Ab 1-1.1 binding in a dose-dependent manner which correlated with the amount of polymerized tubulin visualized in parallel cultures by indirect immunofluorescence, taxol pretreatment alone caused an increase in Ab 1-1.1 binding and prevented in a dose-dependent manner the colchicine-induced decrease in antibody binding, in cells pretreated with colcemid and returned to fresh medium, Ab 1-1.1 binding decreased and recovered in parallel with the depolymerization and regrowth of microtubules in these cells, and comparison of maximal antibody binding per cell between primary mouse embryo, 3T3, and human foreskin fibroblasts correlated with immunofluorescence visualization of microtubules in these cells. Thus, this assay can be used to measure relative changes in the level of polymerized cytoskeletal tubulin. Moreover, by Scatchard-type analysis of the binding data it is possible to estimate the total number of antibody binding sites per cell. Therefore, depending on the stoichiometry of antibody binding, this type of assay may be used for quantitating total cytoskeletal tubulin, specific antigenic subsets of cytoskeletal tubulin, or other cytoskeletal proteins.     

Spatial and temporal colocalization of the Golgi apparatus and microtubules rich in detyrosinated tubulin

The integrity and intracellular distribution of the Golgi apparatus appear to depend upon microtubules. We have found that the microtubules rich in detyrosinated tubulin are located preferentially in the vicinity of the Golgi. Cells were double-stained with antibodies specific for either tyrosinated or detyrosinated tubulin and an antibody to prolactin or wheat germ agglutinin (Golgi markers). Microtubules rich in detyrosinated tubulin showed a close codistribution with the Golgi in three different cultured cell lines GH3, BS-C-1, and AtT20. Disruption of microtubules with nocodazole in GH3 cells resulted in fragmentation and dispersal of the Golgi apparatus as reported previously. During recovery of the microtubules and the Golgi complex after removal of the nocodazole, there was a spatial and temporal colocalization of the Golgi apparatus and microtubules rich in detyrosinated tubulin. Our results suggest that a functional relationship may exist between the structure and organization of the Golgi complex and the detyrosination of alpha- tubulin in microtubules.     

Markedly decreased binding of vincristine to tubulin in vinca alkaloid-resistant Chinese hamster cells is associated with selective overexpression of alpha and beta tubulin isoforms

Vinca alkaloids are among a number of cytotoxic agents which target tumor cell microtubules. Studies described herein document the basis for one form of acquired resistance to these plant alkaloids involving an alteration of tubulin in a variant (DC-3F/VCRd-5L) of DC-3F Chinese hamster cells. Our results revealed a markedly decreased binding of [(3)H]vincristine (VCR) to tubulin extracted from this variant compared to tubulin extracted from wild-type DC-3F cells. This was quantitated as a 10- to 15-fold decrease in on-rate in the presence of GTP for the [(3)H]VCR associating with tubulin in cell-free cytosol and a 10-fold increase in off-rate for GTP-dependent dissociation of the [(3)H]VCR-tubulin complex. Quantitative RT-PCR and nucleotide sequencing of poly(A)(+) RNA also carried out with variant and wild-type DC-3F cells documented a different pattern of relative expression, but no base pair differences in the open reading frame of the three alpha and beta tubulin isoforms detected in each cell type. This was accounted for by selective overexpression of one alpha tubulin (alphaII) and two beta tubulin (betaI and betaIV) isoforms in the variant cells. These results would appear to provide an underlying basis for the large decrease in [(3)H]VCR binding by tubulin in these variant Chinese hamster cells and a major component of their acquired resistance to this vinca alkaloid.     

Spatial distribution of posttranslationally modified tubulins in polarized cells of developing Artemia

In many differentiated cells, posttranslationally modified tubulins exhibit restricted subcellular distribution, leading to the proposal that they are required for the production and maintenance of polarity. To study this possibility, we used immunological approaches to examine tubulin isoforms in developing Artemia larvae and to determine their location in several types of cells within the organism. The amount of tubulin in relation to total protein remained relatively constant during early larval development while detyrosinated tubulin increased, an event correlated with the differentiation of larval gut muscle cells. Except for epidermal cells of the developing thorax, each type of cell within the Artemia larvae exhibited characteristic staining patterns which were very similar for each antitubulin antibody. Within epidermal cells, microtubules containing acetylated tubulin appeared patchy or punctate in their distribution, an image not seen with the other antibodies. In most polarized cells, staining for tubulin and actin colocalized in discrete areas, demonstrating enrichment of both proteins within the same cellular compartment and suggesting functional interactions. Mitotic figures were stained with qualitatively equal intensity by all of the antitubulin antibodies, but asters were not observed. Midbodies were intensely stained with phalloidin as well as the antibodies to tubulin. It was clear that microtubules exhibited a preferential localization in cells of Artemia but in no case was a tubulin isoform found exclusively in one area of a cell. The results support the contention that microtubules influence the organization of polarized cell structure and function but they do not permit the conclusion that this capability is dependent on the localization of posttranslationally modified tubulins to restricted subcellular positions.     

Localization of tektin filaments in microtubules of sea urchin sperm flagella by immunoelectron microscopy

Extraction of doublet microtubules from the sperm flagella of the sea urchin Strongylocentrotus purpuratus with sarkosyl (0.5%)-urea (2.5 M) yields a highly pure preparation of "tektin" filaments that we have previously shown to resemble intermediate filament proteins. They form filaments 2-3 nm in diameter as seen by negative stain electron microscopy and are composed of approximately equal amounts of three polypeptide bands with apparent molecular weights of 47,000, 51,000, and 55,000, as determined by SDS PAGE. We prepared antibodies to this set of proteins to localize them in the doublet microtubules of S. purpuratus and other species. Tektins and tubulin were antigenically distinct when tested by immunoblotting with affinity-purified antitektin and antitubulin antibodies. Fixed sperm or axonemes from several different species of sea urchin showed immunofluorescent staining with antitektin antibodies. We also used antibodies coupled to gold spheres to localize the proteins by electron microscopy. Whereas a monoclonal antitubulin (Kilmartin, J.V., B. Wright, and C. Milstein, 1982, J. Cell Biol. 93:576-582) decorates intact microtubules along their lengths, antitektins labeled only the ends of intact microtubules and sarkosyl-insoluble ribbons. However, if microtubules and ribbons attached to electron microscope grids were first extracted with sarkosyl-urea, the tektin filaments that remain were decorated by antitektin antibodies throughout their length. These results suggest that tektins form integral filaments of flagellar microtubule walls, whose antigenic sites are normally masked, perhaps by the presence of tubulin around them.     

Visualization of assembled and disassembled microtubule protein by double label fluorescence microscopy

Indirect immunofluorescence with rhodamine labelled antibodies and fluoresceinated colchicine (FC) are used to simultaneously localize microtubules and soluble tubulin in cultured ovarian granulosa cells. FC labelled tubulin is most concentrated in regions of the cell occupied by antitubulin stained microtubule bundles. Pretreatment of granulosa cells with colchicine results in a central accumulation of FC and antibody labelled tubulin that coincides with the disposition of 10-nm filament cables. In contrast, the microtubule disrupting agent nocodazole produces a diffuse tubulin distribution as detected with both FC and antibody probes. Taxol treatment, which enhances microtubule assembly, results in a striking concentration of microtubule bundles associated with the nucleus that avidly bind FC. These results suggest that disassembled tubulin is preferentially associated with cytoplasmic microtubules and possibly other formed elements of the cytoskeleton.