The interphase microtubule cytoskeleton of five different phenotypes of microvessel endothelial cell cultures derived from bovine corpus luteum.

The interphase microtubule cytoskeleton of five different microvessel endothelial cell cultures, recently established from bovine corpus luteum, was analysed using anti-tubulin immunofluorescence. An antibody against acetylated microtubules detected four cell types each of which possessed a single cilia. The length of the cilia were up to 10 microns for cell types 1 and 2. Ciliary stubs had a length of up to 0.37 microns in cell types 4 and 5. Cilia were missing in cell type 3. Long and short cilia were located in the perinuclear region from where cytoplasmic microtubules radiated. Cell type 3 displayed straight microtubules rather than the wavy path seen in the other cell types. The amount of tyrosinated microtubules visualized by a specific antibody was consistently higher than that of posttranslationally acetylated microtubules. The latter were more apparent in cell types 4 and 5 than in the other cell types. We conclude: Differences in the cytoplasmic microtubule inventory of each microvessel endothelial cell type points at individual functions maintained in culture.     

Microtubules containing acetylated alpha-tubulin in mammalian cells in culture

The subcellular distribution of microtubules containing acetylated alpha-tubulin in mammalian cells in culture was analyzed with 6-11B-1, a monoclonal antibody specific for acetylated alpha-tubulin. Cultures of 3T3, HeLa, and PtK2 cells were grown on coverslips and observed by immunofluorescence microscopy after double-staining by 6-11B-1 and B-5-1-2, a monoclonal antibody specific for all alpha-tubulins. The antibody 6-11B-1 binds to primary cilia, centrioles, mitotic spindles, midbodies, and to subsets of cytoplasmic microtubules in 3T3 and HeLa cells, but not in PtK2 cells. These observations confirm that the acetylation of alpha-tubulin is a modification occurring in different microtubule structures and in a variety of eukaryotic cells. Some features of the acetylation of cytoplasmic microtubules of mammalian cells are also described here. First, acetylated alpha-tubulin is present in microtubules that, under depolymerizing conditions, are more stable than the majority of cytoplasmic microtubules. In addition to the specific microtubule frameworks already mentioned, cytoplasmic microtubules resistant to nocodazole or colchicine, but not cold-resistant microtubules, contain more acetylated alpha-tubulin than the rest of cellular microtubules. Second, the alpha-tubulin in all cytoplasmic microtubules of 3T3 and HeLa cells becomes acetylated in the presence of taxol, a drug that stabilizes microtubules. Third, acetylation and deacetylation of cytoplasmic microtubules are reversible in cells released from exposure to 0 degrees C or antimitotic drugs. Fourth, the epitope recognized by the antibody 6-11B-1 is not absolutely necessary for cell growth and division. This epitope is absent in PtK2 cells. The acetylation of alpha-tubulin could regulate the presence of microtubules in specific intracellular spaces by selective stabilization.     

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.     

Acetylated and detyrosinated alpha-tubulins are co-localized in stable microtubules in rat meningeal fibroblasts

We have examined the distribution of acetylated alpha-tubulin using immunofluorescence microscopy in fibroblastic cells of rat brain meninges. Meningeal fibroblasts showed heterogeneous staining patterns with a monoclonal antibody against acetylated alpha-tubulin ranging from staining of primary cilia or microtubule-organising centers (MTOCs) alone to extensive microtubule networks. Staining with a broad spectrum anti-alpha-tubulin monoclonal indicated that all cells possessed cytoplasmic microtubule networks. From double-labeling experiments using an antibody against acetylated alpha-tubulin (6-11B-1) and antibodies against either tyrosinated or detyrosinated alpha-tubulin, it was found that acetylated alpha-tubulin and tyrosinated alpha-tubulin were often segregated to different microtubules. The microtubules containing acetylated but not tyrosinated alpha-tubulin were cold stable. Therefore, it appeared that in general meningeal cells possessed two subset of microtubules: One subset contained detyrosinated and acetylated alpha-tubulin and was cold stable, and the other contained tyrosinated alpha-tubulin and was cold labile. These results are consistent with the idea that acetylation and detyrosination of alpha-tubulin are involved in the specification of stable microtubules.     

Gamma-tubulin distribution in interphase and mitotic cells upon stabilization and depolymerization of microtubules

Indirect immunofluorescence and digital videomicroscopy were used to study gamma-tubulin distribution in normal mitotic and interphase HeLa cells and after their treatment with microtubule-stabilizing (taxol) and depolymerizing (nocodazole) drugs. In interphase HeLa cells, the affinity-purified antibodies against gamma-tubulin and monoclonal antibodies against acetylated tubulin stain one or two neighboring dots, centrioles. The gamma-tubulin content in two centrioles from the same cell differs insignificantly. Mitotic poles contain fourfold amount of gamma-tubulin as compared with the centrioles in interphase. The effect of nocodazole (5 microg/ml) on interphase cells resulted in lowering the amount of gamma-tubulin in the centrosome, and in 24 h it was reduced by half. Treatment with nocodazole for 2 h caused a fourfold decrease in the gamma-tubulin content in mitotic poles. Besides, the mitotic poles were unevenly stained, the fluorescence intensity in the center was lower than at the periphery. Upon treatment with taxol (10 microg/ml), the gamma-tubulin content in the interphase cell centrosome first decreased, then increased, and in 24 h it doubled as compared with control. In the latter case, bright dots appeared in the cell cytoplasm along the microtubule bundles. However, after 24 h treatment with taxol, the total amount of intracellular gamma-tubulin did not change. Treatment with taxol for 2-4 h halved the gamma-tubulin content in the centrosome as compared with normal mitosis. In some cells, antibodies against gamma-tubulin revealed up to four microtubule convergence foci. Other numerous microtubule convergence foci were not stained. Thus, the existence of at least three gamma-tubulin pools is suggested: (1) constitutive gamma-tubulin permanently associated with centrioles irrespective of the cell cycle stage and of their ability to serve as microtubule organizing centers; (2) gamma-tubulin unstably associated with the centrosome only during mitosis; (3) cytoplasmic gamma-tubulin that can bind to stable microtubules.     

Vertebrate primary cilia: a sensory part of centrosomal complex in tissue cells,but a “sleeping beauty” in cultured cells?

Primary cilium development along with other components of the centrosome in mammalian cells was analysed ultrastructurally and by immunofluorescent staining with anti-acetylated tubulin antibodies. We categorized two types of primary cilia, nascent cilia that are about 1microm long located inside the cytoplasm, and true primary cilia that are several microm long and protrude from the plasma membrane. The primary cilium is invariably associated with the older centriole of each diplosome, having appendages at the distal end and pericentriolar satellites with cytoplasmic microtubules emanating from them. Only one cilium per cell is formed normally through G(0), S and G(2)phases. However, in some mouse embryo fibroblasts with two mature centrioles, bicilates were seen. Primary cilia were not observed in cultured cells where the mature centriole had no satellites and appendages (Chinese hamster kidney cells, line 237, some clones of l-fibroblasts). In contrast to primary cilia, striated rootlets were found around active and non-active centrioles with the same frequency. In proliferating cultured cells, a primary cilium can be formed several hours after mitosis, in fibroblasts 2-4 h after cell division and in PK cells only during the S-phase. In interphase cells, formation of the primary cilium can be stimulated by the action of metabolic inhibitors and by reversed depolymerization of cytoplasmic microtubules with cold or colcemid treatments. In mouse renal epithelial cells in situ, the centrosome was located near the cell surface and mature centrioles in 80% of the cells had primary cilium protruding into the duct lumen. After cells were explanted and subcultured, the centrosome comes closer to the nucleus and the primary cilium was depolymerized or reduced. Later primary cilia appeared in cells that form islets on the coverslip. However, the centrosome in cultured ciliated cells was always located near the cell nucleus and primary cilium never formed a characteristic distal bulb. A sequence of the developmental stages of the primary cilium is proposed and discussed. We also conclude that functioning primary cilium does not necessarily operate in culture cells, which might explain some of the contradictory data on cell ciliation in vitro reported in the literature.     

Distribution of tyrosinated and acetylated tubulin in centrioles during mitosis of 3T3 and SV40-3T3 cells

We performed a comparative electron microscopic analysis of centriolar and cytoplasmic microtubules stained with antibodies to acetylated or tyrosinated α-tubulin during the cell cycle of mouse nonmalignant Balb 3T3 (clone A31) and virus-transformed heteroploid SV40-3T3 cell lines. It was shown that the pattern of centriole immunostaining changed during the cell cycle in 3T3 (A31) cells, but not in tumorigenic SV40-3T3 cells. Remarkable changes in the centriole immunostaining pattern were observed during interphase-mitosis or mitosis-interphase transitions when the microtubule system and protein organization of centrosomes underwent drastic rearrangements. A high level of tyrosinated tubulin in centrioles was observed at all stages of the cell cycle except when entering mitosis, whereas a high level of acetylated tubulin was visualized in centrioles at all stages of the cell cycle except at the end of mitosis.     

Electron microscope demonstration of tubulin in cilia and basal bodies of rat tracheal epithelium by the use of an antitubulin antibody

It has been previously demonstrated that both cytoplasmic microtubules and the microtubules of cilia, flagella, and sperm tail contain tubulin. Although the morphology of cytoplasmic microtubules and that of axonemes differs in cells from which they have been isolated, the tubulin of the two structures shares physical and chemical properties. In some mammalian tissues, such as tracheal epithelium, cilia and basal bodies are difficult to isolate and characterize. The use of an enzyme- labeled immunoglobulin probe would facilitate identification and in situ localization of such proteins. Tubulin prepared from porcine brain by ion-exchange chromatography and from rat brain by the method of cyclic polymerization and depolymerization with subsequent disk gel electrophoresis with SDS were injected intravenously into rabbits. The animals were intermittently bled and the antisera extracted. The specificity of the antisera was proved by indirect immunofluorescence staining of the mitotic spindle, specific blocking of spindle staining by purified tubulin and not by other proteins, staining of 3T3 cytoplasmic microtubules, single line on immunoelectrophoresis, failure of control antisera to show any of these, and precipitation of antibody with all tubulin preparations and not with actin. We have shown by electron microscopy of ciliated cells of the tracheal epithelium stained with antitubulin by the indirect enzyme-labeled antibody method that the basal bodies, outer doublets, and central pair of the cilia contain tubulin. This indicates that tubulin in microtubules of cilia and basal bodies of rat tracheal epithelium is antigenically similar to tubulin extracted from cytoplasmic neurotubules of brains from the same species and from a different mammalian species. No other axonemal structures stained with the antitubulin. Three different preparations of tubulin from pigs and rats were used to immunize rabbits. All elicited similar antisera which gave identical staining patterns. The specificity of the staining was demonstrated by the absence of staining with immune serum absorbed with purified tubulin, the absence of staining with preimmune serum, and the absence of staining if any of the reagents were omitted during the staining reaction.     

Monoclonal antibodies to kinesin heavy and light chains stain vesicle- like structures, but not microtubules, in cultured cells

Kinesin, a microtubule-activated ATPase and putative motor protein for the transport of membrane-bounded organelles along microtubules, was purified from bovine brain and used as an immunogen for the production of murine monoclonal antibodies. Hybridoma lines that secreted five distinct antikinesin IgGs were cloned. Three of the antibodies reacted on immunoblots with the 124-kD heavy chain of kinesin, while the other two antibodies recognized the 64-kD light chain. When used for immunofluorescence microscopy, the antibodies stained punctate, cytoplasmic structures in a variety of cultured mammalian cell types. Consistent with the identification of these structures as membrane-bounded organelles was the observation that cells which had been extracted with Triton X-100 before fixation contained little or no immunoreactive material. Staining of microtubules in the interphase cytoplasm or mitotic spindle was never observed, nor were associated structures, such as centrosomes and primary cilia, labeled by any of the antibodies. Nevertheless, in double-labeling experiments using antibodies to kinesin and tubulin, kinesin-containing particles were most abundant in regions where microtubules were most highly concentrated and the particles often appeared to be aligned on microtubules. These results constitute the first direct evidence for the association of kinesin with membrane-bounded organelles, and suggest a molecular mechanism for organelle motility based on transient interactions of organelle-bound kinesin with the microtubule surface.     

Small heat shock protein 27 (HSP27) associates with tubulin/microtubules in HeLa cells

One of the monoclonal antibodies raised against mitotic HeLa cells (termed as mH3) recognized a 27-kDa protein and stained microtubules in the mitotic spindles of HeLa cells. Immunoscreening of a HeLa cDNA library revealed that mH3 antigen is a small heat shock protein, HSP27. Immunoprecipitation analysis using mH3 suggested that both alpha- and beta-tubulin are associated with HSP27. Further, sucrose-cushioned ultra centrifugation revealed that HSP27 is co-sedimented with taxol-stabilized microtubules. These results indicate that HSP27 associates with tubulin/microtubules in HeLa cells.     

The cytoskeleton of hydatid cyst cultured cells and its sensitivity to inhibitors

Cell cultures obtained from the germinal layer of hydatid cysts of the parasitic tapeworm Echinococcus granulosus were characterized with respect to their microtubule and microfilament systems. These were stained using monospecific antibodies against tubulin from sea urchin spermatozoa or sheep brain and against Dictyostelium discoideum actin as well as rhodamine conjugated phalloidin. The results show that the distribution of microtubules nad actin containing fibres of these cells is remarkably similar to that of mammalian cells both during interphase and mitosis. Hydatid cells, however, could not be stained with a specific antivimentin antibody. Indirect immunofluorescence with antitubulin antibodies of inhibitor treated cells shows that hydatid cell microtubules are sensitive to several antimicrotubular drugs including benzimidazole derivatives, colchicine, vinblastine, and griseofulvin.     

CONFOCAL ANALYSIS OF PRIMARY CILIA STRUCTURE AND COLOCALIZATION WITH THE GOLGI APPARATUS IN CHONDROCYTES AND AORTIC SMOOTH MUSCLE CELLS

Detyrosinated and acetylated α-tubulins represent a stable pool of tubulin typically associated with microtubules of the centrosome and primary cilium of eukaryotic cells. Although primary cilium—centrosome and centrosome—Golgi relationships have been identified independently, the precise structural relationship between the primary cilium and Golgi has yet to be specifically defined. Confocal immunohistochemistry was used to localize detyrosinated (ID5) and acetylated (6-11B-1) tubulin antibodies in primary cilia of chondrocytes and smooth muscle cells, and to demonstrate their relationship to the Golgi complex identified by complementary lectin staining with wheat germ agglutinin. The results demonstrate the distribution and inherent structural variation of primary cilia tubulins, and the anatomical interrelationship between the primary cilium, the Golgi apparatus and the nucleus. We suggest that these interrelationships may form part of a functional feedback mechanism which could facilitate the directed secretion of newly synthesized connective tissue macromolecules.     

Acetylated alpha-tubulin in Physarum: immunological characterization of the isotype and its usage in particular microtubular organelles

We have used monoclonal antibodies specific for acetylated and unacetylated alpha-tubulin to characterize the acetylated alpha-tubulin isotype of Physarum polycephalum, its expression in the life cycle, and its localization in particular microtubular organelles. We have used the monoclonal antibody 6-11B-1 (Piperno, G., and M. T. Fuller, 1985, J. Cell Biol., 101:2085-2094) as the probe for acetylated alpha-tubulin and have provided a biochemical characterization of the monoclonal antibody KMP-1 as a probe for unacetylated tubulin in Physarum. Concomitant use of these two probes has allowed us to characterize the acetylated alpha-tubulin of Physarum as the alpha 3 isotype. We have detected this acetylated alpha 3 tubulin isotype in both the flagellate and in the myxameba, but not in the plasmodium. In the flagellate, acetylated tubulin is present in both the flagellar axonemes and in an extensive array of cytoplasmic microtubules. The extensive arrangement of acetylated cytoplasmic microtubules and the flagellar axonemes are elaborated during the myxameba-flagellate transformation. In the myxameba, acetylated tubulin is not present in the cytoplasmic microtubules nor in the mitotic spindle microtubules, but is associated with the two centrioles of this cell. These findings, taken together with the apparent absence of acetylated alpha-tubulin in the ephemeral microtubules of the plasmodium suggest a natural correspondence between the presence of acetylated alpha-tubulin and microtubule organelles that are intrinsically stable or cross-linked.     

Microtubules in mouse embryo fibroblasts extracted with Triton X-100

Treatment of mouse embryo fibroblasts with 1% Triton X-100 at 37 degrees C in the presence of 4M glycerol and 1 mM EGTA results in the extraction of about 80% cellular proteins. Indirect immunofluorescent staining with monospecific antibodies against tubulin showed that extracted cultures contained a well developed system of cytoplasmic microtubules, indistinguishable from a system of control non-extracted cells. Microtubules in extracted cells were sensitive to Ca2+ ions, and to cold or prolonged incubation in a glycerol-free buffer. Sodium dodecylsulphate-polyacrilamide gel electrophoresis revealed proteins co-electrophoresed with tubulin and actin in Triton-treated cultures. Electron microscopy demonstrated the presence of both microtubules and microfilament bundles in the extracted cells, but complete dissolution of plasma and intracellular membranes.     

Post-translational modifications and multiple tubulin isoforms in Nicotiana tabacum L. cells

Distribution of post-translationally modified tubulins in cells of Nicotiana tabacum L. was analysed using a panel of specific antibodies. Polyglutamylated, tyrosinated, nontyrosinated, acetylated and Δ2-tubulin variants were detected on α-tubulin subunits; polyglutamylation was also found on β-tubulin subunits. Modified tubulins were detected by immunofluorescence microscopy in interphase microtubules, preprophase bands, mitotic spindles as well as in phragmoplasts. They were, however, located differently in the various microtubule structures. The antibodies against tyrosinated, acetylated and polyglutamylated tubulins gave uniform staining along all microtubules, while antibodies against nontyrosinated and Δ2-tubulin provided dot-like staining of interphase microtubules. Additionally, immunoreactivity of antibodies against acetylated and Δ2-tubulins was strong in the pole regions of mitotic spindles. High-resolution isoelectric focusing revealed 22 tubulin charge variants in N. tabacum suspension cells. Immunoblotting with antibodies TU-01 and TU-06 against conserved antigenic determinants of α- and β-tubulin molecules, respectively, revealed that 11 isoforms belonged to the α-subunit and 11 isoforms to the β-subunit. Whereas antibodies against polyglutamylated, tyrosinated and acetylated tubulins reacted with several α-tubulin isoforms, antibodies against nontyrosinated and Δ2-tubulin reacted with only one. The combined data demonstrate that plant tubulin is extensively post-translationally modified and that these modifications participate in the generation of plant tubulin polymorphism. Received: 2 May 1996 / Accepted: 16 September 1996     

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.     

Cytoplasmic microtubules containing acetylated alpha-tubulin in Chlamydomonas reinhardtii: spatial arrangement and properties

A monoclonal antibody, 6-11B-1, specific for acetylated alpha-tubulin (Piperno, G., and M. T. Fuller, 1985, J. Cell Biol., 101:2085-2094) was used to study the distribution of this molecule in interphase cells of Chlamydomonas reinhardtii. Double-label immunofluorescence was performed using 6-11B-1, and 3A5, an antibody specific for all alpha-tubulin isoforms. It was found that acetylated alpha-tubulin is not restricted to the axonemes, but is also present in basal bodies and in a subset of cytoplasmic microtubules that radiate from the basal bodies just beneath the plasma membrane. Immunoblotting experiments of basal body polypeptide components using 6-11B-1 as a probe confirmed that basal bodies contain acetylated alpha-tubulin. In the cell body, 6-11B-1 stained an average of 2.2 microtubules/cell, while 3A5 stained an average of 6.5 microtubules. Although exposure to 0 degrees C depolymerized both types of cytoplasmic microtubules, exposure to various concentrations of colchicine or nocodazole showed that the acetylated microtubules are much more resistant to drug-induced depolymerization than nonacetylated microtubules. Axonemes and basal bodies are already known to be colchicine-resistant. All acetylated microtubules appear, therefore, to be more drug-resistant than nonacetylated microtubules. The acetylation of alpha-tubulin may be part of a mechanism that stabilizes microtubules.     

The orientation of primary cilia during the wound response in 3Y1 cells

Summary— The behavior of the primary cilia of 3Y1 cells in the interphase was investigated by indirect immunofluorescence microscopy and transmission electron microscopy, using an antibody for tubulin. At 4.5 h after scraping a part of a confluent cell sheet, the primary cilia of cells facing the wound were located predominantly forward of the nucleus on the wounded side, and were oriented in the direction of the leading lamellae. Cytoplasmic microtubules (MTs), emanating from around the base of the cilia, were well developed in the leading lamellae on the wounded side. On the other hand, in the cells of an unperturbed area away from the wounded edge, the primary cilia remained randomly distributed near the nucleus. The position and a certain well-defined orientation of a pair of centrioles seem to play an important role for the development of cytoplasmic MTs, and consequently the orientation of the centrioles is controlled by the primary cilia.     

Differential Distribution of Posttranslationally Modified Microtubules in Osteoclasts

The differential distribution of microtubules in osteoclasts in culture was examined by using antibodies against acetylated, tyrosinated, or detyrosinated tubulins. Tyrosinated tubulin was found throughout the cytoplasmic microtubules in all cells examined. An expanding protrusion that contained tyrosinated tubulin but none of the detyrosinated or acetylated form was seen in the immature osteoclasts. Detyrosinated or acetylated tubulin was detectable in the peripheral cytoplasm of the mature osteoclasts displaying the loss of the expanding protrusion. Although most of the microtubules were derived from the centrosome, noncentrosomal microtubules were distributed in the expanding protrusion, which was predominantly positive for tyrosinated tubulin. By tracing single microtubules, the authors found that their growing ends were always rich in tyrosinated tubulin subunits. End binding protein 1 bound preferentially to the microtubule ends. Both acetylated and tyrosinated microtubules were shown to be closely associated with podosomes. Microtubules appeared to grow over or into the podosomes; in addition, the growing ends of single microtubules could be observed to target the podosomes. Moreover, a microtubule-associated histone deacetylase 6 was localized in the podosomes of the osteoclast. On the basis of these results, the authors conclude that posttranslational modifications of microtubules may correlate with characteristic changes in podosome dynamics in osteoclasts.     

Posttranslational modifications of alpha tubulin: detyrosination and acetylation differentiate populations of interphase microtubules in cultured cells

Subsets of microtubules enriched in posttranslationally detyrosinated (Gundersen, G. G., M. H. Kalnoski, and J. C. Bulinski. 1984. Cell. 38:779) or acetylated (Piperno, G., M. Le Dizet, and X. Chang. 1987. J. Cell Biol. 104:298), alpha tubulin have previously been described in interphase cultured cells. In this study an immunofluorescence comparison of these minor populations of microtubules revealed that, in African green monkey kidney epithelial cells (TC-7 line), the population of microtubules enriched in detyrosinated tubulin was virtually coincident with the population enriched in acetylated alpha tubulin. In some cell types, however, such as human HeLa or marsupial PtK-2 cells, only one posttranslationally modified form of tubulin, i.e., acetylated or detyrosinated, respectively, was detectable in microtubules. In TC-7 cells, although both modifications were present, dissimilar patterns and kinetics of reappearance of microtubules enriched in detyrosinated and acetylated tubulin were observed after recovery of cells from microtubule-depolymerizing treatments or from mitosis. Thus, a minor population of microtubules exists in cultured cells that contains an elevated level of tubulin modified in either one or two ways. While these two modifications occur primarily on the same subset of microtubules, they differ in their patterns of formation in vivo.