Immunocytochemical localization of tubulins in spermatids and spermatozoa of Euptoieta hegesia (Lepidoptera: Nymphalidae)

A comparative analysis of the distribution of tubulin types in apyrene and eupyrene sperm of Euptoieta hegesia butterflies was carried out, also verifying the presence of tubulin in lacinate appendages of the eupyrene sperm. Ultrathin sections of LR White embedded spermatids and spermatozoa were labeled for alpha, beta, gamma, alpha-acetylated and alpha-tyrosinated tubulins. Apyrene and eupyrene spermatids show the same antibody recognition pattern for tubulins. All tubulin types were detected in axonemal microtubules. Alpha and gamma tubulins were also detected on the cytoplasmic microtubules. However, for beta and tyrosinated tubulins only scattered labeling was detected on cytoplasmic microtubules and acetylated tubulin was not detected. In apyrene and eupyrene spermatozoa only the axoneme labeling was analyzed since cytoplasmic microtubules no longer exist in these cells. Alpha, beta and tyrosinated tubulins were easily detected on the apyrene and eupyrene axoneme; gamma tubulin was strongly marked on eupyrene axonemes but was scattered on the apyrene ones. Acetylated tubulin appeared with scattered labeling on the axoneme of both sperm types. Our results demonstrate significant differences in tubulin distribution in apyrene and eupyrene axonemal and cytoplasmic microtubules. Extracellular structures, especially the lacinate appendages, were not labeled by antibodies for any tubulin.     

Composition and organization of tubulin isoforms reveals a variety of axonemal models

In the flagellum of mammalian spermatozoa, glutamylated and glycylated tubulin isoforms are detected according to longitudinal gradients and preferentially in axonemal doublets 1-5-6 and 3-8, respectively. This suggested a role for these tubulin isoforms in the regulation of flagellar beating. In the present work, using antibodies directed against various tubulin isoforms and quantitative immunogold analysis, we aimed at investigating whether the particular accessibility of tubulin isoforms in the mammalian sperm flagellum is restricted to this model of axoneme surrounded with periaxonemal structures or is also displayed in naked axonemes. In rodent lung ciliated cells, all studied tubulin isoforms are uniformly distributed in all axonemal microtubules with a unique deficiency of glutamylated tubulin in the transitional region. A similar distribution of tubulin isoforms is observed in cilia of Paramecium, except for a decreasing gradient of glutamylated tubulin labeling in the proximal part of axonemal microtubules. In the sea urchin sperm flagellum, predominant labeling of tyrosinated and detyrosinated tubulin in 1-5-6 and 3-8 doublets, respectively, were observed together with decreasing proximo-distal gradients of glutamylated and polyglycylated tubulin labeling and an increasing gradient of monoglycylated tubulin labeling. In flagella of Chlamydomonas, the glutamylated and glycylated tubulin isoforms are detected at low levels. Our results show a specific composition and organization of tubulin isoforms in different models of cilia and flagella, suggesting various models of functional organization and beating regulation of the axoneme.     

The acetylation of alpha-tubulin and its relationship to the assembly and disassembly of microtubules

A tight association between Chlamydomonas alpha-tubulin acetyltransferase (TAT) and flagellar axonemes, and the cytoplasmic localization of both tubulin deacetylase (TDA) and an inhibitor of tubulin acetylation have been demonstrated by the use of calf brain tubulin as substrate for these enzymes. A major axonemal TAT of 130 kD has been solubilized by high salt treatment, purified, and characterized. Using the Chlamydomonas TAT with brain tubulin as substrate, we have studied the effects of acetylation on the assembly and disassembly of microtubules in vitro. We also determined the relative rates of acetylation of tubulin dimers and polymers. The acetylation does not significantly affect the temperature-dependent polymerization or depolymerization of tubulin in vitro. Furthermore, polymerization of tubulin is not a prerequisite for the acetylation, although the polymer is a better substrate for TAT than the dimer. The acetylation is sensitive to calcium ions which completely inhibit the acetylation of both dimers and polymers of tubulin. Acetylation of the dimer is not inhibited by colchicine; the effect of colchicine on acetylation of the polymer can be explained by its depolymerizing effect on the polymer.     

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.     

A monoclonal antibody against the dynein IC1 peptide of sea urchin spermatozoa inhibits the motility of sea urchin, dinoflagellate, and human flagellar axonemes.

To investigate the role of axonemal components in the mechanics and regulation of flagellar movement, we have generated a series of monoclonal antibodies (mAb) against sea urchin (Lytechinus pictus) sperm axonemal proteins, selected for their ability to inhibit the motility of demembranated sperm models. One of these antibodies, mAb D1, recognizes an antigen of 142 kDa on blots of sea urchin axonemal proteins and of purified outer arm dynein, suggesting that it acts by binding to the heaviest intermediate chain (IC1) of the dynein arm. mAb D1 blocks the motility of demembranated sea urchin spermatozoa by modifying the beating amplitude and shear angle without affecting the ATPase activity of purified dynein or of demembranated immotile spermatozoa. Furthermore, mAb D1 had only a marginal effect on the velocity of sliding microtubules in trypsin-treated axonemes. This antibody was also capable of inhibiting the motility of flagella of Oxyrrhis marina, a primitive dinoflagellate, and those of demembranated human spermatozoa. Localization of the antigen recognized by mAb D1 by immunofluorescence reveals its presence on the axonemes of flagella from sea urchin spermatozoa and O. marina but not on the cortical microtubule network of the dinoflagellate. These results are consistent with a dynamic role for the dynein intermediate chain IC1 in the bending and/or wave propagation of flagellar axonemes.     

Acetylation of lysine 40 in alpha-tubulin is not essential in Tetrahymena thermophila

In Tetrahymena, at least 17 distinct microtubule structures are assembled from a single primary sequence type of alpha- and beta- tubulin heterodimer, precluding distinctions among microtubular systems based on tubulin primary sequence isotypes. Tetrahymena tubulins also are modified by several types of posttranslational reactions including acetylation of alpha-tubulin at lysine 40, a modification found in most eukaryotes. In Tetrahymena, axonemal alpha-tubulin and numerous other microtubules are acetylated. We completely replaced the single type of alpha-tubulin gene in the macronucleus with a version encoding arginine instead of lysine 40 and therefore cannot be acetylated at this position. No acetylated tubulin was detectable in these transformants using a monoclonal antibody specific for acetylated lysine 40. Surprisingly, mutants lacking detectable acetylated tubulin are indistinguishable from wild-type cells. Thus, acetylation of alpha- tubulin at lysine 40 is non-essential in Tetrahymena. In addition, isoelectric focusing gel analysis of axonemal tubulin from cells unable to acetylate alpha-tubulin leads us to conclude that: (a) most or all ciliary alpha-tubulin is acetylated, (b) other lysines cannot be acetylated to compensate for loss of acetylation at lysine 40, and (c) acetylated alpha-tubulin molecules in wild-type cells contain one or more additional charge-altering modifications.     

Axoneme-dependent tubulin modifications in singlet microtubules of the Drosophila sperm tail

Drosophila melanogaster sperm tubulins are posttranslationally glutamylated and glycylated. We show here that axonemes are the substrate for these tubulin C-terminal modifications. Axoneme architecture is required, but full length, motile axonemes are not necessary. Tubulin glutamylation occurs during or shortly after assembly into the axoneme; only glutamylated tubulins are glycylated. Tubulins in other testis microtubules are not modified. Only a small subset of total Drosophila sperm axoneme tubulins have these modifications. Biochemical fractionation of Drosophila sperm showed that central pair and accessory microtubules have the majority of poly-modified tubulins, whereas doublet microtubules have only small amounts of mono- and oligo-modified tubulins. Glutamylation patterns for different beta-tubulins experimentally assembled into axonemes were consistent with utilization of modification sites corresponding to those identified in other organisms, but surrounding sequence context was also important. We compared tubulin modifications in the 9 + 9 + 2 insect sperm tail axonemes of Drosophila with the canonical 9 + 2 axonemes of sperm of the sea urchin Lytichinus pictus and the 9 + 0 motile sperm axonemes of the eel Anguilla japonica. In contrast to Drosophila sperm, L. pictus sperm have equivalent levels of modified tubulins in both doublet and central pair microtubule fractions, whereas the doublets of A. japonica sperm exhibit little glutamylation but extensive glycylation. Tubulin C-terminal modifications are a prevalent feature of motile axonemes, but there is no conserved pattern for placement or amount of these     

Accessory tubules and axonemal microtubules of Apis mellifera sperm flagellum differ in their tubulin isoform content

In the insect sperm flagellum, an extra set of nine additional microtubules, named accessory tubules, is present surrounding the axoneme. Using a sarcosyl/urea extraction, we were able to fractionate the microtubular cytoskeleton of the sperm flagellum of the insect Apis mellifera resulting in the dissociation of the axonemal microtubule protein components and the accessory tubules. This has allowed us to compare the tubulin isoform content of axonemal microtubules and accessory tubules by immunoelectron microscopy and immunoblotting using a panel of monoclonal antibodies directed against different tubulin post-translational modifications (PTMs). All the PTMs occurring in axonemal tubulin are also present in accessory tubules, which indicates the close relativeness of accessory tubules to axonemal rather than to cytoplasmic microtubules. However, our results demonstrate the presence of significant differences in the tubulin isoform content of axonemal microtubules and accessory tubules. First, the tubulin tyrosination extent of accessory tubules is far lower than that of axonemal microtubules, thus confirming at the molecular level their morphogenetic origin as outgrowths from the B-subtubule of each microtubular doublet. Second, although polyglycylation seems to occurr at the same extent in both microtubular systems, alpha-tubulin exhibits a larger amount of monoglycylated sites in axonemal microtubules than in accessory tubules. Third, a greater amount of beta-tubulin molecules is glutamylated in axonemal microtubules than in accessory tubules. Moreover, highly acidic isoforms, likely molecules with longer polyglutamate side chains, are present only in axonemal microtubules. Taken together, our data are indicative of a higher level of tubulin heterogeneity in axonemal microtubules than in accessory tubules. They also show a segregation of post-translationally modified isoforms between accessory tubules and axonemal microtubules and suggest the implication of PTMs in the functional specialization of the two microtubular systems.     

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.     

Tubulin evolution: Two major types of α-tubulin

Summary Tubulin subunits have been isolated from a variety of protists and marine invertebrates. The sources were: sperm tails of a tunicate (Ciona intestinalis), an abalone (Haliotis rufescens) and a sea anemone (Tealia crassicornis), the gill cilia of a clam (Mercenaria mercenaria), the cilia of a ciliate (Tetrahymena pyriformis) and the cytoplasm of a slime mold (Physarum polycephalum). All the -tubulins, as characterised by their electropherograms after limited proteolytic cleavage withStaphylococcus aureus protease, were fairly similar. In contrast, two markedly different peptide patterns were found for the -tubulins of (a) metazoan axonemes and (b) protistan axonemes, plant axonemes and slime mold cytoplasm.Metazoan axonemal -tubulin peptide patterns could be further divided into two similar but distinct subtypes which did not correlate with the taxonomic divisions of deuterostomia and protostomia, or to different tubulins within an axoneme, or to different tubulins of flagella and cilia. We have postulated that these small differences may be accounted for by a simple glutamicaspartic acid exchange at a particular position in the -tubulin sequence. Identical peptide patterns were observed for sea urchin and sea anemone sperm tail tubulins, proving that the metazoan type of axonemal tubulin arose before the divergence of bilateral and radial symmetric organisms.The close similarity of the slime mold cytoplasmic -tubulin peptide pattern to protistan and plant axonemal -tubulin patterns suggests that the same type of tubulin might be used to form both axonemal and cytoplasmic types of microtubules in protists and plants. The large structural constraints imposed upon this tubulin molecule probably allowed very little change in its primary structure, thus explaining the similarity of tubulins from organisms which diverged at such an early time in eukaryote history. Duplication and modification of the tubulin gene may then have led to the development of specific axonemal and cytoplasmic microtubules during the evolution of the metazoa.     

Microtubule diversity in ciliated cells: evidence for its generation by post-translational modification in the axonemes of Paramecium and quail oviduct cells.

The diversity of microtubular networks was analyzed in quail oviduct and in Paramecium cells using conventional and confocal immunofluorescence as well as pre- and post-embedding EM immunocytochemistry with a variety of anti-tubulin antibodies. The 6-11B-1 monoclonal antibody, specific for the post-translational acetylation of Lys 40 of alpha-tubulin, and a polyclonal antibody raised against Paramecium axonemal tubulin (anti-PA tubulin antibody) both decorated stable microtubular arrays in Paramecium ie ciliary axonemes and a set of microtubular bundles associated with the cortex, suggesting that the two antibodies may be directed against the same epitope. However, several differences in the immunocytological patterns yielded by each antibody on the two cell types were evident. For example, in quail, as in all other Metazoa, the anti-PA tubulin antibody only decorated axonemes enclosed in normal ciliary membrane while it was unreactive on cytoplasmic tubulins. Immunoblotting of peptide maps of axonemal tubulins demonstrated that the epitopes of the two antibodies were indeed completely different. Double immunolabelling of dividing paramecia using a universal anti-tubulin antibody and the anti-PA tubulin one revealed that all newly assembled microtubular arrays were first detected by the universal antibody and, only shortly afterwards, by the anti-PA tubulin one. This provided a strong indication that the anti-PA tubulin antibody is directed against a post-translational modification taking place on already assembled microtubules (MTs) (as previously known to be the case for acetylation and detyrosination). In taxol-treated quail cells undergoing ciliogenesis, massive assembly of MTs and even axonemes occurred in the cytoplasm. These MTs were not decorated by the anti-PA tubulin antibody however, suggesting that in Metazoa the post-translational modification can only take place within the ciliary lumen. The present work provides one further mechanism for generating MT immunological and biochemical diversity post-translationally; this may account for the high multiplicity of tubulin isoforms observed in ciliates which contain very little if any genetic diversity of tubulin genes.     

Acetylated alpha-tubulin in microtubules during mouse fertilization and early development

alpha-Tubulin in the microtubules of mouse oocytes and embryos is acetylated in a specific spatial and temporal sequence. In the unfertilized oocyte, a monoclonal antibody to the acetylated form of alpha-tubulin is bound predominantly at the poles of the arrested metaphase meiotic spindle. The labeling intensity of the spindle microtubules is weaker as observed by immunofluorescence using oocytes double-labeled for total tubulin and acetylated alpha-tubulin, and as measured by immuno high-voltage electron microscopy (immunoHVEM) with colloidal gold; cytasters are not acetylated. At meiotic anaphase, the spindle becomes labeled, and by telophase and during second polar body formation only the meiotic midbody is acetylated. The sperm axoneme retains its acetylation after incorporation though the interphase microtubules are not detected. First mitosis follows a pattern similar to that observed at the second meiosis and during interphase only the mitotic midbodies are acetylated. After treatment with cold, colcemid, or griseofulvin, the remaining stable microtubules are acetylated, but immunoHVEM observations suggest that these fibers might not have been acetylated prior to microtubule disruption. Taxol stabilization does not alter acetylation patterns. Acetylated microtubules are not necessarily old microtubules since acetylated fibers are observed at 30 sec after cold recovery. These results show the presence of acetylated microtubules during meiosis and mitosis and demonstrate a cell-cycle-specific pattern of acetylation, with acetylated microtubules found at the centrosomes at metaphase, an increase in spindle labeling at anaphase, and the selective deacetylation of all but midbody microtubules at telophase.     

Posttranslational modifications of tubulin in teleost photoreceptor cytoskeletons

1. Posttranslational modifications of tubulin by acetylation and detyrosination have been correlated previously with microtubule stability in numerous cell types. 2. In this study, posttranslational modifications of tubulin and their regional distribution within teleost photoreceptor cones and rods are demonstrated immunohistochemically using antibodies specific for acetylated, detyrosinated, or tyrosinated tubulin. 3. Immunolocalization was carried out on isolated whole cones and mechanically detached rod and cone inner/outer segments. 4. Acetylated tubulin within rods and cones is found only in microtubules of the ciliary axoneme of the outer segment. Detyrosinated tubulin is also enriched in axonemes of both rod and cone outer segments. 5. Distributions of tyrosinated and detyrosinated cytoplasmic microtubules differ within cones and rods. In cones, detyrosinated and tyrosinated tubulins are both abundant throughout the cell body. In rods, the ellipsoid and myoid contain much more tyrosinated tubulin than detyrosinated tubulin. Comparisons between whole cones and cone fragments suggest that detyrosinated microtubules are more stable than tyrosinated microtubules in teleost photoreceptors. 6. Our findings provide further evidence that microtubules of teleost cones differ from rod microtubules in their stabilities and rapidity of turnover within the photoreceptor inner segment.     

Molecular chaperones in cilia and flagella: implications for protein turnover

The mechanisms of protein incorporation and turnover in 9+2 ciliary axonemes are not known. Previous reports of an HSP70-related protein, first in Chlamydomonas flagella and then in sea urchin embryonic cilia, suggested a potential role in protein transport or incorporation. The present study further explores this and other chaperones in axonemes from a representative range of organisms. Two-dimensional gel electrophoresis proved identity between the sea urchin ciliary 78 kDa HSP and a constitutive cytoplasmic HSP70 cognate (pI = 5.71). When isolated flagella from mature sea urchin sperm were analyzed, the same total amount and distribution of 78 kDa protein as in cilia were found. Antigens of similar size were detected in ctenophore comb plate, molluscan gill, and rabbit tracheal cilia. Absent from sea urchin sperm flagella, TCP-1alpha was detected in sea urchin embryonic and rabbit tracheal cilia; the latter also contained HSP90, detected by two distinct antibodies. Tracheal cilia were shown to undergo axonemal protein turnover while tracheal cells mainly synthesized ciliary proteins. TCP-1alpha progressively appeared in regenerating embryonic cilia only as their growth slowed, suggesting a regulatory role in incorporation or turnover. These results demonstrate that chaperones are widely distributed ciliary and flagellar components, potentially related to axonemal protein dynamics.     

Glutamylated tubulin: diversity of expression and distribution of isoforms

Glutamylation of alpha and beta tubulin isotypes is a major posttranslational modification giving rise to diversified isoforms occurring mainly in neurotubules, centrioles, and axonemes. Monoglutamylated tubulin isoforms can be differentially recognized by two mAbs, B3 and GT335, which both recognize either polyglutamylated isoforms. In the present study, immunoelectron microscopy and immunofluorescence analyses were performed with these two mAbs to determine the expression and distribution of glutamylated tubulin isoforms in selected biological models whose tubulin isotypes are characterized. In mouse spermatozoa, microtubules of the flagellum contain polyglutamylated isoforms except in the tip where only monoglutamylated isoforms are detected. In spermatids, only a subset of manchette microtubules contain monoglutamylated tubulin isoforms. Cytoplasmic microtubules of Sertoli cells are monoglutamylated. Mitotic and meiotic spindles of germ cells are monoglutamylated whereas the HeLa cell mitotic spindle is polyglutamylated. Three models of axonemes are demonstrated as a function of the degree and extent of tubulin glutamylation. In lung ciliated cells, axonemes are uniformly polyglutamylated. In sea urchin sperm and Chlamydomonas, flagellar microtubules are polyglutamylated in their proximal part and monoglutamylated in their distal part. In Paramecium, cilia are bi- or monoglutamylated only at their base. In all cells, centrioles or basal bodies are polyglutamylated. These new data emphasize the importance of glutamylation in all types of microtubules and strengthen the hypothesis of its role in the regulation of the intracellular traffic and flagellar motility.     

Comparative structural analysis of eukaryotic flagella and cilia from Chlamydomonas, Tetrahymena, and sea urchins

Although eukaryotic flagella and cilia all share the basic 9+2 microtubule-organization of their internal axonemes, and are capable of generating bending-motion, the waveforms, amplitudes, and velocities of the bending-motions are quite diverse. To explore the structural basis of this functional diversity of flagella and cilia, we here compare the axonemal structure of three different organisms with widely divergent bending-motions by electron cryo-tomography. We reconstruct the 3D structure of the axoneme of Tetrahymena cilia, and compare it with the axoneme of the flagellum of sea urchin sperm, as well as with the axoneme of Chlamydomonas flagella, which we analyzed previously. This comparative structural analysis defines the diversity of molecular architectures in these organisms, and forms the basis for future correlation with their different bending-motions.     

Subpellicular and flagellar microtubules of Trypanosoma brucei brucei contain the same alpha-tubulin isoforms

The cytoskeleton of the parasitic hemoflagellate Trypanosoma brucei brucei essentially consists of two microtubule-based structures: a subpellicular layer of singlet microtubules, which are in close contact with the cell membrane, and the flagellar axoneme. In addition, the cells contain a small pool of soluble tubulin. Two-dimensional gel electrophoretic analysis of the tubulins present in these subcellular compartments revealed two distinct electrophoretic isoforms of alpha-tubulin, termed alpha 1 and alpha 3. alpha 1-Tubulin most likely represents the primary translation product, while alpha 3-tubulin is a posttranslationally acetylated derivative of alpha 1-tubulin. In the pool of soluble cytoplasmic tubulin, alpha 1 is the predominant species, while the very stable flagellar microtubules contain almost exclusively the alpha 3-tubulin isoform. The subpellicular microtubules contain both isoforms. Neither of the two alpha-tubulin isoforms is organelle specific, but the alpha 3 isoform is predominantly located in stable microtubules.     

Exposure of lumenal microtubule sites after mild fixation

High-resolution analysis of tubulin structure and docking the structure of tubulin dimer into a map of microtubules led to a prediction that sites for tubulin acetylation are in the interior of microtubules. This is somehow difficult to reconcile with their susceptibility to proteases and acetylation in assembled microtubules. To assess the availability of acetylated alpha-tubulin for antibodies, immunofluorescence on detergent-extracted cells, on cells fixed under various conditions and in microinjected cells was performed with monoclonal antibodies of known epitope locations. The presented data indicate that acetylated alpha:Lys40 is not exposed on unfixed microtubules but that this region of lumenal microtubule surface becomes easily exposed under mild fixation conditions.     

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.     

Inner arm dynein ATPase fraction of sea urchin sperm flagella causes active sliding of axonemal outer doublet microtubule.

In order to clarify the role of the inner arms of the axoneme in sperm flagellar movement, we prepared an ATPase fraction (12S) from the outer arm-depleted axonemes of sea urchin sperm flagella. When both arm-depleted axonemes were incubated with the 12S ATPase, they exhibited the sliding disintegration of outer doublet microtubules. Electron microscopy revealed that the ATPase rebound to the original inner arm sites of the axoneme. Therefore, it is quite likely that the 12S ATPase is one of the components of the inner arms. We referred to it as "inner arm dynein".