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.     

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.     

Evaluation of various methods of measurement of the microtubule length in the cytoplasm of cultured cells

It is generally assumed that microtubules in tissue culture cells extend from the centrosome to cell periphery, and the length of individual microtubules averages several dozens of microns. However, direct electron-microscopic measurements have cast some doubt on this assumption. In this study, the average length of microtubules in cultured Vero cells was estimated using a combined approach. The length of free cytoplasmic and centrosomal microtubules was determined by means of electron microscopy in serial sections; concurrently, the length of free microtubules in the lamella was measured in preparations stained with tubulin antibodies (an indirect immunofluorescent method), by tracing saltatory particle movements along the microtubules in living cells. According to the data of immunofluorescent microscopy, microtubule length in the lamella averaged 4.57 +/- 3.69 microns. However, since two or more microtubules can overlap, their length may be slightly overestimated by this method. On the other hand, saltatory movements are easy to monitor and measure fairly accurately, but their range may be shorter than the actual microtubule length because of a limited processiveness of motors (kinesin and dynein). On average, the trajectories of saltatory movements in living cells were 3.85 +/- 0.72 microns long. At the electron-microscopic level, microtubule length was analyzed using pseudo-three-dimensional reconstructions of the microtubule systems around the centrosome and in the lamella. The length of free microtubules in the lamella reached 18 microns, averaging 3.33 +/- 2.43 microns; the average length of centrosomal microtubules was 1.49 +/- 0.82 microns. Good correspondence between the data on microtubule length and arrangement obtained by different methods allows the conclusion that most of free microtubules in Vero cells actually have a length of 2-5 microns; i.e., they are much shorter than the cell radius (about 25 microns). Microtubules extending from the centrosome are shorter still and do not reach the cell periphery. Thus, most microtubules in the lamella of Vero cells are free and their ordered arrangement is not associated with their attachment to the centrosome.     

Protoplast isolation,development, and regeneration in different strains ofPilayella littoralis (L.) Kjellm. (Phaeophyceae)

Summary Using an antibody against tyrosinated tubulin and epifluorescence microscopy, mitosis was studied in three different microvessel endothelial cell types recently isolated from bovine corpus luteum. Dividing cells were flat and at certain stages individual microtubules could be followed for considerable lengths. The structure of the spindle apparatus and the course of mitosis were conventional. Microtubule asters were small from prophase until metaphase in all three cell types. However, whereas in two cell types telophase asters remained inconspicuous, prominent asters, of mostly straight microtubules, formed in telophase cells of a third cell type. Thus, aster size is heterogeneous between different endothelial cell types. Large microtubule asters are not regularly found in dividing cultured mammalian cells. The microendothelial cell types present themselves as appropriate systems for spindle research and especially for the study of aster microtubule dynamics and function.     

Experimental model for studying the primary cilia in tissue culture cells.

In HeLa, PK, 3T3, PtK1 cells and rat embryo fibroblasts (REF), antibodies against acetylated tubulin stained centrioles, primary cilia, some cytoplasmic microtubules and microtubule bundles of the mid-body. The primary cilia were stained more intensively than cytoplasmic microtubules and could easily be distinguished. This makes it possible to detect the primary cilia in cultured cells and to estimate their number by light microscopy. The four cultures studied had 1/4 to 1/3 of interphase cells with detectable primary cilia, and only in HeLa cells the primary cilia were very rare. Comparison of electron microscopic and immunofluorescence data showed that the frequencies of occurrence of the primary cilia in four tissue cultures determined by these two methods were the same. Therefore, antibodies against acetylated tubulin can be used to study the primary cilia. In synchronized mitotic fibroblasts (3T3 and REF) the primary cilia appeared first 2 h after the cells had been plated on coverslips, which is 1 h after the cells had entered the interphase. Four hours after plating the number of ciliated cells reached the average level for nonsynchronous population. This model can be used for further studies of the expression of primary cilia.     

Posttranslational modification of distinct microtubule subpopulations during cell polarization and differentiation in the mouse preimplantation embryo

During the course of preimplantation development, the cells of the mouse embryo undergo both a major subcellular reorganization (at the time of compaction) and, subsequently, a process of differentiation as the phenotypes of trophectoderm and inner cell mass cell types diverge. We have used antibodies specific for tyrosinated (Kilmartin, J. V., B. Wright, and C. Milstein. 1982. J. Cell Biol. 93:576-582) and acetylated (Piperno, G., and M. T. Fuller. 1985. J. Cell Biol. 101:2085-2094) alpha-tubulin in immunofluorescence studies and found that subsets of microtubules can be distinguished within and between cells during the course of these events. Whereas all microtubules contained tyrosinated alpha-tubulin, acetylated alpha-tubulin was detected only in a subpopulation, located predominantly in the cell cortices. Striking differences developed between the distribution of the two populations during the course of development. Firstly, whereas the microtubule population as a whole tends to redistribute towards the apical domain of cells as they polarize during compaction (Houliston, E., S. J. Pickering, and B. Maro. 1987. J. Cell Biol. 104:1299-1308), the microtubules recognized by the antiacetylated alpha-tubulin antibody became enriched in the basal part of the cell cortex. After asymmetric division of polarized cells to generate two distinct cell types (termed inside and outside cells) we found that, despite the relative abundance of microtubules in outside cells, acetylated microtubules accumulated preferentially in inside cells. Treatment with nocodazole demonstrated that within each cell type acetylated microtubules were the more stable ones; however, the difference in composition of the microtubule network between cell types was not accompanied by a greater stability of the microtubule network in inside cells.     

Temporal and spatial pattern of differences in microtubule behaviour during Drosophila embryogenesis revealed by distribution of a tubulin isoform

Immunofluorescence staining of Drosophila embryos with a monoclonal antibody specific for acetylated alpha-tubulin has revealed that acetylated and nonacetylated alpha-tubulin isoforms have different patterns of distribution during early development. Acetylated alpha-tubulin was not detected in either interphase or mitotic spindle microtubules during the rapid early cleavage or syncytial blastoderm divisions. Acetylated alpha-tubulin was first observed as interphase lengthened at the end of syncytial blastoderm, and at cycle 14 was localized to a ring of structures clustered around the interphase nuclei. These structures probably represent a set of stable microtubules involved in nuclear elongation. Absence of detectable acetylated alpha-tubulin prior to cellular blastoderm seems to be due to rapid turnover of microtubule arrays rather than to lack of the enzyme required for modification, since acetylated alpha-tubulin appeared in early embryos when micro-tubules were stabilized by taxol treatment or anoxia. Because acetylated alpha-tubulin seems to be characteristic of stable microtubule arrays, the appearance of the antigen at cycle 14 represents a fundamental change in microtubule behaviour in the somatic cells of the embryo. Acetylated alpha-tubulin was not detected in pole cells during the blastoderm or early gastrula stages, indicating that acetylation of alpha-tubulin is not merely a consequence of cellularization. After the onset of gastrulation, interphase microtubule arrays in most cell types contain acetylated alpha-tubulin. However, cells in mitosis lack antibody staining. The resulting unstained patches reveal the stereotyped spatial pattern of cell division during gastrulation. Although the cells that give rise to the amnioserosa have acetylated alpha-tubulin in their interphase arrays at early gastrulation, by germ band elongation these large, plastic cells completely lack staining with anti-acetylated alpha-tubulin. In contrast, differentiated cell types such as neurones, which have arrays of stable axonal microtubules, stain brightly with the specific antibody. Although acetylated and nonacetylated alpha-tubulin are present in roughly equal amounts by the late stages of embryogenesis, acetylated alpha-tubulin is partitioned into the pellet during centrifugation of extracts of embryos homogenized at 4 degrees C.     

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.     

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.     

Dynamics of microtubule depolymerization in monocytes

Human monocytes, which contain few interphase microtubules (35.+/- 7.7), were used to study the dynamics of microtubule depolymerization. Steady-state microtubule assembly was abruptly blocked with either high concentrations of nocodazole (10 micrograms/ml) or exposure to cold temperature (3 degrees C). At various times after inhibition of assembly, cells were processed for anti-tubulin immunofluorescence microscopy. Stained cells were observed with an intensified video camera attached to the fluorescence microscope. A tracing of the entire length of each individual microtubule was made from the image on the television monitor by focusing up and down through the cell. The tracings were then digitized into a computer. All microtubules were seen to originate from the centrosome, with an average length in control cells of 7.1 +/- 2.7 microns (n = 957 microtubules). During depolymerization, the total microtubule polymer and the number of microtubules per cell decreased rapidly. In contrast, there was a slow decrease in the average length of the persisting microtubules. The half-time for both the loss of total microtubule polymer and microtubule number per cell was approximately 40 s for nocodazole-treated cells. The rate-limiting step in the depolymerization process was the rate of initiation of disassembly. Once initiated, depolymerization appeared catastrophic. Further kinetic analysis revealed two classes of microtubules: 70% of the microtubule population was very labile and initiated depolymerization at a rate approximately 23 times faster than a minor population of persistent microtubules. Cold treatment yielded qualitatively similar characteristics of depolymerization, but the initiation rates were slower. In both cases there was a significant asynchrony and heterogeneity in the initiation of depolymerization among the population of microtubules.     

Three-dimensional ultrastructural analysis of the Saccharomyces cerevisiae mitotic spindle

The three dimensional organization of microtubules in mitotic spindles of the yeast Saccharomyces cerevisiae has been determined by computer- aided reconstruction from electron micrographs of serially cross- sectioned spindles. Fifteen spindles ranging in length from 0.6-9.4 microns have been analyzed. Ordered microtubule packing is absent in spindles up to 0.8 micron, but the total number of microtubules is sufficient to allow one microtubule per kinetochore with a few additional microtubules that may form an interpolar spindle. An obvious bundle of about eight interpolar microtubules was found in spindles 1.3- 1.6 microns long, and we suggest that the approximately 32 remaining microtubules act as kinetochore fibers. The relative lengths of the microtubules in these spindles suggest that they may be in an early stage of anaphase, even though these spindles are all situated in the mother cell, not in the isthmus between mother and bud. None of the reconstructed spindles exhibited the uniform populations of kinetochore microtubules characteristic of metaphase. Long spindles (2.7-9.4 microns), presumably in anaphase B, contained short remnants of a few presumed kinetochore microtubules clustered near the poles and a few long microtubules extending from each pole toward the spindle midplane, where they interdigitated with their counterparts from the other pole. Interpretation of these reconstructed spindles offers some insights into the mechanisms of mitosis in this yeast.     

Three-dimensional analysis and ultrastructural design of mitotic spindles from the cdc20 mutant of Saccharomyces cerevisiae.

The three-dimensional organization of mitotic microtubules in a mutant strain of Saccharomyces cerevisiae has been studied by computer-assisted serial reconstruction. At the nonpermissive temperature, cdc20 cells arrested with a spindle length of approximately 2.5 microns. These spindles contained a mean of 81 microtubules (range, 56-100) compared with 23 in wild-type spindles of comparable length. This increase in spindle microtubule number resulted in a total polymer length up to four times that of wild-type spindles. The spindle pole bodies in the cdc20 cells were approximately 2.3 times the size of wild-type, thereby accommodating the abnormally large number of spindle microtubules. The cdc20 spindles contained a large number of interpolar microtubules organized in a "core bundle." A neighbor density analysis of this bundle at the spindle midzone showed a preferred spacing of approximately 35 nm center-to-center between microtubules of opposite polarity. Although this is evidence of specific interaction between antiparallel microtubules, mutant spindles were less ordered than the spindle of wild-type cells. The number of noncore microtubules was significantly higher than that reported for wild-type, and these microtubules did not display a characteristic metaphase configuration. cdc20 spindles showed significantly more cross-bridges between spindle microtubules than were seen in the wild type. The cross-bridge density was highest between antiparallel microtubules. These data suggest that spindle microtubules are stabilized in cdc20 cells and that the CDC20 gene product may be involved in cell cycle processes that promote spindle microtubule disassembly.     

Distribution of acetylated alpha-tubulin in retina and in vitro-assembled microtubules

We have used the mouse monoclonal antibody 6-11 B-1, specific for acetylated alpha-tubulin, to determine the distribution of acetylated alpha-tubulin in in vitro-assembled microtubules and retinal tissue. Analysis by immunoblots revealed that microtubules assembled from bovine brain extracts contain both acetylated and nonacetylated alpha-tubulin. Immunofluorescence, using 6-11 B-1 and antitubulin B-5-1-2, a monoclonal antibody specific for alpha-tubulin, demonstrated the colocalization of both alpha-tubulin species in neurons of the retina and that acetylated microtubules are relatively abundant in neurons. However, analysis at higher resolution revealed that rod photoreceptors contain spatially distinct microtubule arrays which differ in content of acetylated alpha-tubulin and differ in stability. Acetylated microtubules which composed those of the rod outer segment and connecting cilium were resistant to depolymerization in nocodazole or colchicine. In contrast, the nonacetylated microtubules which composed those of the rod-inner segment were depolymerized in nocodazole or colchicine. Therefore, these acetylated microtubules are more resistant to depolymerization than non-acetylated microtubules.     

A role for Tctex-1 (DYNLT1) in controlling primary cilium length

The microtubule motor complex cytoplasmic dynein is known to be involved in multiple processes including endomembrane organization and trafficking, mitosis, and microtubule organization. The majority of studies of cytoplasmic dynein have focused on the form of the motor that is built around the dynein-1 heavy chain. A second isoform, dynein heavy chain-2, and its specifically associated light intermediate chain, LIC3 (D2LIC), are known to be involved in the formation and function of primary cilia. We have used RNAi in human epithelial cells to define the cytoplasmic dynein subunits that function with dynein heavy chain 2 in primary cilia. We identify the dynein light chain Tctex-1 as a key modulator of cilia length control; depletion of Tctex-1 results in longer cilia as defined by both acetylated tubulin labeling of the axoneme and Rab8a labeling of the cilia membrane. Suppression of dynein heavy chain-2 causes concomitant loss of Tctex-1 and this correlates with an increase in cilia length. Compared to individual depletions, double siRNA depletion of DHC2 and Tctex-1 causes an even greater increase in cilia length. Our data show that Tctex-1 is a key regulator of cilia length and most likely functions as part of dynein-2.     

Kinesin-13 regulates the quantity and quality of tubulin inside cilia

Kinesin-13, an end depolymerizer of cytoplasmic and spindle microtubules, also affects the length of cilia. However, in different models, depletion of kinesin-13 either lengthens or shortens cilia, and therefore the exact function of kinesin-13 in cilia remains unclear. We generated null mutations of all kinesin-13 paralogues in the ciliate Tetrahymena. One of the paralogues, Kin13Ap, localizes to the nuclei and is essential for nuclear divisions. The remaining two paralogues, Kin13Bp and Kin13Cp, localize to the cell body and inside assembling cilia. Loss of both Kin13Bp and Kin13Cp resulted in slow cell multiplication and motility, overgrowth of cell body microtubules, shortening of cilia, and synthetic lethality with either paclitaxel or a deletion of MEC-17/ATAT1, the α-tubulin acetyltransferase. The mutant cilia assembled slowly and contained abnormal tubulin, characterized by altered posttranslational modifications and hypersensitivity to paclitaxel. The mutant cilia beat slowly and axonemes showed reduced velocity of microtubule sliding. Thus kinesin-13 positively regulates the axoneme length, influences the properties of ciliary tubulin, and likely indirectly, through its effects on the axonemal microtubules, affects the ciliary dynein-dependent motility.     

Observation and quantification of individual microtubule behavior in vivo: microtubule dynamics are cell-type specific

Recent experiments have demonstrated that the behavior of the interphase microtubule array is cell-type specific: microtubules in epithelial cells are less dynamic than microtubules in fibroblasts (Pepper-kok et al., 1990; Wadsworth and McGrail, 1990). To determine which parameters of microtubule dynamic instability behavior are responsible for this difference, we have examined the behavior of individual microtubules in both cell types after injection with rhodamine-labeled tubulin subunits. Individual microtubules in both cell types were observed to grow, shorten, and pause, as expected. The average amount of time microtubules remained within the lamellae of CHO fibroblasts, measured from images acquired at 10-s intervals, was significantly shorter than the average amount of time microtubules remained within lamellae of PtK1 epithelial cells. Further analysis of individual microtubule behavior from images acquired at 2-s intervals reveals that microtubules in PtK1 cells undergo multiple brief episodes of growth and shortening, resulting in little overall change in the microtubule network. In contrast, microtubules in lamellae of CHO fibroblasts are observed to undergo fewer transitions which are of longer average duration, resulting in substantial changes in the microtubule network over time. A small subset of more stable microtubules was also detected in CHO fibroblasts. Quantification of the various parameters of dynamic instability behavior from these sequences demonstrates that the average rates of both growth and shortening are significantly greater for the majority of microtubules in fibroblasts than for microtubules in epithelial cells (19.8 +/- 10.8 microns/min, 32.2 +/- 17.7 microns/min, 11.9 +/- 6.5 microns/min, and 19.7 +/- 8.1 microns/min, respectively). The frequency of catastrophe events (1/interval between catastrophe events) was similar in both cell types, but the frequency of rescue events (1/time spent shrinking) was significantly higher in PtK1 cells. Thus, individual microtubules in PtK1 lamellae undergo frequent excursions of short duration and extent, whereas most microtubules in CHO lamellae undergo more extensive excursions often resulting in the appearance or disappearance of microtubules within the field of view. These observations provide the first direct demonstration of cell-type specific behavior of individual microtubules in living cells, and indicate that these differences can be brought about by modulation of the frequency of rescue. These results directly support the view that microtubule dynamic instability behavior is regulated in a cell-type specific manner.     

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.     

Microtubular system during spermiogenesis and in the spermatozoon of Convoluta saliens (platyhelminthes,acoela): Tubulin immunocytochemistry and electron microscopy

Spermiogenesis and the spermatozoon were studied in Convoluta saliens, an acoel platyhelminth, by transmission electron microscopy, labelling of nuclei and immunocytochemistry of tubulin with various antibodies. Spermiogenesis involves formation of a long spermatid shaft containing two axonemes. It is established that the nucleus, after a stage of elongation, does not migrate up to the distal extremity of the spermatid, and that the centriolar derivatives are located at the distal extremity of the shaft. This contrasts with the parasitic Platyhelminthes. The mature spermatozoon, 180 μm in length, comprises a nuclear region, 50 μm in length, and a cytoplasmic region, with a short region of overlap. The cytoplasmic region contains two lateral axonemes with a 9 + 2 pattern of microtubules, granules of two different sizes, and two rows of longitudinal microtubules in the center. Each row consists of 5–6 singlet microtubules, with links between them. Whereas the two axonemes are labelled by antibodies against alpha, acetylated‐alpha, and beta tubulin, the microtubule rows are labelled only by the anti‐beta‐tubulin antibody. This suggests that acetylation does not occur in this part of the cytoskeleton, and that the epitope recognized by the anti‐alpha‐tubulin antibody (DM1A) is different in these units. Mol. Reprod. Dev. 52:74–85, 1999. © 1999 Wiley‐Liss, Inc.     

Posttranslationally modified tubulins and microtubule organization in hemocytes of the brine shrimp, Artemia franciscana

Crustaceans possess blood cells (hemocytes) that mediate organismal defense and are analogous to vertebrate leukocytes. In order to more fully characterize these types of cells, hemocytes of the branchiopod crustacean, Artemia franciscana, were analyzed. The data indicate that Artemia have one type of hemocyte, ranging in morphology from compact and spherical to flat and spreading when examined in vitro. Electron microscopy revealed many cytoplasmic granules in the hemocytes and only a limited number of other membrane-bound organelles. Centrioles and microtubules were also visible in thin sections of chemically fixed samples. The cytoplasm of spherical hemocytes was completely labeled by general antitubulin antibodies, but in flattened hemocytes packing of cytoskeletal elements was less tight and individual microtubules were observed. Probing of Western blots disclosed acetylated, tyrosinated, and detyrosinated tubulin isoforms in hemocyte homogenates, the first characterization of posttranslationally modified tubulins in this cell type. Acetylated tubulin was restricted to a subset of microtubules, whereas tyrosinated microtubules were displayed more abundantly. Staining obtained with antibody to detyrosinated tubulin was unusual because it was limited to the perinuclear region of hemocytes. Incubation of blood cells with a monoclonal antibody to gamma-tubulin yielded fluorescent dots sometimes in pairs, a pattern characteristic of centrosomes. The findings support the conclusion that Artemia hemocytes undergo rapid morphogenesis in vitro accompanied by extensive rearrangement of their microtubules, the latter probably indicative of cytoskeletal changes that occur during cell movement and phagocytosis. Additionally, the hemocytes contain posttranslationally modified alpha-tubulins and centrosome-associated gamma-tubulin, both with the potential to influence microtubule organization and function.     

Posttranslational modification and microtubule stability

We have probed the relationship between tubulin posttranslational modification and microtubule stability, using a variation of the antibody-blocking technique. In human retinoblastoma cells we find that acetylated and detyrosinated microtubules represent congruent subsets of the cells' total microtubules. We also find that stable microtubules defined as those that had not undergone polymerization within 1 h after injection of biotin-tubulin were all posttranslationally modified; furthermore dynamic microtubules were all unmodified. We therefore conclude that in these cells the stable, acetylated, and detyrosinated microtubules represent the same subset of the cells' total network. Posttranslational modification, however, is not a prerequisite for microtubule stability and vice versa. Potorous tridactylis kidney cells have no detectable acetylated microtubules but do have a sizable subset of stable ones, and chick embryo fibroblast cells are extensively modified but have few stable microtubules. We conclude that different cell types can create specific microtubule subsets by modulating the relative rates of posttranslational modification and microtubule turnover.