Control of microtubule nucleation and stability in Madin-Darby canine kidney cells: the occurrence of noncentrosomal, stable detyrosinated microtubules.

The microtubule-nucleating activity of centrosomes was analyzed in fibroblastic (Vero) and in epithelial cells (PtK2, Madin-Darby canine kidney [MDCK]) by double-immunofluorescence labeling with anti-centrosome and antitubulin antibodies. Most of the microtubules emanated from the centrosomes in Vero cells, whereas the microtubule network of MDCK cells appeared to be noncentrosome nucleated and randomly organized. The pattern of microtubule organization in PtK2 cells was intermediate to the patterns observed in the typical fibroblastic and epithelial cells. The two centriole cylinders were tightly associated and located close to the nucleus in Vero and PtK2 cells. In MDCK cells, however, they were clearly separated and electron microscopy revealed that they nucleated only a few microtubules. The stability of centrosomal and noncentrosomal microtubules was examined by treatment of these different cell lines with various concentrations of nocodazole. 1.6 microM nocodazole induced an almost complete depolymerization of microtubules in Vero cells; some centrosome nucleated microtubules remained in PtK2 cells, while many noncentrosomal microtubules resisted that treatment in MDCK cells. Centrosomal and noncentrosomal microtubules regrew in MDCK cells with similar kinetics after release from complete disassembly by high concentrations of nocodazole (33 microM). During regrowth, centrosomal microtubules became resistant to 1.6 microM nocodazole before the noncentrosomal ones, although the latter eventually predominate. We suggest that in MDCK cells, microtubules grow and shrink as proposed by the dynamic instability model but the presence of factors prevents them from complete depolymerization. This creates seeds for reelongation that compete with nucleation off the centrosome. By using specific antibodies, we have shown that the abundant subset of nocodazole-resistant microtubules in MDCK cells contained detyrosinated alpha-tubulin (glu tubulin). On the other hand, the first microtubules to regrow after nocodazole removal contained only tyrosinated tubulin. Glu-tubulin became detectable only after 30 min of microtubule regrowth. This strongly supports the hypothesis that alpha-tubulin detyrosination occurs primarily on "long lived" microtubules and is not the cause of the stabilization process. This is also supported by the increased amount of glu-tubulin that we found in taxol-treated cells.

Microtubules are stabilized in confluent epithelial cells but not in fibroblasts.

Rhodamine-tagged tubulin was microinjected into epithelial cells (MDCK) and fibroblasts (Vero) to characterize the dynamic properties of labeled microtubules in sparse and confluent cells. Fringe pattern fluorescence photobleaching revealed two components with distinct dynamic properties. About one-third of the injected tubulin diffused rapidly in the cytoplasm with a diffusion coefficient of 1.3-1.6 x 10(-8) cm2/s. This pool of soluble cytoplasmic tubulin was increased to greater than 80% when cells were treated with nocodazole, or reduced to approximately 20% upon treatment of cells with taxol. Fluorescence recovery of the remaining two-thirds of labeled tubulin occurred with an average half-time (t1/2) of 9-11 min. This pool corresponds to labeled tubulin associated with microtubules, since it was sensitive to treatment of cells with nocodazole and since taxol increased its average t1/2 to greater than 22 min. Movement of photobleached microtubules in the cytoplasm with rates of several micrometers per minute was shown using very small interfringe distances. A significant change in the dynamic properties of microtubules occurred when MDCK cells reached confluency. On a cell average, microtubule half-life was increased about twofold to approximately 16 min. In fact, two populations of cells were detected with respect to their microtubule turnover rates, one with a t1/2 of approximately 9 min and one with a t1/2 of greater than 25 min. Correspondingly, the rate of incorporation of microinjected tubulin into interphase microtubules was reduced about twofold in confluent MDCK cells. In contrast to the MDCK cells, no difference in microtubule dynamics was observed in sparse and confluent populations of Vero fibroblasts, where the average microtubule half-life was approximately 10 min. Thus, microtubules are significantly stabilized in epithelial but not fibroblastic cells grown to confluency.

Cytoskeletal control of centrioles movement during the establishment of polarity in Madin-Darby canine kidney cells.

The two centrioles that are localized close to each other and to the nucleus in single Madin-Darby Canine kidney cells (MDCK) move apart by distances as large as 13 microns after the establishment of extensive cellular junctions. Microfilaments, and possibly microtubules appear to be responsible for this separation. In fully polarized cells, the centrioles are localized just beneath the apical membrane. After disruption of intercellular junctions in low calcium medium, the centrioles move back towards the cell center. This process requires intact microtubules but happens even in the absence of microfilaments. These results indicate that the position of centrioles is determined by opposing forces produced by microtubules and microfilaments and suggest that the balance between these forces is modulated by the assembly of cellular junctions. Centriole separation appears to be an early event in the process that precedes their final positioning in the apical-most region of the polarized cell.

Regulation of microtubule dynamics and nucleation during polarization in MDCK II cells.

MDCK cells form a polarized epithelium when they reach confluence in tissue culture. We have previously shown that concomitantly with the establishment of intercellular junctions, centrioles separate and microtubules lose their radial organization (Bacallao, R., C. Antony, C. Dotti, E. Karsenti, E.H.K. Stelzer, and K. Simons. 1989. J. Cell Biol. 109:2817-2832. Buendia, B., M.H. Bré, G. Griffiths, and E. Karsenti. 1990. 110:1123-1136). In this work, we have examined the pattern of microtubule nucleation before and after the establishment of intercellular contacts. We analyzed the elongation rate and stability of microtubules in single and confluent cells. This was achieved by microinjection of Paramecium axonemal tubulin and detection of the newly incorporated subunits by an antibody directed specifically against the Paramecium axonemal tubulin. The determination of newly nucleated microtubule localization has been made possible by the use of advanced double-immunofluorescence confocal microscopy. We have shown that in single cells, newly nucleated microtubules originate from several sites concentrated in a region localized close to the nucleus and not from a single spot that could correspond to a pair of centrioles. In confluent cells, newly nucleated microtubules were still more dispersed. The microtubule elongation rate of individual microtubules was not different in single and confluent cells (4 microns/min). However, in confluent cells, the population of long lived microtubules was strongly increased. In single or subconfluent cells most microtubules showed a t1/2 of less than 30 min, whereas in confluent monolayers, a large population of microtubules had a t1/2 of greater than 2 h. These results, together with previous observations cited above, indicate that during the establishment of polarity in MDCK cells, microtubule reorganization involves both a relocalization of microtubule-nucleating activity and increased microtubule stabilization.

Polyglycylation of tubulin is essential and affects cell motility and division in Tetrahymena thermophila.

We analyzed the role of tubulin polyglycylation in Tetrahymena thermophila using in vivo mutagenesis and immunochemical analysis with modification-specific antibodies. Three and five polyglycylation sites were identified at glutamic acids near the COOH termini of alpha- and beta-tubulin, respectively. Mutants lacking all polyglycylation sites on alpha-tubulin have normal phenotype, whereas similar sites on beta-tubulin are essential. A viable mutant with three mutated sites in beta-tubulin showed reduced tubulin glycylation, slow growth and motility, and defects in cytokinesis. Cells in which all five polyglycylation sites on beta-tubulin were mutated were viable if they were cotransformed with an alpha-tubulin gene whose COOH terminus was replaced by the wild-type COOH terminus of beta-tubulin. In this double mutant, beta-tubulin lacked detectable polyglycylation, while the alpha-beta tubulin chimera was hyperglycylated compared with alpha-tubulin in wild-type cells. Thus, the essential function of polyglycylation of the COOH terminus of beta-tubulin can be transferred to alpha-tubulin, indicating it is the total amount of polyglycylation on both alpha- and beta-tubulin that is essential for survival.

Polyglycylation of tubulin: a posttranslational modification in axonemal microtubules.

A posttranslational modification was detected in the carboxyl-terminal region of axonemal tubulin from Paramecium. Tubulin carboxyl-terminal peptides were isolated and analyzed by Edman degradation sequencing, mass spectrometry, and amino acid analysis. All of the peptides, derived from both alpha and beta tubulin subunits, were modified by polyglycylation, containing up to 34 glycyl units covalently bound to the gamma carboxyl group of glutamyl residues. This modification, present in one of the most stable microtubular systems, may influence microtubule stability or axoneme function, or both.

Tubulin polyglycylation: differential posttranslational modification of dynamic cytoplasmic and stable axonemal microtubules in paramecium.

Polyglycylation, a posttranslational modification of tubulin, was discovered in the highly stable axonemal microtubules of Paramecium cilia where it involves the lateral linkage of up to 34 glycine units per tubulin subunit. The observation of this type of posttranslational modification mainly in axonemes raises the question as to its relationship with axonemal organization and with microtubule stability. This led us to investigate the glycylation status of cytoplasmic microtubules that correspond to the dynamic microtubules in Paramecium. Two anti-glycylated tubulin monoclonal antibodies (mAbs), TAP 952 and AXO 49, are shown here to exhibit different affinities toward mono- and polyglycylated synthetic tubulin peptides. Using immunoblotting and mass spectrometry, we show that cytoplasmic tubulin is glycylated. In contrast to the highly glycylated axonemal tubulin, which is recognized by the two mAbs, cytoplasmic tubulin reacts exclusively with TAP 952, and the alpha- and beta- tubulin subunits are modified by only 1-5 and 2-9 glycine units, respectively. Our analyses suggest that most of the cytoplasmic tubulin contains side chain lengths of 1 or 2 glycine units distributed on several glycylation sites. The subcellular partition of distinct polyglycylated tubulin isoforms between cytoplasmic and axonemal compartments implies the existence of regulatory mechanisms for glycylation. By following axonemal tubulin immunoreactivity with anti-glycylated tubulin mAbs upon incubation with a Paramecium cellular extract, the presence of a deglycylation enzyme is revealed in the cytoplasm of this organism. These observations establish that polyglycylation is reversible and indicate that, in vivo, an equilibrium between glycylating and deglycylating enzymes might be responsible for the length of the oligoglycine side chains of tubulin.

A massive new posttranslational modification occurs on axonemal tubulin at the final step of spermatogenesis in Drosophila.

Using two antibodies raised against Paramecium axonemal tubulin, a monoclonal antibody, AXO 49 (Callen et al., Biol. Cell 81, 95-119 (1994)), and a polyclonal antibody, PAT (Cohen et al., Biol. Cell 44, 35-44 (1982)), which have been shown elsewhere to detect a new posttranslational modification of tubulin presumably corresponding to polyglycylation, we have analyzed the occurrence of this modification during spermatogenesis in Drosophila. Results obtained by immunofluorescence on cysts isolated by laceration of testes showed that the antibodies reacted on axonemal microtubules of several species within the genus. Observation of different stages of differentiation of D. obscura sperm cells indicated, first, that the epitopes reactive with both antibodies appeared at late stages, and secondly, that they were detected simultaneously along all axonemes within a cyst. Immunofluorescence on semithin sections and electron microscopic immunocytochemistry on ultrathin sections confirmed that the appearance of the epitope recognized by the monoclonal antibody occurred at the time of the individualization process of spermatids in D. melanogaster. These results indicate that the posttranslational modification occurs as a very late event, after complete assembly of axonemal microtubules, and that the axonemal tubulin becomes modified when axonemal microtubules become coupled with the membrane, suggesting that the modification may in some way be induced by the microtubule-membrane interaction.

Cytochemical studies of nuclear basic proteins in control and vitamin B12 starved Euglena.

In avitaminosis B12, Euglena gracilis Z is blocked in the cell cycle in the S/G2 phase. In these blocked cells, transcription and traduction go on and the amount of DNA is less than doubled and remains constant during the blockage. Chromatin clumps observed in situ with classical electron microscopic methods are always condensed in control cells but are not visualized in B12 starved cells. Two cytochemical reactions, ethanolic phosphotungstic acid and ammoniacal silver reaction, specific for lysine- or arginine-rich residues, are performed to reveal basic nuclear proteins of chromatin. With these two methods, control chromatin in situ always shows a condensed aspect, whereas the starved chromatin appears dispersed. These cytochemical differences might be considered to result from a different supramolecular organization of the two kinds of chromatin.

Effects of brain microtubule-associated proteins on microtubule dynamics and the nucleating activity of centrosomes.

In this paper, we report on the effect of brain microtubule-associated proteins (MAPs) on the dynamic instability of microtubules as well as on the nucleation activity of purified centrosomes. Under our experimental conditions, tau and MAP2 have similar effects on microtubule nucleation and dynamic instability. Tau increases the apparent elongation rate of microtubules in proportion to its molar ratio to tubulin, and we present evidence indicating that this is due to a reduction of microtubule instability rather than to an increase of the on rate of tubulin subunits at the end of growing microtubules. Increasing the molar ratio of tau over tubulin leads also to an increase in the average number of microtubules nucleated per centrosome. This number remains constant with time. This suggests that the number of centrosome-nucleated microtubules at steady state can be determined by factors that are not necessarily irreversibly bound to centrosomes but, rather, affect the dynamic properties of microtubules.

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.

Tubulin polyglycylation: a morphogenetic marker in ciliates.

The occurrence of the tubulin post-translational modification, polyglycylation, in stable microtubular structures was investigated during morphogenesis in two ciliates, Paramecium and Frontonia atra, belonging to the Epiplasmata group. This analysis was carried out by means of immunofluorescence and post-embedding immunoelectron microscopy using two monoclonal antibodies, TAP 952 and AXO 49, respectively recognizing mono- and polyglycylated sites in alpha- and beta-tubulin. In the course of cell division, the TAP 952 epitope is detected in all microtubular structures including the newly assembled ones, such as cortical and oral basal bodies and cilia. In contrast, the AXO 49 epitope is only present in 'old' microtubular structures such as parental cortical and oral basal bodies and cilia. Our observations show that, in ciliates: 1) this tubulin post-translational modification takes place early in the course of morphogenesis; and 2) the lengthening of the polyglycine chains occurs after a great delay following addition of the first glycine residues on the tubulin glycylation sites, and following microtubule assembly. Thus, a sequential mechanism of polyglycylation is shown to take place in the tubulin molecule and during morphogenesis in Paramecium and Frontonia atra. Accordingly, polyglycylation, through a time-dependent polyglycine chain elongation process, appears to be a morphogenetic marker in ciliates.

Cellular interactions and tubulin detyrosination in fibroblastic and epithelial cells.

In mammalian cells most microtubules are enriched in tyrosinated alpha-tubulin (tyr-tubulin). Other subclasses of microtubules are present in variable amounts and some are enriched in detyrosinated alpha-tubulin (glu-tubulin). We examined the effect of cell-cell interactions on the level of glu-tubulin in microtubules. This was studied by quantitative immunofluorescence using antibodies against tyr- and glu-tubulin. We found that in cells which have established cell-cell contacts, the ratio of glu-/tyr-tubulin is higher than in isolated cells. We also examined the effect of cell-cell interactions on the glu-/tyr-tubulin ratio by using the antibody blocking method of Schulze and Kirschner [42]. Microtubules containing mainly tyr-tubulin had been blocked first by a polyclonal antibody against tyr-tubulin and several layers of secondary antibodies. The unblocked microtubules were then labeled by a monoclonal antibody against alpha-tubulin. Since the coating efficiency of microtubules by the anti-tyr tubulin depends on the amount of tyr-tubulin in each microtubule, this procedure allows the visualization of microtubules enriched or depleted in tyr-tubulin in specific domains of each cell. Microtubules were more extensively blocked in subconfluent than in confluent cells and preferentially at the periphery of the cytoplasm. In cells present at the margin of an artificial wound produced in a confluent monolayer, the amount of blocked microtubules increased slowly with time (between 2 and 4 h). These results are consistent with the hypothesis that cell-cell contacts lead to increased tubulin dytyrosination both in fibroblastic and epithelial cells.

Modulation of fibroblast motility by a cytosolic extract of Cyanobacteria.

Proliferation and migratory behavior of L929 murine fibroblasts were shown to be modified in the presence of a cytosolic extract of Phormidium sp. (Cyanobacteria). The addition of Phormidium extract to the growth medium (Dulbecco's modified Eagle's medium) supplemented with 0.5% newborn calf serum increased cell proliferation. The effect was shown to be cell line specific. A quantitative analysis performed according to De Laat, Tertoolen, and Bluemink (1981, Eur. J. Cell Biol., 23, 273-279), showed that Phormidium extract was a potential aggregative effector for fibroblasts. Heating (100 degrees C, 4 min) inactivated the clustering effect of the extract, but the effect on cell proliferation was retained. A video analysis of cells after divisions showed that the extract activated cell migration in the same way as 5% serum did during the first 24 h of treatment. Between 24 and 48 h of treatment, cell migration in the presence of the extract was inhibited when compared to migration in 0.5 or 5% serum. We have shown that Phormidium extract may contain two or three kinds of effectors which acted as exogenous growth factors (allowing attachment and proliferation) and as modulator(s) of the cell migratory behavior (activator of migration in early times of the growth and inhibitor later).

A flow cytometric study of DNA staining in situ in exponentially growing and stationary Euglena gracilis.

DNA stainability by different fluorochromes has been compared in exponentially dividing and stationary Euglena cells. With the intercalating fluorochromes, ethidium bromide, acridine orange and DAPI, a decrease of fluorescence intensity of the G1 cells is observed when cells enter stationary stage. However this decrease of fluorescence is not obtained with the nonintercalating fluorochrome Hoechst 33258. If nuclear basic proteins are extracted, however, the intensity of staining by either Hoechst 33258 or ethidium-bromide is comparable in stationary and dividing cells. Therefore, the decrease of fluorescence intensity of the G1 cells observed during the transition from exponential to stationary phase is not due to a loss of DNA but is related to the exposure of chromatin binding sites for ethidium bromide. In Euglena cells, DNA accessibility for intercalating fluorochromes depends upon chromatin structure and consequently upon cell age.

DNA flow cytometry of control Euglena and cell cycle blockade of vitamin B12-starved cells.

Vitamin B12 starvation in Euglena induces a cell cycle arrest that leads to unbalanced growth. Microfluorometry and flow cytometry analyses of cellular DNA fluorescence after Hoechst 33258 staining were performed on control and vitamin B12-deficient cells. Convergent results are obtained with both methods. Histograms that represent arrested cells are unimodal, with a mode channel value nearly twice that of the G1 control cell peak. Dispersion of fluorescence values is great, and values from 2C and over 4C are observed and discussed. It appears that vitamin B12 starvation in Euglena leads to defective DNA synthesis. Blocked cells have different DNA content, corresponding to blockade of DNA replication during the S phase. A second block prevents the onset of mitosis even for 4C cells.

Euglena plasma membrane during normal and vitamin B12 starvation growth.

Freeze-fracture and optical diffraction techniques were used to study the organization of the Euglena pellicle during the normal and replicative stages of the cell cycle and during vitamin B12 starvation. It was shown that the diffuse layer underlying the tripartite structure has a fibrillar structure. Despite the absence of homology in the 2 fracture faces of the pellicle, the EF striated and the PF particulate ones appear complementary as shown by optical diffraction studies; it must therefore be considered as a true membrane. The grooves are free from such particles and striations. They appear as a specific pattern of the cortex, different from the ridges in their structural organization and their replicative capacity as observed during vitamin B12 starvation. This notion is confirmed by the mode of pellicular growth which is characterized by 2 steps. The first occurs during the early replicative stage (pre-mitotic phase of the cell cycle) when the formation of a new ridge is correlated with the appearance of the 'minor' orientation of a 2-dimensional lattice on the EF and the PF faces and the spread of the particles over the PF face of the space between the old ridges. The second takes place during the lengthening of the ridges from the initiating posterior side (non-replicative stage). During this second step, the 'major' orientation of the lattice is preferentially observed in control cells and exclusively in starved cells. The striking differences between the grooves and the ridges is discussed, as well as the 2 modes of growth and their significance in morphogenesis.

[Analyses of histones and of nucleosomal DNA of normal and vitamin B 12 starved Euglena]. / Analyses des histones et de l'ADN nucléosomal de l'euglène normale et carencée en vitamine B12.

An extraction and electrophoresis of histones from normal and starved Euglena cell nuclei allows us to show the presence of five types of histones in the two cases but a lower degree of acetylation appears in H4 histone from starved cells. It was shown that DNA repeat length from the two kinds of chromatin is comparable (225 b.p.).

Glutamylated tubulin probed in ciliates with the monoclonal antibody GT335.

Microtubular networks are extensively developed in many ciliate species. In several of them, we investigate the occurrence of the post-translational glutamylation of tubulin [Eddé et al., 1990: Science 247:82-85; Eddé et al., 1991: J. Cell. Biochem. 46:134-142] using as a probe for such modified tubulin, the monoclonal antibody GT335 [Wolff et al., 1992: Eur. J. Cell Biol. 59:425-432]. Results obtained in Paramecium strongly suggest that both axonemal and cytoplasmic tubulin are glutamylated. As in the vertebrate brain tubulin so far tested, the GT335 epitope is located at the carboxy-terminal fragment of cytoplasmic tubulin removed by subtilisin treatment. Immunoblotting and immunofluorescence experiments reveal that, unlike tubulin acetylation, glutamylation is not restricted to cold-resistant microtubules. In addition, immunofluorescence studies performed on dividing cells show that glutamylation takes place soon after the polymerization of microtubules. Finally, glutamylated tubulin is also detected in the ciliate species Euplotes, Tetrahymena, and Paraurostyla. Together with results obtained on flagellate species, this suggests that tubulin glutamylation came out early in the course of eukaryotic evolution and has been widely exploited in various cellular strategies.

Expression of glycylated tubulin during the differentiation of spermatozoa in mammals.

Using quantitative immunogold analyses of tubulin isoforms we previously demonstrated a unique differential expression of glutamylated tubulin in the flagellum of mouse and man spermatozoa [Fouquet et al., 1997: Tissue Cell 29:573-583]. We have performed similar analyses for glycylated tubulin using two monoclonal antibodies, TAP 952 and AXO 49, directed to mono- and polyglycylated tubulin respectively. Glycylated tubulin was not found in centrioles and cytoplasmic microtubules (manchette) of germ cells. In mouse and man, axonemal tubulin was first monoglycylated and uniformly distributed in all doublets at all levels of the flagellum in elongating spermatids. In human mature spermatozoa axonemal microtubules were enriched in monoglycylated tubulin from the base to the tip of the flagellum. In mouse sperm flagellum a similar gradient of monoglycylated tubulin was also observed in addition to an opposite gradient of polyglycylated tubulin. In both species, monoglycylated tubulin labeling predominated in doublets 3-8 whereas glutamylated tubulin labeling [Fouquet et al., 1997] predominated in doublets 1-5-6. These differential labelings were suppressed after motility inhibition of mouse spermatozoa by sodium azide treatment and in non-motile human spermatozoa lacking dynein arms. The unique distribution of these tubulin isoforms and the known inhibition of motility induced by their specific antibodies are consistent with a complementary role of tubulin glycylation and glutamylation in the regulation of flagellar beating in mammalian spermatozoa.