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M Bornens

Publications and source records attributed to M Bornens.

At least 19 recordsLinked to original sources
PubMed2004

Suppression of nuclear oscillations in Saccharomyces cerevisiae expressing Glu tubulin.

In most eukaryotic cells, the C-terminal amino acid of alpha-tubulin is aromatic (Tyr in mammals and Phe in Saccharomyces cerevisiae) and is preceded by two glutamate residues. In mammals, the C-terminal Tyr of alpha-tubulin is subject to cyclic removal from the peptide chain by a carboxypeptidase and readdition to the chain by a tubulin-Tyr ligase. There is evidence that tubulin-Tyr ligase suppression and the resulting accumulation of detyrosinated (Glu) tubulin favor tumor growth, both in animal models and in human cancers. However, the molecular basis for this apparent stimulatory effect of Glu tubulin accumulation on tumor progression is unknown. Here we have developed S. cerevisiae strains expressing only Glu tubulin and used them as a model to assess the consequences of Glu tubulin accumulation in cells. We find that Glu tubulin strains show defects in nuclear oscillations. These defects are linked to a markedly decreased association of the yeast ortholog of CLIP170, Bik1p, with microtubule plus-ends. These results indicate that the accumulation of Glu tubulin in cells affects microtubule tip complexes that are important for microtubule interactions with the cell cortex.

Amino Acid Substitution↗
PubMed2001

Two splice variants of Golgi-microtubule-associated protein of 210 kDa (GMAP-210) differ in their binding to the cis-Golgi network.

GMAP-210 (Golgi-microtubule-associated protein of 210 kDa) is a peripheral Golgi protein that interacts with the minus end of microtubules through its C-terminus and with cis-Golgi network membranes through its N-terminus; it participates in the maintenance of the structural integrity of the Golgi apparatus [Infante, Ramos-Morales, Fedriani, Bornens and Rios (1999) J. Cell Biol. 145, 83--98]. We report here the cloning of a new isoform of GMAP-210 that lacks amino acid residues 105--196. On the basis of the analysis of the gmap-210 genomic sequence, we propose that the small isoform, GMAP-200, arises from alternative splicing of exon 4 of the primary transcript. Overexpression of GMAP-200 induces perturbations in both the Golgi apparatus and the microtubule network that are similar to those previously reported for GMAP-210 overexpression. We show that both isoforms are able to oligomerize under overexpression conditions. Analysis in vitro and in vivo, with the green fluorescent protein as a marker, reveals that the binding of the N-terminal domain of GMAP-200 to the cis-Golgi network membranes is lower than that of the N-terminal domain of GMAP-210. Implications for the regulation of interaction between the cis-Golgi network and microtubules are discussed.

Alternative Splicing↗
PubMed2001

Differential effect of two stathmin/Op18 phosphorylation mutants on Xenopus embryo development.

Stathmin/Op18 destabilizes microtubules in vitro and regulates microtubule polymerization in vivo. Both a microtubule catastrophe-promoting activity and a tubulin sequestering activity were demonstrated for stathmin in vitro, and both could contribute to microtubule depolymerization in vivo. Stathmin activity can be turned down by extensive phosphorylation on its four phosphorylatable serines, and down-regulation of stathmin activity by phosphorylation is necessary for cells to proceed through mitosis. We show here that microinjection of a nonphosphorylatable Ser to Ala (4A) quadruple mutant in Xenopus two-cell stage embryos results in cell cleavage arrest in the injected blastomeres and aborted development, whereas injection of a pseudo-phosphorylated Ser to Glu quadruple mutant (4E) does not prevent normal development. Addition of these mutants to mitotic cytostatic factor-arrested extracts in which spindle assembly was induced led to a dramatic reduction of spindle size with 4A stathmin, and to a moderate increase with 4E stathmin, but both localized to spindle poles. Interestingly, the microtubule assembly-dependent phosphorylation of endogenous stathmin was abolished in the presence of 4A stathmin, but not of 4E stathmin. Altogether, this shows that the phosphorylation-mediated regulation of stathmin activity during the cell cycle is essential for early Xenopus embryonic development.

Animals↗
PubMed2001

Centrosome-dependent exit of cytokinesis in animal cells.

As an organelle coupling nuclear and cytoplasmic divisions, the centrosome is essential to mitotic fidelity, and its inheritance could be critical to understanding cell transformation. Investigating the behavior of the centrosome in living mitotic cells, we documented a transient and remarkable postanaphase repositioning of this organelle, which apparently controls the release of central microtubules from the midbody and the completion of cell division. We also observed that the absence of the centrosome leads to defects in cytokinesis. Together with recent results in yeasts, our data point to a conserved centrosome-dependent pathway that integrates spatial controls into the decision of completing cell division, which requires the repositioning of the centrosome organelle.

3T3 Cells↗
PubMed2001

Structural and functional effects of hydrostatic pressure on centrosomes from vertebrate cells.

In an attempt to better understand the role of centrioles in vertebrate centrosomes, hydrostatic pressure was applied to isolated centrosomes as a means to disassemble centriole microtubules. Treatments of the centrosomes were monitored by analyzing their protein composition, ultrastructure, their ability to nucleate microtubules from pure tubulin, and their capability to induce parthenogenetic development of Xenopus eggs. Moderate hydrostatic pressure (95 MPa) already affected the organization of centriole microtubules in isolated centrosomes, and also impaired microtubule nucleation. At higher pressure, the protein composition of the peri-centriolar matrix (PCM) was also altered and the capacity to nucleate microtubules severely impaired. Incubation of the treated centrosomes in Xenopus egg extract could restore their capacity to nucleate microtubules after treatment at 95 MPa, but not after higher pressure treatment. However, the centriole structure was in no case restored. It is noteworthy that centrosomes treated with mild pressure did not allow parthenogenetic development after injection into Xenopus eggs, even if they had recovered their capacity to nucleate microtubules. This suggested that, in agreement with previous results, centrosomes in which centriole architecture is impaired, could not direct the biogenesis of new centrioles in Xenopus eggs. Centriole structure could also be affected by applying mild hydrostatic pressure directly to living cells. Comparison of the effect of hydrostatic pressure on cells at the G1/S border or on the corresponding cytoplasts suggests that pro-centrioles are very sensitive to pressure. However, cells can regrow a centriole after pressure-induced disassembly. In that case, centrosomes eventually recover an apparently normal duplication cycle although with some delay.

Animals↗
PubMed2001

Basal body replication in green algae--when and where does it start?

Basal body duplication in the green alga Spermatozopsis similis was reinvestigated using GT335, an antibody binding to polyglutamylated tubulins, and antibodies directed to p210, a component of the flagellar transition region which represents the distal border of the basal body. p210 was also detected in small spots at the base of each basal body which increased in size prior to mitosis. The presence of p210 on one of the microtubular flagellar roots suggested a transport of basal body material along these tracks. Immunogold electron microscopy confirmed the presence of p210 in the probasal bodies. Further, small probasal bodies are apparently connected to the mature basal bodies by centrin fibers as observed after artificially induced basal body separation in Xenopus egg extract. While basal bodies grew, most of the p210 remained at the tip of elongating basal bodies, but two or four additional spots were observed in distinct patterns near the base of the basal bodies. In cytokinesis, basal body pairs separated and p210 was observed in a strong signal at the tip and a weaker one in the vicinity of the proximal end of each basal body. We interpret the data as indicating that a new p210-containing structure forms near the proximal end of the basal bodies during basal body elongation, representing the precursor of the next generation of basal bodies. Thus, basal bodies appear to seed the succeeding generation already during their own development, a mechanism which could ensure the correct number and position of basal bodies.

Animals↗
PubMed2001

Stathmin/Op18 phosphorylation is regulated by microtubule assembly.

Stathmin/Op 18 is a microtubule (MT) dynamics-regulating protein that has been shown to have both catastrophe-promoting and tubulin-sequestering activities. The level of stathmin/Op18 phosphorylation was proved both in vitro and in vivo to be important in modulating its MT-destabilizing activity. To understand the in vivo regulation of stathmin/Op18 activity, we investigated whether MT assembly itself could control phosphorylation of stathmin/Op18 and thus its MT-destabilizing activity. We found that MT nucleation by centrosomes from Xenopus sperm or somatic cells and MT assembly promoted by dimethyl sulfoxide or paclitaxel induced stathmin/Op18 hyperphosphorylation in Xenopus egg extracts, leading to new stathmin/Op18 isoforms phosphorylated on Ser 16. The MT-dependent phosphorylation of stathmin/Op18 took place in interphase extracts as well, and was also observed in somatic cells. We show that the MT-dependent phosphorylation of stathmin/Op18 on Ser 16 is mediated by an activity associated to the MTs, and that it is responsible for the stathmin/Op18 hyperphosphorylation reported to be induced by the addition of "mitotic chromatin." Our results suggest the existence of a positive feedback loop, which could represent a novel mechanism contributing to MT network control.

Animals↗
PubMed2000

The respective contributions of the mother and daughter centrioles to centrosome activity and behavior in vertebrate cells.

We have generated several stable cell lines expressing GFP-labeled centrin. This fusion protein becomes concentrated in the lumen of both centrioles, making them clearly visible in the living cell. Time-lapse fluorescence microscopy reveals that the centriole pair inherited after mitosis splits during or just after telophase. At this time the mother centriole remains near the cell center while the daughter migrates extensively throughout the cytoplasm. This differential behavior is not related to the presence of a nucleus because it is also observed in enucleated cells. The characteristic motions of the daughter centriole persist in the absence of microtubules (Mts). or actin, but are arrested when both Mts and actin filaments are disrupted. As the centrioles replicate at the G1/S transition the movements exhibited by the original daughter become progressively attenuated, and by the onset of mitosis its behavior is indistinguishable from that of the mother centriole. While both centrioles possess associated gamma-tubulin, and nucleate similar number of Mts in Mt repolymerization experiments. during G1 and S only the mother centriole is located at the focus of the Mt array. A model, based on differences in Mt anchoring and release by the mother and daughter centrioles, is proposed to explain these results.

3T3 Cells↗
PubMed2000

Tissue-specific expression and subcellular localisation of mammalian delta-tubulin.

The properties of the microtubule network are regulated at various levels including tissue-dependent isotype switching, post-translational modification of alpha- and beta-tubulin, and by a variety of microtubule-associated molecules (for reviews, see [1-3]). Microtubule nucleation is attributed to gamma-tubulin, which is present in protein complexes at the centrosome and in the cytoplasm [4,5]. A screen for flagellar mutants in the green alga Chlamydomonas reinhardtii has led to the identification of a fourth member of the tubulin gene superfamily, delta-tubulin. In this unicellular organism, the lack of a functional delta-tubulin gene copy causes aberrant numbers of flagella, depending on the age of the corresponding basal bodies; mutants also show abnormal ultrastructure of the basal bodies and a misplacement of the cleavage furrow at mitosis [6]. Here, we report the isolation of the mouse delta-tubulin homologue and show that the gene is highly expressed in testis. In the elongating spermatid, delta-tubulin associated with the manchette, a specialised microtubule system present during reshaping of the sperm head. The protein specifically localised at the perinuclear ring of the manchette, at the centriolar vaults and along the principal piece of the sperm flagellum. In somatic cell lines, unlike most other tubulins, mammalian delta-tubulin was both cytoplasmic and nuclear and did not colocalise with microtubules. The protein was enriched at the spindle poles during mitosis and we found that gamma-tubulin coimmunoprecipitated with delta-tubulin. Together, the data indicate a specialised role for mammalian delta-tubulin that is distinct from other known tubulins.

Animals↗
PubMed2000

A role for centrin 3 in centrosome reproduction.

Centrosome reproduction by duplication is essential for the bipolarity of cell division, but the molecular basis of this process is still unknown. Mutations in Saccharomyces cerevisiae CDC31 gene prevent the duplication of the spindle pole body (SPB). The product of this gene belongs to the calmodulin super-family and is concentrated at the half bridge of the SPB. We present a functional analysis of HsCEN3, a human centrin gene closely related to the CDC31 gene. Transient overexpression of wild-type or mutant forms of HsCen3p in human cells demonstrates that centriole localization depends on a functional fourth EF-hand, but does not produce mitotic phenotype. However, injection of recombinant HsCen3p or of RNA encoding HsCen3p in one blastomere of two-cell stage Xenopus laevis embryos resulted in undercleavage and inhibition of centrosome duplication. Furthermore, HsCEN3 does not complement mutations or deletion of CDC31 in S. cerevisiae, but specifically blocks SPB duplication, indicating that the human protein acts as a dominant negative mutant of CDC31. Several lines of evidence indicate that HsCen3p acts by titrating Cdc31p-binding protein(s). Our results demonstrate that, in spite of the large differences in centrosome structure among widely divergent species, the centrosome pathway of reproduction is conserved.

Animals↗
PubMed2000

Microtubule minus-end anchorage at centrosomal and non-centrosomal sites: the role of ninein.

The novel concept of a centrosomal anchoring complex, which is distinct from the gamma-tubulin nucleating complex, has previously been proposed following studies on cochlear epithelial cells. In this investigation we present evidence from two different cell systems which suggests that the centrosomal protein ninein is a strong candidate for the proposed anchoring complex. Ninein has recently been observed in cultured fibroblast cells to localise primarily to the post-mitotic mother centriole, which is the focus for a classic radial microtubule array. We show here by immunoelectron microscopical analyses of centrosomes from mouse L929 cells that ninein concentrates at the appendages surrounding the mother centriole and at the microtubule minus-ends. We further show that localisation of ninein in the cochlear supporting epithelial cells, where the vast majority of the microtubule minus-ends are associated with apical non-centrosomal sites, suggests that it is not directly involved in microtubule nucleation. Ninein seems to play an important role in the positioning and anchorage of the microtubule minus-ends in these epithelial cells. Evidence is presented which suggests that ninein is released from the centrosome, translocated with the microtubules, and is responsible for the anchorage of microtubule minus-ends to the apical sites. We propose that ninein is a non-nucleating microtubule minus-end associated protein which may have a dual role as a minus-end capping and anchoring protein.

Animals↗
PubMed2000

Differential expression and cellular distribution of centrin isoforms during human ciliated cell differentiation in vitro.

Centrin protein is an ubiquitously expressed cytoskeletal component and is a member of the EF-hand superfamily of calcium-binding proteins. It was first discovered in the flagellar apparatus of unicellular green algae where it is involved in contraction of Ca(2+)-sensitive structures. Centrin protein is associated with centrosome-related structures such as spindle pole body in yeast, and centriole/basal bodies in flagellar and ciliated cells. Three centrin genes have been cloned in human cells. In this work, we have performed a comparative biochemical and functional analysis of centrin isoforms using a primary culture of human nasal epithelial cells which provides an efficient way to obtain a complete ciliated cell differentiation process. RT-PCR experiments show that the expression of the three human centrin genes increases during cell differentiation, and that only centrin 2 and 3 are expressed during cell proliferation. Using polyclonal antibodies raised against recombinant human centrin 2 and 3, we show a specific pattern of protein expression. Ultrastructural immunolocalization suggests that centrin proteins are involved in the early process of centriole assembly, as they are concentrated within the precursor structures of centriole/basal bodies. It also shows a differential localisation of centrin proteins in mature centriole/basal bodies, suggesting different functions for centrins 1/2 and centrin 3. This is also supported by functional analyses showing that centrin 1 and/or centrin 2 are involved in ciliary beating.

Calcium-Binding Proteins↗
PubMed1999

Tubulin binding sites on gamma-tubulin: identification and molecular characterization.

gamma-Tubulin is essential to microtubule organization in eukaryotic cells. It is believed that gamma-tubulin interacts with tubulin to accomplish its cellular functions. However, such an interaction has been difficult to demonstrate and to characterize at the molecular level. gamma-Tubulin is a poorly soluble protein, not amenable to biochemical studies in a purified form as yet. Therefore basic questions concerning the existence and properties of tubulin binding sites on gamma-tubulin have been difficult to address. Here we have performed a systematic search for tubulin binding sites on gamma-tubulin using the SPOT peptide technique. We find a specific interaction of tubulin with six distinct domains on gamma-tubulin. These domains are clustered in the central part of the gamma-tubulin primary amino acid sequence. Synthetic peptides corresponding to the tubulin binding domains of gamma-tubulin bind with nanomolar K(d)s to tubulin dimers. These peptides do not interfere measurably with microtubule assembly in vitro and associate with microtubules along the polymer length. On the tertiary structure, the gamma-tubulin peptides cluster to surface regions on both sides of the molecule. Using SPOT analysis, we also find peptides interacting with gamma-tubulin in both the alpha- and beta-tubulin subunits. The tubulin peptides cluster to surface regions on both sides of the alpha- and beta- subunits. These data establish gamma-tubulin as a tubulin ligand with unique tubulin-binding properties and suggests that gamma-tubulin and tubulin dimers associate through lateral interactions.

Amino Acid Sequence↗
PubMed1999

GMAP-210, A cis-Golgi network-associated protein, is a minus end microtubule-binding protein.

We report that a peripheral Golgi protein with a molecular mass of 210 kD localized at the cis-Golgi network (Rios, R.M., A.M. Tassin, C. Celati, C. Antony, M.C. Boissier, J.C. Homberg, and M. Bornens. 1994. J. Cell Biol. 125:997-1013) is a microtubule-binding protein that associates in situ with a subpopulation of stable microtubules. Interaction of this protein, now called GMAP-210, for Golgi microtubule-associated protein 210, with microtubules in vitro is direct, tight and nucleotide-independent. Biochemical analysis further suggests that GMAP-210 specifically binds to microtubule ends. The full-length cDNA encoding GMAP-210 predicts a protein of 1, 979 amino acids with a very long central coiled-coil domain. Deletion analyses in vitro show that the COOH terminus of GMAP-210 binds to microtubules whereas the NH2 terminus binds to Golgi membranes. Overexpression of GMAP-210-encoding cDNA induced a dramatic enlargement of the Golgi apparatus and perturbations in the microtubule network. These effects did not occur when a mutant lacking the COOH-terminal domain was expressed. When transfected in fusion with the green fluorescent protein, the NH2-terminal domain associated with the cis-Golgi network whereas the COOH-terminal microtubule-binding domain localized at the centrosome. Altogether these data support the view that GMAP-210 serves to link the cis-Golgi network to the minus ends of centrosome-nucleated microtubules. In addition, this interaction appears essential for ensuring the proper morphology and size of the Golgi apparatus.

Amino Acid Sequence↗