Bernard Eddé

Glutamylation of centriole and cytoplasmic tubulin in proliferating non-neuronal cells

We have examined the distribution of glutamylated tubulin in non-neuronal cell lines. A major part of centriole tubulin is highly modified on both the alpha- and beta-tubulin subunits, whereas a minor part of the cytoplasmic tubulin is slightly modified, on the beta-tubulin only. Furthermore, we observed that tubulin glutamylation varies during the cell cycle: an increase occurs during mitosis on both centriole and spindle microtubules. In the spindle, this increase appears more obvious on the pole-to-pole and kinetochore microtubules than on the astral microtubules. The cellular pattern and the temporal variation of this post-translational modification contrast with other previously described tubulin modifications. The functional significance of this distribution is discussed.

Polyglutamylation of Nucleosome Assembly Proteins

Polyglutamylation is an original posttranslational modification, discovered on tubulin, consisting in side chains composed of several glutamyl units and leading to a very unusual protein structure. A monoclonal antibody directed against glutamylated tubulin (GT335) was found to react with other proteins present in HeLa cells. After immunopurification on a GT335 affinity column, two prominent proteins of ∼50 kDa were observed. They were identified by microsequencing and mass spectrometry as NAP-1 and NAP-2, two members of the nucleosome assembly protein family that are implicated in the deposition of core histone complexes onto chromatin. Strikingly, NAP-1 and NAP-2 were found to be substrates of an ATP-dependent glutamylation enzyme co-purifying on the same column. We took advantage of this property to specifically label and purify the polyglutamylated peptides. NAP-1 and NAP-2 are modified in their C-terminal domain by the addition of up to 9 and 10 glutamyl units, respectively. Two putative glutamylation sites were localized for NAP-1 at Glu-356 and Glu-357 and, for NAP-2, at Glu-347 and Glu-348. These results demonstrate for the first time that proteins other than tubulin are polyglutamylated and open new perspectives for studying NAP function.

Evolution of tubulin heterogeneity during mouse brain development

In this report, we have characterized tubulin subunit heterogeneity and its evolution during mouse brain development, from embryonic to adult stages. A modification of the two-dimensional protein analysis was used to specify these events. The number of isotubulins increases from 6 (4 alpha and 2 beta), in the embryonic brain, to 11 (6 alpha and 5 beta), in the adult. The changes occurring in tubulin heterogeneity are developmentally controlled but it seems that alpha and beta isotubulins are independently regulated: changes in alpha tubulin occur only just before birth whereas the major evolution is concerned with the appearance and accumulation of acidic beta isotubulins throughout development.

Polyglutamylation Is a Post-translational Modification with a Broad Range of Substrates

Polyglutamylation is a post-translational modification that generates lateral acidic side chains on proteins by sequential addition of glutamate amino acids. This modification was first discovered on tubulins, and it is important for several microtubule functions. Besides tubulins, only the nucleosome assembly proteins NAP1 and NAP2 have been shown to be polyglutamylated. Here, using a proteomic approach, we identify a large number of putative substrates for polyglutamylation in HeLa cells. By analyzing a selection of these putative substrates, we show that several of them can serve as in vitro substrates for two of the recently discovered polyglutamylases, TTLL4 and TTLL5. We further show that TTLL4 is the main polyglutamylase enzyme present in HeLa cells and that new substrates of polyglutamylation are indeed modified by TTLL4 in a cellular context. No clear consensus polyglutamylation site could be defined from the primary sequence of the here-identified new substrates of polyglutamylation. However, we demonstrate that glutamate-rich stretches are important for a protein to become polyglutamylated. Most of the newly identified substrates of polyglutamylation are nucleocytoplasmic shuttling proteins, including many chromatin-binding proteins. Our work reveals that polyglutamylation is a much more widespread post-translational modification than initially thought and thus that it might be a regulator of many cellular processes.

Characterisation of PGs1, a subunit of a protein complex co-purifying with tubulin polyglutamylase.

Polyglutamylation is a post-translational modification initially discovered on tubulin. It has been implicated in multiple microtubule functions, including neuronal differentiation, axonemal beating and stability of the centrioles, and shown to modulate the interaction between tubulin and microtubule associated proteins. The enzymes catalysing this modification are not yet known. Starting with a partially purified fraction of mouse brain tubulin polyglutamylase, monoclonal antibodies were raised and used to further purify the enzyme by immunoprecipitation. The purified enzyme complex (Mr 360×103) displayed at least three major polypeptides of 32, 50 and 80×103, present in stochiometric amounts. We show that the 32×103 subunit is encoded by the mouse gene GTRGEO22, the mutation of which has recently been implicated in multiple defects in mice, including male sterility. We demonstrate that this subunit, called PGs1, has no catalytic activity on its own, but is implicated in the localisation of the enzyme at major sites of polyglutamylation, i.e. neurones, axonemes and centrioles.

Ilf3 and NF90 associate with the axonal targeting element of Tau mRNA

In neurons, the selective translocation of Tau mRNA toward axons is due to the presence of a nucleotide sequence located in its 3′ untranslated region and serving as axonal targeting element. Using this RNA sequence as a probe by a Northwestern approach, we have detected several proteins that interact with the targeting RNA element and could potentially be involved in Tau mRNA translocation, translation halting, and/or stabilization. Among them, two proteins were identified as the interleukin enhancer binding factor 3 (Ilf3) and NF90, two isoforms derived from a single gene product through alternative splicing. Each protein comprises two double‐stranded RNA binding motifs that can interact with the predicted stem‐loop secondary structure of the axonal targeting element. Specific antibodies raised against common or specific peptide sequences showed that both Ilf3 and NF90 are polymorphic proteins that are detected in neuronal nuclei and cell bodies, as well as in the proximal neuritic segments. This observation favors the idea that Ilf3 and NF90 are part of a protein complex that escorts Tau mRNA toward the axon.

Posttranslational modifications of tubulin in cultured mouse brain neurons and astroglia

Posttranslational modifications of tubulin were analyzed in mouse brain neurons and glia developing in culture. Purified tubulin was resolved by isoelectric focusing. After 3 weeks of culture neurons were shown to express a high degree of tubulin heterogeneity (8 α and 10 β isoforms), similar to that found in the brain at the same developmental stage. Astroglial tubulin exhibits a less complex pattern consisting of 4 α and 4 β isoforms.

One β-tubulin subunit accumulates during neurite outgrowth in mouse neuroblastoma cells

Abstract Tubulin heterogeneity was analyzed during morphological differentiation of a mouse neuroblastoma clone (C 1300). One form of β-tubulin was found to be specific of the differentiated cells. The synthesis of this isoform is strictly related to neurite outgrowth process and has been shown to be regulated at the post-translational level.

Changes in some cytoskeletal proteins during neuroblastoma cell differentiation.

After in vitro microtubule assembly of mouse neuroblastoma crude extracts, six protein species migrate in the tubulin region of two-dimensional electrophoregrams. The evolution of these forms after morphological cell differentiation of the clone NIE115 shows two major modifications. Form 5 decreased drastically while form 6 increases during neurite formation. Peptide mapping analysis reveals that forms 5 and 6 are vimentin, a component of intermediate filaments, and beta-tubulin subunit, respectively. Sodium butyrate treatment of NIE115 cells or serum starvation of NIA103 cells, conditions blocking cell division and failing to induce morphological differentiation, prevent any modifications in the relative proportion of these proteins. It is concluded that the changes in the distribution of the tubulin isoforms and vimentin are directly related to neurite formation.

Inhibition of flagellar beat frequency by a new anti-β-tubulin antibody

A panel of monoclonal antibodies (mAbs) has been generated against sea urchin sperm axonemes and selected for their ability to inhibit the motility of sea urchin sperm models. The mAb C9 recognized a 50 kDa protein on blots of sea urchin sperm axonemes. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that C9 recognized isoforms of beta-tubulin. Low concentrations of C9 (0.1-1.0 microgram/ml) blocked the motility of sea urchin sperm models by decreasing the sliding velocity and frequency of flagellar beating to less than 1 Hz and by modifying the shear angle along the axoneme, especially the distal end. Other antitubulin antibodies had little effect on motility at concentrations 100-fold higher than those effective for C9. The effects on motility were not restricted to flagella of sea urchin spermatozoa. Flagellar beating of the dinoflagellate Oxyrrhis marina was completely blocked by C9 in a manner reminiscent of that of sea urchin sperm flagella. The mAb also inhibited the motility of human spermatozoa and Chlamydomonas reinhardtii. Immunofluorescence techniques revealed that C9 stains the whole axoneme of sea urchin spermatozoa and O. marina flagella together with the cortical network of O. marina cell body. C9 is the first antitubulin antibody reported to interfere with flagellar beat frequency. The observation that this antibody arrests the motility of flagella from sea urchin sperm along with that of dinoflagellate, algae, and human sperm flagella suggests that the epitope recognized by C9 is conserved over a long period of evolution and plays an important role in sperm motility.