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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.

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The proteins carried by the slow axonal transport in the rat sciatic motor axons were radiolabeled by injecting 35S-methionine into the spinal cord, and the distribution of their solubility through the 2 main components of slow transport (SCa and SCb) was considered. For this purpose, a cytoskeleton-stabilizing buffer was designed in which a pellet enriched in macromolecular and polymeric structures was separated from the solubilized proteins. The monomer/polymer ratios for tubulin were quantified in the 2 rate components. Our results indicate that 90% of the total tubulin was carried with SCa. Of this, 75% was in a polymeric state, versus only 50% of the tubulin carried with SCb. The monomeric tubulin recovered in the soluble fraction was concomitantly transported with the polymerized microtubules, suggesting that it might represent metastable regions of these microtubules. The insoluble and soluble fractions of the transported actin were measured. Actin was mostly (70%) transported with SCb. Of this, more than 80% was recovered in the soluble fraction, but we cannot say whether it was in a monomeric or polymeric state, nor if it was transported free or bound to a structure solubilized during fractionation. The other 30% of the actin, most of it transported with SCa, was recovered in the polymer-enriched fraction, probably bound to a stabilized polymer, such as the microtubules.(ABSTRACT TRUNCATED AT 250 WORDS)

Three distinct mRNAs have been shown to be produced by alternative splicing from the unique mouse peripherin gene. They generate three translation products, one major form, Pe-58, and two minor forms, Pe-56 which possess a shorter C-terminal sequence, and Pe-61 in which an additional sequence has been inserted in the central rod domain (Landon et al., 1989, EMBO J. 8, 1719-1726). In this study, the simultaneous occurrence of multiple transcripts in murine nervous tissues and neuroblastoma cell lines was shown by PCR amplification of fragments overlapping the sites of alternative splicing. Recombinant peripherin isoforms were purified from E. coli expressing full-length cDNAs. Rabbit antisera were raised against synthetic peptides mimicking parts of the two C-terminal sequences and of the inserted sequence of Pe-61 and were immunoadsorbed until they became monoreactive. By western blot analysis, the peripherin isoforms were localised in neuroblastoma NB2a cell lysates and detergent insoluble fractions separated by two-dimensional electrophoresis. In addition, each isoform was resolved into several charge variants. At the cellular level, each antibody decorated the filament array of the NB2a cells, suggesting the participation of the minor peripherin isoforms in the intermediate filament network.

Polyglutamylation, a posttranslational modification which consists of the sequential addition of one to six glutamyl units in the carboxy-terminal domain of both tubulin subunits, is a major event in neurons. Its structure has been investigated by using monoreactive polyclonal antibodies directed against distinct glutamylation motifs, ie alpha- and gamma-linkages between glutamyl units. It is shown that, beside alpha-linkages previously characterized, gamma-linkages also occur in glutamyl chains of brain tubulin. The co-existence of these two basic motifs leads to a conception of the polyglutamyl chain with a very sophisticated structure which could, through its complexity, help the microtubule to reach its structure and fulfil its functions.

The discovery that the dominant X-linked form of Charcot-Marie-Tooth disease (CMTX), a genetic disease of the peripheral nervous system (PNS), is associated with mutations in connexin32 (Cx32) has brought attention to the importance of connexins in glial cell biology. To gain further insight into the consequences of Cx32 deficiency, we have undertaken a detailed characterization of the gene expression profile of Schwann cells isolated from the sciatic nerve of wild-type and Cx32-null mice. Schwann cells exhibit two distinct phenotypes, myelinating and nonmyelinating, which are defined by their different morphology with respect to axons and by their unique profile of gene expression. Our findings show that, regardless of the mouse genotype, cultured Schwann cells express similar levels of messages for a number of connexins and for genes characteristic of both the myelinating and the nonmyelinating phenotypes. Furthermore, we have identified Cx36, a member of the gamma subclass of connexins, which are preferentially expressed in neuronal cells of mouse brain and retina, as an additional connexin present in Schwann cells. Mice lacking Cx32, however, exhibited a marked up-regulation of glial fibrillary acidic protein (GFAP), a cytoskeletal protein usually synthesized only by nonmyelinating Schwann cells. This observation was extended to the PNS in vivo and did not reflect a general perturbation of the expression of other nonmyelinating Schwann cell genes. These findings demonstrate that the absence of Cx32 results in a distinct pattern of gene dysregulation in Schwann cells and that Schwann cell homeostasis is critically dependent on the correct expression of Cx32 and not just any connexin. Identifying the relationship between increased GFAP expression and the absence of Cx32 could lead to the definition of specific roles for Cx32 in the control of myelin homeostasis and in the development of CMTX.