Altered gene expression in Schwann cells of connexin32 knockout animals.

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.

Mouse peripherin isoforms.

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.

alphaB-crystallin interacts with cytoplasmic intermediate filament bundles during mitosis.

The small heat-shock protein alphaB-crystallin interacts with intermediate filament proteins. Using cosedimentation assay, we showed previously that in vitro binding of alphaB-crystallin to peripherin and vimentin was temperature-dependent. Furthermore, when NIH 3T3 cells were submitted to different stress conditions a dynamic reorganization of the intermediate filament network was observed concomitantly with the recruitment of alphaB-crystallins on the intermediate filament proteins. Thus, the intracellular state of alphaB-crystallin correlated directly with the remodeling of the intermediate filament network in response to stress. Here, we show data suggesting that alphaB-crystallin is implicated in remodeling of intermediate filaments during cell division. We investigated the intracellular distribution of alphaB-crystallin in naturally occurring mitotic NIH 3T3 cells and in neuroblastoma N2a and N1E115 cells. In NIH 3T3 cells, alphaB-crystallin remained diffused throughout the cell cycle. Subcellular fractionation of alphaB-crystallin showed that alphaB-crystallin remained in the cytosolic compartment during mitosis. Furthermore, alphaB-crystallin accumulated in mitotically arrested NIH 3T3 cells. This increased level of alphaB-crystallin protein was due to an increased level of alphaB-crystallin mRNA in mitotic NIH 3T3 cells. In the neuroblastoma cells, the intermediate filaments were rearranged into thick cable-like structures and alphaB-crystallin was recruited onto them. In neuroblastoma N2a cells the level of expression did not change during the cell cycle. However, a small fraction of alphaB-crystallin switched onto the insoluble fraction in mitotically arrested N2a cells. Our results suggested that depending on the state of rearrangement of the intermediate filament network during mitosis alphaB-crystallin was either recruited onto the intermediate filaments or upregulated in the cytosolic compartment.

Glial fibrillary acidic protein expression in a new human glioma cell line in culture before and after xenogenic transplantation into nude mice.

A human glioma cell line, SA146, was initiated on precoated extracellular matrix from a stereotactic biopsy of a glioblastoma. We report modulation in the expression of glial fibrillary acidic protein (GFAP) by SA146 passed in vitro before or after xenogenic transplantation into nude mice. Immunofluorescence data show a decrease in the percentage of GFAP-expressing cells with increasing in vitro passages but a full reexpression (100% of GFAP-positive cells among vimentin-positive cells) was observed in cultures just derived from the xenotransplanted tumor. These changes are correlated with the mRNA content (Northern blot probed with a cDNA for GFAP) and with the protein level (cytoskeletal fraction analyzed by two-dimensional gel electrophoresis and Western blots probed with a monoclonal antibody). At the optimal level of GFAP expression, a large range of micro-heterogeneity in GFAP isoforms is reached for which post-translational events are clearly involved since mRNA translation in cell free system would provide at best three isomers. We suggest that SA146 would be an appropriate model to study the regulation of GFAP expression in the context of human glial tumor biology.

AlphaB-crystallin interacts with intermediate filaments in response to stress.

The small heat shock protein alphaB-crystallin interacts with intermediate filament proteins. Using a co-sedimentation assay, we showed that in vitro binding of alphaB-crystallin to peripherin and vimentin was temperature-dependent. Specifically, a synthetic peptide representing the first ten residues of alphaB-crystallin was involved in this interaction. When cells were submitted to different stress conditions such as serum starvation, hypertonic stress, or heat shock, we observed a dynamic reorganisation of the intermediate filament network, and concomitant recruitment of alphaB-crystallins on intermediate filament proteins. Under normal conditions alphaB-crystallin was extracted from cells by detergent. In stressed cells, alphaB-crystallin colocalised with intermediate filament proteins, and became resistant to detergent extraction. The intracellular state of alphaB-crystallin seemed to correlate directly with the remodelling of the intermediate filament network in response to stress. This suggested that alphaB-crystallin functions as a molecular chaperone for intermediate filament proteins.

Melanoblast/melanocyte early marker (MelEM) is a glutathione S-transferase subunit.

We have biochemically characterized the antigen recognized by the melanoblast/melanocyte early marker (MelEM) monoclonal antibody (Mab) which labels early melanoblasts and melanocytes in the avian embryo [1]. While among the neural crest derivatives MelEM Mab is strictly specific for the melanocytic lineage, some endodermal derivatives such as hepatocytes react with this Mab. Since MelEM Mab immunoprecitates a protein of the same relative mass from both liver extracts and melanocytes, we immunopurified MelEM protein from liver extract of quail embryos at 10 days of incubation. The N-terminal sequence of this protein being blocked, we determined three internal peptide sequences. Our study reveals that the MelEM protein is an Alpha class subunit of glutathione S-transferase which is common to hepatocytes and to neural crest-derived pigment cells during their differentiation.

Plasma protein adsorption to artificial ligament fibers.

The adsorption of plasma proteins onto biomedical polymers is an important factor in the biocompatibility of biomaterials. We identified the plasma proteins adsorbed onto four polymeric fibers used for synthetic ligament replacement: polyarylamide, polylactic acid, polyester, and polypropylene. The adsorbed proteins were eluted and analyzed by one-dimensional and two-dimensional gel electrophoresis. Fibrinogen, albumin, IgG, high molecular weight kininogen (HMWK), and lipoproteins ApoA-1 and ApoE were the major proteins adsorbed onto polyarylamide. The three others biomaterials bound albumin, fibrinogen, ApoA-1, and ApoE; however, the proportions of proteins bound to each polymer differed. There was an inverse relationship between ApoA-1 and fibrinogen binding for all four biomaterials; polyarylamide bound a high percentage of fibrinogen, but little ApoA-1; polylactic acid, polyester, and polypropylene bound a high percentage of ApoA-1, but little fibrinogen. These results support suggestions that low fibrinogen adsorption might be due to the preferential adsorption of Apo-1. High fibrinogen binding to polyarylamide ligaments may favor fibroblast adherence, growth, and tissue repair.

The network organization and the phosphorylation of cytokeratins are concomitantly modified by forskolin in the enterocyte-like differentiated Caco-2 cell line.

Confluent Caco-2 cells, originating from a human colon carcinoma, display morphological and functional characteristics of differentiated enterocytes such as the presence of a polarized monolayer covered by an apical brush border that express several hydrolases. The adaptation of these cells to grow in the continuous presence of forskolin, a drug known to stimulate adenylyl cyclase permanently, has been previously shown to result in a decreased apical expression of hydrolases and in morphological alterations including the disappearance of intercellular spaces and shortening of microvilli. In the present work we have analyzed the possibility that cytoskeletal proteins may be the target of forskolin in living Caco-2 cells. We show that forskolin initiates dramatic changes in the spatial organization of the cytokeratin network that correlate with an increased phosphorylation of cytokeratin molecules, whereas microtubules, microfilaments and vimentin remain mainly unaffected. Indirect immunofluorescence studies show that the cytokeratin network is redistributed from the cell periphery to the cytoplasm. Biochemical experiments indicate that forskolin doesn't interfere with the cytokeratin profile, since the three cytokeratins normally found in intestine (CK 8, CK 18, CK 19) are similarly expressed in both control and forskolin-Caco-2 cells. Analysis of 32P-labeled cytokeratin extracted from the two cell populations demonstrates that forskolin quantitatively increases the phosphorylation of type I cytokeratin (CK 18 and CK 19), whereas the phosphorylation of type II cytokeratin (CK 8) is altered both quantitatively and qualitatively with the emergence of a new phosphorylation site. These results provide a new cell system in which it is possible to control the subcellular distribution of cytokeratin by changing their phosphorylation status and therefore to study their potential cellular functions.

Structure of the polyglutamyl chain of tubulin: occurrence of alpha and gamma linkages between glutamyl units revealed by monoreactive polyclonal antibodies.

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.

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.

Distribution of glutamylated alpha and beta-tubulin in mouse tissues using a specific monoclonal antibody, GT335.

A monoclonal antibody (GT335) directed against polyglutamylated tubulin was obtained by immunization with a synthetic peptide which mimics the structure of the polyglutamylated site of alpha-tubulin. This peptide corresponds to the C-terminal sequence Glu441-Gly448 and was chemically modified by the addition of two glutamyl units at Glu445. The specificity of GT335 was assayed by direct and competitive enzyme-linked immunosorbent assay (ELISA) against tubulin and several synthetic peptides differing either by the structure of the added polyglutamyl chain or by their amino acid sequence. Further characterization was carried out by immunoblotting detection after one- or two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The epitope appears to be formed by at least two constituents: a basic motif of monoglutamylation which is retained in the polyglutamylated forms independent of their degree of glutamylation, and some elements of the polypeptide chain close to the site of glutamylation. Given the specificity of GT335 and the delineation of its epitope, our results indicate that, in addition to alpha and beta' (class III)-tubulin, other beta-tubulin isotypes are also glutamylated. This antibody has been used to analyze the cell and tissue distributions of glutamylated tubulin. In mouse brain extracts, GT335 reacts strongly with alpha-tubulin and, to a lesser extent, with beta' (class III) and beta-tubulin. The same reactivity is also observed with cultured neurons whereas astroglial cells exhibit only low levels of glutamylated tubulin. In non-nervous mouse tissues such as spleen, lung or testis, glutamylation was shown to involve only beta-tubulin, but at far lower levels than in brain.

Comparison of biochemical properties of DNA-topoisomerase I from normal and regenerating liver.

Biochemical properties of topoisomerase I from normal and regenerating rat liver were analysed using crude or fractionated nuclear extracts. We could not detect significative change in topoisomerase I content or activity (magnesium stimulation and inhibition by ATP) during the course of liver regeneration. Topoisomerase I can be resolved into two species of 97 kDa and 100 kDa, with the same pI of 8.2-8.6 as shown by two dimensional gel electrophoresis. The two polypeptides contained a non-phosphorylated precursor and others forms with variable degrees of phosphorylation. In-vitro dephosphorylation with alkaline phosphatase leads to the disappearance of the phosphorylated forms and inactivation of the enzyme. The affinity of topoisomerase I for chromatin (measured by salt elution) differs markedly between normal and regenerating liver: nearly 50% of topoisomerase I remained bound to the chromatin from normal liver at 250 mM NaCl whereas it was completely eluted from 24-h-regenerating-liver nuclei. The biological significance of these results is discussed.

Calcium-activated neutral protease (CANP) in normal and dysmyelinating mutant paralytic tremor rabbit myelin.

Calcium-activated neutral protease (CANP) in normal and dysmyelinating mutant, paralytic tremor (PT) rabbit myelin and premyelin fractions was studied using immature (4-5 wk) or adult animals. The enzyme was estimated by determination of its catalytic activity as well as by using immunoblot analysis after SDS-PAGE separation. The presence of two forms of CANP--one activated by calcium in the micromolar concentration (mu CANP) range and the other exhibiting low calcium sensitivity in the millimolar concentration range (m-CANP)--was found in the myelin and premyelin fractions. The developmental pattern of the enzyme activity was different for each of these two enzyme isoforms depending on the fraction studied. The higher activity on CANP (both isoforms) found in PT myelin and premyelin could be related to delayed myelination and/or to the higher turnover rate of already formed myelin. These results suggest complex and specific roles for these isoenzymes during myelin formation as is discussed further in this article. Our results confirm the extensive degradation of myelin basic protein (MBP), proteolipid protein (PLP), and, to a lesser extent, the other myelin proteins by endo- and exogenous CANP. This degradation process was significantly elevated in PT rabbit myelin. Moreover as was shown by two-dimensional gel electrophoresis, calcium-controlled proteolysis in nonmutant rabbits affected the net-charge of MBP in a manner similar to that reported for PT myelin, suggesting the possible involvement of CANP in the generation of charge isomers of MBP.

Expression of glial fibrillary acidic protein and vimentin in mouse lens epithelial cells during development in vivo and during proliferation and differentiation in vitro: comparison with the developmental appearance of GFAP in the mouse central nervous system.

Analysis of glial fibrillary acidic protein (GFAP) and vimentin in mouse lens epithelial cells (MLEC) during ontogenesis revealed a two-step developmental expression similar to that observed in astrocytes. Vimentin was first immunostained at E11 corresponding with the closure of the lens vesicle, whereas GFAP was detected only after a further 7 days (E18); this protein appeared simultaneously in the mouse lens and CNS. In the latter case, it was present in the hypothalamic tanycytes and spinal cord. This similarity in the timing of appearance of GFAP in the non-neural MLEC and in fetal astrocytes suggests a common mechanism for its expression in tissues of different embryological origin. However, it has previously been observed that, in contrast to the situation in astrocytes, GFAP disappears from differentiating MLEC in vivo. We have shown that in vitro this protein also disappears rapidly from MLEC in the presence of fetal calf serum (FCS). However, the use of mouse serum instead of FCS inhibited the migration of MLEC out of the explant, and in these cells GFAP persisted.

Differential axonal transport of isotubulins in the motor axons of the rat sciatic nerve.

The axonal transport of the diverse isotubulins in the motor axons of the rat sciatic nerve was studied by two-dimensional polyacrylamide gel electrophoresis after intraspinal injection of [35S]methionine. 3 wk after injection, the nerve segments carrying the labeled axonal proteins of the slow components a (SCa) and b (SCb) of axonal transport were homogenized in a cytoskeleton-stabilizing buffer and two distinct fractions, cytoskeletal (pellet, insoluble) and soluble (supernatant), were obtained by centrifugation. About two-thirds of the transported-labeled tubulin moved with SCa, the remainder with SCb. In both waves, tubulin was found to be associated mainly with the cytoskeletal fraction. The same isoforms of tubulin were transported with SCa and SCb; however, the level of a neuron-specific beta-tubulin subcomponent, termed beta', composed of two related isotubulins beta'1 and beta'2, was significantly greater in SCb than in SCa, relative to the other tubulin isoforms. In addition, certain specific isotubulins were unequally distributed between the cytoskeletal and the soluble fractions. In SCa as well as in SCb, alpha''-isotubulins were completely soluble in the motor axons. By contrast, alpha''' and beta'2-isotubulins, both posttranslationally modified isoforms, were always recovered in the cytoskeletal fraction and thus may represent isotubulins restricted to microtubule polymers. The different distribution of isotubulins suggests that a recruitment of tubulin isoforms, including specific posttranslational modifications of defined isoforms (such as, at least, phosphorylation of beta' and acetylation of alpha'), might be involved in the assembly of distinct subsets of axonal microtubules displaying differential properties of stability, velocity and perhaps of function.

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 alpha and 10 beta isoforms), similar to that found in the brain at the same developmental stage. Astroglial tubulin exhibits a less complex pattern consisting of 4 alpha and 4 beta isoforms. After incubation of neuronal and glial cells with 3H-acetate in the presence of cycloheximide, a major posttranslational label was found associated with alpha-tubulin and a minor one with beta-tubulin. The acetate-labeled isotubulins of neurons were resolved by isoelectric focusing into as many as 6 alpha and 7 beta isoforms, while those of astroglia were resolved into only 2 alpha and 2 beta isoforms. The same alpha isoforms were also shown to react with a monoclonal antibody recognizing selectively the acetylated form(s) of alpha-tubulin. Whether acetate-labeling of alpha-tubulin in these cells corresponds to the acetylation of Lys40, as reported for Chlamydomonas reinhardtii, is discussed according to very recent data obtained by protein sequence analysis. Tubulin phosphorylation was analyzed by incubation of cell cultures with 32PO4. No phosphorylation of alpha-tubulin isoforms was detected. A single beta-tubulin isoform (beta'2), expressed only in neurons, was found to be phosphorylated. This isoform is similar to that previously identified in differentiated mouse neuroblastoma cells.

Stable and metastable cytoskeletal polymers carried by slow axonal transport.

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)

Myelin composition and activities of CNPase and Na+,K+-ATPase in hypomyelinated "pt" mutant rabbit.

A disorder of CNS myelination was found in paralytic tremor ("pt") rabbits. The condition is inherited in a sex-linked recessive mode. Ultrastructurally, an obvious myelin deficiency with aberration of myelin sheath formation is observed. The yield of myelin isolation was reduced to 20-30% of control. Myelin isolated from 4-week-old "pt" rabbits contained reduced amounts of galactosphingolipids and of several myelin protein markers. Moreover, myelin basic protein, analyzed by two-dimensional gel electrophoresis, showed a deficit in its more basic components. All these facts suggest a delay in myelin maturation. Ganglioside content was increased as well as Na+,K+-ATPase specific activity. 2',3'-Cyclic nucleotide phosphodiesterase (CNPase) specific activity was the same in "pt" as in control myelin but differed by having greater sensitivity to detergent activation.

The aminoterminal domain of alpha-tubulin probed by monoclonal antibodies. Recognition of a rarely exposed epitope by the monoclonal antibody 111 B52 C2.

The production and identification of a monoclonal antibody, 111 B52 C2, raised against fragments obtained after limited proteolysis of purified tubulin is described. The recognized epitope is located on the aminoterminal domain of the alpha-tubulin subunit and differs from the antigenic sites reacting with the presently existing panel of available monoclonal antibodies. This monoclonal antibody thus constitutes a potentially useful tool to explore interactions between tubulin and other specific ligands.

Control of isotubulin expression during neuronal differentiation of mouse neuroblastoma and teratocarcinoma cell lines.

Mouse neuroblastoma and teratocarcinoma constitute adequate cellular systems to study the expression of tubulin isoforms during early as well as later steps of neuronal differentiation. Tubulin heterogeneity is extensively analyzed using both isoelectric focusing and two-dimensional electrophoresis. Multipotential embryonal carcinoma cells express mainly one alpha-tubulin isoform (alpha 1) and three beta-tubulin isoforms: a major one (beta 3) and two minor ones (beta 4 and beta 5). Early events of neuronal differentiation are shown to induce the expression of an additional beta-tubulin isoform, beta'1, which is encoded by a specific mRNA. Neurite extension further increases tubulin heterogeneity and leads to the appearance of post-translationally modified isoforms: beta'2 in neuroblastoma and alpha 2 in teratocarcinoma cells. beta' 2 is shown to derive from the above mentioned beta'1 by phosphorylation, while alpha 2 is probably an acetylated form of the common alpha 1-tubulin. These results show that specific changes in tubulin heterogeneity are induced at different steps of neuronal differentiation and are controlled both at the transcriptional (or post-transcriptional) and post-translational levels.