Philippe Denoulet

Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons.

Recently, we and others reported that the doublecortin gene is responsible for X-linked lissencephaly and subcortical laminar heterotopia. Here, we show that Doublecortin is expressed in the brain throughout the period of corticogenesis in migrating and differentiating neurons. Immunohistochemical studies show its localization in the soma and leading processes of tangentially migrating neurons, and a strong axonal labeling is observed in differentiating neurons. In cultured neurons, Doublecortin expression is highest in the distal parts of developing processes. We demonstrate by sedimentation and microscopy studies that Doublecortin is associated with microtubules (MTs) and postulate that it is a novel MAP. Our data suggest that the cortical dysgeneses associated with the loss of Doublecortin function might result from abnormal cytoskeletal dynamics in neuronal cell development.

INTERACTION OF KINESIN MOTOR DOMAINS WITH ALPHA - AND BETA -TUBULIN SUBUNITS AT A TAU-INDEPENDENT BINDING SITE : REGULATION BY POLYGLUTAMYLATION

Interaction of rat kinesin and Drosophila nonclaret disjunctional motor domains with tubulin was studied by a blot overlay assay. Either plus-end or minus-end-directed motor domain binds at the same extent to both α- and β-tubulin subunits, suggesting that kinesin binding is an intrinsic property of each tubulin subunit and that motor directionality cannot be related to a preferential interaction with a given tubulin subunit. Binding features of dimeric versus monomeric rat kinesin heads suggest that dimerization could drive conformational changes to enhance binding to tubulin. Competition experiments have indicated that kinesin interacts with tubulin at a Tau-independent binding site. Complementary experiments have shown that kinesin does not interact with the same efficiency with the different tubulin isoforms. Masking the polyglutamyl chains with a specific monoclonal antibody leads to a complete inhibition of kinesin binding. These results are consistent with a model in which polyglutamylation of tubulin regulates kinesin binding through progressive conformational changes of the whole carboxyl-terminal domain of tubulin as a function of the polyglutamyl chain length, thus modulating the affinity of tubulin for kinesin and Tau as well. These results indicate that microtubules, through tubulin polymorphism, do have the ability to control microtubule-associated protein binding.

High level of tubulin microheterogeneity in the mouse brain

Using a high resolution isoelectric focusing method, we have demonstrated a high level of tubulin heterogeneity in mouse brain: 21 isotubulins were identified. Determination of the apparent molecular weight and analysis of the peptide map of each isotubulin species allowed us to identify 7 alpha and 14 beta tubulin subunits. Furthermore, variations in this tubulin population during brain development were confirmed; in particular, an increase in the number of beta isotubulins was observed at the beginning of the postnatal period.

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.

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.

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

Summary— 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 α‐ and γ‐linkages between glutamyl units. It is shown that, beside α‐linkages previously characterized, γ‐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.

Heterogeneity of Tau proteins during mouse brain development and differentiation of cultured neurons.

Tau microtubule-associated proteins constitute a group of developmentally regulated neuronal proteins. Using the high-resolution two-dimensional polyacrylamide gel electrophoresis system, we have resolved more than 60 distinct Tau isoforms in the adult mouse brain. Tau protein heterogeneity increases drastically during the second week of brain development. In neuronal primary cell cultures, some of these developmental changes can be observed. The increase of Tau heterogeneity in culture is more limited and reaches a plateau after a period corresponding to the second week of development. Most, if not all, of the vast Tau heterogeneity can be attributed to intensive post-translational phosphorylation, which may affect the structure of the proteins.

Association of Estramustine Resistance in Human Prostatic Carcinoma Cells with Modified Patterns of Tubulin Expression

Estramustine (EM) is an antimicrotubule drug used in the treatment of hormone refractory advanced prostate cancer. To investigate the mechanism of resistance to EM, we compared its effects on human prostate cancer cells (DU145) and an estramustine-resistant derived cell line (E4). Immunofluorescence demonstrated that EM caused depolymerization of microtubules and blocked cells in mitosis in DU145 cells, with less effect in E4 cells. Using tubulin isotype-specific antibodies, a threefold increase in betaIII and approximately twofold increase in betaI + II isotype in E4 cells compared to DU145 cells were observed. A most interesting observation concerned an increase in the posttranslational modification of alpha-tubulin of both polyglutamylation and acetylation in the E4 cells. Significant to this observation, using direct EM photoaffinity labeling of tubulin, drug binding to the most acidic posttranslationally modified forms of alpha-tubulin was shown to be minimal. Taken together, these results indicate that the modification of the tubulin expression pattern may be responsible for estramustine resistance by both lowering the amount of drug bound to microtubules and inducing more stable microtubules.

Estramustine resistance correlates with tau over-expression in human prostatic carcinoma cells

Estramustine (EM) is an anti‐microtubule drug used in the treatment of hormone‐refractory advanced prostate cancer. Since microtubules are the targets for EM cytotoxicity, we investigated the effects of EM on the microtubule‐associated protein tau to determine what role it may play in drug resistance. We have compared tau expression in human prostate cancer cells (DU145) and an EM‐resistant derived cell line (E4). Reverse transcriptase polymerase chain reaction has established that tau is expressed in both cell lines but increased 1.9‐fold in E4 compared with DU145 cells. This result was confirmed at the protein level by Western blotting. Tau is a phosphoprotein, most of its reported phosphorylation sites being serine or threonine residues. We have shown, however, that tau is also phosphorylated at tyrosine residues in DU145 cells and that the phosphotyrosine level of tau is significantly increased in E4 cells. Moreover, DU145 cells exposed to short term micromolar drug concentrations enter a phase of microtubule depolymerization, display an increased level of tau phosphorylation and follow a pattern similar to that observed in EM‐resistant E4 cells. EM is therefore able to induce a very rapid change in the post‐translational state of tau. Our results show that the acquisition of EM resistance in E4 cells, which is accompanied by changes at the tubulin level, is also associated with important changes in tau expression and phosphorylation. Int. J. Cancer 77:626–631, 1998.