The role of microtubule-associated proteins in the assembly of tubulin to microtubules in vitro has been studied.1. It has been confirmed that pure tubulin obtained by phosphocellulose column chromatography does not significantly assemble in vitro in the absence of minor components which co-polymerize with tubulin. Although tubulin aggregates in a morpholino-ethanesulfonate buffer containing high Mg2 + concentrations, this process was neither inhibited by Ca2+ or colchicine, nor reversed by cold exposure.2. Microtubule-associated proteins were prepared, either by phosphocellulose column chromatography or by a direct method based on boiling reassembled microtubules in the presence of 2 mM dithiothreitol and 0.75 M NaCl. From each of these preparations two protein fractions were purified, either by Ultrogel ACAW chromatography or by sucrose gradient ultracentrifugation. The first one, with a high molecular weight, did not promote tubulin assembly; ageing of this material did not induce any activity. On the other hand, the second fraction, with an apparent molecular weight of 70000 (z protein), when almost completely purified, was active in promoting assembly.Thus a single specific protein is able to promote assembly of pure tubulin.Increasing evidence is available showing that in several systems where the microtubules are not stable as in cilia, specific signals induce a rapid massive polymerization of microtubules from a preexisting store of their basic subunit 6-S tubulin [l -41.For instance, in previous preliminary publications [5,6] we have shown that the amount of tubulin is not a limiting factor during rat brain development. However, the rate of polymerization in vitro and the overall extent of microtubule assembly is much lower with crude supernatants prepared from young rat brain than with control adult rat brain preparations. These results suggested that a 'factor' or 'signal' is needed for microtubule assembly and that the amount of this factor is limiting in the young brain.The nature of these signals or factors remains controversial.

Developmental changes in the composition of brain microtubule-associated proteins have been studied in three species : the rat and the mouse, which are characterized by post-natal brain development, and the guinea-pig, whose brain is mature at birth.1. At an adult stage, and whatever the species, two major microtubule-associated proteins, which have been referred to MAP2 and z, have been identified by polyacrylamide gel electrophoresis. Rat z is composed of four closely spaced bands; mouse z contains only three components with one of them being present in higher proportion than the others; adult guinea-pig z is essentially present as a single band.2. Microtubule-associated proteins were also prepared at different stages of brain development. In the three species only two bands were seen in the z region at immature stages of development (fast z and slow z). However adult z factors progressively replace the young entities. In contrast, only small changes were seen in the proportion of MAP2.3. Peptide mapping analysis of the purified z entities confirmed that the four adult rat proteins are very similar. In contrast, peptide mapping of the two young rat z proteins were very different from each other and from those of the adult ones. Peptide mappings of young and adult MAP2 were only slightly different. 4. The activities of young z proteins and young MAP2 in promoting pure tubulin assembly were much lower than those of the adult ones. Young fast z and young slow z were purified and both show to be active in promoting pure tubulin polymerization.5 . These data demonstrate the existence of two types of heterogeneity of microtubule-associated proteins : plurality of protein species at every stage of brain development and changes in composition and activity dependent on development.

Chronic treatments with antidepressants active on major depressive disorders influence pathways involved in cell survival and plasticity. As astrocytes seem to play a key role in the protection of brain cells, we investigated in these cells the rapid effects of the antidepressant fluoxetine (Prozac) on signaling cascades and gene induction, which probably play a role in neuroprotection. We show here that fluoxetine alone activates the extracellular signal-regulated-protein kinase (Erk) and p38 mitogen-associated protein (MAP) kinase cascades. RT-PCR revealed that genes, modulated in brain by long-term fluoxetine treatment, are rapidly induced by fluoxetine in cultured astrocytes: brain-derived nerve factor (BDNF) and its receptors, glial-derived nerve factor (GDNF) and deiodinase 3 (D3). Induction of D3 by fluoxetine is inhibited by U0126 and SB203580, suggesting that Erk and p38 MAP kinases are involved. Glial-derived nerve factor (GDNF) induction by fluoxetine is prevented by U0126, suggesting that Erk is implicated. Brain-derived nerve factor (BDNF) induction seems mediated by other signaling pathways. In conclusion, we show that fluoxetine alone rapidly activates mitogen activated protein (MAP) kinase cascades in rat astrocytes and that genes involved in neuroprotection are induced in a few hours in a MAP kinase-dependent or -independent manner.

Phagosomes are complex organelles that form after ingestion by phagocytic cells of pathogens, dying cells, or cell debris. Highly dynamic interactions of phagosomes first with endosomes and then with lysosomes lead to the maturation of phagosomes into phagolysosomes. Contrary to other phagocytes, which degrade ingested particles to amino acids, dendritic cells only partially degrade ingested proteins, preserving short peptides for the onset of adaptive immune responses. We have modified a series of latex bead-based techniques, previously reported, in order to analyze phagosome maturation using flow cytometry. The analysis of the phagosomal pH, degradation, or oxidation relies on techniques based on the fate of specific probes bound to particles to be phagocytosed. These techniques are very sensitive and quantitative.