Showing papers by "Javier Díaz-Nido published in 1991"

The distribution and phosphorylation of the microtubule-associated protein MAP 1B in growth cones

Abstract: Primary cultures of dissociated embryonic day 18 rat cerebral cortices were labelled by immunofluorescence with antibodies directed either against phosphorylated and non-phosphorylated MAP 1B (antibody 81) or against phosphorylated MAP 1B (antibody 150). Both antibodies stain cortical neurons, including their neurites and growth cones, in early (18 h) cultures, whereas only antibody 81 stained glial cells. By 4 days in culture, phosphorylated MAP 1B is largely restricted to axonal processes and growth cones, where it is often distributed in a gradient that is highest distally. In axonal processes and growth cones after 18 h and 4 days in culture, the phosphorylated form of MAP 1B is present both in a soluble form and bound to microtubules. Growth cones isolated from postnatal day 5 rat forebrain were labelledin vitro with32P-orthophosphate and detergent soluble and insoluble (cytoskeleton) fractions prepared. SDS-PAGE analysis revealed several major phosphoproteins in isolated growth cone cytoskeletons, including MAP 1B. Phosphorylated MAP 1B was also present in the detergent soluble fraction of growth cones. Immunoblotting and immunoprecipitation with MAP 1B antibodies confirmed the identification of MAP 1B and that the protein is phosphorylated in growth cones. These data show that MAP 1B, in particular the phosphorylated isoform, is present in growth cones and suggest that phosphorylation of MAP 1B may play an important role in neurite elongation.

Microtubule protein phosphorylation in neuroblastoma cells and neurite growth.

Abstract: The development of highly asymmetrical neurones from undifferentiated neuroblasts involves the extension of processes (axon and dendrites), that depends on the assembly of an inner microtubule scaffolding. Clonal cell lines of neuronal origin, N2A and NIE-115 neuroblastoma cells, have been chosen as model systems to study the modifications of microtubule protein which accompany the outgrowth of axon-like processes (neurites). Neuroblastoma cells grow as proliferating and undifferentiated cells in standard culture medium but can be considered as committed neuronal precursors. Thus, they are characterized by a high content of tubulin, including the minor neuronal-specific beta 3 isoform, and of MAPs including MAP1B and tau-like proteins. Serum withdrawal from the culture medium results in the extension of axon-like processes which is paralleled by a net increase in the amount of assembled tubulin. However, there is not any increase in the total amount of either tubulin or major MAPs which suggests an involvement of other regulatory factors in the promotion of microtubule assembly. Of relevance in this respect is the fact that beta 3-tubulin, MAP1B, and tau-like proteins become phosphorylated during neurite extension. A casein kinase II-like enzyme may be involved in some of these phosphorylation events. This enzyme is primarily localized to the nuclei in undifferentiated neuroblastoma cells, whereas a wider distribution of the enzyme between the nucleus and the cytoplasm is found in differentiating neuroblastoma cells. It thus appears plausible that a modified sorting of casein kinase II into the nucleus and the cytoplasm may be involved in the triggering of the phosphorylation of microtubule proteins during neuroblastoma cell differentiation.

Addition of protease inhibitors to culture medium of neuroblastoma cells induces both neurite outgrowth and phosphorylation of microtubule-associated protein MAP-1B

Abstract: The addition of two synthetic peptides with antiprotease activity to the culture medium of mouse neuroblastoma cells results in the promotion of neurite outgrowth. One of these peptides has a sequence corresponding to the reactive center of protease nexin-1 and inhibits both trypsin and thrombin. Its effect on neuroblastoma cells is similar to that found on serum withdrawal from the culture medium, giving rise to cells with one or two long neurites, and is reversed upon the addition of thrombin to the culture medium. The sequence of the other peptide is present in one of the precursor proteins of the main component of the amyloid plaques of Alzheimer's disease patients' brains, and corresponds to protease nexin-2. It can inhibit trypsin but fails to inhibit thrombin at low doses. Its effect on neuroblastoma cells is slightly different from that observed after serum deprivation, as a significant proportion of stellate cells, with short and branched neurites, is observed. An increase in the phosphorylation of microtubule-associated protein MAP-1B, which accompanies neurite outgrowth induced by serum deprivation, is also observed upon addition of the two antiprotease synthetic peptides, although the nexin-2 (amyloid) peptide induces a less marked increase in phosphorylated MAP-1B than does the nexin-1 peptide. These results may be correlated with the different antiprotease activities of both synthetic peptides, thus suggesting a role for a balance between trypsin-like and thrombin-like proteases and their inhibitors in eliciting neurite outgrowth under normal and pathological conditions.