Partial characterization of the cytoskeleton of CAD cells

BISIG, C.G.; PURRO, S.A.; BARRA, H.S. Y ARCE, C.A.

Villa Carlos Paz, Argentina

Congreso; Sociedad Argentina de Investigaciones Bioquímicas y Biología Molecular (SAIB); 2002

CAD is a cell line derived from tumors originated in catecholaminergic regions of the brain. These cells accumulate L-dopa because they lack dopa-decarboxylase. An interesting and useful property of these cells is that they can divide in culture medium containing fetal serum and can differentiate (they emit neurites) in the absence of serum. This phenomenon is reversible. In this work, cytoskeletal proteins of these cells were compared with those of brain tissue and other cells lines possessing neuronal characteristics. By using immunoblot with specific antibodies, it was determinated that CAD cells are devoid of the principal microtubules-stabilizing proteins: Tau, MAP1b, Map2 and N-STOP. This observation coincides with the high dynamics shown by microtubules in these cells as judge by disassembly with nocodazol (t1/2 <5 min.). In spite of the lack of stabilizing proteins, microfilamnet intermediate filaments and microtubules are present in these cells according to immunoblot and immunofluorescence with antibodies specific to actin, 200K-neurofilamnet polypeptide and tubulin. Tubulin was shown to be compose mainly by the ?tyrosinated? form, while the ?detyrosinated? form was scarce and ?delt2? tubulin was null. This tyrosination state is similar to that of embryonic brain tissue. It is possible that the high dynamics of microtubules explains the fast retraction of neurites of differentiated CAD cells (total retraction in 15 min.) when fetal serum is added.