EFFECTS OF S100A1 AND S100B ON MICROTUBULE STABILITY. AN IN VITRO STUDY USING TRITON-CYTOSKELETONS FROM ASTROCYTE AND MYOBLAST CELL LINES G. SORCI, A. L. AGNELETTI and R. DONATO* Section of Anatomy, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Via del Giochetto, C.P. 81 Succ. 3, 06122 Perugia, Italy Abstract —S100A1 and S100B are members of a multigenic family of Ca 2+ -binding proteins of the EF-hand type highly abundant in astrocyte and striated muscle cells that have been implicated in the Ca 2+ -dependent regulation of several intracellular activities including the assembly and disassembly of microtubules and type III intermediate filaments. In the present work we tested S100A1 and S100B for their ability to cause microtubule and/or intermediate filament disassembly in situ using triton-cytoskeletons obtained from U251 glioma cells and rat L6 myoblasts. Our results indicate that: (i) both proteins cause a Ca 2+ -dependent disassembly of cytoplasmic microtubules in a dose-dependent manner; (ii) the S100A1- and S100B-inhibitory peptide, TRTK-12, blocks the S100A1 and S100B effects on microtubules; (iii) S100A1D88-93, an S100A1 mutant lacking the C-terminal extension, does not affect microtubule stability; and (iv) no obvious S100A1- or S100B-dependent intermediate filament disassembly could be observed under the experimental conditions used in the present study, but S100A1- and S100B-dependent microtubule disassembly results in a tendency of vimentin intermediate filaments to aggregate into bundles and/or to condense. Together, these results suggest that S100A1 and S100B probably cause microtubule disassembly by interacting with the microtubule wall, and that the two proteins do not affect intermediate filament stability via interaction with preformed intermediate filaments, in agreement with previous biochemical investigation. Our present data lend support to the possibility that S100A1 and S100B might have a role in the in vivo regulation of the state of assembly of microtubules in a Ca 2+ -regulated manner and, potentially, on microtubule-based activities in astrocytes and myoblasts. Also, these data suggest that the both S100 proteins use their C-terminal extension for interacting with microtubules.  2000 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: S100A1, S100B, microtubules, calcium, disassembly. Microtubules (MTs), a major constituent of the cytoskeleton in eukaryotes, are essential for cell division, the polarization of motile cells, the organization of the Golgi complex (and related secretory events), and the spatial organization of signal transduction. 22,34,53,57 The highly dynamic nature of MTs depends on the existence of stabilizing and destabilizing factors acting on tubulin, the backbone element of MTs, in an orchestrated manner as well as on post-translational modifi- cations of MT-associated proteins. Ca 2+ is long known as a MT-destabilizing agent, causing disassembly of preformed MTs and inhibition of MT assembly. Based on the observa- tion that the presence of a few micromolar amounts of Ca 2+ in cell homogenates caused inhibition of MT assembly, whereas much higher concentrations of Ca 2+ were required for inhibi- tion of assembly of purified microtubule protein, 59 factors were hypothesized to exist within cells with ability to mediate Ca 2+ effects on MT stability by, e.g. increasing the Ca 2+ sensitivity of MTs. S100A1 and S100B, two members of a multigenic family of Ca 2+ -modulated proteins of the EF-hand type, have been proposed to play a role in the regulation of protein phosphorylation, Ca 2+ homeostasis, the activity of a number of enzymes and transcription factors, and the dynamics of cytoskeleton constituents. 11,47,64 In particular, S100A1 and S100B were shown to inhibit MT assembly in a Ca 2+ - and pH-dependent manner by binding to and sequestering un- assembled tubulin, and to cause disassembly of preformed MTs by binding to the MT wall thereby increasing the Ca 2+ sensitivity of MTs, in vitro. 8,10 S100B is highly abundant in, although not restricted to astrocytes, and S100A1 is mostly expressed in neurons and striated muscle cells. 11,47,64 Further studies have shown that S100B is found associated with MTs in tissue cells (e.g. ependymal cells, olfactory vesicles and tracheal cells) and cell lines (cultured Schwann cells, astro- cytes, myoblasts). 6,41,42,50,51,55 Although direct association of S100A1 with MTs has not been documented, 50,51,61 published studies do not rule out the association of a small portion of the endogenous S100A1 pool with MTs. A link between S100A1 and MTs was recently inferred from results showing that inhibition of expression of S100A1 in a neuronal cell line results in an increase in unpolymerized tubulin at constant levels of polymerized tubulin. 65 These observations suggest that S100A1 and S100B might have a role in the regulation of the assembly of MTs in vivo, possibly with some differences as to their mechanism of action. Besides, S100A1 and S100B were shown to inhibit type III [glial fibrillary acidic protein (GFAP) and desmin] intermediate filament (IF) assembly and cause IF disassembly by binding to and sequestering un- assembled IF subunits in a Ca 2+ -dependent manner, in vitro, 4,16 and again, S100B, but not S100A1, was found asso- ciated with IFs in cell lines. 40,51,61 Other members of the S100 S100A1- and S100B-induced disassembly of cytoplasmic microtubules 773 773 Neuroscience Vol. 99, No. 4, pp. 773–783, 2000  2000 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00 PII: S0306-4522(00)00238-4 Pergamon www.elsevier.com/locate/neuroscience *To whom correspondence should be addressed. Tel.: +39-075-585-7453, Fax: +39-075-585-7451. E-mail address: donato@unipg.it (R. Donato). Abbreviations: BSA, bovine serum albumin; DDT, dithiothreitol; EGTA, ethyleneglycolbis(aminoethylether)tertracetate; FBS, fetal bovine serum; GFAP, glial fibrillary acidic protein; IFs, intermediate filaments; MTs, microtubules; PAGE polyacrylamide gel electrophoresis; PBS, phosphate buffered saline; SDS, sodium dodecyl sulphate; TBS, Tris- buffered saline; TW, Tween-20.