INTRODUCTION Cell migration involves interactions of the cell with the surrounding extracellular matrix and requires a coordinated series of steps, including attachment to the substrate, generation of force and subsequent detachment from the substrate (Lee et al., 1993). Cell attachment to the substrate is mediated by adhesion receptors such as integrins (for review on adhesion receptors see Aplin et al., 1998). The nature of integrin-induced transmembrane signals controlling cell migration is still unclear. Changes of [Ca 2+ ] i are of particular interest, since such modifications may well modulate various stages of the migration process. Indeed, many of the key proteins involved in migration can be regulated by [Ca 2+ ] i , including protein kinase C, myosin light chain kinase, actin cytoskeleton regulating proteins such as gelsolin (Cunningham et al., 1991) or fodrin (Harris and Morrow, 1990) and integrins, whose affinity for extracellular matrix proteins can be decreased by increases in [Ca 2+ ] i (Hendey et al., 1992). The relationship between changes in [Ca 2+ ] i and cell migration has been studied only in a few cell types. Multiple transient increases in [Ca 2+ ] i have been observed in response to the chemoattractant f-Met-Leu-Phe (fMLP) in neutrophils migrating on vitronectin or fibronectin (Jaconi et al., 1991; Marks et al., 1991). Similar calcium transients during migration were observed in the MDCK-F cell line (Wojnowski et al., 1994) or in granule cells migrating from cerebellar microexplant cultures (Komuro and Rakic, 1996). The migration of vascular smooth muscle cells (VSMC) is one important component in the formation of atherosclerotic plaques (Ross, 1993). The mechanisms that govern this function in this cell type are largely unexplored. Collagens are the dominant surrounding extracellular matrix proteins for VSMC in blood vessels and α 2 β 1 integrin has been shown to be the adhesion receptor implicated in in vitro migration on collagen (Pickering et al., 1997). However, the role of [Ca 2+ ] i changes in any cell type migrating on collagen has, to our knowledge, never been studied, although it has been reported that α 2 β 1 integrin-mediated adhesion of human platelets to collagen is associated with an increase of [Ca 2+ ] i (Poole and Watson, 1995). We have thus investigated the importance of changes in [Ca 2+ ] i in human VSMC migrating on type I collagen. MATERIALS AND METHODS Materials Cell culture media RPMI 1640 and M199, Hepes, L-glutamine, penicillin, streptomycin, amphotericin B and trypsin-EDTA solution (trypsin 0.5 g/l, EDTA 0.2 g/l) were from Gibco (Paisley, UK). 653 Journal of Cell Science 113, 653-662 (2000) Printed in Great Britain © The Company of Biologists Limited 2000 JCS0822 Migration of vascular smooth muscle cells (VSMC) is a key event in the formation of neointima during atherosclerosis. Fura-2 loaded VSMCs were used to investigate calcium homeostasis during cell migration. Multiple spontaneous transient increases in cytosolic free calcium [Ca 2+ ] i were observed in single human VSMCs migrating on type I collagen. Such [Ca 2+ ] i transients were dependent on the presence of serum or PDGF-BB. Removal of serum, or loading cells with BAPTA, abolished the transients and decreased cell migration speed. The transients were not affected by disruption of cell polarization by dihydrocytochalasin B. Adhesion was used to investigate the specific role of cell-substrate interactions in the generation of transients. Transients are seen in VSMCs adhering either on collagen or on poly-L-lysine, suggesting that generation of transients is not strictly dependent on integrins. Buffering [Ca 2+ ] i with BAPTA led to accumulation of β1 integrins at the cellular tail, and to increased release of integrin on the extracellular matrix. These results demonstrate a role for [Ca 2+ ] i transients in the rapid, serum-dependent migration of VSMCs. These [Ca 2+ ] i transients are present in migrating VSMCs only when two simultaneous events occur: (1) substrate independent spreading and (2) stimulation of cells by serum components such as PDGF-BB. Key words: Migration, Smooth muscle, Integrin, Calcium, Collagen SUMMARY Migration of human vascular smooth muscle cells involves serum-dependent repeated cytosolic calcium transients Arnaud Scherberich, Manuel Campos-Toimil, Philippe Rondé, Kenneth Takeda and Alain Beretz* Pharmacologie et Physico-Chimie des Interactions Cellulaires et Moléculaires, UMR CNRS 7034, Faculté de Pharmacie, Université Louis Pasteur de Strasbourg, Illkirch, France *Author for correspondence (e-mail: aberetz@pharma.u-strasbg.fr) Accepted 2 December 1999; published on WWW 31 January 2000