INTRODUCTION Calcium ions acting as second messengers transduce extracellular signals into a wide variety of intracellular responses and thereby regulate many different biological processes such as secretion, proliferation, differentiation, transcription, apoptosis, and muscle contraction (for reviews see Parekh and Penner, 1997; Berridge, 1997). In recent years it has become clear that a number of human diseases, including cardiomyopathies, hypertension, neurodegenerative and neoplastic disorders are linked to altered Ca 2+ handling mediated by Ca 2+ -binding proteins (Heizmann and Braun, 1992, 1995; van Eldik and Griffin, 1994; Richard et al., 1995; Polans et al., 1996; Heizmann, 1996). The largest family of Ca 2+ -binding proteins shares a common structural motif, the EF-hand, named after the E- and F-helices of parvalbumin. The EF-hand domain consists of a helix-loop-helix motif that binds Ca 2+ selectively and with high affinity (Kretsinger, 1980; Nakayama and Kretsinger, 1994). Whereas calmodulin, the most prominent member of the EF- hand protein family, is ubiquitously expressed and multifunctional (Cohen and Klee, 1988; James et al., 1995), most of the other EF-hand proteins are expressed in a tissue- and cell-specific manner (Heizmann and Hunziker, 1991). This is also true for the S100 proteins representing the largest subfamily of EF-hand Ca 2+ -binding proteins (Donato, 1991; Hilt and Kligman, 1991; Fano et al., 1995; Schäfer and Heizmann, 1996). S100 proteins are acidic proteins of low molecular mass (10- 12 kDa), containing two distinct EF-hands with significantly different affinities for Ca 2+ . Both EF-hands are flanked by hydrophobic regions at either terminal and are separated by a 2043 Journal of Cell Science 111, 2043-2054 (1998) Printed in Great Britain © The Company of Biologists Limited 1998 JCS3760 Changes in cytosolic Ca 2+ concentration control a wide range of cellular responses, and intracellular Ca 2+ -binding proteins are the key molecules to transduce Ca 2+ signaling via interactions with different types of target proteins. Among these, S100 Ca 2+ -binding proteins, characterized by a common structural motif, the EF-hand, have recently attracted major interest due to their cell- and tissue-specific expression pattern and involvement in various pathological processes. The aim of our study was to identify the subcellular localization of S100 proteins in vascular smooth muscle cell lines derived from human aorta and intestinal smooth muscles, and in primary cell cultures derived from arterial smooth muscle tissue under normal conditions and after stimulation of the intracellular Ca 2+ concentration. Confocal laser scanning microscopy was used with a specially designed colocalization software. Distinct intracellular localization of S100 proteins was observed: S100A6 was present in the sarcoplasmic reticulum as well as in the cell nucleus. S100A1 and S100A4 were found predominantly in the cytosol where they were strongly associated with the sarcoplasmic reticulum and with actin stress fibers. In contrast, S100A2 was located primarily in the cell nucleus. Using a sedimentation assay and subsequent electron microscopy after negative staining, we demonstrated that S100A1 directly interacts with filamentous actin in a Ca 2+ -dependent manner. After thapsigargin (1 μM) induced increase of the intracellular Ca 2+ concentration, specific vesicular structures in the sarcoplasmic reticulum region of the cell were formed with high S100 protein content. In conclusion, we demonstrated a distinct subcellular localization pattern of S100 proteins and their interaction with actin filaments and the sarcoplasmic reticulum in human smooth muscle cells. The specific translocation of S100 proteins after intracellular Ca 2+ increase supports the hypothesis that S100 proteins exert several important functions in the regulation of Ca 2+ homeostasis in smooth muscle cells. Key words: Smooth muscle cell, Calcium-binding S100 protein, Actin, Confocal microscopy, Immunohistochemistry SUMMARY Distinct subcellular localization of calcium binding S100 proteins in human smooth muscle cells and their relocation in response to rises in intracellular calcium Anna Mandinova 1 , Dan Atar 2 , Beat W. Schäfer 3 , Martin Spiess 1 , Ueli Aebi 1 and Claus W. Heizmann 3, * 1 Maurice E. Müller-Institute, Biocentrum, University of Basel, 4056 Basel, Switzerland 2 Division of Cardiology, Department of Internal Medicine, University Hospital, 8032 Zürich, Switzerland 3 Division of Clinical Chemistry and Biochemistry, Department of Pediatrics, University of Zürich, 8032 Zürich, Switzerland *Author for correspondence (e-mail: heizmann@kispi.unizh.ch) Accepted 19 May; published on WWW 30 June 1998