sign in sign up
<<
Home , Calgranulin, S100A1, S100A4, S100 protein

Journal of Cell Science 111, 2043-2054 (1998) 2043 Printed in Great Britain © The Company of Biologists Limited 1998 JCS3760

Distinct subcellular localization of binding S100 in human smooth muscle cells and their relocation in response to rises in intracellular calcium

Anna Mandinova1, Dan Atar2, Beat W. Schäfer3, Martin Spiess1, Ueli Aebi1 and Claus W. Heizmann3,* 1Maurice E. Müller-Institute, Biocentrum, University of Basel, 4056 Basel, Switzerland 2Division of Cardiology, Department of Internal Medicine, University Hospital, 8032 Zürich, Switzerland 3Division of Clinical Chemistry and Biochemistry, Department of Pediatrics, University of Zürich, 8032 Zürich, Switzerland *Author for correspondence (e-mail: [email protected])

Accepted 19 May; published on WWW 30 June 1998

SUMMARY

Changes in cytosolic Ca2+ concentration control a wide associated with the and with actin range of cellular responses, and intracellular Ca2+-binding stress fibers. In contrast, was located primarily in proteins are the key molecules to transduce Ca2+ signaling the . Using a sedimentation assay and via interactions with different types of target proteins. subsequent electron microscopy after negative staining, we Among these, S100 Ca2+-binding proteins, characterized by demonstrated that directly interacts with a common , the EF-hand, have recently filamentous actin in a Ca2+-dependent manner. After attracted major interest due to their cell- and tissue-specific thapsigargin (1 µM) induced increase of the intracellular expression pattern and involvement in various pathological Ca2+ concentration, specific vesicular structures in the processes. The aim of our study was to identify the sarcoplasmic reticulum region of the cell were formed with subcellular localization of S100 proteins in vascular smooth high S100 content. In conclusion, we demonstrated muscle cell lines derived from human aorta and intestinal a distinct subcellular localization pattern of S100 proteins smooth muscles, and in primary cell cultures derived from and their interaction with actin filaments and the arterial smooth muscle tissue under normal conditions and sarcoplasmic reticulum in human smooth muscle cells. The after stimulation of the intracellular Ca2+ concentration. specific translocation of S100 proteins after intracellular Confocal laser scanning microscopy was used with a Ca2+ increase supports the hypothesis that S100 proteins specially designed colocalization software. Distinct exert several important functions in the regulation of Ca2+ intracellular localization of S100 proteins was observed: homeostasis in smooth muscle cells. was present in the sarcoplasmic reticulum as well as in the cell nucleus. S100A1 and were found Key words: Smooth muscle cell, Calcium-binding , predominantly in the cytosol where they were strongly Actin, Confocal microscopy, Immunohistochemistry

INTRODUCTION F-helices of . The EF-hand domain consists of a helix-loop-helix motif that binds Ca2+ selectively and with high Calcium ions acting as second messengers transduce affinity (Kretsinger, 1980; Nakayama and Kretsinger, 1994). extracellular signals into a wide variety of intracellular Whereas , the most prominent member of the EF- responses and thereby regulate many different biological hand protein family, is ubiquitously expressed and processes such as secretion, proliferation, differentiation, multifunctional (Cohen and Klee, 1988; James et al., 1995), transcription, , and (for reviews most of the other EF-hand proteins are expressed in a tissue- see Parekh and Penner, 1997; Berridge, 1997). In recent years and cell-specific manner (Heizmann and Hunziker, 1991). This it has become clear that a number of human diseases, including is also true for the S100 proteins representing the largest , hypertension, neurodegenerative and subfamily of EF-hand Ca2+-binding proteins (Donato, 1991; neoplastic disorders are linked to altered Ca2+ handling Hilt and Kligman, 1991; Fano et al., 1995; Schäfer and mediated by Ca2+-binding proteins (Heizmann and Braun, Heizmann, 1996). 1992, 1995; van Eldik and Griffin, 1994; Richard et al., 1995; S100 proteins are acidic proteins of low molecular mass (10- Polans et al., 1996; Heizmann, 1996). 12 kDa), containing two distinct EF-hands with significantly The largest family of Ca2+-binding proteins shares a different affinities for Ca2+. Both EF-hands are flanked by common structural motif, the EF-hand, named after the E- and hydrophobic regions at either terminal and are separated by a 2044 A. Mandinova and others central hinge region. The carboxy-terminal EF-hand contains were visualized by immunofluorescence using confocal laser- the canonical Ca2+-binding loop encompassing 12 amino acids, scanning microscopy. Our results indicate that S100 Ca2+- whereas the amino-terminal loop consisting of 14 amino acids binding proteins exhibit a distinct subcellular localization is specific for S100 proteins. To date, some 17 different pattern in human vascular smooth muscle cell lines and in proteins have been assigned to the S100 protein family that primary cell cultures derived from smooth muscle tissue. We display various degrees of amino acid in provide evidence that the S100A1 protein specifically interacts the range of 25% to 65%. At least thirteen S100 were with filamentous actin (F-actin) in smooth muscle cells, thus found to be clustered on human 1q21 (Engelkamp possibly representing a new regulatory system together with et al., 1993; Wicki et al., 1996a,b), leading to the introduction calponin and caldesmon in modulating smooth muscle of a new S100 nomenclature (Schäfer et al., 1995). The contraction. In addition, they specifically translocate within localization of the S100 cluster on human chromosome these cells after an increase of the intracellular Ca2+ 1q21 is of special interest since a number of rearrangements concentration. These experiments provide new insights into the (deletions, translocations, duplications) have been found to function of S100 proteins through their interaction with occur in this chromosomal region in cancer cells (Gendler et different target proteins in the Ca2+- cascade. al., 1990; Hoggard et al., 1995; Forus et al., 1995; Bartoli et al., 1996; Weterman et al., 1996). Alterations in S100 expression have been demonstrated in MATERIALS AND METHODS diseases such as Down’s syndrome (Allore et al., 1988), Alzheimer’s disease (van Eldik and Griffin, 1994), chronic Cell cultures inflammation (Rammes et al., 1997), and cardiomyopathies All experiments were performed with two stable smooth muscle cell (Remppis et al., 1996). lines derived from human aorta (HVSMC ATCC-13145 CRL-1999) S100 proteins are thought to exert their effect through Ca2+- and from human jejunum (HISM ATCC CRL-1692). Cells obtained regulated interactions with specific intracellular target proteins. from the American Type Culture collection (Maryland, USA) were Some intracellular S100 target proteins are myosin (Burgess et grown in Dulbecco’s modified Eagle’s medium (DMEM), al., 1984), (Donato et al., 1989), - supplemented with 1% L-glutamine, 10% fetal bovine serum, 100 units/µl penicillin and streptomycin (all from Gibco BRL). The cells associated tau-proteins (Baudier and Cole, 1988), glial were incubated in a humidified incubator (Heraeus, Switzerland) at fibrillary acidic protein (Bianchi et al., 1996), tropomyosin 5% CO2and 37°C. Cells in passages 3 through 5 were used for (Gimona et al., 1997), phosphoglutamase (Landar et al., 1996), analysis. twitchin (Heierhost et al., 1996), cytosolic Primary cultures derived from human arterial smooth muscle tissue phospholipase A2 (Wu et al., 1997), Ca2+ release channel were a kind gift from Dr Z. Yang, University of Zurich, Switzerland. () of the sarcoplasmic reticulum (SR) Briefly, cells were prepared from the left internal mammary artery of (Treves et al., 1997), and (Garbuglia et al., 1996). patients undergoing aorto-coronary bypass surgery. The work was Here we demonstrate that smooth muscle cells represent a performed in accordance with the requirements of the institutional valuable model system to investigate the distinct subcellular review committee for the use of human material. Immediately upon localization and the functional role of S100 proteins since these tissue removal samples were taken to the laboratory and processed within 30 minutes. Following removal of the adventitia, tissues were cells co-express S100A1, S100A2, S100A4, and S100A6. minced into blocks of approximately 1 mm3. Tissue blocks were S100A1, found to be expressed in muscle tissues transferred to a sterile 25 cm2 culture flask covered with fresh DMEM (Engelkamp et al., 1992; Pedrocchi et al., 1993; Song and plus 10% fetal bovine serum, 1% L-glutamine, and 100 units/µl Zimmer, 1996; Remppis et al., 1996), was also shown to penicillin and streptomycin. Flasks were placed in a humidified activate invertebrate giant protein involved in the incubator for 6 hours to allow time for the tissue blocks to attach to regulation of muscle function (Heierhorst et al., 1996), and the culture surface. After 6 hours, fresh DMEM containing L- modulates adenylate cyclase activity (Fano et al., 1989a) and glutamine, antibiotics, and FBS was added and flasks were returned the Ca2+-induced Ca2+ release in different types of muscle cells to the incubator for the next 48 hours. Confluent cells between passage (Fano et al., 1989b; Treves et al., 1997). S100A2, expressed in 0 and 2 were used for experiments. a number of tissues, is of particular interest because it is down- Antibodies regulated in tumor cells, suggesting it may be a candidate Polyclonal antisera against human recombinant S100A1, S100A4, tumor suppressor gene (Lee et al., 1992; Wicki et al., 1997). and S100A6 proteins were raised in goats, and the polyclonal S100A4 has been identified as playing a causal role in the antiserum against human recombinant S100A2 in rabbits. These metastatic behavior of different cancer cells in rodents (Davies primary polyclonal antibodies are specific and only recognize the et al., 1996). Moreover, it has been shown that S100A4 appears corresponding antigen. They do not cross-react either with other S100 in association with tropomyosin as well as with actin stress or Ca2+-binding proteins or with other cellular proteins (Ilg et al., fibers (Takenaga et al., 1994). Finally, S100A6 is expressed in 1996). The mouse monoclonal antibody (mAb) G1/296 raised against the of epithelial cells, fibroblasts, neuronal and a novel 63 kDa membrane protein (p63), which identifies the SR in muscle cells, and is overexpressed in a number of tumor tissues the cytoplasm of primate cells (Schweizer et al., 1993), was a kind (for review, see Schäfer and Heizmann, 1996), and it has been gift from Dr H. P. Hauri, Biocenter of the University of Basel. shown to specifically interact with annexins (Watanabe et al., A fluorescent derivative of phalloidin, a mushroom toxin recognizing all isoforms of F-actin (FITC-phalloidin), was purchased 1993a), caldesmon (Skripnikova and Gusev, 1989), and other from Sigma (Biosciences, USA). The secondary Cy3-coupled rabbit proteins (Filipek et al., 1996; Filipek and Wojda, 1996; anti-goat and Cy3-coupled goat anti-rabbit antibodies were from Tokumitsu et al., 1992). Sigma (Biosciences, USA), and the donkey Cy2-coupled anti-mouse In the present work, we investigated the distinct subcellular antibody was purchased from Amersham (Life Science, USA). CyDye localizations of S100 proteins. For this purpose, S100 proteins is a mixture of fluorescent dyes (cyanine fluors) for labeling proteins. Ca2+-binding S100 proteins in smooth muscle 2045

Immunofluorescence labeling images were prepared with the ‘Imaris’, ‘Voxel Shop’, and HVSMC and freshly isolated arterial human smooth muscle cells were ‘Colocalization’ software products (Messerli et al., 1993), available transferred into 24-well cell culture plates containing 1 ml/well of from Bitplane AG (Zürich, Switzerland). growth medium. For further processing, this medium was replaced by modified Ca2+-free Hanks’ buffer (MHB), containing 2 mM EGTA and Sedimentation assay 5 mM MES (2-morpholino-ethanesulfonic acid), pH 6.2 to 6.4 (Small The interaction of S100A1 and S100A6 with F-actin was evaluated and Celis, 1978), and then quickly substituted with ‘permeabilization by high-speed centrifugation. To this end, 5 µM G-actin (purified from buffer’, i.e. MHB containing 2% octyl-POE (n-octylpolyoxyethylene; rabbit as described by Bremer et al., 1994) at a protein ALEXIS Corporation, Switzerland) and 0.125% glutaraldehyde concentration of 1 mg/ml (i.e. 24 µM) was polymerized to F-actin in (Electron Microscopy Sciences, USA). Time from detachment to imidazole-buffer A (2.5 mM imidazole, 0.005% NaN3, 0.2 mM permeabilization was kept under 3 minutes. After 5 minutes of CaCl2, 0.2 mM ATP, pH 7) by adding KCl to 50 mM and 1 mM MgCl2 permeabilization and prefixation, cells were fixed for 20 minutes with (120 minutes, at room temperature). F-actin was then incubated for MHB containing 1% glutaraldehyde. The HVSMC and the primary cell 120 minutes at room temperature with 25 µM of human recombinant cultures were then washed 3 to 4 times with MHB. Aldehyde groups S100A1 or S100A6 in buffer A containing either 1 µM Ca2+ or 1 µM were reduced by treating the cells twice for 10 minutes each with EGTA. For controls, the proteins were incubated in buffer A, also 2+ NaHB4 (0.5 mg/ml) in MHB on ice. Immunofluorescence labeling was containing either 1 µM Ca or 1 µM EGTA. After incubation the carried out as described (Baschong et al., 1997) by incubating the cells mixtures were centrifuged at 100,000 g for 15 minutes. Supernatants with appropriate concentrations of primary and fluorochrome- and pellets were analyzed by SDS-PAGE. conjugated secondary antibodies. Finally, the cells were washed with MHB, mounted bottom-up (i.e. inverted prior to mounting) in Mowiol Electron microscopy 4-88 (Hoechst, Germany) containing 0.75% n-propyl-gallate as an anti- The interaction of S100A1 with F-actin filaments was visualized by bleaching agent. Mounted slides were left to dry for 24 hours at room negative staining. Briefly, a 5 µl aliquot of the sample was absorbed temperature in the dark, and then stored at 4°C in the dark until viewed. for 1 minute onto glow-discharged (Aebi and Pollard, 1987) carbon- For controls, the cells were incubated either with pre-immune sera coated, 400-mesh/inch copper grids. The grid was washed on two or with polyclonal anti-S100 antibodies preabsorbed with the drops of deionized water and then placed on two drops of 0.75% corresponding antigens. For this purpose 50 µg of recombinant S100 uranyl formate (pH 4.25) for 15 seconds each. The specimens were protein (Pedrocchi et al., 1994) was added to 100 µl of undiluted inspected in a Zeiss 910 TEM operated at 100 kV, and electron polyclonal anti-S100 antibodies and incubated for 10 hours at 4°C. micrographs were recorded on Kodak S0-163 electron image film After centrifugation at 14,000 g the supernatant was taken for control (Eastman Kodak Co.) at a nominal magnification of ×50,000. The stainings. exact magnification was calibrated according to the method of Wrigley (1968). Intracellular calcium stimulation A rise of intracellular [Ca2+] in smooth muscle cells was achieved by treatment with 1 µM thapsigargin (12 minutes at 37°C; Molecular Probes, USA). Cells were then washed 3 times with MHB for 2 RESULTS minutes each, fixed, permeabilized, and immunolabeled as described above. Distinct localization of S100A1, S100A2, S100A4, and S100A6 Confocal laser scanning microscopy To investigate the subcellular localization of human S100 Micrographs were taken with a confocal microscope consisting of a proteins in HVSMC, immunofluorescence staining using Zeiss Axiovert fluorescence microscope with a Zeiss Plan Apo 63/1.4 oil objective lens and an Odyssey XL confocal laser-scanning unit S100-specific polyclonal antibodies and fluorescent secondary (NORAN, USA), driven by the Intervision software package run on antibodies was applied to smooth muscle cells from aorta. The an INDY workstation (Silicon Graphics Inc., USA). The light source fluorescence micrographs in Fig. 1 display two different optical was an Argon laser tuned so that the exitation wave length for Cy3 sections through a cell that was stained with an anti-S100A1 was 529 nm, and that for FITC and Cy2 488 nm. The colocalization primary antibody. The section taken close to the tip of the cell

Fig. 1. Intracellular immunolocalization of S100A1. The localization of S100A1 in HVSMC is visualized using confocal laser scanning microscopy (A,B). The cells were stained with a primary goat anti-S100A1 antibody (dilution 1:20) and a secondary Cy3- coupled anti-goat antibody (dilution 1:1,000). Control staining (C) was performed with an antibody preabsorbed with the corresponding recombinant antigen. The first fluorescence image of a HVSMC (A) was optically sectioned at a distance of 1.76 µm from the coverslip. S100A1 resides in the cytosol around the cell nucleus and is organized in a reticular network-like structure. The second fluorescence image (B) of the same cell was sectioned at a distance of 0.80 µm from the substrate. Here too, S100A1 is located exclusively in the cytosol where it appears to be associated with stress fiber-like structures in the periphery of the cell. Control labeling (C) with an anti- S100A1 antibody preabsorbed with the recombinant antigen is negative. 2046 A. Mandinova and others

Fig. 2.Intracellular immunolocalization of S100A4. The cells (A,B) were stained with a primary goat anti-S100A4 antibody (dilution 1:50) and a Cy3-conjugated anti-goat antibody (dilution 1:1,000). Control staining (C) was performed with an anti-S100A4 antibody preabsorbed with the corresponding antigen. The distance of the optical section from the substrate is indicated in µm. The optical sections (A,B) represent two basically different regions within the cell: the first section, recorded close to the top of the cell (A), depicts a central region around the cell nucleus, where S100A4 is organized in a reticular network-like structure. The second section, recorded close to the substrate (B), represents a more peripheral region of the cell, where S100A4 appears to be associated with the stress fiber- like structures. The control staining (C) indicates only weak background labeling.

(distal face of the cell in Fig. 1A) exhibits distinct reticular nucleus similar to the S100A1 and S100A4 antibodies. In network-like staining around the cell nucleus. In contrast, in contrast to S100A1 and S100A4, S100A6 showed no stress the optical section close to the glass coverslip (proximal face fiber-like staining and was found in the cell nucleus in 10% of of the cell in Fig. 1B) S100A1-specific labeling is labeled cells (Fig. 3B). On the other hand, no stress fiber-like predominantly revealed in the peripheral regions of the cell, staining was revealed for S100A6. yielding a stress fiber-like pattern. No significant S100A1 When cells were immunofluorescence-stained for S100A2, staining was found in the cell nuclei. These initial results yet another localization pattern was observed (Fig. 4). suggested a distinct localization of S100A1 in HVSMC, i.e. the Different optical sections through the same cell (Fig. 4A,B) protein was found exclusively in the cytoplasm of the cells showed that S100A2 was found only in the cell nucleus. where it was strongly associated with stress fibers and the SR. Reconstruction of the complete 3D distribution of S100A2 Fig. 2 shows the subcellular distribution of S100A4 in after recording of 30 optical sections through the labeled cells HVSMC. For the best possible comparison with S100A1, confirmed that localization of S100A2 was confined to the cell sections were recorded at the same cross-sectional level within nucleus (data not shown). These staining patterns revealed a the cells as in Fig. 1A and B. Accordingly, the subcellular dispersed organization of punctate stained structures, which localization of S100A4 appears to be very similar to that of were prominent in the center of the nucleus. S100A1. The anti-S100A4 antibody produced a stress fiber- The intracellular localization pattern of S100 proteins in like labeling in the cell periphery simultaneously with an HVSMC were identical to those found in human smooth extended SR-like membraneous network labeling pattern muscle cells derived from jejunum (HISM) or human vascular around the cell nucleus. These findings prompted us to make smooth muscle tissue (data not shown). a detailed qualitative and quantitative analysis of the S100 In order to compare the specific localization patterns of S100 protein colocalization patterns within distinct cell structures proteins in stable smooth muscle cell lines with the in vivo (see below). situation, freshly isolated human arterial smooth muscle cells Fig. 3 reveals two smooth muscle cells after were labeled with the S100-specific antibodies as already immunofluorescence staining for S100A6. The optical sections described. The fluorescence micrographs in Fig. 5 display the (Fig. 3A and B) were recorded equivalent through two different subcellular distribution of S100A1 and S100A4 in primary cells. In 90% of the cells (Fig. 3A) the S100A6 antibodies cultures of human smooth muscle cells. The reticular network- labeled a reticular network-like structure around the cell like staining around the cell nucleus (Fig. 5A and C) and the

Fig. 3.Intracellular immunolocalization of S100A6. Localization of S100A6 in HVSMC revealed by a polyclonal goat anti-S100A6 antibody (dilution 1:100) and a secondary Cy3-conjugated anti- goat antibody (dilution 1:1,000). The distance of the optical section from the substrate is indicated in µm. Optical sections (A,B) are recorded of two different cells at the same distance from the substrate. In 90% of the cells (A), S100A6 is localized exclusively in the cytosol, where it is associated with a network-like structure around the cell nucleus. Approximately 10% of the remainder of the cells show a prominent nuclear S100A6 staining and a weak cytoplasmatic labeling. Control staining (C) with an anti-S100A6 antibody that had been the corresponding antigen demonstrates very little unspecific reactivity. Ca2+-binding S100 proteins in smooth muscle 2047

Fig. 4. Intracellular immunolocalization of S100A2. Two different optical sections through a HVSMC stained with a polyclonal rabbit anti-S100A2 polyclonal antibody (dilution 1:200) and a secondary Cy3-coupled anti- rabbit antibody (dilution 1:500) (A,B). S100A2 is localized exclusively in the cell nucleus, where it appears in punctate stained structures in the center of the nucleus. Control staining (C) with an anti-S100A2 antibody preabsorbed with the recombinant antigen reveals an unspecific background. stress fiber-like structures in the periphery of the cells (Fig. 5B with F-actin. Fig. 6B reveals an optical section of a double- and D) are identical to those found in subcultured HVSMC. stained cell close to the substrate. The fluorescence signal from The staining pattern of S100A2 and S100A6 in primary cell the green channel (FITC) indicates actin staining and that from cultures and cell lines is also identical (not shown). the red channel (Cy3) S100A1 labeling. The overlap of both fluorescence signals produces a yellow signal, demonstrating a S100A1 and S100A4 colocalize with actin stress high degree of colocalization between F-actin and S100A1. fibers Fig. 6A represents a 2-D histogram of the simultaneously As documented in Figs 1 and 2, S100A1 and S100A4 appear recorded green and red channels. To establish this 2-D to be associated with stress fiber- and SR-like structures. To histogram, a collection of voxels (volume elements defined by positively identify these structures, colocalization studies were the confocal scanning procedures) representing the two performed. A specifically designed ‘colocalization’ software fluorescence channels, was analyzed. For each voxel a point for confocal images (Bitplane, AG; Switzerland) was applied was generated in the 2-D histogram with the x-coordinate for qualitative and quantitative analysis of the degree of representing the intensity of the voxel in the green channel (for colocalization of two individually labeled proteins in the cell. actin-staining) and the y-coordinate representing the intensity For double immunofluorescence, first the respective S100 of the voxel in the red channel (for S100A1 staining). A region protein was labeled with the primary and secondary Cy3- of interest excluding the background staining was defined coupled antibody. Second, actin filaments were visualized by (yellow frame), and the selected voxels were used for further FITC-conjugated phalloidin, known to recognize F-actin such statistical analysis and visualization. Starting from this data set, as in stress fiber-like structures, independently of the actin all voxels with identical intensity for both fluorescence signals isotype. Fig. 6 illustrates the specific colocalization of S100A1 were identified. Hence, this finite set of voxels comprises only

Fig. 5. Intracellular immunolocalization of S100A1 and S100A4 in primary cultures derived from human arterial smooth muscle tissue. Freshly isolated human arterial smooth muscle cells were stained with a primary goat anti-S100A1 antibody (A,B), a primary goat anti-S100A4 antibody (C,D), and a secondary Cy3-conjugated anti-goat antibody. The optical sections recorded at a distance of 1.76 µm from the coverslip (A,C) display a central region around the cell nucleus, where S100A1 and S100A4 are organized in a reticular network-like structure. In the sections obtained close to the substrate (B,D) S100A1 and S100A4 appear to be associated with stress fiber-like structures in the periphery of the cells. 2048 A. Mandinova and others

Fig. 6.Colocalization of S100A1 (A-C) and S100A4 (D- F) with actin stress fibers. S100A1 and S100A4 were visualized using primary polyclonal antibodies and Cy3- conjugated secondary antibodies. Aligned and superimposed microscope images (B,E) identified association of S100 proteins with actin stress fibers. 2-D histograms (A,D) were computed for quantitative estimation of the degree of colocalization of S100A1 (C) and S100A4 (F) with the actin stress fiber system (see also Table 1). signals arising from image elements that contain information further investigated using a sedimentation assay (Fig. 7). from both fluorescence channels. The true colocalization (i.e. S100A1 in F-actin filament promoting buffer (see Materials and inters off all volume elements harboring the same intensity Methods) by itself did not sediment upon centrifugation at fluorescence signal in the two channels) of the labeled proteins 100,000 gfor 30 minutes in the presence or absence of 1 µM can in this fashion be reliably demonstrated. As a result, a Ca2+ (Fig. 7A). However, when S100A1 and highly purified F- colocalization image consisting of S100A1- and actin- actin from rabbit skeletal muscle were mixed at appropriate containing structures is generated in each of the observed ratios in the presence or absence of Ca2+ for 120 minutes, Ca2+- optical sections (Fig. 6C). The statistical analysis of the dependent co-sedimentation after high-speed centrifugation was colocalization image demonstrates a significant colocalization observed (Fig. 7B, lane 4), indicating a direct interaction of between S100A1 and actin filaments in this optical section. S100A1 with F-actin. For comparison, the same sedimentation In order to investigate the complete 3-D association between assay was also performed with F-actin and S100A6, the S100A1 and actin, 20 optical sections through several stained subcellular localization of which is distinct from that of actin cells were recorded at an interval of 0.12 µm in the z-direction, stress fibers. As expected, S100A6 remained in the supernatant and 20 colocalization data sets for each cell were generated both in the presence and absence of Ca2+ (Fig. 7C, lanes 1-4). (data not shown). Table 1A represents the colocalization Unfortunately, sedimentation experiments could not be measurements obtained in a single cell. S100A1 and F-actin performed with S100A4, which is known to form oligomers appeared with different degrees of association between each (Ilg et al., 1996) and was found to aggregate by itself in F-actin other, due to the irregular distribution of the two proteins in the forming buffer. cells. The tips of the cells contain no stress fibers and therefore The direct interaction of F-actin (Fig. 8A) with S100A1 was the integrated spatial analysis indicates very low colocalization further confirmed by electron microscopy of negatively stained ratios for the layers in these regions (layers 12-20). specimens. As documented in Fig. 8B, S100A1 was associated Fig. 6 (D-F) shows the colocalization data of S100A4 with with F-actin filaments in the presence of 1 µM Ca2+. S100A1/F- F-actin. This is very similar to the data obtained with S100A1. actin complexes were visualized as partially decorated filaments The results demonstrate that there is significant colocalization with a low background, representing unbound S100A1. Since in the spatial arrangement of S100A4 and actin filaments in the S100A1 has a low molecular mass (11 kDa), it is not surprising peripheral regions of cells (for details see Table 1b). that the decoration pattern of the actin filaments is not Colocalization is highest close to the substrate (i.e. near the immediately obvious when compared to native F-actin. When bottom of the cell layers 1-7), again reflecting the intracellular the same experiments were performed in the absence of Ca2+, distribution of actin and S100A4. Taken together, these results S100A1 was unable to bind to actin filaments and remained as suggest that S100A4, like S100A1, is closely associated with monomers in the background (Fig. 8C), consistent with the the actin-stress fiber systems in cultured vascular smooth results obtained above. These data indicate that S100A1 has the muscle cells. intrinsic capacity to directly interact with F-actin. S100A1 interacts in vitro with F-actin in a Ca2+- Association of S100A1, S100A4, and S100A6 with dependent manner the SR The ability of some S100 proteins to bind directly to F-actin was A reticular network-like distribution of S100A1, S100A4, and Ca2+-binding S100 proteins in smooth muscle 2049

Table 1. Statistical analysis* of the colocalization between Table 2. Statistical analysis* of the colocalization between S100A1(a)/S100A4(b) and F-actin S100A1(a)/S100A4(b)/S100A6(c) and the SR a. S100A1 b. S100A4 a. S100A1 b. S100A4 c. S100A6 colocalization [%]** colocalization [%] colocalization [%]** colocalization [%] colocalization [%] Layer S100A1:actin S100A4:actin Layer S100A1:SR S100A4:SR S100A6:SR 1 2.1171 5.7129 1 16.2838 2.4702 20.2410 2 11.4323 6.2360 2 16.6524 6.7178 24.8448 3 23.4097 9.0321 3 16.0275 15.5763 39.3835 4 38.2400 35.6290 4 17.2575 24.8626 43.8811 5 47.2455 45.7478 5 18.1292 29.6781 45.5202 6 45.2946 39.3479 6 18.9343 32.1597 45.6484 7 35.4227 16.5480 7 20.5629 33.3634 45.5747 8 22.4369 7.7015 8 37.7927 33.0480 44.5010 9 11.6939 3.2220 9 48.1293 31.0533 43.0249 10 5.0408 0.3209 10 49.7057 27.6670 41.3939 11 1.9090 0.0650 11 50.5547 24.0544 39.4459 12 0.6442 0.0094 12 50.5173 20.4294 37.0080 13 0.2113 0.0015 13 50.0242 17.0400 33.5275 14 0.0696 0.0000 14 49.0297 13.8088 28.0748 15 0.0223 0.0000 15 43.3162 10.2400 19.7476 16 0.0058 0.0000 16 38.1031 6.5258 15.2340 17 0.0027 0.0000 17 33.6444 3.4333 14.4535 18 0.0004 0.0000 18 30.4385 1.4356 14,0193 19 0.0004 0.0000 19 27.6954 0.4475 13.0931 20 0.0000 0.0000 20 25.3859 0.1014 12.0959

*The analysis was performed using information from all 20 sections *The analysis was performed using information from all 20 sections through the stained cell. through the stained cell. **Information about the colocalization degree between both fluorescence **Information about the colocalization degree between both fluorescence channels. channels.

S100A6 proteins around the cell nuclei was demonstrated in S100A1, S100A4, and S100A6 with the SR-membrane bound HVSMC, indicating a possible association of these proteins p63 in HVSMC. The optical sections displayed here depict a with the SR in addition to their association with actin stress central region around the cell nucleus. Data concerning the fibers. To date it is known that Ca2+ homeostasis in smooth spatial relationship of the S100 proteins and the SR were muscle cells is regulated by two membrane systems: the obtained by acquiring a series of 20 optical sections of each plasma membrane and the SR. Since S100 proteins have been cell spaced at 0.16 µm in the z-direction. Statistical analysis of found to stimulate adenylate cyclase in the SR (Fano et al., these optical sections revealed a similar, high degree of 1989a) and Ca2+-induced Ca2+ release from the SR in muscle colocalization of S100A1, S100A4, and S100A6 with the SR. cells (Fano et al., 1989b; Treves et al., 1997), it has been The section by section numeric evaluation from this speculated that they accumulate in the SR of smooth muscle quantitative colocalization analysis is presented in Table 2. The cells. To investigate the association between S100 proteins and colocalization ratio here indicates that for all these S100 the SR in more details, a colocalization approach was used as proteins the highest degree of colocalization is observed in the described above. Visualization of the SR was achieved by middle of the cell (for S100A1 in layers 9-16; for S100A4 in staining the cells with a primary monoclonal antibody directed layers 4-12, and for S100A6 in layers 2-14). These findings against a novel 63 kDa SR-membrane bound protein (p63), suggest that the reticular-like organization of these S100 which recognizes the SR membranes in human cells proteins is due to their association with the SR surrounding the (Schweizer et al., 1993). Fig. 9 shows the colocalization of cell nucleus.

Fig. 7. Sedimentation assay of S100A1 and S100A6 with F-actin. SDS-PAGE of (A) human recombinant S100A1 (lanes 1-4) and S100A6 (lanes 5-8) in the presence of Ca2+ (lanes 1, 2 and 5, 6) and in the absence of Ca2+ (lanes 3, 4 and 7, 8). Supernatant: lanes 1, 3, 5, 7; pellet: lanes 2, 4, 6, 8. (B) Human recombinant S100A1 (lanes 1-4) incubated with F-actin in the presence of Ca2+ (lanes 3, 4) and in the absence of Ca2+ (lanes 1, 2). (C) Human recombinant S1006 (lanes 1-4) incubated with F-actin in the presence of Ca2+ (lanes 3, 4) and in the absence of Ca2+ (lanes 1, 2). For details see Materials and Methods. 2 µg F-actin and 3 µg of S100A1 or S100A6 were applied in each lane. 2050 A. Mandinova and others

In summary, we conclude that S100A1, S100A4, and Fig. 10 shows relocation of S100A1, S100A4, and S100A6 S100A6 are specifically located at the SRof human vascular but not of nuclear S100A2 upon thapsigargin exposure.When smooth muscle cells. the cytosolic Ca2+ concentration rose, the SR-associated S100A1 (Fig. 10A), S100A4 (Fig. 10B), and S100A6 (Fig. Relocation of S100A1, S100A4, and S100A6 but not 10C) relocated to vesicle-like structures in the central region of S100A2 after thapsigargin-induced rise of around the cell nucleus. The intracellular localization pattern intracellular [Ca2+] of S100A2 (Fig. 10D), the nuclear localization pattern of In order to investigate whether a rise of intracellular [Ca2+] S100A6, and the S100A1 and S100A4 associated with F-actin affects the subcellular localization of S100 proteins, HVSMC (data not shown), however, were unaffected. Individual were treated with thapsigargin (a sesquiterpine lactone) known vesicles with high content of S100A1, S100A4, and S100A6 to elevate cytosolic Ca2+ by blocking the SR-Ca2+-ATPase in proteins could be clearly distinguished by serial sectioning many cells, including vascular smooth muscle cells (Thastrup along the z-axis through the cells (data not shown). et al., 1990). From these observations we conclude that local Ca2+ release leads to relocation only of the SR-associated S100 protein moiety.

DISCUSSION

Impairment of Ca2+ homeostasis and altered expression of Ca2+-binding proteins have been associated with a number of muscle disorders. Members of the S100 protein family deserve particular scrutiny because of their cell-specific expression pattern and their association with a variety of proteins known to modulate muscle contraction. Previous investigations into the physiological role of S100 proteins and their intracellular localization in muscle cells have been hampered by the lack of an appropriate in vitro model system. The present study: (i) demonstrates that smooth muscle cells (cell lines and primary cultures) are a suitable model for investigating intracellular distribution of co-expressed S100 proteins, and (ii) it provides new information regarding the specific interaction of S100 proteins with distinct intracellular structures and their physiological functions. First, the distinct intracellular localization of S100 protein in smooth muscle cells and their association with specific intracellular structures is reported. Specifically, S100A1 and S100A4 were found only in the cytoplasm, where they were strongly associated with the SR and with actin stress fibers. S100A6 was also localized in the SR, but in addition, it also resided in the cell nucleus. S100A2 was located only in the cell nucleus. To the best of our knowledge, these data are the first to demonstrate a differential localization pattern of S100 proteins in smooth muscle cells. S100 proteins have been reported to interact with numerous cytoskeletal proteins. Among others, tubulin, , and junctional membrane protein (Donato, 1991; Garbuglia et al., 1996) have been identified as targets of S100 proteins. Recent studies have also demonstrated distinct localization patterns of S100 proteins in muscle cells (Haimoto and Kato, 1988; Zimmer and Landar, 1995). However, these localization and interaction experiments were performed with protein samples that were extracted from whole organs, thus containing different types of S100 proteins according to tissue distribution in the corresponding organ. As was demonstrated by Ilg et al. (1996), antibodies raised against these proteins as well as the commercially available anti-S100 antibodies crossreact with Fig. 8.Electron microscopy. Negatively stained F-actin filaments (A) other family members. Hence, the use of such isolated and and F-actin/S100A1 complexes (arrowheads) in the presence (B) and purified proteins and antibodies for interaction and localization absence (C) of Ca2+ imaged by TEM. Bars: 150 nm (overviews); 60 studies limits interpretation of these previously published nm (insets). results. Ca2+-binding S100 proteins in smooth muscle 2051

Only the recent development of human recombinant S100 manner with a molar ratio of approximately five S100A1 proteins together with the generation of specific anti-S100 molecules per one actin molecule. This finding is in line with antibodies allowed us to demonstrate unambiguously the the results of Watanabe et al. (1993b), who have previously distinct intracellular distribution patterns of different S100 described a Ca2+-dependent interaction of F-actin with proteins in human smooth muscle cells. calvasculin, a protein that is highly homologous to S100A4. Both single- and double-fluorescence experiments combined Our present results demonstrating the association of S100A1 with an integrated colocalization analysis clearly demonstrated and S100A4 with actin stress fibers in vivo strengthen the notion that S100A1 and S100A4 appear to be strongly associated with that members of the S100 protein family are involved in the actin stress fibers (see Fig. 6). The functional significance of regulation of actin filament polymerization. Takenaga et al. the association of S100 proteins with actin filaments, however, (1994) described a direct association of S100A4 with remains elusive, although several possibilities are being tropomyosin, thus proposing a direct regulatory function of evaluated. It has been suggested that interaction of S100 S100A4 in the tropomyosin-actin interaction. Heierhorst et al. proteins with caldesmon and calponin modulates their (1996) reported that a myosin-associated giant protein kinase, interaction with F-actin and therefore smooth muscle present in striated and smooth muscle along with sarcomeric contraction (Pritchard and Marston, 1991; Fuji et al., 1994), proteins, is activated by S100 in a Ca2+-dependent manner, but direct evidence for such interactions in living cells has not thereby possibly constituting a novel, third level of Ca2+ been reported. regulation in muscle. Taking these results together with our Another intriguing piece of evidence underscoring the present findings, we propose that S100 proteins play an important observed in situ association between S100 proteins and the role in the organization of the actin cytoskeleton via a Ca2+- actin cytoskeleton using immunofluorescence was the ability sensitive interaction with F-actin and actin-associated proteins. In of S100 proteins to directly bind to F-actin in vitro, as assessed vitro reconstitution experiments with native muscle thin filaments by a sedimentation assay and subsequent electron microscopy are in progress to systematically investigate this possibility. (Figs 7, 8). This set of data clearly demonstrates that S100A1 The results from our colocalization experiments with a novel can interact directly with F-actin filaments in a Ca2+-dependent marker protein for human SR (Schweizer et al., 1993) indicate

Fig. 9. Colocalization of S100A1 (A-C), S100A4 (D-F), and S100A6 (G-I) with SR. S100 proteins were stained with the respective primary polyclonal anti-S100 antibodies and secondary Cy3-conjugated antibodies. The SR was stained with a monoclonal antibody against p63 (a marker protein for human SR) and a secondary Cy2-conjugated antibody. Optical sections through the double-labeled cells (B,E,H) were used for computing of 2-D histograms (A,D,G) for quantitative estimation of the degree of colocalization in images C,F,I. See also Table 2. 2052 A. Mandinova and others

Fig. 10.Specific relocation of S100A1, S100A4, and S1006 after increasing the intracellular calcium concentration. Persistent increase of the cytosolic calcium concentration was induced by exposing HVSMC to 1 µM thapsigargin for 12 minutes. Changes in S100A1 (A), S100A4 (B), S100A6 (C), and S100A2 (D) were imaged using immunofluorescence labeling and confocal laser-scanning microscopy. All fluorescence images were sectioned at the same distance (1.76 µm) from the substrate. that S100A1, S100A4, and S100A6 are also closely associated and S100A6 in smooth muscle cells implies that these proteins with the SR of human smooth muscle cells. These findings do take part in fundamental nuclear functions. correlate with previously described interactions of S100 It is widely believed that cell function and survival are proteins with contraction-modulating enzymes located in the critically dependent on the precise regulation of the intracellular SR of muscle cells. For example, Fano et al. (1989a,b) and Ca2+ content. This intracellular Ca2+ regulation in vascular Treves et al. (1997) reported that S100 proteins stimulate the smooth muscle cells is crucial because it is a primary factor in Ca2+-induced release of Ca2+ from the SR as well as the basal the regulation of muscle contraction via activation of Ca2+- Mg2+-activated SR-associated adenylate cyclase. Both Ca2+- binding proteins. However, as yet the exact nature of the dependent intracellular effects play a central role in the activation mechanism for S100 proteins is only poorly pathophysiology of muscle disorders with an altered understood. Therefore, we investigated whether treatment of contractile activity. Whether S100A1, S100A4, and S100A6 vascular smooth muscle cells with thapsigargin, which is known interact with modulators of muscle contraction, and whether to increase the cytosolic [Ca2+] within the physiological range, these proteins regulate the activity of such modulators, requires affects the intracellular localization of S100 proteins. In the further examination. Nevertheless, at this stage we can only course of these investigations, we found that prolonged (12 speculate that the observed distribution of S100 proteins in minutes) rise in the intracellular [Ca2+] causes distinct human smooth muscle cells suggests their participation in relocation of the cytoplasmic but not of the nuclear S100 fundamental Ca2+-dependent cell activities. proteins, thereby yielding vesicles with high S100 content in the In contrast to the distribution of S100A1 and S100A4, a region of the SR. This observation is in agreement with previous completely different localization pattern was revealed for studies by Subramanian and Meyer (1997) who also S100A6, and particularly, for S100A2. These two proteins demonstrated that the structural integrity of the SR remained were found to be localized in the cell nucleus. There is now preserved during short Ca2+ transients or Ca2+ store depletion. compelling evidence that Ca2+ ions play an important role in In contrast, persistent Ca2+ increase lasting longer than 10 the regulation of several nuclear functions (for review, see minutes leads to fragmentation of the SR into vesicles. Such Gilchrist et al., 1994; Bachs et al., 1994). This regulation persistent Ca2+ overload in muscle cells is thought to be the system acts through activation of Ca2+-binding proteins, basic pathophysiological phenomenon in a variety of cardiac located in the cell nucleus. However, another important and smooth muscle disorders related to altered contraction. 2+ function of Ca -binding proteins may be the regulation of Therefore, we speculate that specific relocation of S100 proteins 2+ 2+ intracellular Ca concentrations via perinuclear Ca stores in response to rise of intracellular [Ca2+] is involved in the residing in the lumen of the nuclear envelope and the SR. alterations of fundamental cellular functions in smooth muscle. Whether S100 proteins are directly involved in these events in the cell nucleus and the nuclear envelope is still not known. We are very much indebted to Dr M. Steinmetz for teaching us the However, the distinct nuclear localization pattern of S100A2 actin-binding assay. A. Hefti is gratefully acknowledged for providing Ca2+-binding S100 proteins in smooth muscle 2053

2+ 2+ help with electron-microscopy. We are also indebted to Dr H. P. Hauri R. (1989b). S-100ao protein stimulates Ca -induced Ca release from for kindly providing antibodies against p63, and to Dr A. Rowlerson isolated sarcoplasmic reticulum vesicles. FEBS Lett. 255, 381-384. and M. Killen for critical reading of the manuscript. This study was Fano G., Biocca, S., Fulle, S., Mariggio, M. A., Belia, S. and Calissano, P. supported by the Swiss National Science Foundation (grant no. 31- (1995). The S-100: A protein family in search of a function. Progr. 50510.97 to C.W.H. and no. 32-49126.96 to D.A.); BIOMED 2 Neurobiol. 46, 71-82. (European Union grant no. BMH4CT950319/BBW grant no. Filipek, A. and Wojda, U. (1996). Chicken gizzard calcyclin – Distribution and potential target proteins. Biochem. Biophys. Res. Commun. 225, 151- 95.0215-1, Switzerland); the Swiss Heart Foundation; the Swiss 154. Society of Cardiology; the Ciba-Geigy Foundation; and the Sandoz Filipek, A., Zasada A., Wojda U., Makuch, R. and R. Dabrowska. (1996). Foundation. D.A. is a recipient of a career development award from Characterization of chicken gizzard calcyclin and examination of its the Foundation Professor Max Cloëtta, Zurich. interaction with caldesmon. Comp. Biochem. Physiol. 113B, 745-752. Forus, A., Weghuis, D. O., Smeets, D., Fodstad, O., Myklebost, O. and van Kessel, A. G. (1995). Comparative genomic hybridization analysis of REFERENCES human : I. Occurrence of genomic imbalances and identification of a novel major amplicon at 1q21-q22 in soft tissue sarcomas. Genes Cancer 14, 8-14. Aebi, U. and Pollard, T. D. (1987). A glow discharge unit to render electron Fujii, T., Oomatsuzawa, A., Kuzumaki, N. and Kondo, Y. (1994). Calcium- microscope grids and other surfaces hydrophilic. J. Electron Microsc. Tech. dependent regulation of vascular smooth muscle caldesmon by S100 and 7, 29-33. related smooth muscle proteins. Biochem. J. 277, 819-824. Allore, R., O’Hanlon, D., Price, R., Neilson, K., Willard, H. F., Cox, D. R., β Garbuglia, M., Verzini, M., Dimlich, R. V., Jamieson, G. A., Jr and Marks, A. and Dunn, R. J. (1988). Gene encoding the subunit of S100 Donato, R. (1996). Characterization of type III intermediate filament protein is on chromosome 21: implications for Down’s syndrome. Science regulatory protein target epitopes: S-100 (α and/or β) binds the N-terminal 239, 1311-1313. head domain; II2-p11 (2) binds the rod domain. Biochim. Biophys. Bachs, O., Agell, N. and Carafoli, E. (1994). Calmodulin and calmodulin- Acta. 1313, 268-276. binding proteins in the nucleus. Cell Calcium 16, 289-296. Gendler, S. J., Cohen, E. P., Craston, A., Duhig, T., Johnstone, G. and Bartoli, C., Bono, A., Clemente, C., Prato, I. D., Zurrida, S. and Cascinelli, Barnes, D. (1990). The locus of the polymorphic epithelial mucin (PEM) N. (1996). Clinical diagnosis and therapy of cutaneous in situ. tumor antigen on chromosome 1q21 shows a high frequency of alteration Cancer 77, 888-892. in primary human tumours. Int. J. Cancer 45, 431-435. Baschong, W., Sütterlin, R. and Aebi, U. (1997). Punch-wounded, fibroblast Gilchrist, J. S., Czubryt, M. P. and Pierce, G. N. (1994). Calcium and populated collagen matrices: a novel approach for studying cytoskeletal calcium-binding proteins in the nucleus. Mol. Cell. Biochem. 135, 79-88. changes in three dimensions by confocal laser scanning microscopy. Eur. J. Gimona, M., Lando, Z., Dolginov, Y., Vandekerckhove, J., Kobayashi, R., Cell Biol. 72, 189-201. Sobieszek, A. and Helfman, D. M. (1997). Ca2+-dependent interaction of Baudier, J. and Cole, R. D. (1988). Interactions between the microtubule- S100A2 with muscle and nonmuscle tropomyosins. J. Cell Sci. 110, 611- associated tau proteins and regulate tau by the 621. Ca2+/calmodulin-dependent protein kinase II. J. Biol. Chem. 263, 5876- Haimoto, H. and Kato, K. (1988). S100a (αα) protein in . 5883. o Berridge, M. J. (1997). Elementary and global aspects of calcium signalling. Isolation from human cardiac muscle and ultrastructural localization. Eur. J. Exp. Biol. 200, 315-319. J. Biochem. 171, 409-415. Heierhorst, J., Kobe, B., Feil, S. C., Parker, M. W., Benian, G. M., Weiss, Bianchi, R., Garbuglia, M., Verzini, M., Giambanco, I., Ivanenkov, V. V., 2+ Dimlich, R. V., Jamieson, G. A. Jr and Donato, R. (1996). S-100 (α and β) K. R. and Kemp, B. E. (1996). Ca /S100 regulation of giant protein binding peptide (TRTK-12) blocks S-100/GFAP interaction: identification kinases. Nature 380, 636-639. of a putative S-100 target epitope within the head domain of GFAP. Biochim. Heizmann, C. W. and Hunziker, W. (1991). Intracellular calcium-binding Biophys. Acta 1313, 258-267. proteins: more sites than insights. Trends Biochem. Sci. 16, 98-103. 2+ Bremer, A., Henn, C., Goldie, K. N., Engel, A., Smith, P. R. and Aebi, U. Heizmann, C. W. and Braun, K. (1992). Changes in Ca -binding proteins (1994). Towards atomic interpretation of F-actin filament three-dimensional in human neurodegenerative disorders. Trends Neurosci. 15, 259-264. reconstructions. J. Mol. Biol. 242, 683-700. Heizmann, C. W. and Braun, K. (1995). Calcium Regulation by Calcium- Burgess, W. H., Watterson, D. M. and Van Eldik, L. J. (1984). Identification Binding Proteins in Neurodegenerative Disorders. Springer-Verlag, R. G. of calmodulin-binding proteins in chicken embryo fibroblasts. J. Cell Biol. Landes, Berlin, Austin. 133 pp. 99, 550-557. Heizmann, C. W. (1996). Multistep in health and disease. Cohen, P. and Klee, C. B., editors (1988). Calmodulin. Molecular Aspects of Mol. Cells 6, 629-636. Cellular Regulation, vol. 5. Elsevier, Amsterdam. Hilt, D. C. and Kligman, D. (1991). The S-100 protein family: A biochemical Davies, M. P. A., Rudland, P. S., Robertson, L., Parry, E. W., Jolicoeur P. and functional overview. In Novel Calcium-Binding Proteins. Fundamentals and Barraclough, R. (1996). Expression of the calcium-binding protein and Clinical Implications (ed. C. W. Heizmann), pp. 65-103. Springer- S100A4 (p9Ka) in MMTV-neu transgenic mice induces metastasis of Verlag, Berlin Heidelberg. mammary tumours. Oncogene 13, 1631-1637. Hoggard, N., Brintnell, B., Howell, A., Weissenbach, J. and Varley, J. Donato, R., Giambanco, I. and Aisa, M. C. (1989). Molecular interaction of (1995). Allelic imbalance on in human . II. S-100 proteins with microtubule proteins in vitro. J. Neurochem. 53, 566- Microsatellite repeat analysis. Genes Chromosomes Cancer 12, 24-31. 571. Ilg, E. C., Schäfer, B. W. and Heizmann, C. W. (1996). Expression pattern Donato, R. (1991). Perspectives in S-100 protein biology. Cell Calcium 12, of S100 calcium-binding proteins in human tumors. Int. J. Cancer 68, 325- 713-726. 332. Engelkamp, D., Schäfer, B. W., Erne, P. and Heizmann, C. W. (1992). James, P., Vorherr, T. and Carafoli, E. (1995). Calmodulin-binding domains: S100α, CAPL, and CACY: molecular cloning and expression analysis of just two faced or multi-faceted? Trends Biochem. Sci. 20, 38-42. three calcium-binding proteins from human heart. Biochemistry 31, 10258- Kretsinger, R. H. (1980). Structure and evolution of calcium-modulated 10264. proteins. CRC Crit. Rev. Biochem. 8, 119-174. Engelkamp, D., Schäfer, B. W., Mattei, M. G., Erne, P. and Heizmann, C. Landar, A., Caddell, G., Chessher, J. and Zimmer, D. B. (1996). W. (1993). Six S100 genes are clustered on human chromosome 1q21: Identification of an S100A1/S100B target protein: phosphoglucomutase. identification of two genes coding for the two previously unreported Cell Calcium 20, 279-285. calcium-binding proteins S100D and S100E. Proc. Nat. Acad. Sci. USA 90, Lee, S. W., Tomasetto, C., Swisshelm, K., Keyomarsi, K. and Sager, R. 6547-6551. (1992). Down-regulation of a member of the S100 gene family in mammary Fano, G., Angelella, P., Mariggio, D., Aisa, M. C., Giambanco, I. and cells and reexpression by azadeoxycytidine treatment. Proc. Nat. 2+ Donato, R. (1989a). S-100ao protein stimulates the basal (Mg -activated) Acad. Sci. USA 89, 2504-2508. adenylate cyclase activity associated with skeletal muscle membranes. FEBS Messerli, J. M., van der Voort, H. T., Rungger-Brändle, E. and Perriard, Lett. 248, 9-12. J. C. (1993). Three-dimensional visualization of multi-channel volume data: Fano, G., Marsili, V., Angelella, P., Aisa, M. C., Giambanco, I. and Donato the amSFP algorithm. Cytometry 14, 725-735. 2054 A. Mandinova and others

Nakayama, S. and Kretsinger, R. H. (1994). Evolution of the EF-hand family Takenaga, K., Nakamura, Y., Sakiyama, S., Hasegawa, Y., Sato, K. and of proteins. Biophys. Biomol. Struct. 23, 473-507. Endo, H. (1994). Binding of pEL98 protein, an S100-related calcium- Parekh, A. B. and Penner, R. (1997). Store depletion and calcium influx. binding protein, to nonmuscle tropomyosin. J. Cell Biol. 124, 757-768. Physiol. Rev. 77, 901-930. Thastrup, O., Cullen, P. J., Dobrak, B. K., Hanley, M. R. and Dawson, A. Pedrocchi, M., Hauer, C. R., Schäfer, B. W., Erne, P. and Heizmann, C. P. (1990). Thapsigargin, a tumor promoter, discharges intracellular Ca2+ W. (1993). Analysis of Ca2+-binding S100 proteins in human heart by stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. HPLC-electrospray mass spectrometry. Biochem. Biophys. Res. Commun. Proc. Nat. Acad. Sci. USA87, 2466-2470. 197, 529-535. Tokumitsu, H., Mizutani, A., Minami, H., Kobayashi, R. and Hidaka, H. Pedrocchi, M., Schäfer, B. W., Müller, H., Eppenberger, U. and Heizmann, (1992). A calcyclin-associated protein is a newly identified member of the C. W. (1994). Expression of Ca2+-binding proteins of the S100 family in Ca2+/phospholipid-binding proteins, annexin family. J. Biol. Chem. 267, malignant human breast-cancer cell lines and biopsy samples. Int. J. Cancer 8919-8924. 57, 684-690. Treves, S., Scutari, E., Robert, M., Groh, S., Ottolia, M., Prestipino, G., Polans, A., Baehr, W. and Palczewski, K. (1996). Turned on by Ca2+? The Ronjat, M. and Zorzato, F. (1997). Interaction of S100A1 with the Ca2+ physiology and pathology of Ca2+-binding proteins in the retina. Trends release channel (ryanodine receptor) of skeletal muscle. Biochemistry 36, Neurosci. 19, 547-554. 11496-11503. Pritchard, K. and Marston, S. B. (1991). Ca2+-dependent regulation of van Eldik, L. J. and Griffin, W. S. (1994). S100βexpression in Alzheimer’s vascular smooth-muscle caldesmon by S100 and related smooth-muscle disease: relation to neuropathology in brain regions. Biochim. Biophys. Acta proteins. Biochem. J. 277, 819-824. 1223, 398-403. Rammes, A., Roth, J., Goebeler, M., Klempt, M., Hartmann, M. and Sorg, Watanabe, M., Ando, Y., Tokumitsu, H. and Hidaka, H. (1993a). Binding C. (1997). Myeloid-related protein (MRP) 8 and MRP14, calcium-binding site of annexin XI on the calcyclin molecule. Biochem. Biophys. Res. proteins of the S100 family, are secreted by activated monocytes via a novel, Commun. 196, 1376-1382. tubulin-dependent pathway. J. Biol. Chem. 279, 9496-9502. Watanabe, Y., Usada, N., Minami, H., Morita, T., Tsugane, S., Ishikawa, Remppis, A., Greten, T., Schäfer, B. W., Hunziker, P., Erne, P., Katus, H. R., Kohama, K., Tomida, Y. and Hidaka, H. (1993b). Calvasculin, as a A. and Heizmann, C. W. (1996). Altered expression of the Ca2+-binding factor affecting the microfilament assemblies in rat fibroblasts transfected protein S100A1 in human . Biochim. Biophys. Acta 1313, by src gene. FEBS Lett. 324, 51-55. 253-257. Weterman, M. A. J., Wilbrink, M., Dijkhuizen, T., van den Berg, E. and Richard, I., Broux, O., Allamand, V., Fougerousse, F., Chiannilkulchai, N., van Kessel, A. G. (1996). Fine mapping of the 1q21 breakpoint of the Bourg, N., Brenguier, L., Devaud, C., Pasturaud, P., Roudaut, C. et al. papillary renal cell carcinoma-associated (X;1) translocation. Hum. Genet. (1995). Mutations in the proteolytic enzyme 3 cause limb-girdle 98, 16-21. muscular dystrophy type 2A. Cell81, 27-40. Wicki, R., Schäfer, B. W., Erne, P. and Heizmann, C. W. (1996a). Schäfer, B. W., Wicki, R., Engelkamp, D., Mattei, M. G. and Heizmann, Characterization of the human and mouse cDNAs coding for , a C. W. (1995). Isolation of a YAC clone covering a cluster of nine S100 genes new member of the S100 protein family. Biochem. Biophys. Res. Commun. on human chromosome 1q21: rationale for a new nomenclature of the S100 227, 594-599. calcium-binding protein family. Genomics25, 638-643. Wicki, R., Marenholz, I., Mischke, D., Schäfer, B. W. and Heizmann, C. Schäfer, B. W. and Heizmann, C. W. (1996). The S100 family of EF-hand W. (1996b). Characterization of the human ( C, p6, calcium-binding proteins: function and pathology. Trends Biochem. Sci. 21, CAAF1, CGRP) gene, a new member of the S100 gene cluster on 134-140. chromosome 1q21. Cell Calcium20, 459-464. Schweizer, A., Ericsson, M., Bachi, T., Griffiths, G. and Hauri, H.-P. Wicki, R., Franz, C., Scholl, F. A., Heizmann, C. W. and Schäfer, B. W. (1993). Characterization of a novel 63 kDa membrane protein: Implications (1997). Repression of the candidate tumor suppressor gene S100A2 in breast for the organization of the ER-to-Golgi pathway. J. Cell Sci. 104, 671-683. cancer is mediated by site-specific hypermethylation. Cell Calcium22, 243- Skripnikova, E. V. and Gusev, N. B. (1989). Interaction of smooth muscle 254. caldesmon with S-100 protein. FEBS Lett. 257, 380-382. Wu, T., Angus, C. W., Yao, X.-L., Logun, C. and Shelhamer, J. H. (1997). Small, J. V. and Celis, J. E. (1978). Direct visualization of the 10-nm (100- p11, a unique member of the S100 family of calcium-binding proteins, Å)-filament network in whole and enucleated cultured cells. J. Cell Sci. 31, interacts with and inhibits the activity of the 85-kDa cytosolic phospholipase 393-409. A2. J. Biol. Chem. 272, 17145-17153. Song, W. and Zimmer, D. B. (1996). Expression of the rat S100A1 gene in Wrigley, N. G. (1968). The lattice spacing of crystalline catalase as an internal neurons, glia, and skeletal muscle. Brain Res. 721, 204-216. standard of length in electron microscopy. J. Ultrastruct. Res. 24, 454-464. Subramanian, K. and Meyer, T. (1997). Calcium-induced restructuring of Zimmer, D. B. and Landar, A. (1995). Analysis of S100A1 expression during nuclear envelope and endoplasmic reticulum calcium stores. Cell89, 963- skeletal muscle and neuronal cell differentiation. J. Neurochem. 64, 2727- 971. 2736.